Bioreactor

Curr Opin Biotechnol, 1998 Apr, 9(2), 152 - 6
Apoptosis in cell culture; al-Rubeai M et al.; Most cells can exhibit a biochemical pathway which mediates their own destruction in a highly controlled and genetically defined manner . In animal cells, a morphologically distinct form of this 'programmed cell death' has been identified and extensively characterised . This phenomenon, which has been named apoptosis, accounts for most of the cell deaths that take place during the production of biopharmaceuticals from animal cell lines . In the past few years, the factors responsible for the induction of apoptosis in the bioreactor environment have been identified . Furthermore, a growing number of studies have demonstrated that the suppression of apoptosis by the overexpression of anti-apoptosis genes, most notably bcl-2, result in improved culture productivity.

Biol Pharm Bull, 1998 Apr, 21(4), 330 - 4
Non-antigenic and low allergic gelatin produced by specific digestion with an enzyme-coupled matrix; Sakai Y et al.; Porcine gelatin (heat-denatured collagen) was digested with a bioreactor using an enzyme-coupled matrix (ECM) with purified collagenase . The digested gelatin, FreAlagin type R (M.W . range 200-10000 Da), was further purified by an HPLC system depending upon molecular size . The molecular weight range of the purified fractions, FreAlagin type P and type AD, were 200-500 and 2000-10000 Da, respectively, and glycine was the N-terminal amino acid of both types (> or =93%) . ECM has the capability of digesting gelatin at a specific point in the sequence before glycine, and it was determined that FreAlagin type P consists of a tri-peptide fraction with the amino acid sequence Gly-X-Y . No types of FreAlagin exhibited any reactivity with gelatin-specific IgG antibody raised in guinea pigs, and they also possessed an extremely low reactivity with gelatin-specific IgE antibody from the sera of patients who had experienced an anaphylactic reaction against gelatin after vaccination or after eating gelatin-containing foods . From these results, it was determined that FreAlagin types R and AD were non-antigenic, low-allergic gelatins . FreAlagin type R, and especially type AD, had strong adsorption-blocking activity comparable to the level of bovine serum albumin, whereas type P and glycine had virtually no adsorption-blocking activity . Therefore, the new types of gelatin, FreAlagin types R and AD, are suitable for pharmaceutical use to avoid gelatin allergy.

Protein Expr Purif, 1998 Apr, 12(3), 390 - 8
Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells; Zhang J et al.; Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia . Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated . In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors . Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors . Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps . The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers . The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars . In an in vivo bioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent . The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation . Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined .

Protein Expr Purif, 1998 Apr, 12(3), 323 - 30
Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein; Schmidt M et al.; The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus . The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography . Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight . The final yield was 5 mg of purified protein per liter of cell culture . Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study . A specific interaction between the two proteins was shown . Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies .

J Leukoc Biol, 1998 May, 63(5), 550 - 62
Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C; Cooper D et al.; Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished . Activation marker expression was significantly reduced in RWV cultures . Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells . Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness . However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction . Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture . Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture . Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Transfus Clin Biol, 1998 Feb, 5(1), 80 - 7
{Hepatocytes in cell therapy}; Clement B et al.; Cell-based therapy could represent an alternative treatment to orthotopic liver transplantation in acute liver failures and for the correction of genetic defects of various enzymatic functions . Several recent studies indicate that hepatocytes injected either in the spleen or in portal vein can restore liver-specific function(s) in animal model systems . Alternatively, an extracorporal hybrid bioartificial liver might provide liver-specific functions, maintain the patient alive and allow spontaneous recovery of the patient's own liver, or act as a bridge toward liver transplantation in acute liver failures . Various drawbacks of devices such as flat culture substrates, hollow-fiber bioreactors or microcarriers led us to develop a reliable extracorporeal bioartificial liver based on alginate-entrapped hepatocytes . This system was used successfully for the correction of the Gunn rat genetic defect which results in the lack of bilirubin conjugation . The development of this system for clinical purposes requires large yields of functional hepatocytes . We isolated porcine hepatocytes by collagenase perfusion of the liver and cells were immobilized within alginate beads which were subsequently inoculated in a bioreactor . Porcine hepatocytes expressed liver-functions at high levels, particularly those involved in detoxification and biotransformation processes; they were immunoisolated from immunoglobins and could be cryopreserved . This system represents a promising tool for the design of an extracorporeal bioartificial liver in human beings.

Biotechnol Prog, 1998 Mar, 14(2), 265 - 9
Enhancing xanthan fermentations by different modes of glucose feeding
Amanullah A, Satti S, Nienow AW.
This paper is the fourth in a series aimed at improving the understanding and operation of conventional agitated fermenters for the production of the commercially important gum, Xanthan . In the first, reproducible fermentations were established and this protocol was used in studies of different agitator types and of bulk mixing and dissolved oxygen concentration in the next two . Here, building on the previous work, the influence of different glucose feeding strategies on Xanthan production in a 20-L agitated fermenter under equivalent conditions of agitation and dissolved oxygen is reported . The biological performances in three types of fed-batch cultures (a two-step glucose addition, multiple glucose-pulse feeding and continuous feeding of glucose) are compared to two batch fermentations with different initial glucose concentrations . The work confirmed that improved performance cannot be achieved by increasing the initial glucose concentration above 50 g/L nor by a single 10 g/L pulse addition (initial glucose concentration of 40 g/L) while significant nitrogen is still present . On the other hand, the simple pulse and continuous feeding strategies, after nitrogen has been essentially exhausted and under conditions of nonlimiting dissolved oxygen and similar bulk mixing, can result in a greatly enhanced performance compared to batch fermentations . Using the final Xanthan gum concentration, the yield on glucose and the overall productivity as performance indices, values of 62 g/L, 0.82 g of Xanthan/g of glucose, and 0.72 g/(L.h), respectively, were obtained compared to literature values for conventional stirred bioreactors of 15-30 g/L, 0.27-0.86 g of Xanthan/g of glucose, and 0 . 12-0.43 g/(L.h).

Biotechnol Prog, 1998 Mar, 14(2), 203 - 9
Screening tool for hollow-fiber bioreactor process development
Gramer MJ, Poeschl DM.
Fundamental research of factors affecting cell growth in hollow-fiber bioreactors is hindered by the lack of an efficient screening tool . To address this issue, a hollow-fiber micro-bioreactor has been developed . Hollow fibers with 10 kDa molecular weight cutoffs are housed within a piece of silicone tubing . Cells are inoculated within the hollow fibers which provides a 0.2-mL culture volume . The space between the fibers and silicone tubing (5 or 16 mL) is used as a medium reservoir sufficient to feed the cells for at least 24 h . Oxygenation is provided directly through the silicone tubing so that a pump for medium recirculation is not required . As a result, many conditions can be tested simultaneously in a single incubator . Three days after inoculation at 5 x 10(6) cells/mL in the micro-bioreactor, the rho 1D4 murine hybridoma cell line reached 2.8 x 10(7) cells/mL with an antibody concentration of 0.17 mg/mL . When inoculated at 5 x 10(7) cells/mL, the cell concentration reached 1.8 x 10(8)/mL after 3 days with an antibody concentration of 1.0 mg/mL . Results from a series of experiments with the micro-bioreactor suggested that the initial growth phase of this cell line in a hollow-fiber system is dependent on the serum concentration in the medium reservoir . This prediction was tested by simultaneously inoculating two production-scale hollow-fiber bioreactor systems . The cell side of the membrane for each bioreactor contained 10% serum, but serum was added to the reservoir side of only one of the bioreactors . The cells with only basal medium in the reservoir died after a few days, while the cells with 10% serum in the medium reservoir grew rapidly . These results demonstrate that the micro-bioreactor developed here can support good cell growth and that it can be used as a research tool to predict the performance of large-scale hollow-fiber systems.

J Biotechnol, 1998 Feb 5, 60(1-2), 47 - 54
Recombinant gene expression in Escherichia coli cultivation using lactose as inducer; Gombert AK et al.; The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli . Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase . Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses . The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein.

Exp Cell Res, 1998 Apr 10, 240(1), 58 - 65
Chondrogenesis in a cell-polymer-bioreactor system; Freed LE et al.; Chondrogenesis was studied under controlled in vitro conditions using a cell-polymer-bioreactor system . Bovine calf articular chondrocytes were seeded onto biodegradable polymer scaffolds and cultured in rotating bioreactor vessels . Concomitant increases in the amounts of glycosaminoglycan (GAG) and type II collagen resulted in cell-polymer constructs with continuous cartilaginous matrix over their entire cross sections (6.7 mm diameter x 5 mm thick) after 40 days of cultivation . As compared to natural calf cartilage, constructs had comparable cellularities, 68% as much GAG and 33% as much type II collagen per gram wet weight . The progression of chondrogenesis in chondrocyte-polymer constructs was similar to that suggested previously for precursor cells in vitro and developing limbs in vivo . In particular, the polymer scaffold provided a three-dimensional structure that could be seeded with chondrocytes at high cell densities in order to establish cell-to-cell contacts and initiate cartilage tissue development, whereas the bioreactor vessel provided a permissive microenvironment for chondrogenesis . This work demonstrates the promise of using tissue engineered constructs for in vitro studies of cell interactions and differentiation.

Int J Artif Organs, 1998 Jan, 21(1), 43 - 8
Positive biochemical effects of a bioartificial liver support system (BALSS) in a porcine fulminant hepatic failure (FHF) model; Sheil AG et al.; This study describes biochemical changes in the plasma and blood of pigs with devascularised livers treated in a bioartificial liver support system (BALSS) . Porcine hepatic cells were incubated with collagen-coated dextran microspheres (CDM) for 3 hours and the medium tested to determine cellular metabolic activity . Incubation continued for a further 18 hours during which the hepatic cells attach to the CDM . The CDM-attached cells were inoculated into a hollow fibre bioreactor which was part of an extracorporeal support system . Hepatic cell content of the bioreactor was 6 x 10(9) cells . The system was tested in a controlled trial in pigs prepared in a surgical model of fulminant hepatic failure (FHF) . When plasma from FHF pigs was circulated through the device containing hepatic cells, there was significantly less increase in the accumulation of ammonia and most amino acids, together with a decrease in plasma lactate and of one amino acid, compared to control experiments when hepatic cells were excluded . We conclude that primary porcine hepatocytes can contribute beneficial metabolic function in a BALSS.

Pharm Dev Technol, 1996 Apr, 1(1), 63 - 8
Novel technology for the preparation of sterile alginate-poly-l-lysine microcapsules in a bioreactor; Abraham SM et al.; The purpose of this study was to develop a method that may be suitable for the commercial manufacture of sterile alginate-polylysine-alginate microcapsules in a bioreactor . A Turbotak atomizing device in conjunction with a Bellco Bioreactor was used to prepare sterile microcapsules . Aseptic procedures were followed using sterilized equipment and materials . Sodium alginate solution was sprayed into calcium chloride solution using the Turbotak, with nitrogen as the atomizing gas . The resultant gelled alginate microcapsules were coated with polylysine and alginate to produce alginate-poly-l-lysine microcapsules . In-process contamination of the atomizing gas and microcapsules was investigated using modified USP sterility tests . Microcapsule size was determined using a light blockage technique (Accusizer) which measures both number and volume weighted mean diameters . The microcapsules prepared passed a modified USP sterility test, and the Bellco Bioreactor was found to minimize the possibilities of environmental contamination and therefore enhanced operator safety . The flow rate of the atomizing gas was determined to significantly alter number and volume weighted mean microcapsule diameters . Statistical analysis indicated that the number weighted mean diameters in conjunction with the volume weighted mean diameters can be used to detect batch-to-batch changes in microcapsule diameters . In conclusion, the modified Bellco Bioreactor offers a novel approach for producing sterile alginate-polylysine microcapsules on a laboratory scale.

Br Med Bull, 1997, 53(4), 730 - 44
Bioartificial liver support: developments in hepatocyte culture and bioreactor design; Riordan S et al.; Bioartificial liver devices aim to support patients with acute liver failure (ALF) until orthotopic liver transplantation (OLT) or spontaneous recovery due to hepatic regeneration can occur . However, initial clinical experiences with two devices have indicated that functional efficacy in this setting may be less than in the experimental situation . Several fundamental issues remain unresolved, including the cell mass required to provide meaningful support and which of those hepatocyte components and bioreactor designs so far proposed is best able to do this . In particular, further studies of the efficacy of devices incorporating human hepatocyte lines transformed by either cultural conditions or genetic engineering and those based on multi-channel or flat bed bioreactor designs in which hepatocytes are co-cultured with non-parenchymal cells are awaited . Controlled trials on a multicentre basis in well-defined patient groups and with standardised outcome measures will be required to properly evaluate the clinical value of these devices . A better understanding of factors promoting recovery and regeneration of the native liver and to what extent these can be provided by extracorporeal devices will be essential to the further development of effective bioartificial liver support systems.

Hybridoma, 1998 Feb, 17(1), 69 - 72
Generation of murine monoclonal antibodies in serum-free medium; Liu RS et al.; Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells . Serum is also required for subcloning of hybridoma cells to support low density cell growth . IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells . We found that the serum requirement in media used for generation of hybridomas can be totally eliminated by substituting with 300 units/ml of IL-6 . Stable hybridoma cell lines were generated to peptide and protein antigens using serum-free adapted P3.653 myelomas as the fusion partner and medium containing IL-6 . Our results indicate that, in general, the fusion efficiencies of serum-free IL-6 supplemented fusions are lower than the fusions employing serum containing media (40%-60% vs . 80%-100%) . However, in spite of the lower fusion efficiency, the number of antigen-specific clones generated using IL-6 was equal to or greater than fusions using serum supplements . The use of IL-6 instead of serum in the generation of monoclonal antibodies (MAbs) has several advantages . We are able to eliminate the costly need for serum in media by using IL-6 that is prepared in house . In addition, we eliminate the need for time-consuming serum-free adaptation of hybridoma cell lines prior to transfer to hollow fiber bioreactors.

Sci Total Environ, 1997 Dec 3, 208(1-2), 49 - 57
Estimation of the environmental contamination by phthalic acid esters leaching from household wastes; Bauer MJ et al.; Phthalic acid esters (PAE) have been used as plasticizers in many products . Here we estimate the contaminating potential of PAE codisposed with domestic wastes . In order to determine the maximum threat to the environment, we analysed several fractions of different household wastes . A maximum of 2.6% of di-(2-ethylhexyl) phthalate (DEHP) was measured in 'compound materials' . More than 90% of all the PAE present in the refuse was due to DEHP . Assuming defined compositions of the refuse we calculated that approx . 1 kg of phthalate esters per ton of dry waste may be codisposed . The actual amount of PAE that could be eluted by the leachate was estimated using laboratory bioreactors which were filled with waste of exactly known composition . Only approx . 1 g of PAE per ton of dry waste was leached . However, the elution of hydrophobic DEHP by the leachate is dependent on the composition of the waste which gave rise to different concentrations of dissolved organic carbon . Because of the experimental setup the amounts of leached PAE represent minimum levels of possible environmental contamination . Hence, there will be a constant output of PAE from unlined municipal landfills for decades.

J Biotechnol, 1997 Dec 17, 59(1-2), 39 - 52
Plant cell suspension cultures: some engineering considerations; Kieran PM et al.; Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances . For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes . Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances . However, the industrial application of plant cell suspension cultures has, to date, been limited . Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations . This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures . Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells . Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated.

J Clin Lab Anal, 1998, 12(1), 6 - 13
Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: difference between fPSA from LNCaP cell and seminal plasma; Wu JT et al.; We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate . By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained . We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation . More than 90% of the PSA from LNCaP cell cultures are fPSA . Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography . Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms . This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid . We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.

Indian J Exp Biol, 1997 Aug, 35(8), 886 - 9
Production of L-phenylacetylcarbinol by free and immobilized yeast cells; Tripathi CK et al.; Production of L-phenylacetylcarbinol (L-PAC) through biotransformation of benzaldehyde by free and immobilized cells of the yeast Saccharomyces cerevisiae has been attempted . L-PAC production was found to be maximum (0.4 microliter/ml) when anaerobically grown free cells were used as biocatalyst during aerobic biotransformation for two hours with magnetically stirred bioreactor . Growth under oxygen limited conditions led to accumulation of higher amount of pyruvate decarboxylase enzyme and co-substrate, pyruvate, resulting in higher L-PAC formation . L-PAC yield was low when biotransformations were carried out anaerobically either for aerobically or anaerobically grown free cells . Free cells were found to be more efficient biocatalyst for L-PAC production, as compared with the immobilized cells, with the investigated benzaldehyde concentration (0.3% v/v) and cell density (17.5% w/v) . The study has explored and indicated the possibility of optimizing the yield of L-PAC by growing the yeast cells under oxygen limited condition for suitable aerobic mode of benzaldehyde biotransformation.

Analyst, 1997 Dec, 122(12), 1539 - 41
Enzymic flow injection determination of free L-carnitine in infant formulae; Ferreira IM et al.; Infant formula samples were analysed to determine their free L-carnitine content by using flow injection (FI) with an incorporated immobilised carnitine acetyltransferase bioreactor . The methodology was based on the spectrophotometric determination through its reaction with carnitine acetyltransferase coupled with acetyl coenzyme A (acetyl-CoA) and dithiobenzoate . The merging zones technique was used to minimise acetyl CoA consumption . Linearity was observed over the concentration range 10-80 mg l-1 with L-carnitine as standard (r = 0.9998) and the rate of analysis was 50 h-1 infant formula samples . The enzymic FI method afforded a low RSD (2.2%) . Comparisons were made with other methods of known accuracy . The enzymic reactors were stable for 3 months when used daily at the optimum pH.

Ann N Y Acad Sci, 1997 Nov 21, 829, 83 - 96
Treatment of trichloroethene-contaminated water with a fluidized-bed bioreactor; Segar RL Jr et al.; Biological systems are a potentially attractive means of treatment for groundwater contaminated with chlorinated solvents . In this research, a bench-scale fluidized-bed biological reactor (FBBR) was used to treat water contaminated by trichloroethene (TCE) . The bed volume was 2.4 L and the flow rate was 0.8 L/min . Applications of the reactor are primarily for treatment of groundwater extracted during aquifer remediation . Reactor feed water was amended with phenol and nutrients to stimulate the growth of bioparticles with the ability to cometabolize TCE . The FBBR was operated in a bed growth mode for two weeks followed by a TCE removal period of two weeks during which TCE and phenol were fed simultaneously . Experiments were conducted to determine the removal of 0.1 mg/L TCE for phenol loading rates ranging from 3 to 9 mg/min . Higher phenol loading inhibited TCE degradation and resulted in phenol not being completely removed by the reactor . Phenol was not detected in the reactor effluent at loadings below 6 mg/min (3.6 g/L/d) . A phenol to TCE mass ratio of 75:1 provided an average 60% removal of 0.1 mg/L TCE at an empty bed contact time of 3 minutes . Higher removal of TCE may be possible in field-scale reactors where the FBBR height (bed-depth) may be extended.

Biologicals, 1997 Dec, 25(4), 415 - 9
A rapid and simple procedure to detect the presence of MVM in conditioned cell fluids or culture media; Chang A et al.; During the manufacture of biopharmaceuticals, numerous adventitious agents have been detected in Master Cell Banks, end-of-production cells as well as bulk harvest fluid . Recently, a number of large-scale production bioreactors have become infected with Minute Virus of Mice (MVM) during cGMP (current good manufacturing practices) operations, and this has resulted in both the loss of product and the need for major cleaning validation procedures to be put in place . We have developed a simple DNA extraction/PCR assay to detect the presence of MVM in cell culture supernatant (conditioned cell fluids) . This highly specific assay can detect 10 or fewer genome equivalents (copies) of MVM following PCR and gel electrophoresis visualization . For routine high-throughput detection, 300-100 copies could be consistently detected . The extraction procedure was shown to reliably detect MVM at a concentration of 1 TCID50/ml . The combination of the extraction/PCR procedure establishes a powerful, sensitive, specific assay that can detect the presence of MVM sequences with a 1-day turnaround time.

Int J Artif Organs, 1997 Nov, 20(11), 644 - 9
Immunoisolation of hybrid liver support systems by semipermeable membranes; Gleissner M et al.; Immunoisolation of hybrid liver support systems (LSS) utilizing suitable semipermeable membranes as an immune barrier enables neither immunocompetent cytotoxic factors to cause damage to the hepatocytes in the bioreactor nor xenogenic hepatocyte products to cause immunological side effects in patients . To determine the capability of membranes as an immune barrier, 6 flat membranes were investigated: Cuprophan (C-100), cut-off MW 1000, Cuprophan (C-240), cut-off MW 10,000, Polypropylen hydrophilic and hydrophobic (PPhi, PPho), cut-off MW 500,000-1,000,000, Polysulfon (PS), cut-off MW 1,000,000, Polyamid (PA), cut-off beyond MW 1,000,000 . The permeability of the membranes to plasma factors and liver protein fractions (LP) was studied by routine biochemical methods and gel electrophoresis . In a second study, pigs (n=7) were immunised by LP after membrane passage . The results showed PA, PS, and PPhi to be completely permeable for plasma factors and LP C-100 and C-240 for urophanic substances, and C-240 again for LP under MW 14.000 . All 7 pig sera studied by Western blot discovered pre-formed xenoreactive natural IgG-antibodies (NAB) against human liver antigen (AG) with MW 26.000 . AB de-novo-synthesis was demonstrated for AG with MW 45.000 . No AB-synthesis was induced for epitopes under MW 26,000 . These results suggest that limiting the cut-off of bioreactor outflow membranes to MW < 26,000 could avoid immunological side effects to patients.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13885 - 90
Tissue engineering of cartilage in space; Freed LE et al.; Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth . Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10(-4)-10(-6) g) or Earth (1 g) . This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue . Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen . Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior . The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues . These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13554 - 8
A direct electrode-driven P450 cycle for biocatalysis; Reipa V et al.; The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles . Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode . Required oxygen was produced at a Pt counter electrode by water electrolysis . A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers . The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor/nmol of CYP101 per min.

Biotechnol Prog, 1997 Sep-Oct, 13(5), 583 - 9
Biodesulfurization of flue gases and other sulfate/sulfite waste streams using immobilized mixed sulfate-reducing bacteria; Selvaraj PT et al.; Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere that cause acid rain . Microbial processes for reducing SO2 to hydrogen sulfide (H2S) have previously been demonstrated by utilizing mixed cultures of sulfate-reducing bacteria (SRB) with municipal sewage digest as the carbon and energy source . To maximize the productivity of the bioreactor for SO2 reduction in this study, various immobilized cell bioreactors were investigated: a stirred tank with SRB flocs and columnar reactors with cells immobilized in either potassium-carrageenan gel matrix or polymeric porous BIO-SEP beads . The maximum volumetric productivity for SO2 reduction in the continuous stirred-tank reactor (CSTR) with SRB flocs was 2.1 mmol of SO2/(h.L) . The potassium-carrageenan gell matrix used for cell immobilization was not durable at feed sulfite concentrations greater than 2000 mg/L (1.7 mmol/(h.L)) . A columnar reactor with mixed SRB cells that had been allowed to grow into highly stable BIO-SEP polymeric beads exhibited the highest sulfite conversion rates, in the range 16.5 mmol/(h.L) (with 100% conversion) to 20 mmol/(h.L) (with 95% conversion) . The average specific activity for sulfite reduction in the column, in terms of dry weight of SRB biomass, was 9.5 mmol of sulfite/(h.g) . In addition to flue gas desulfurization, potential applications of this microbial process include the treatment of sulfate/sulfite-laden wastewater from the pulp and paper, petroleum, mining, and chemical industries.

Appl Microbiol Biotechnol, 1997 Dec, 48(6), 745 - 52
Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil; Barbeau C et al.; The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil . A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass . Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass . Within 30 days, PCP-degrading bacteria increased from 10(5) cfu/g to 10(8) cfu/g soil . Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution . The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l . This high level of tolerance was attributed to the beneficial effect of the soil particles . Once produced, the activated soil biomass remained active for 5 weeks at 20 degrees C and for up to 3 months when kept at 4 degrees C . The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms . Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration . The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar.

Artif Organs, 1997 Nov, 21(11), 1177 - 81
Semipermeable hollow fiber membranes in hepatocyte bioreactors: a prerequisite for a successful bioartificial liver?
Flendrig LM, te Velde AA, Chamuleau RA.
Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure . Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor . The gold standard remains the intact liver . Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer . These conditions are not met in bioreactors based on hollow fiber membranes . Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient . We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion . The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates . The efficacy of this original design was tested in rats with liver ischemia . Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments.

Folia Microbiol (Praha), 1997, 42(4), 349 - 52
Characterization of phytase produced by Aspergillus niger; Dvorakova J et al.; The extracellular activity of Aspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor . The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55 degrees C and high pH and temperature stability . The Km for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively . The enzyme is noncompetively inhibited by inorganic monophosphate (Ki = 2.85 mmol/L) and by Cu2+, Zn2+, Hg2+, Sn2+, Cd2+ ions and strongly by F- ones; it is activated by Ca2+, Mg2+ and Mn2+ ions . The substrate specificity of phytase is broad with the highest affinity to calcium phytate.

J Immunol Methods, 1997 Oct 13, 208(1), 65 - 73
Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography; Manzke O et al.; A method for large scale production and single-step purification of bispecific antibodies is described . Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month . Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step . Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays . Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA . Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step . Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.

Chin J Biotechnol, 1997, 13(3), 169 - 76
Extractive L-lactic acid fermentation with immobilized Rhizopus oryzae in a three-phase fluidized bed; Lu Y et al.; The Rhizopus oryzae was immobilized by the calcium alginate entrapment method . A three-phase fluidized-bed bioreactor was designed to perform the immobilized-cell L-lactic acid fermentation . A solvent extraction column was coupled with the bioreactor to remove L-lactic acid from the fermentation broth . The TRPO was selected as solvent and sulfonated kerosene as diluent . The results indicated that the pH value in the broth was regulated above pH 3.5 and the fermentation rate was as high as 11 g L-lactic acid per hour per liter of beads . A mathematical model was proposed to describe the concentration of L-lactic acid in the extractive fermentation.

Biotechnol Appl Biochem, 1997 Dec, 26 ( Pt 3), 203 - 12
Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: evaluation of performance in vivo; Jean-Francois J et al.; The L-asparaginase of Escherichia coli (ASNase) is currently used in combination with antineoplastic drugs to treat various lymphoblastic leukaemias . However, its use is limited by severe immunological reactions and the short serum half-life associated with the enzyme . Immobilization of ASNase into a biocompatible matrix can greatly decrease the immunogenicity of the enzyme, increase its half-life in vivo and its therapeutic index . Thus the E . coli ASNase was immobilized in a biocompatible hydrogel made of rat serum albumin and poly(ethylene glycol) (PEG; molecular mass 10 kDa) . The effectiveness of this enzymic bioreactor to deplete serum L-asparagine was evaluated after its peritoneal implantation in rats . Seven units of immobilized ASNase/rat depleted serum asparagine to an undetectable level (< 1 microM) during 6 days, while 5 units of immobilized ASNase/rat decreased the level of serum asparagine by 85-90% during at least 2 days . Under both conditions asparagine levels returned to normal about 10 days after surgery, and hydrogels still retained 80% of their enzymic activity when assayed in vitro . After 10-14 days in vivo, hydrogels became opaque and surrounded by a fibrotic capsule with a few inflammatory sites . Nevertheless, the enzymic hydrogel showed great stability in vivo, and, after 4 months of implantation, 12% of the initial ASNase activity was still present . At 6 months, histological analysis showed stabilization of the fibrotic capsule thickness . Assays on the levels of ASNase and asparagine synthetase indicated an induction of the latter activity, mainly in the pancreas when compared with the level observed in spleen or liver . ELISA tests at 28 days and 120 days showed the presence of anti-ASNase (and, in lower amounts, anti-PEG) antibodies in sera of implanted rats . As observed with other enzyme-immobilization systems used in vivo, the formation of fibroblast-like cell layers around the implant, which block the translocation of the substrate into the enzymic matrix, is the major factor affecting the performance and longevity of the bioreactor.

Anal Chem, 1997 Nov 15, 69(22), 4601 - 7
Improving the activity of immobilized subtilisin by site-specific attachment to surfaces; Huang W et al.; Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors . The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties . To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis . This cysteine residue was designed to be away from the active site of the enzyme . The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports . This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase . Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Biotechnol Prog, 1997 Nov-Dec, 13(6), 810 - 3
Enzymatic large-scale production of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid in enzyme membrane reactors; Salagnad C et al.; The enzymatic synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) starting from D-mannose and pyruvic acid using Neu5Ac-aldolase has been scaled up . A repetitive batch ultrafiltration bioreactor was used for the KDN synthesis on 100 g scale with a conversion of up to 85% . Furthermore, a 440 mL pilot-scale enzyme membrane reactor (EMR) was performed for the continuous production of KDN . Conversion of mannose was 75% at a space--time yield of 375 g/(L d) . KDN was advanteageously isolated by crystallization with an overall yield of 75%.

Biotechnol Prog, 1997 Nov-Dec, 13(6), 799 - 804
Kinetics of growth and ribosome-inactivating protein production from Trichosanthes kirilowii plant cell cultures in a 5-L bioreactor; Stoner MR et al.; Ribosome-inactivating proteins, named for their ability to inhibit protein translation in cell-free systems, are an important class of natural plant defense proteins with potential human therapeutic and agricultural applications . The kinetics of growth, nutrient consumption, and extracellular protein translation inhibitory activity are presented for Trichosanthes kirilowii plant cell suspensions in 5-L bioreactors at two agitation rates (50 and 100 rpm) . The cultures had a 7-9.5 day lag phase followed by exponential growth with a doubling time of less than 2 days . Biomass concentrations reached levels of approximately 19 g (dry weight)/L . Protein translation inhibitory activity was observed in the culture broths during the exponential growth phase and reached levels of approximately 50-60 units . No detrimental effects of agitation were observed at 100 rpm . These studies demonstrate the potential for plant cell culture production of ribosome-inactivating proteins in bioreactor systems.

Am J Kidney Dis, 1997 Nov, 30(5 Suppl 4), S28 - 31
The bioartificial renal tubule assist device to enhance CRRT in acute renal failure; Humes HD et al.; Current therapy for acute tubular necrosis (ATN) continues to have an exceedingly high mortality rate, exceeding 50% even with dialytic or hemofiltrative support . Current renal replacement therapy in ATN only substitutes for filtration function of the kidney but not its cellular metabolic functions . Replacing these metabolic functions may optimize current therapy for this devastating disease process . In this regard, a renal tubule assist device (RAD) has been developed to be placed in an extracorporeal continuous hemoperfusion circuit in series with a hemofilter . The RAD consists of porcine renal proximal tubule cells grown as confluent monolayers of a multifiber bioreactor with a membrane surface area from 0.4 to 1.6 m2 . The cells along the inner surface of the hollow fibers are immunoprotected from the patient's blood by the hollow fiber membrane . In preliminary experiments in uremic dogs, this device has been shown to tolerate a uremic environment while providing reabsorptive, metabolic, and endocrinologic activity . Pilot human trials of the RAD are anticipated within the next year to improve current renal replacement therapy in ATN.

Braz J Med Biol Res, 1997 Aug, 30(8), 923 - 8
Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain; Miranda EA et al.; A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system . On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath . Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Curr Microbiol, 1997 Dec, 35(6), 356 - 62
Stable Transformation of Chlorella: Rescue of Nitrate Reductase-Deficient Mutants with the Nitrate Reductase Gene
Dawson HN, Burlingame R, Cannons AC.
Unicellular green algae, like Chlorella, offer a potentially useful system for the expression of heterologous proteins . However, the development of Chlorella as a bioreactor has been delayed owing to the lack of a stable transformation technique . Here we report on the use of micro-projectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants . The stable transformants were able to grow on nitrate medium after repeated passages between selective and nonselective medium and exhibited inducible nitrate reductase activity comparable to that of wild-type cells . Southern analysis suggests homologous recombination occurs with insertion of the wild type gene into the mutated gene and that the genes of the two Chlorellaspecies used are very similar . Specific RNase protection assays, selecting for a poorly conserved region of the gene, identified the presence of the C . vulgaris NR transcript only in the transformed C . sorkiniana mutant and not in the mutant.

Appl Microbiol Biotechnol, 1997 Sep, 48(3), 424 - 30
Removal of tetrachloroethylene in an anaerobic column bioreactor; Noftsker C et al.; Removal of tetrachloroethylene (perchloroethylene; C2Cl4) by microbial consortia from two sites with different C2Cl4 exposure histories was examined in a bench-scale anaerobic column bioreactor . It was hypothesized that optimal removal would be observed in the reactor packed with sediments having an extensive exposure history . Microbial consortia were enriched from hyporheic-zone (HZ) sediments from the Portneuf aquifer near Pocatello, Idaho, and from industrial-zone (IZ) sediments from a highly contaminated aquifer in Portland, Oregon . Lactate and acetate were the electron donors during experiments conducted over 9 and 7 months for HZ and IZ sediments, respectively . In the HZ bioreactor, the retention time ranged from 31 h to 81 h, and inlet C2Cl4 concentrations ranged from 0.1 ppm to 1.0 ppm . Dechlorination of C2Cl4 averaged 60% and reached a maximum of 78% . An increase in C:N from 27:1 to 500:1 corresponded to an 18% increase in removal efficiency . Trichloroethylene production corresponded to decreased effluent C2Cl4; further intermediates were not detected . In the IZ bioreactor, the retention time varied from 34 h to 115 h; the inlet C2Cl4 concentration was 1.0 ppm . C2Cl4 removal averaged 70% with a maximum of 98% . Trichloroethylene and cis-dichloroethylene were detected in the effluent . Increases in C:N from 50:1 to 250:1 enhanced dechlorination activity.

Appl Environ Microbiol, 1997 Oct, 63(10), 4090 - 2
Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium; Fang A et al.; Production of the antibacterial polypeptide microcin B17 (MccB17) by Escherichia coli ZK650 was inhibited by simulated microgravity . The site of MccB17 accumulation was found to be different, depending on whether the organism was grown in shaking flasks or in rotating bioreactors designed to establish a simulated microgravity environment . In flasks, the accumulation was cellular, but in the reactors, virtually all the microcin was found in the medium . The change from a cellular site to an extracellular one was apparently not a function of gravity, since extracellular production occurred in these bioreactors, irrespective of whether they were operated in the simulated microgravity or normal gravity mode . More probably, excretion is due to the much lower degree of shear stress in the bioreactors . Addition of even a single glass bead to the 50-ml medium volume in the bioreactor created enough shear to change the site of MccB17 accumulation from the medium to the cells.

Cell Transplant, 1997 Sep-Oct, 6(5), 537 - 40
An attempt to add biological functions by genetic engineering in order to produce high-performance bioreactor cells for hybrid artificial liver: transfection of glutamine synthetase into Chinese hamster ovary (CHO) cell; Enosawa S et al.; In the course of immortalization, hepatocyte cell lines lose their original differentiated functions, such as ammonia removal and urea formation, drug metabolism, serum protein synthesis, etc . (Enosawa et al., Cell Transplant . 5:S39-S40; 1996) . With the aim of adding lost or deficient functions and producing cell lines for the bioreactor of a hybrid artificial liver, rat glutamine synthetase (GS) gene was transfected into Chinese hamster ovary (CHO) cells, because it is able to lower the ammonia level . The GS gene-inserted pSV2 plasmid was transfected into the CHO-K1 line by electroporation . Transfected CHO (GS-CHO) cells were cultured in a glutamine-free medium containing ammonia, glutamic acid, and the GS inhibitor methionine sulfoximine (MSX) . The MSX concentration was increased stepwise from 25 mumol/L to 1600 mumol/L to amplify the GS gene . In several GS-CHO sublines resistant to 300-1600 mumol/L of MSX, the specific activities of GS were increased from 0.2 x 10(4) to 1.7-2.9 x 10(4) unit/10(6) cells . When the amplified GS-CHO cells were cultured in the ammonia-containing medium, a slow but steady decrease of the ammonia level was observed when the level was high . Finally, the prospect of genetically modulated cells for bioreactors in the hybrid artificial liver is discussed.

Mol Reprod Dev, 1997 Nov, 48(3), 324 - 31
Cre-mediated recombination at the murine whey acidic protein (mWAP) locus; Rucker EB et al.; The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4 . Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4) . Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette . Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion . Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus . The frequency of this event was 23% (11 of 48) of that obtained with random integration . This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells . Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells . Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal "bioreactor" models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements . In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner . It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland.

Enzyme Microb Technol, 1997 Oct, 21(5), 355 - 60
The influence of pH and aeration rate on the fermentation of D-xylose by Candida shehatae; Sanchez S et al.; The effects of the initial pH and air supply on the production of ethanol from D-xylose using the yeast Candida shehatae in a batch reactor were investigated . The initial pH was altered within the range of 2.5-6.5 and the specific aeration rate from 0.0-0.3 vv-1 min-1 . The results showed that the most favorable initial pH for ethanol production was 4.5 and aeration via the stirring vortex of the bioreactor was sufficient . Under these conditions, the maximum specific growth rate (mu(m)) was 0.329 h-1; biomass production rate (b), 0.024 kg m-3 h-1; overall biomass yield (YGx/s), 0.036 kg kg-1; the specific uptake rate of D-xylose (qs), 2.0 kg kg-1 h-1; and the specific ethanol production rate (qE), 0.72 kg kg-1 h-1 (both at 20 h culture time) . The average xylitol yield (Yxy/s) was 0.078 kg kg-1 and the overall ethanol yield (YGE/s), 0.41 kg kg-1 . Both qs and qE diminished once the exponential growth phase was over.

J Dairy Sci, 1997 Sep, 80(9), 2213 - 24
Transgenic dairy cattle: genetic engineering on a large scale; Wall RJ et al.; Amid the explosion of fundamental knowledge generated from transgenic animal models, a small group of scientists has been producing transgenic livestock with goals of improving animal production efficiency and generating new products . The ability to modify mammary-specific genes provides an opportunity to pursue several distinctly different avenues of research . The objective of the emerging gene "pharming" industry is to produce pharmaceuticals for treating human diseases . It is argued that mammary glands are an ideal site for producing complex bioactive proteins that can be cost effectively harvested and purified . Consequently, during the past decade, approximately a dozen companies have been created to capture the US market for pharmaceuticals produced from transgenic bioreactors estimated at $3 billion annually . Several products produced in this way are now in human clinical trials . Another research direction, which has been widely discussed but has received less attention in the laboratory, is genetic engineering of the bovine mammary gland to alter the composition of milk destined for human consumption . Proposals include increasing or altering endogenous proteins, decreasing fat, and altering milk composition to resemble that of human milk . Initial studies using transgenic mice to investigate the feasibility of enhancing manufacturing properties of milk have been encouraging . The potential profitability of gene "pharming" seems clear, as do the benefits of transgenic cows producing milk that has been optimized for food products . To take full advantage of enhanced milk, it may be desirable to restructure the method by which dairy producers are compensated . However, the cost of producing functional transgenic cattle will remain a severe limitation to realizing the potential of transgenic cattle until inefficiencies of transgenic technology are overcome . These inefficiencies include low rates of gene integration, poor embryo survival, and unpredictable transgene behavior.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 198 - 203
The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture; Dunster CA et al.; The CHO320 cell line, engineered to produce human interferon gamma was investigated with regard to its susceptibility to oxidative stress . Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20% . High concentrations of hydrogen peroxide (in excess of 200 microM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 microns and 100 microM had no effect on recombinant protein production . Buthionine sulphoximine (50 microM and 100 microM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen . It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress.

Cell Biol Toxicol, 1997 Jul, 13(4-5), 357 - 64
Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver; Sielaff TD et al.; A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure . The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure . The device is an automated mammalian cell culture system supporting 6-7 x 10(9) porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors . Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium . This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application . The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack . The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes . These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.

Cell Biol Toxicol, 1997 Jul, 13(4-5), 349 - 55
Long-term liver cell cultures in bioreactors and possible application for liver support; Gerlach JC; Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy . A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems . An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells . The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix . Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network . Cells are perfused via independent capillary membrane compartments . Decentralized oxygen supply and carbon dioxide removal with low gradients is possible . There is a decentralized co-culture compartment for nonparenchymal liver cells . The use of identical parallel units to supply a few hepatocytes facilitates scale-up.

J Immunol Methods, 1997 Jul 14, 205(2), 109 - 14
Measurement of glucose consumption by hybridoma cells growing in hollow fiber cartridge bioreactors: use of blood glucose self-monitoring devices; Nayak RC et al.; Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media . The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration . The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl . The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated . A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration . Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading . The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor.

Appl Microbiol Biotechnol, 1997 Jul, 48(1), 47 - 52
Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme; Schafer A et al.; By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4 . The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1 . Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 bioreactor at 37 degrees C and pH 7.0 . Overexpression of MDH in Escherichia coli BL21(DE3) {pASFG1} was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis . Under standard growth conditions, E . coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low . Decreasing the growth temperature to 27 degrees C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26,000 U/l, which corresponds to 0.38 g/l MDH . This was an 87-fold overexpression of MDH compared to that of the natural host R . sphaeroides Si4, and a 236-fold improvement of the volumetric yield . MDH was purified from E . coli BL21(DE3) {pASFG1} with 67% recovery, using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration . Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.

Transfusion, 1997 Jul, 37(7), 685 - 90
Retroviral transduction of peripheral blood leukocytes in a hollow-fiber bioreactor; Shankar R et al.; BACKGROUND: Peripheral blood white cells (leukocytes) (PBLs) have been used as effective targets for genetic manipulation by transduction with retroviruses in open systems . A semi-closed hollow-fiber bioreactor was tested for culturing and transducing lymphocytes . STUDY DESIGN AND METHODS: PBLs were isolated from five healthy donors, and 5 x 10(7) cells were cultured in hollow-fiber bioreactors for 4 days after stimulation with anti-CD3 in medium containing 200 units per mL of recombinant interleukin 2 . Transduction with retroviral vector containing the gene for iduronate-2-sulfatase and G 418 resistance, L2SN, was performed daily on Days 4, 5, 6, and 7, and the cells were expanded for an additional 3 days . RESULTS: PBLs from three donors were harvested from the bioreactor after transduction and expansion, and 4.5 x 10(9), 7.0 x 10(9), and 2.9 x 10(9) cells were recovered, representing 90-, 136-, and 58-fold expansions . The transduction frequency of L2SN was 10, 5, and 1 percent, respectively . For additional expansion of PBLs, in two cases the bioreactor was reinoculated with 5 x 10(7) cells, which were expanded again for 16 and 8 days, respectively, yielding 1.4 x 10(9) and 3.1 x 10(9) cells, which reflected an additional 28- and 62-fold expansion of cells . PBLs from two other donors were transduced and expanded in the bioreactor, and then 0.8 mg per mL of G 418 was added to the medium in an attempt to enrich the transduced population . Although 2.5 and 10 percent of the cells were transduced, cell death and absence of expansion in the presence of G 418 resulted in final cell lots with viabilities of only 4 and 8 percent . In all cases, the harvested cells tested negative in bacterial and fungal cultures . CONCLUSION: Hollow-fiber bioreactors are an efficient and effective system for the retroviral transduction and expansion of PBLs for clinical gene therapy.

Thromb Haemost, 1997 Jul, 78(1), 543 - 7
The past, present and future of transgenic bioreactors; Drohan WN; Hybrid genes can control the tissue-specific synthesis of human proteins in transgenic animals . Thus, it is now possible to produce proteins of biomedical value in the body fluids or cells of transgenic livestock . In fact, the first transgenically produced protein, antithrombin III, is now in clinical trials and others will soon follow.

Gene Ther, 1997 Jun, 4(6), 600 - 10
Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer; Forestell SP et al.; We report increased transduction of human hematopoietic progenitor cells through a combination of novel retroviral vector packaging cell lines, and improved vector supernatant production . The new ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic particles (ProPak-A cells), and ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing . Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells . All vector types transduced primary human CD34-positive or CD4-positive cells, regardless of tropism . However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector . The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines . This ping-pong amplification yielded supernatant containing vector targeted to two distinct receptors present on human cells, and did not result in detectable RCR formation . In addition, we describe conditions for improved vector supernatant production in a packed-bed bioreactor.

J Hepatol, 1997 Jun, 26(6), 1379 - 92
In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates; Flendrig LM et al.; BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy . We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli . METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e . a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal . Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact . Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion . RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo . The biochemical performance of the bioreactor remained stable over the investigated period . The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures . Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size . CONCLUSIONS: The novel bioreactor showed encouraging efficiency . The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.

In Vitro Cell Dev Biol Anim, 1997 Jun, 33(6), 459 - 66
Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor; Ingram M et al.; Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated . The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture . Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm . Architecture of spheroids varied with cell type . Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures . Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs . Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells . Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

Appl Environ Microbiol, 1997 Jun, 63(6), 2366 - 71
Silencing MIG1 in Saccharomyces cerevisiae: effects of antisense MIG1 expression and MIG1 gene disruption; Olsson L et al.; Silencing of MIG1, a transcription factor imposing carbon catabolite repression on invertase, was attempted, either by disrupting the gene or by expressing antisense copies of the gene . The performance of the recombinant strains in bioreactor batch cultivations on sucrose, in the presence of glucose, was compared with that of the wild-type strain under the same conditions . In the delta migI strain, the rate of sucrose utilization was independent (10 mmol/g/h) of the glucose concentration . During the cultivations with the wild-type strain and the antisense strains, two distinct phases were observed . The rates of sucrose hydrolysis were < 1 mmol/g/h and 9 to 10 mmol/g/h in the first and second phases, respectively . Entry into the second cultivation phase was characterized by a decline in glucose concentration below 12 mmol/liter . As expected, disruption of MIG1 resulted in a relief of glucose repression . However, silencing of MIG1 expression was not achieved by expressing antisense MIG1, even though antisense MIG1 RNA was sufficiently stable to be detected . In the wild-type and delta migI strains, the specific growth rate was 0.32 to 0.33 h-1, whereas it was lower in the antisense strains, 0.25 to 0.30 h-1.

J Biotechnol, 1997 May 23, 55(1), 31 - 41
Plasmid instability kinetics in continuous culture of a recombinant Saccharomyces cerevisiae in airlift bioreactor; Zhang Z et al.; Plasmid instability of a recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) was examined during continuous culture in a nonselective medium in an airlift bioreactor . The recombinant strain contained a 2-micron based shuttle vector pGAC9 and expresses Aspergillus awatnori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter . The changes in the fraction of plasmid-bearing cells and glucoamylase activity followed first-order kinetics . Expressed as a function of time, the decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates . If expressed as a function of cell generations, the decay rates were nearly constant over the dilution rates tested . The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible . This was probably due to the low copy number of the 2-micron based yeast shuttle vector . Thus the contribution of preferential growth to apparent plasmid instability was negligible . A novel numerical method is proposed to evaluate the parameters related to plasmid stability . The estimated values of probability of plasmid loss (P = 0.0499) were nearly constant at different dilution rates . No significant effect of growth rates on plasmid instability was observed . The proposed kinetics agreed well with experimental observations.

Curr Opin Hematol, 1997 May, 4(3), 157 - 62
Use of stem cell factor to mobilize hematopoietic progenitors; Bearman SI; Stem cell factor (SCF) is a multipotent growth factor that plays a role in the growth and development of hematopoietic cells, spermatocytes, melanocytes, and mast cells . SCF alone has little direct stimulatory activity on hematopoietic progenitors but acts synergistically with other colony-stimulating factors to potentiate their effects on colony growth . SCF also potentiates the mobilization of hematopoietic progenitor cells into the peripheral blood in response to granulocyte colony-stimulating factor (G-CSF), with or without chemotherapy . The combination of SCF plus G-CSF appears to be a particularly effective mobilization regimen for patients who have been heavily pretreated . Whether engraftment of peripheral blood progenitor cells mobilized by SCF plus G-CSF is superior to that of cells mobilized by G-CSF alone remains unclear . SCF appears to be a necessary requirement for ex vivo expansion of hematopoietic progenitors in both liquid and bioreactor culture systems and will be an important component of future cellular therapies, such as expansion of placental cord blood progenitors and retroviral-mediated gene transfer into hematopoietic target cells.

In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 386 - 91
Skeletal muscle satellite cells cultured in simulated microgravity; Molnar G et al.; Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells . Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development . Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish . Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo . Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development . Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells . Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV . Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture . Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems . Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells . Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar . Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate . Aggregation of beads did not occur in the absence of cells . Myotubes were often seen on individual beads or spanning the surface of two beads . In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.

In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 381 - 5
Microgravity tissue engineering; Freed LE et al.; Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity . In particular, vessel rotation speed was adjusted such that 10 mm diameter x 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall . Feasibility was demonstrated for two different cell types: cartilage and heart . Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously . Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes . Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.

In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 352 - 7
Induction of carcinoembryonic antigen expression in a three-dimensional culture system; Jessup JM et al.; MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture . However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue . We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures . To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor . MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers . These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.

In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 337 - 43
Neonatal rat heart cells cultured in simulated microgravity; Akins RE et al.; In vitro characteristics of cardiac cells cultured in simulated microgravity are reported . Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane . Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types . Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system . The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue . We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity . The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension . HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques . Both systems were uniformly inoculated and medium exchanged at standard intervals . Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization . Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type . In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures . Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 332 - 6
Cultivation of fall armyworm ovary cells in simulated microgravity; Francis KM et al.; A methodology is presented to culture Fall Armyworm Ovary cells in simulated micrograviy using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel . In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture . Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectively . Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism . Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture . Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase . These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm2 in shaker culture to under 0.5 dyne/cm2 in the NASA vessel . These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.

Neth J Med, 1997 May, 50(5), 228 - 32
Gastro-entero-hepatology in the next millennium; Tytgat GN; The scope of gastroenterology and hepatology now and its development into the next century are great and expanding . Only some of the many exciting improvements which are expected during the next few years can be discussed here . Progress in the microbial aetiology and novel targeted treatments for inflammatory bowel disease (IBD) will be paralleled by better understanding of functional upper and lower gut disorders, their neural control and neuropharmacological treatment . Technical developments in the pipeline include improvements in endoscopic and endosonographic equipment and techniques, including MR- or CT-based 'virtual colonoscopy' to obviate many invasive diagnostic colonoscopies, and the remarkable self-propelled endoscope, which will worm its way to regions of interest in the bowel . In hepatology, transplantation, and vaccination for hepatitides will increase, and improved treatment of acute liver failure may involve bioreactors or cryopreserved human hepatocytes . Gastrointestinal oncology will progress through more extensive surgery, gene therapy and techniques such as mucosal resection . A target force of about 150 trained gastroenterologist/hepatologists will be needed in the Netherlands--one for every 100,000 of the population.

Biochem Biophys Res Commun, 1997 Apr 7, 233(1), 238 - 43
A radiobiological probe for simultaneous NMR spectroscopy and 192Ir gamma irradiation of Saccharomyces cerevisiae; Magness JE et al.; A novel nuclear magnetic resonance (NMR) spectroscopy probe was designed and constructed for the study of transient metabolic changes in cellular systems during exposure to ionizing radiation . The probe incorporated a bioreactor, a radiation source, and a radiofrequency detection circuit tunable between 100 and 300 MHz for in vivo NMR spectroscopy of 23Na, 13C, and 31P at 11.7 Tesla . The bioreactor system allowed perfusion, oxygenation, and temperature control of cultured cells during irradiation and while performing simultaneous spectroscopic experiments . The concentric design of the bioreactor allowed for the insertion of a 192Ir gamma ray source (E(gamma) = 370 keV) to allow irradiation of the bioreactor system during the acquisition of NMR spectra . Initial results of 31P spectra obtained during simultaneous gamma irradiation of Saccharomyces cerevisiae at approximately 8 Gy/hr show rapid decreases in adenosine triphosphate (ATP) and polyphosphate at the onset of irradiation followed by a slow recovery of polyphosphate.

J Biotechnol, 1997 Apr 4, 54(1), 1 - 14
Regulation of a continuous yeast bioreactor near the critical dilution rate using a productostat; Andersen MY et al.; Regulation of a continuous bioreactor with Saccharomyces cerevisiae is investigated . A number of different sensors are evaluated for this purpose and the process dynamics is investigated around the critical dilution rate . A sensor for reducing gas concentration in exhaust gases is selected for regulating the substrate flow rate . Closed loop identification experiments are carried out to enable identification of the process dynamics near the critical diluton rate . Due to the time-varying nature of this process an adaptive regulator seems to be a promising tool for providing good regulatory and setpoint tracking performance . A simple third order model is used for a model based control design with a Linear Quadratic (LQ)-regulator . The LQ-regulator performs well experimentally, both in an adaptive version where the model parameters are updated on-line, and in a non-adaptive version . During the test the process is exposed to a large disturbance in substrate feed concentration and to a small setpoint disturbance . The proposed regulator is a practical realisation of a productostat where the product in this case is an undesired primary metabolite . Thus, this paper demonstrates a more general principle of utilizing metabolic overflow metabolism for directing fluxes through a desired metabolic pathway . This principle is applicable in the presented form, if a (by-)product can be measured on-line.

Curr Opin Biotechnol, 1997 Apr 1, 8(2), 169 - 74
Partitioning bioreactors; Daugulis AJ; A very wide range of methods has recently been employed to selectively partition fermentation products, substrates, or other metabolites to achieve an overall improvement in bioprocess performance . Techniques ranging from extraction and gas stripping to electrokinetic methods have been used to favorably influence the microenvironment of cells being cultivated in bioreactors . Virtually all types of cells, including animal cells, have benefitted by such selective partitioning and very high value fermentation products, such as monoclonal antibodies and secondary metabolites, have been produced using this process concept.

Curr Opin Biotechnol, 1997 Apr 1, 8(2), 165 - 8
Innovative bioreactors; Deshusses MA et al.; Recent papers have described new bioreactor designs . Most innovations addressed either oxygen transfer, shear induced by stirring, control of water activity in organic phase systems or waste biotreatment . Innovations made during the past year were reported in mainly three areas: bioreactor designs for increases in oxygen transfer and decreases in shear stress; bioreactors for two-phase reactions with water activity control; and environmental bioreactors.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 185 - 94
Mist deposition onto hairy root cultures: aerosol modeling and experiments; Wyslouzil BE et al.; We analyzed the applicability of the standard models for aerosol deposition in randomly packed fibrous filter beds to mist deposition across a bed of hairy roots in the nutrient mist bioreactor . Although the assumptions inherent in the models are met on a local level, the overall structure of the root bed introduces some uncertainty into the correct choice of root packing fraction and gas velocity required by the model . For reasonable parameter values, the minimum in the deposition efficiency curves is close to the peak in the mist number and mass distributions, and good penetration of the root bed is possible . We then measured the deposition of mist across a packed bed of Artemisia annua transformed roots as a function of droplet size, bed length, and gas flow rate at a root packing fraction alpha = 0.5 . We compared the experimental measurements with the predictions of the aerosol deposition model and found good agreement between the measured and predicted values for the diameter where the deposition efficiency across the bed is 50%, D0.5 . Agreement between the model and the experiments broke down when the flow rate was increased to the point where the creeping flow assumptions were no longer valid.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 151 - 5
High-level expression of a heterologous gene in Escherichia coli in response to carbon-nitrogen source and C/N ratio in a batch bioreactor; Bhattacharya SK et al.; Overexpression of the target gene in a recombinant strain of Escherichia coli has been analyzed in view of changes in the carbon-nitrogen source and as a function of C/N ratio . The rate of target gene expression varied over a range of 200%, and the yield coefficient for the target gene product changed over 400% with change in carbon source at 10 gL-1 in M9 medium . With variation in nitrogen source, however, only marginal changes (10-15%) occurred in these parameters of overexpression . Higher concentration, for example 2.5 g L-1, of any nitrogen source adversely affected heterologous expression as rate of target gene expression declined in the range 35%-50% and the yield coefficient of foreign protein decreased between 45% and 60% . A C/N ratio of 15 was found to be optimum for both parameters for overexpression and host specific parameters such as specific growth rate and observed yield coefficient for cellular growth . An analysis with respect to temperature and pH revealed that host and expression parameters were quite susceptible to changes in these factors.

Appl Biochem Biotechnol, 1997 Feb-Mar, 62(2-3), 291 - 302
The culture of chicken embryo fibroblast cells on microcarriers to produce infectious bursal disease virus; Zhang L et al.; The cultures of chicken embryo fibroblast (CEF) cells in flasks, spinner bottles, and bioreactors were studied . The growth and metabolism characteristics of CEF cells and the feasibility of the CEF cell culture in bioreactor were investigated . The plating process of the CEF cells on GT-2 microcarriers in spinner bottles was studied, and a plating kinetic model was presented . The culture of CEF cells in 1.5 L CelliGen bioreactor to produce infectious bursal disease virus (IBDV) had met success . Whereas the additive microcarriers were fed during the culture, the cell density was increased 10 times as against seed cells adhering to microcarriers and the virus titer was as high as 7.5 . All the aforementioned experimental results have laid the foundation for high density culture of CEF cells and process scale-up.

Int J Artif Organs, 1997 Feb, 20(2), 119 - 24
Comparative evaluation of different membranes for the construction of an artificial liver support system; Qiang S et al.; During the past decades, many technological improvements have been made in the construction of extracorporeal liver support systems . Among these achievements, membranes of artificial capillary system, used as substrates of hepatocyte growth, aroused our interest in their application for the construction of bioreactors . The present paper studied the comparison of hepatocyte growth and function on six different membranes . Four of them are cellulose based membranes, Cuprophan, Hemophan, Cellulose acetate, and Bioflux; two are synthetic polymer SPAN and Polysulphone . Human hepatoma cell line SMMC-7721, with moderately differentiated hepatocyte-specific functions, was inoculated into the hollow fiber cartridges . These cells were allowed to attach and to grow over these membranes . It was found that there existed differences in hepatocyte immobilization and growth among these membranes . They influenced the growth and functions of hepatoma cells in vitro to some extent . These results show that membrane is an important factor in the construction of capillary membrane bioreactors for artificial liver support.

Trends Biotechnol, 1997 Feb, 15(2), 45 - 50
Plants as bioreactors for biopharmaceuticals: regulatory considerations; Miele L; Plants are one of several novel hosts that can be used for the production of recombinant biopharmaceuticals such as cytokines, hormones, monoclonal antibodies, enzymes and vaccines . The novelty of this technology and its wide range of potential applications require an assessment of possible regulatory concerns in the clinical development of plant-derived biopharmaceuticals . General principles extrapolated from experience gained with biotechnology products from other sources can serve as a foundation to develop scientifically sound strategies for the large-scale production and clinical development of safe and effective biopharmaceuticals in plant hosts.

J Biomed Mater Res, 1997 Feb, 34(2), 211 - 20
Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts; Grande DA et al.; Injury to articular cartilage predisposes that joint to further degeneration and eventually osteoarthritis . Recent studies have demonstrated the feasibility of using chondrocytes together with different biomaterial carriers as grafts for the repair of cartilage defects . The following study was undertaken to determine the effect of a variety of these materials on chondrocyte growth and extracellular matrix synthesis . We cultured chondrocytes on several commonly used materials and compared their rates of synthesis of proteoglycan and collagen . Additionally, we evaluated them in a closed culture recirculating system on these materials and compared them with standard culture techniques . This was done to see whether such a bioreactor-type system can be used to enhance the quality of in vitro reconstructed tissues . Our results demonstrated marked variability with respect to how chondrocytes responded to culture on the various materials . Bioabsorbable polymers such as polyglycolic acid (PGA)--enhanced proteoglycan synthesis, whereas collagen matrices stimulated synthesis of collagen . The use of the closed culture system, in general, improved the rates of synthesis of collagen and proteoglycan on the different material scaffolds . Exceptions were collagen synthesis on collagen matrices: use of the closed culture system did not enhance the rate of synthesis . Rates of proteoglycan synthesis on PGA scaffold initially was higher in the closed culture system but did not sustain a difference over the entire course of the 3-week culture period . This study demonstrates the importance of carrier material for the purpose of cartilage tissue reconstruction in vitro.

J Immunol Methods, 1997 Jan 15, 200(1-2), 39 - 46
Determination of anti-HBsAg IgM monoclonal antibodies in cell culture media by perfusion immunoassay; Brackett JM et al.; A rapid, specific, perfusion immunoassay for active anti-HBsAg monoclonal IgM is described . The immunoassay requires less than 3.5 min per sample . The precision was found to be 3.6% at an IgM concentration of 17 microg/ml . A detection limit of 1 microg/ml IgM in culture media was determined . Assay results were found to correlate very well with standard size exclusion chromatography and radial immunodiffusion techniques . This perfusion immunoassay was demonstrated to be useful for determining anti-HBsAg IgM in complex matrices such as cell culture media . The utility of the immunoassay for monitoring production of anti-HBsAg IgM in a perfusion bioreactor is demonstrated.

Chin J Biotechnol, 1997, 13(1), 51 - 8
Engineering factors affecting the granulation of sludge in the UASB reactor; Guo Y et al.; The influence of inoculum sludge involving different microbial populations, different modes of stream flow and different flow rates in a bioreactor on the granulation of sludge was studied . In the granulation process there were four periods: formation of microbial floccus, formation of subcore, growth of subcore, and maturity of granules . The production of microfloccus could be attributed to acid-forming bacteria . The hydrodynamic momentum transfer caused by moving phases and the hydraulic shear effect are the crucial factors affecting the formation of the subcore . For this a new concept-the lowest limited flow rate-is proposed, i.e., the lowest flow rate to form an expanded sludge bed . A sufficient feed rate (higher than the lowest flow rate), an appropriate sludge load, and an equal distribution of feed, as well as a suitable alkalinity in the medium are the essential factors for the scaling-up and process-control of the UASB reactor.

Immunogenetics, 1997, 45(6), 379 - 85
Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501; Prilliman K et al.; A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8(+) T-cell response . Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual's CD8(+) cytotoxic T lymphocytes will be presented by a second individual's different class I molecules . Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules . In applying this strategy to HLA-B*1501, we isolated milligram quantities of B*1501-bound peptides and mapped them using mass spectrometry . Repeated analyses consistently assign the same peptide binding signature to B*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures.

J Ind Microbiol Biotechnol, 1997 Jan, 18(1), 22 - 5
Secondary metabolism in simulated microgravity: beta-lactam production by Streptomyces clavuligerus; Fang A et al.; Rotating bioreactors designed at NASA's Johnson Space Center were used to simulate a microgravity environment in which to study secondary metabolism . The system examined was beta-lactam antibiotic production by Streptomyces clavuligerus . Both growth and beta-lactam production occurred in simulated microgravity . Stimulatory effects of phosphate and L-lysine, previously detected in normal gravity, also occurred in simulated microgravity . The degree of beta-lactam antibiotic production was markedly inhibited by simulated microgravity.

Adv Space Biol Med, 1997, 6, 255 - 74
Bioregeneration with maltose excreting Chlorella: system concept, technological development, and experiments; Wolf L; ESA has been studying a small-scale bioregenerative system to support long-term biological experiments on-board spacecraft with oxygen, water and food . Core component of this system is a special photo-bioreactor in which a maltose-producing strain of the green micro alga Chlorella is cultivated . A number of auxiliary system components have been developed and are functioning on the ground according to the design specifications, among them a gas/liquid phase separator operating at the same time as a low shear-stress pneumatic pump, a dehumidifier, a maltose separator, and a liquid transfer system . All components have been designed so that--in principle--they will operate in weightlessness, though this has so far only been verified for the gas/liquid separator . The bioreactor and some of the auxiliary components have been integrated in a prototype system, which has been subjected to preliminary testing . The prototype has been sterilized successfully by autoclaving, except for the liquid transfer unit which is disinfected with isopropyl alcohol . Chlorella 241.80 has been cultured several times under controlled conditions for up to 8 weeks . Algal growth to a biomass concentration of 9 g.l-1 dry weight and maltose production to a concentration of 17 g.l-1 have been achieved . The low shear-stress pneumatic pump works reliably without the mechanical cell damage produced by other types of pumps . Contamination of the algal cultures by other micro-organisms has been avoided in most of the experiment runs . The maximum oxygen production rate observed was 2 ml.min-1, when the culture was aerated with air +0.5% CO2 . This production rate is well below the CO2 gas transfer rate of 5 ml.min-1 under these conditions . It can probably be doubled by increasing the maximum light intensity of the illumination unit (currently 300 micro E.m-2S-1) . In a preliminary closed gas loop experiment with Periplaneta as consumer, the possibility of controlling the Chlorella culture so as to match the needs of the consumer colony has been established . A maltose excreting Chlorella strain has been selected as the photosynthetic producer, because the technique for automatic culturing of this organism and harvesting its products was expected to be much less complex than that required for culturing higher plants . Although the prototype system developed in our laboratory has reached a high level of sophistication, there remain still a number of technical and biological problems to be solved before the feasibility of this concept is definitely demonstrated . The major problem is maintaining sterility, and eventually automatic cleaning and resterilization when contamination occurs during operation . The culture medium, which contains minerals, cell fragments and considerable amounts of sugars, is an ideal substrate for many other microorganisms . Another problem is long term operation . The prototype system contains many tubes and ducts which are perfused with culture medium . These may clog, which may lead to loss of sensor information essential for controlling the culture . Even when we succeed in demonstrating the feasibility of this concept, it will be a difficult task to demonstrate convincingly that the expected advantages of a bioregenerative system can outweigh the simplicity and reliability of a non-regenerative stored resource system in terms of volume, mass and amount of consumables required over the operational time.

Microbiology, 1997 Jan, 143 ( Pt 1), 203 - 18
Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae; Nissen TL et al.; A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived . The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation . Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations . The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH4+, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA) . The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model . All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor . Carbon balances closed within 98% . The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h-1 . The fluxes through reactions located around important branch points of the metabolism were compared, i.e . the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways . Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase . Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose . The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis . It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.

Appl Environ Microbiol, 1997 Jan, 63(1), 122 - 7
Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter; Suarez A et al.; Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough . However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields . To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer . The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg . Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized . The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B . pertussis grown for 24 h . The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure . The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression . Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography . The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test . These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B . bronchiseptica background.

Cell Biol Toxicol, 1996 Dec, 12(4-6), 325 - 9
Cell-based therapy of acute liver failure: the extracorporeal bioartificial liver; Fremond B et al.; The need for an alternative treatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporarily providing liver functions are being actively tested . Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function . An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation . Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines . Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes . This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation . The development of this system for clinical purposes requires large yields of functional hepatocytes . We have isolated normal porcine hepatocytes by collagenase perfusion of the liver . Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor . Porcine hepatocytes expressed liver-specific functions at high levels, particularly protein neosynthesis and enzymatic activities involved in detoxication and biotransformation processes . In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins . This system represents a promising tool for the design of an extracorporeal bioartificial liver in human beings.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14118 - 21
Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice; Yarus S et al.; Tracheal antimicrobial peptide (TAP) is a member of the beta-defensin family of antibiotic peptides found in the tracheal mucosa of the cow . TAP gene expression in the bovine airway is inducible by lipopolysaccharide and inflammatory mediators, suggesting that it functions to protect the upper airway from infection . Limited availability of bovine TAP (bTAP) has precluded investigation of its potential utility in agriculture and medicine . To overcome this problem, transgenic mice expressing bTAP using an expression vector driven by control sequences from the murine whey acidic protein (WAP) gene have been generated . The WAP/bTAP transcript was detected in RNA isolated from mammary tissue of transgenic females . bTAP was purified to homogeneity from milk via acid precipitation, reverse-phase HPLC, and ion-exchange chromatography . This milk-derived bTAP had antimicrobial activity against Escherichia coli . Amino-terminal peptide sequencing confirmed the identity of this material as a bTAP isoform . bTAP available from a mammary gland bioreactor will allow evaluation of bTAP for use as an antibiotic in agriculture and medicine.

Semin Liver Dis, 1996 Nov, 16(4), 435 - 44
Use of bioartificial and artificial liver support devices; Hughes RD et al.; With all the new work and current interest in extracorporeal liver support systems incorporating hepatocytes, the findings with earlier artificial systems need to be reconsidered, particularly as they may constitute a component of some bioartificial devices . Furthermore, new and more effective artificial systems are currently under development . Essential hepatic functions need to be replaced, including excretory (the capability of adsorbents and dialysis) and synthetic and biotransformatory function, but the relative importance of these three functions in terms of promoting recovery of the native liver is as yet unclear . Two bioartificial devices have already been used clinically in the treatment of acute liver failure (ALF): the bioartificial liver (BAL) based on pig hepatocytes attached to microcarriers, and the extracorporeal liver assist device (ELAD) which contains a human liver-derived tumor cell line . As with earlier completely artificial systems, the results so far obtained in man are less impressive than in animal models of ALF . An important question not yet answered relates to quantity of cells and specific function in the new hybrid bioreactor devices required for clinical benefit, as well as the duration of support needed . A better understanding of the effects of these devices on the metabolic function of the damaged liver and the recovery process will be essential in the further development and design of effective systems . Controlled clinical trials on a multicenter basis will be needed for proper evaluation of these new approaches to treatment of ALF . From our own initial experience, the design of these protocols and the selection of biochemical tests will be difficult.

Biotechnol Prog, 1996 Nov-Dec, 12(6), 855 - 64
Use of the Centritech Lab centrifuge for perfusion culture of hybridoma cells in protein-free medium; Johnson M et al.; As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell-separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium . Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor . The centrifuge efficiently separated viable cells from dead ones . Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80% . The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed . This understanding of the separation process inside the centrifuge is important and may apply to other similar devices . Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion . However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case . Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge . This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L-1, equal to the performance of filter-based perfusion systems with the same cell line . It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 x 10(6) cells mL-1) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations.

Biotechnol Prog, 1996 Nov-Dec, 12(6), 837 - 46
Cyclic operation of ceramic-matrix animal cell bioreactors for controlled secretion of an endocrine hormone . A comparison of single-pass and recycle modes of operation; Grampp GE et al.; Controlled secretion processes for the production of secretory proteins in monolayer culture have been described previously (Grampp et al . Adv . Biochem . Eng./ Biotechnol . 1992, 46, 35-62), but little is known about the feasibility of scaling such processes into high-density bioreactors . Two immobilized-cell, ceramic-matrix bioreactor configurations were tested using the beta TC-3 cell model system which, in monolayer culture, can be manipulated to secrete murine insulin in a highly controlled manner . One reactor was configured with an external recirculation reservoir for oxygen transfer and was operated as a conventional immobilized bed/recycle reactor . The other reactor was configured as a single-pass perfusion system with oxygen supplied by diffusion from silicone tubing positioned proximal to the porous walls of the ceramic matrix . After inoculation with beta TC-3 cells, both systems were perfused with serum-supplemented medium to stimulate cell growth, and they ultimately attained high densities (approximately 5 x 10(8) cells/mL of pore volume) . To initiate controlled secretion operations, the reactor cores were washed with a serum-free basal medium, then exposed to a serum-free discharging medium containing secretory stimulants . Following several hours of discharging, the reactors were washed again, then switched to a serum-containing medium designed to quench the regulated secretion process . For the single-pass reactor these cycling operations were simple to implement and were effective in promoting the cyclic discharge and recharge of murine insulin . Because of the ability to reduce the perfusion rate in the single-pass reactor independent of oxygen transfer, the discharged insulin was captured in a relatively small volume (2 reactor core hold-up volumes), yielding a mean product concentration 10-fold greater than in the steady-state perfusate . Cyclic operation of the recirculating reactor was more difficult due to the complexity of switching between recirculation reservoirs, and the introduction of air bubbles during such operations resulted in the loss of biomass from the reactor after one cycle . Even in the first discharging cycle, the insulin yield was much lower than in the perfusate from the single-pass reactor, despite the comparable metabolic rates . The single-pass reactor was cycled successfully through four discharging and recharging episodes and maintained its ability to discharge insulin, albeit at a slower rate after the first discharge . Overall, 50-60% of the insulin secreted during the 48 h cycles was recovered during the brief discharging episodes . When insulin secretion rates and discharging yields were normalized to metabolic activity, neither high-density reactor system performed as well as did identically treated control T-flask cultures . It is hypothesized that the productivity and responsiveness of the high-density, pore-immobilized beta TC-3 cells are lower than in monolayer culture.

Biotechnol Prog, 1996 Nov-Dec, 12(6), 751 - 7
Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin expression for enhancing Escherichia coli growth in a microaerobic bioreactor; Kallio PT et al.; Expression of a gene encoding hemoglobin (VHb) from the aerobic bacterium Vitreoscilla sp . in several organisms, including Escherichia coli, has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation . The suitability of VHb to enhance microaerobic metabolism has been suggested to depend on its unusual oxygen binding characteristics . To examine whether hemoproteins of other origins can also elicit the positive effects VHb exerts in microaerobic E . coli cells, we subcloned the genes encoding Vitreoscilla VHb, horse heart myoglobin (HMb), and yeast flavohemoglobin (YFb) behind the IPTG-inducible tac promoter on a medium-copy-number vector and transformed these globin-expression plasmids into E . coli MG1655 and DH5 alpha . Biologically active VHb, HMb, and YFb were produced from these constructions in E . coli as judged by their ability to abduct carbon monoxide . The presence of HMb increased the growth of wild-type cells during the early stages of fed-batch growth, but the final optical densities of HMb-expressing cultures were comparable with the wild-type control not synthesizing HMb . The presence of VHb increased the cell density by 70% under the same cultivation conditions . The expression of wild-type YFb reduced the final cell density by 30% relative to the non-globin-expressing control.

Int J Artif Organs, 1996 Nov, 19(11), 677 - 91
A carrier-mediated transport of toxins in a hybrid membrane . Safety barrier between a patients blood and a bioartificial liver; Stange J et al.; Combination of detoxifying liver support systems with liver cell bioreactors may have additional benefits for the treatment of liver failure due to the replacement of known and unknown metabolic activities of the liver . However, the problem of side effects and possible risks caused by the use of animal hepatocytes or hepatoma cells remains unsolved which underlines the need of a safety barrier between the patients blood and the extracorporeal bioreactor . Passive filters do not meet the requirements of such membranes, because in liver failure desired and undesired molecules in the patients blood share similar physicochemical properties . That challenges the development of biologically designed separation membranes . A hybrid membrane is formed by implementation of transport proteins into a highly permeable hollow fiber . The transport of free solutes and albumin bound toxins is tested in vitro in comparison with conventional high flux membranes . The transport characteristics for tightly albumin bound toxins are significantly improved for the hybrid membrane . The transport of albumin bound toxins across the membrane is not associated with albumin . The selectivity of the transport is evaluated in vivo . No significant loss of middle molecular weight hormones attached to other carrier proteins was observed . Neither transport of immunologically relevant proteins across the membrane nor loss of valuable proteins was measured . Also in vivo, a significant reduction of protein bound toxins and a transport of metabolically relevant solutes, like amino acids, was shown . The presented hybrid membrane may be used like an "intelligent membrane" as a safety barrier between the patients blood and cell devices.

Int J Artif Organs, 1996 Nov, 19(11), 670 - 6
Importance of the kinetic characterization of liver cell metabolic reactions to the design of hybrid liver support devices; Catapano G et al.; Hybrid liver support devices (HLSDs) developed for the treatment of fulminant hepatic failure often perform well on a laboratory scale but rapidly lose their metabolic functions, or are not therapeutically effective, on a clinical scale . This suggests that the procedures adopted so far for the design of HLSDs are susceptible to improvement . In this paper, we discuss how essential a reliable and thorough kinetic characterization of the liver cell metabolic reactions is to the design of a clinically effective membrane HLSD . The features of the bioreactors used for the kinetic characterization of liver cell reactions are presented and discussed on the basis of the multifactorial nature of such reactions . The relevance of kinetics to the design of a membrane HLSD is also discussed with respect to the effect of the kinetics of oxygen consumption on the performance of the device.

Int J Artif Organs, 1996 Nov, 19(11), 645 - 54
Development of a hybrid liver support system: a review; Gerlach JC; Hybrid liver support systems (LSS) for the use of the detoxifying, metabolic synthetic and regulatory capabilities of liver cells are under development for extracorporeal therapy of acute liver failure and for bridging to liver transplantation . A summary of our development is discussed . A five-step technique for primary liver cell isolation has been introduced in order to address larger scale procurement of hepatocytes . Immobilisation of the cells after isolation appears to be one of the main factors in maintaining hepatocyte function in vitro . Different techniques have been investigated . Using the cell-cell adhesion technique, a culture model was developed for the immobilisation of hepatocytes between capillary membranes . Four separate capillary membrane systems, each forming independent compartments are woven in order to create a three dimensional network . A bioreactor design has been developed . The construction provides different functions, including decentralised cell perfusion . The bioreactor enables 3 dimensional reorganisation of cells, integral oxygenation and decentralised metabolite exchange . The bioreactor has been scaled-up to allow hepatocytes and sinusoidal endothelial cells to be cultured in quantities sufficient for therapeutic application . In a healthy pig model, possible limiting side effects of therapy with this device were excluded . The efficacy of the system has been demonstrated in a hepatectomised pig model . Subsequently, a complete hybrid liver support system for human studies was introduced and applied clinically.

Analyst, 1996 Nov, 121(11), 1695 - 8
Enzymic determinations with rotating bioreactors and continuous-flow-stopped-flow processing . Determination of choline esters in pharmaceuticals; Lopez Ruiz B et al.; The neuromuscular blocking agent, succinylcholine, and the neurotransmitter, acetylcholine, have been determined in pharmaceutical preparations utilizing a sensing unit comprised of a rotating bioreactor and an amperometric detector . The bioreactor consisted of a rotating disk made of Teflon (1 cm id) with cholinesterase (EC 3.1.1.8) and choline oxidase (EC 1.1.3.17) co-immobilized on the top surface of the disk . Amperometric detection of H2O2 and initial rate measurements were employed for quantification.

Virus Res, 1996 Nov, 45(1), 45 - 57
Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells; Hussy P et al.; The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses . The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion . Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA . E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1 . The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns . Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase . A isolated from bovine heart and gamma-{32P}ATP . Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients . Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

J Biotechnol, 1996 Oct 18, 51(1), 1 - 20
Bioreactor studies on temperature induction of the Q- mutant of bacteriophage lambda in Escherichia coli; Chen BY et al.; The parasite-host interactions between bacteriophage lambda (denoted as lambda) and Escherichia coli bacteria were studied in different bioreactor systems . Although the replicated lambda-DNA of Q- mutant remains naked for a longer time to reach a high gene expression, the epidemic of lambda-infection and the coevolutionary host-phage relations limit the temperature induction efficiency . The temperature induction is strongly dependent upon the susceptible population density at which lambda-infection is activated . Maximum beta-galactosidase expression occurs at the threshold of the infection system . According to this concept, the lethal level of parasitic lambda to hosts is approx . 5 x 10(6) pfu/ml . Since a higher phage lambda burden is exerted upon host cells at a low ODsh, the system moves towards virulence reduction for total survival . Prey-predator isocline analysis is used to consider the stability of the outcome of infection . The host growth has a destabilizing effect at lower population densities and a stabilization effect at higher population . Based upon the predictions, a substrate enrichment enhances bacterial growth and reporter protein production . However, the operations still need to follow the trajectory of threshold tie line to guarantee maximal productivity . Since the washout of infected cells reduces induction performance in continuous cultures, a batch mode of operation is better than continuous stirred tank reactor (CSTR) mode to achieve high gene expression . The threshold cell density regulates induction performances and therefore produces the optimal gene expression by maintaining maximal viable cells that provide sufficient resources for lambda expression.

Bull Acad Natl Med, 1996 Oct, 180(7), 1753 - 63; discussion 1764-7
{A step towards a bioartificial liver: temporary extracorporeal replacement using isolated hepatocytes}; Clement B et al.; The mortality rate of fulminant hepatic failure is about 80% . Besides orthotopic liver transplantation, specific therapies are not currently available . Indeed, not only removal of toxins is required, by means of dialysis or hemoperfusion, but specific hepatic functions must be provided to allow spontaneous liver regeneration or as a bridge before liver transplantation . Treatment with an extracorporeal bioartificial liver is an attractive approach . This system was successfully used for the correction of the Gunn rat genetic defect which results in the lack of bilirubin conjugation . The development of this system for clinical purpose requires both a large yield of functional hepatocytes and their immunoprotection in an appropriate device . We have isolated normal porcine hepatocytes by collagenase perfusion of the liver; cells are subsequently entrapped within membrane-coated alginate beads which are inoculated in a bioreactor . Plasma from an animal undergoing fulminant hepatic failure by end-to-site portocaval shunt and whole porta hepatic tightening circulates within the bioreactor . Porcine hepatocytes express liver-specific functions at high levels, particularly secretion of plasma proteins and several enzyme activities involved in the detoxication and biotransformation of xenobiotics . In addition, hepatocytes are immunoseparated from circulating immunoglobulins . Ethical concerns are discussed in the field of physiopathology, immunopathology and public health, as a prerequisite to create departments of Cell Therapy, in the near future.

J Biotechnol, 1996 Oct 1, 50(2-3), 171 - 80
Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein; Johansson T et al.; The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors . The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell . In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes . Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively . Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell . The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM) . Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography . This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.

Int J Artif Organs, 1996 Oct, 19(10), 610 - 6
Comparison of hollow fibre membranes for hepatocyte immobilisation in bioreactors; Gerlach JC et al.; Various hollow fibre membranes of polyamide, cellulose and polypropylene were investigated as potential substrata for hepatocyte immobilisation in bioreactors for hybrid liver support systems . Membranes were subjected to a cytocompatibility test in which the attachment and morphology of primary hepatocytes were evaluated . The effect of coating with collagen and fibronectin was also studied . Adequate cell immobilisation was possible on polypropylene and polyamide membranes even without coating . The flattening process of the cells was dependent on the material and the coating . The incorporation of porous polypropylene hollow fibres in hybrid liver cell bioreactors and their specific permeability properties could also offer means for cell oxygenation, metabolite distribution and immuno-isolation purposes.

Int J Artif Organs, 1996 Oct, 19(10), 605 - 9
Comparisons of porcine hepatocyte spheroids and single hepatocytes in the non-woven fabric bioartificial liver module; Naruse K et al.; We previously developed a new bioreactor of the bioartificial liver composed of non-woven fabric . We have also experimented with hepatocyte spheroids, with the aim of improving the efficiency of this NWF bioreactor . In this study, we compared the efficiencies of NWF bioreactors employing hepatocyte spheroids versus single hepatocytes . Hepatocytes were isolated from a whole pig liver by Seglen's method . 1.0 x 10(10) single hepatocytes were immobilized in the NWF bioreactor . Another 1.0 x 10(10) hepatocytes were allowed to form spheroids by 24 hr suspension culture in a 4-L culture vessel, before being immobilized in the bioreactor . Hepatocyte spheroids were found to be functionally superior, on a per-cell basis, to single hepatocytes in the NWF bioreactor . However, the NWF bioreactor employing hepatocyte spheroids exhibited lower efficiency than thaT employing single hepatocytes, because the total number of the hepatocytes had decreased during the 24 hr suspension culture.

J Hematother, 1996 Oct, 5(5), 475 - 83
Production of human natural killer cells for adoptive immunotherapy using a computer-controlled stirred-tank bioreactor; Pierson BA et al.; Large-scale ex vivo expansion of human natural killer (NK) cells for adoptive immunotherapy requires assurance of good manufacturing practices . However, maximal expansion of NK is also desired to facilitate clinical trials with large numbers of IL-2-activated NK (ANK) . A closed-system stirred-tank bioreactor is amenable to computer control of culture variables, thereby reducing the risk of contamination . We demonstrate that NK cultured in 250-ml spinner flasks expand 2.5-fold more than NK cultured in stationary tissue culture wells . We further show that during 33 days of culture, it is feasible to control the pH between 7.0 and 7.2 and the dissolved oxygen concentration at 40% of air saturation via direct on-line computer control in a 750-ml stirred-tank bioreactor . On-line measurement of optical density by a laser turbidity sensor, as a measure of cell concentration, correlated well with actual cell count data . NK expansion in the 750-ml bioreactor was 7-fold greater than in stationary tissue culture controls and 3-fold greater than in spinner flask controls . Consumption rates of glucose and oxygen and the production rate of lactate were measured and will be used to develop a nutrient feeding strategy to convert the batch reactor experiment into closed-system fed-batch or continuous flow modes . Computer-controlled stirred-tank bioreactors may facilitate clinical trials with high-purity ANK populations for adoptive immunotherapy.

J Lab Clin Med, 1996 Oct, 128(4), 408 - 16
Biochemical and 31P-NMR spectroscopic evaluation of immobilized perfused rat Sertoli cells; Farghali H et al.; Cell immobilization and perfusion are used for physiologic studies of Sertoli cells with phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy and biochemical methods . In this study the 31P NMR spectra of Sertoli cells isolated from 18-to 21-day-old rats and immobilized in agarose threads continuously perfused with oxygenated Dulbecco's modifed Eagle medium were obtained at 81 MHz on an NMR system . Cytosolic Ca2+, intracellular Mg2+, lactate and pyruvate, and oxygen consumption were measured with standard biochemical methods . Perfused Sertoli cells maintain a stable intracellular adenosine triphosphate concentration for more than 10 hours . Sertoli cells placed in cold storage overnight and then subjected to perfusion partially regenerate cellular adenosine triphosphate levels . Sertoli cells consume an average of 4.8 +/- 0.4 nmol O2/min/10(6) cells and maintain average ambient lactate and pyruvate levels of 7.1 +/- 0.8 mg/dl and 0.65 +/- 0.05 mg/dl, respectively, with a lactate/pyruvate ratio in the range 8 to 12 . The basal Ca2+(i) of Sertoli cells is 98 +/- 0.7 nmol/L (n = 58), which declines to a level less than 10 nmol/L when the Sertoli cells are perfused with a calcium-free medium . Perfusion of Sertoli cells with a sodium-free medium, with 10(-6) mol/L carbonyl cyanide P-trifluoromethoxy-thenylhydrozone, or with Ca2+ ionophore A23187 at a concentration of 10(-6) mol/L increases the Ca2+(i) to a level of 426 +/- 107 nmol/L, 274 +/- 29 nmol/L, or 282 +/- 57 nmol/L, respectively . A bioreactor for physiologic studies of Sertoli cells in real time with NMR spectroscopy has been developed . These data demonstrate that isolated, immobilized, and perfused Sertoli cells are stable for prolonged periods . In addition, these data suggest that Sertoli cells possess a functional Na+-Ca2+ antiporter and that they sequester extracellular Ca2+ in one or more intracellular compartments.

J Immunol Methods, 1996 Sep 9, 195(1-2), 93 - 101
High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies; Kretzschmar T et al.; We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h . The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix . This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98% . The final yield was 32 mg/l (10(9) cells/l) . Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion . The dissociation constant of the scFv monomers is about 1 x 10(-4) M . By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M . Marks et al . (1991) J . Mol . Biol . 222, 581-597: Marks et al . (1992) Bio/Technology 10, 779-783) . This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E . coli expression.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Sep-Oct, (5), 49 - 53
{The prospects for using the genetically engineered surface antigen of the hepatitis B virus (HBsAg) expressed by recombinant poxvirus strains as the basis for vaccines against hepatitis B}; Zheleznova NV et al.; The use of a recombinant poxvirus (RPV) strain, expressing HBsAg in the process of reproduction in different bioreactor systems under stationary and bioreactor conditions of cultivation, made it possible to obtain highly purified HBsAg . The identity and purity of HBsAg was confirmed by the analysis of its amino acid composition, SDS electrophoresis in polyacrylamide gel, electron microscopy and high-performance liquid chromatography . Good prospects of the use of RPV-expressed gene engineering HBsAg as the basis vaccines against hepatitis B was demonstrated in 10 experimental batches of vaccine . All batches of the preparation had pronounced immunogenicity and were safe and nontoxic in animal experiments . The ID50 of experimental batches did not exceed 211 ng/ml, which, according to the data of comparative experiments, was lower than, or equal to, corresponding values of analogous foreign commercial preparations, based on plasma or yeast HBsAg.

Enzyme Microb Technol, 1996 Sep, 19(4), 261 - 6
Control of pellet morphology of filamentous fungi in fluidized bed bioreactors by means of a pulsing flow . Application to Aspergillus niger and Phanerochaete chrysosporium; Moreira MT et al.; The application of a pulsing flow to fluidized-bed bioreactors in order to control pellet morphology of filamentous fungi was investigated . The operation at an optimum pulsation frequency allowed two effects: a narrower pellet size distribution which improves fluidization quality, and an enhanced production of citric acid by Aspergillus niger and manganese peroxidase by Phanerochaete chrysosporium . In the case of A . niger, the pellet diameter corresponding to the pulsed system operated at 0.35 s-1 was kept between 3.3 +/- 0.1 mm after 22 days of operation; however, in the nonpulsed bioreactor which was operative for only 11 days, pellets with a diameter of 6.7 +/- 0.3 mm were produced . Similar results were obtained in the case of P . chrysosporium, since with a pulsing frequency of 0.0625 s-1, a pellet diameter of 2.1 +/- 0.4 mm after 34 days of operation was maintained . On the contrary, the system without pulsation presented great conglomerates of mycelia with an average diameter of 3 cm surrounded by free pellets with a diameter distribution of 2.75 +/- 0.5 mm . The nonpulsed bioreactor was only operative for 14 days . Both citric acid and manganese peroxidase production attained higher values and were maintained longer in the pulsed bioreactor.

Enzyme Microb Technol, 1996 Sep, 19(4), 256 - 60
Kinetics of taxol production and nutrient use in suspension cultures of Taxus cuspidata in shake flasks and a Wilson-type bioreactor; Pestchanker LJ et al.; Suspension cultures of Taxus cuspidata were grown in shake flasks and Wilson-type reactors where bubbled air provided both agitation and mixing . Taxol titers of 22 mg l-1 were achieved for both configurations in 20 days for a volumetric productivity of 1.1 mg l-1 day-1 . This productivity is many-fold higher than reported for other Taxus sp . suspension cultures . Taxol was released to the extracellular medium as it was produced with little intracellular retention (< or = 10%) . Taxol production occurred during the last seven days of the cultivation period and was not growth-associated . Although the same taxol titers could be obtained in both reactor types, nutrient uptake rates were faster in the Wilson-type reactor than in shake flasks . Formation of a growth ring in the Wilson-type reactor reduced measured cell mass yields.

Appl Biochem Biotechnol, 1996 Sep, 60(3), 303 - 13
Continuous wine making by delignified cellulosic materials supported biocatalyst . An attractive process for industrial applications; Iconomou L et al.; The continuous making of wine by a delignified cellulosic (DC) material-supported biocatalyst is reported . It was prepared by immobilizing the alcohol resistant strain AXAZ-1 on DC material . The product was found suitable for the continuous process in industrial applications . The operational stability was maintained for 2 mo with monitoring the ethanol concentration, wine, and alcohol productivities as well as the stability of oBe density at the outlet . Wine productivity was three- to sixfold higher than obtained by natural fermentation, alcohol concentrations of the wine was in the range of 9.3-11.2% v/v and low volatile acidities of 0.15-0.36 g acetic acid/L were obtained . The effect of total acidity and flow rate of must were also examined . To demonstrate that the operational stability of the bioreactor is due to DC material that promotes the fermentation, and it takes place at even higher ethanol levels, an analogous system of kissiris supported biocatalyst was studied . Likewise, the tolerance in the alcohol concentration, as compared with free cells, were studied by their stability of the activity in the repeated batch fermentation of must.

Cell Transplant, 1996 Sep-Oct, 5(5 Suppl 1), S65 - 9
Application of a novel B cell line MIN6 to a mesh-reinforced polyvinyl alcohol hydrogel tube and three-layer agarose microcapsules: an in vitro study; Hayashi H et al.; This study examines the function of a novel B cell line (MIN6) enclosed in hybrid bioartificial pancreas with mesh-reinforced polyvinyl alcohol hydrogel tube (MRPT) or with improved, three-layer agarose microcapsules . MIN6 was established from insulinomas obtained by targeted expression of the simian virus 40 T-antigen gene in transgenic mice . MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations . The MRPT and the three-layer agarose microcapsules, which were developed to act as an artificial pancreas, were readily permeated by insulin, glucose, and other nutrients . Both can immunoisolate enclosed MIN6 cells from the recipient's humoral and cellular immunosystems, which causes a xenogeneic rejection response . MIN6 cells (5.0 x 10(6) or 1.5 x 10(6)) were enclosed in MRPT or in a hundred three-layer microcapsules and subjected to an in vitro perifusion study or a static incubation study to observe the insulin release from each bioartificial pancreas in response to glucose stimulation . In vitro study revealed that the insulin secretion in response to 16.7 mM glucose stimulation was twice that with 3.3 mM glucose stimulation with both MRPT and the three-layer agarose microcapsules . The present study demonstrates that MIN6 effectively functions as a bioreactor for the hybrid bioartificial pancreas . The application of MIN6 cells to the hybrid bioartificial pancreas may offer a solution to the current serious dearth of organs.

Cell Transplant, 1996 Sep-Oct, 5(5 Suppl 1), S35 - 7
Immobilization of rat hepatocytes on multiporous microcarriers with larger pores and their metabolic activity; Kino Y et al.; We have investigated the availability of multiporous microcarriers (MCs) for immobilizing isolated rat hepatocytes, but the pore size of MCs was too small (35 microns) for hepatocyte immobilization . In this study, we immobilized isolated rat hepatocytes on MCs with larger pores, and evaluated their metabolic activity . Isolated hepatocytes were immobilized on MCs precoated with collagen by the intermittent stirring method and by aspiration, and the cell-protein content per 100 mg MCs was determined for comparison of these methods . Metabolic activity was evaluated by analyzing NH3 metabolism, urea nitrogen synthesis and glucose synthesis . The aspiration method immobilized significantly more of hepatocytes on MCs than the intermittent stirring method (p < 0.05) . A stationary culture of hepatocytes immobilized on MCs showed a similar NH3 metabolism to monolayer cultured hepatocytes, and hepatocytes immobilized on MCs in a floating culture showed significantly higher NH3 metabolism than those in a stationary culture (p < 0.01) . However, monolayer cultured hepatocytes showed higher glucose synthesis than hepatocytes immobilized on MCs in a stationary culture (p < 0.01) . In conclusion, hepatocytes immobilized on MCs proved to be useful as a bioreactor in a hybrid artificial liver.

Trends Biotechnol, 1996 Sep, 14(9), 341 - 9
Hematopoietic cell culture therapies (Part I): Cell culture considerations; McAdams TA et al.; Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, is a rapidly growing area of tissue engineering with many potential applications in bone-marrow transplantation, gene therapy and the production of blood products . Hematopoietic cultures are considerably more complex than established animal cell culture technologies owing to the presence of many cell types at various differentiation stages, and stringent medium and growth factor requirements . Culture parameters, such as pH, oxygen tension, seeding density and feeding schedules, significantly affect the proliferation and differentiation of hematopoietic cells . A number of bioreactor systems are currently under development.

Appl Environ Microbiol, 1996 Sep, 62(9), 3292 - 7
Biodegradation of organic wastes containing surfactants in a biomass recycle reactor; Konopka A et al.; The microbial biodegradation of simulated graywater, containing 21.5 mg of linear alkylbenzene sulfonate liter-1, was investigated with a continuous-flow bioreactor with 100% biomass recycle . Low concentrations of organic matter in the ultrafiltration eluate were achieved by hydraulic residence times as short as 1.6 h and for periods of up to 74 days at a hydraulic residence time of 6 h . Upon a shift from the chemostat to the biomass recycle mode, the increase in biomass with time approximated a linear rather than an exponential function . Biomass densities as high as 6.8 g of cell protein liter-1 were reached; this was 50-fold higher than the steady-state biomass level in chemostats fed the same medium . We assessed physiological changes in the microbial community after a switch from the chemostat to the biomass recycle mode . Over 150 h, there was a two- to fourfold decrease in the respiratory potential of the microbes . After this decrease, respiratory potentials were relatively constant up to 74 days of operation . A decline in reactivity was also indicated by increasing lag periods before growth in response to organic nutrient inputs and by a decrease in the proportion of cells able to reduce tetrazolium dye . However, the bioreactor system was still capable of rapidly metabolizing inputs of organic matter, because of the very high biomass concentrations . It appears that < 10% of the organic carbon inputs accumulate as biomass.

Microbiology, 1996 Aug, 142 ( Pt 8), 2299 - 310
Physiological effects of nitrogen starvation in an anaerobic batch culture of Saccharomyces cerevisiae; Schulze U et al.; The effects of nitrogen starvation on the anaerobic physiology of Saccharomyces cerevisiae were studied in cells cultivated in a bioreactor . The composition of the mineral medium was designed such that the nitrogen source became depleted while there was still ample glucose left in the medium . The culture was characterized by acoustic gas analysis, flow injection analysis and HPLC analysis of extracellular substrates and metabolites . During the cultivation, the macromolecular composition of the cells was analysed with respect to the cellular content of RNA, protein, trehalose and glycogen . During exponential growth under anaerobic conditions, the maximum specific growth rate conditions . Depletion of ammonium in the medium led to an abrupt decrease (mumax) of S . cerevisiae CBS 8066 (0.46 h-1) was identical to the mumax determined under aerobic in the flux through glycolysis . Subsequently, a continuous decrease in the carbon dioxide evolution rate, caused by catabolite inactivation of the hexose-transport system, was observed . The apparent half-life of the transport system under nitrogen starvation was 13 h . During the exponential growth phase, the cellular content of RNA and protein was 15% (w/w) and 60% (w/w), respectively . At the end of the cultivation where the cells had been starved of nitrogen for 18 h, the cellular content of RNA and protein had decreased to 4% (w/w) and 22% (w/w), respectively . The intracellular carbohydrate content increased dramatically as trehalose and glycogen accumulated to final concentrations of 7% (w/w) and 25% (w/w), respectively . Glycerol formation during nitrogen starvation was higher than that accounted for by the formation of organic acids, suggesting a protein turnover of approximately 6% h-1 . The growth energetics of S . cerevisiae CBS 8066 also changed as a result of nitrogen starvation, and YxATP was observed to increase from 80 mmol g-1 during the exponential growth phase to more than 130 mmol g-1 towards the end of the cultivation . The presented results illustrate the effect of nitrogen starvation on glycerol formation, protein turnover, catabolite inactivation of the sugar-transport system, the cellular composition, the cell cycle and growth energetics.

Aust N Z J Surg, 1996 Aug, 66(8), 547 - 52
Preclinical trial of a bioartificial liver support system in a porcine fulminant hepatic failure model; Sheil AG et al.; BACKGROUND: This study describes the pre-clinical trials of an extracorporeal bioartificial liver support system (BALSS) . It includes the biochemical changes which occur in the plasma and blood of pigs with devascularized livers when the plasma is treated in a BALSS, and the testing of the system for presence or absence of infective agents, pyrogens and for toxicity . METHODS: Hepatic cells were prepared from littermate juvenile white landrace pigs with a double-step collagenase digest technique . The cell preparations were incubated with collagen-coated dextran microspheres (CDM) for 3 h and the medium was tested to determine cellular metabolic activity . Incubation continued for a further 20 h during which the hepatic cells attach to the CDM . The CDM-attached cells were inoculated into a hollow fibre bioreactor which was part of an extracorporeal liver support system . RESULTS: Hepatic cell content of the bioreactor was 6 x 10(9) +/- 3 x 10(8) cells, equivalent to those present in half a pig's liver . The system was tested in a controlled trial with the plasma of pigs with fulminant hepatic failure (FHF) due to devascularized livers . When plasma from FHF pigs was circulated through the device there was significantly less of an increase in the accumulation of ammonia, lactate and most amino acids when hepatic cells were included in the circuit compared with those in control experiments when they were excluded . Similar changes occurred in procine blood . There were few infections diagnosed and an absence of pyrogens, endotoxins and toxicity in the bioreactor contents or in the terminating reservoir or animal blood samples . CONCLUSIONS: We believe that the results, demonstrating function of the porcine hepatic cells in the circuit, together with low risks, justify a clinical trial of use of the BALSS in Australia.

Appl Environ Microbiol, 1996 Aug, 62(8), 2919 - 25
Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene; Saito Y et al.; A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid . Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity . The activity of the enzyme was reduced to 76% of the original by oxidation . From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity . To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid . In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system . These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.

Transplantation, 1996 Jul 27, 62(2), 224 - 8
Is a clinical application of hybrid liver support systems limited by an initial disorder in cellular amino acid and alpha-keto acid metabolism, rather than by later gradual loss of primary hepatocyte function?
Gerlach JC, Fuchs M, Smith MD, Bornemann R, Encke J, Neuhaus P, Riedel E.
The in-vitro amino acid (AA) and alpha-keto acid (KA) metabolism of bioreactors initially seeded with 2.5 x 10(9) pig hepatocytes was investigated with a perfusion technique . Considerable changes in the culture medium concentrations of AA and KA were measured during the first days in culture . This is indicative of dynamic cellular metabolism in the initial phase . While the concentration of pyruvate decreased starting on the first day, alpha-ketoglutarate, alpha-ketoisocaproate, alpha-ketoisovalerate, and alpha-keto-beta-methyl-n-valerate were synthesized . The long term use of hepatocyte cultures in bioreactors and thus a desirable clinical hybrid liver support therapy appears to be possible since the hepatocytes switched, after 15 days in culture, to steady-state conditions with a stable amino acid turnover featuring general AA uptake accompanied by KA release . The release of branched chain KA, in particular that of alpha-ketoisocaproate, reflects an effective transamination activity in the bioreactor system . Primary pig hepatocytes cultivated in hybrid liver support systems for therapy of acute liver failure or as devices for bridging to liver transplantation can sustain amino acid metabolism for at least 30 days in vitro . However, an initial disorder following the cell isolation that is demonstrated may limit immediate utilization of the systems prior to the reorganisation of the cells to tissue-like structures in bioreactors.

J Biotechnol, 1996 Jul 18, 48(1-2), 153 - 9
Construction of a bioreactor to produce special breakdown products of phytate; Greiner R et al.; Escherichia coli phytase was covalently immobilized on NHS-activated Sepharose High Performance . The pH dependence of the phytase activity was not influenced by immobilization, whereas stability against heat treatment was enhanced as a consequence of immobilization . Compared to the free phytase the immobilized enzyme exhibits the same excellent substrate specifity, but showed an increased Km-value . Using the immobilized phytase in a packed-bed bioreactor makes special isomers of the lower myo-inositol phosphate esters available . The major isomers formed were identified as I(1,2,3,4,5)P5, I(2,3,4,5)P4, I(2,4,5)P3 and I(2,5)P2.

J Biotechnol, 1996 Jul 18, 48(1-2), 107 - 16
Kinetics of continuous GM-CSF production by recombinant Saccharomyces cerevisiae in an airlift bioreactor; Shu CH et al.; Continuous production of murine GM-CSF by recombinant Saccharomyces cerevisiae in an airlift bioreactor was studied at three different dilution rates . The reactor was initially fed with a selective medium to increase cell concentration, and then was fed with a rich, nonselective medium for GM-CSF production . Ethanol was used as the main carbon source to provoke GM-CSF expression . In continuous culture, GM-CSF production was maintained for over 150 h, even though the fraction of plasmid-carrying cells continuously dropped to lower than 20% . The stable GM-CSF production during the later phase of the continuous culture was attributed to increased specific cell productivity possibly resulting from an increase in the plasmid copy number in plasmid-carrying cells . This also indicated the possibility of natural selection of high-copy number cells in continuous culture . Plasmid stability was found to be growth rate (dilution rate) dependent; it increased with the dilution rate . Reactor productivity and specific productivity also increased with the dilution rate . A two-parameter kinetic equation was used to model the plasmid stability kinetics . The growth rate ratio between plasmid-carrying and plasmid-free cells increased from 0.996 to 0.998 while the segregational instability or the probability of plasmid loss from each cell division increased from 1.1% to 16% as the dilution rate decreased from 0.11 h-1 to 0.05 h-1 . Oscillation of the dilution rate between 0.05 h-1 and 0.11 h-1 stabilized the plasmids and gave a higher productivity than that achieved without oscillation.

J Biotechnol, 1996 Jul 18, 48(1-2), 43 - 9
Morphological analysis of yeast cells using an automated image processing system; Zalewski K et al.; In order to control the growth of the yeast Saccharomyces cerevisiae during cultivations, a fully-automated sampling and analysing system has been developed . Based on the microscopic appearance of cells, a detailed characterization of yeast suspensions from the bioreactor is available, presenting data of current cell concentrations, distributions of process-dependent cell structures and parts of cells containing vacuoles . This measuring system combines quantitative and qualitative analysis methods in a digital image processing technique, which offers the possibility of on-line estimation of cell development that approximates real-time . Perturbations within the process are detectable in time and regulative actions can be done without any delay.

J Ind Microbiol, 1996 Jul, 17(1), 53 - 5
Effect of hydromechanical stress on cellular antigens of Bordetella pertussis; Rodriguez ME et al.; Cells of Bordetella pertussis grown in a bioreactor under stirring conditions were studied to investigate the effect of shear stress on cellular-bound filamentous haemagglutinin (FHA) . FHA attached to the bacterial surface, unlike extracellular FHA, was not affected at the shear levels tested . Moreover, no other cellular immunogen involved in the whole-cell protective activity seemed to be affected by hydromechanical forces.

Biotechnol Prog, 1996 Jul-Aug, 12(4), 449 - 56
Kinetics and stability of GM-CSF production by recombinant yeast cells immobilized in a fibrous-bed bioreactor; Yang ST et al.; The continuous production of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by recombinant yeast cells immobilized in a fibrous-bed bioreactor was studied . A high cell density of approximately 68 g/L and a GM-CSF productivity of approximately 3.5 mg/L.h were attained in the fibrous-bed bioreactor-fed with a rich (nonselective, pH 6.7) medium at a dilution rate of 0.16 h-1 . The GM-CSF production was stable even though the fraction of plasmid-carrying cells in the reactor effluent gradually dropped below 5% over a period of 2 weeks . At the end of that period, the immobilized cells in the fibrous matrix still had a high fraction, approximately 26%, of plasmid-carrying cells . Similar results were obtained with reactors operated at 0.05 h-1 dilution rate and pH 4.0 . Although the GM-CSF production was lower at pH 4, the reactor was stably operated for over 4 weeks without contamination or significant loss of productivity . The stable long-term GM-CSF production from the fibrous-bed bioreactor was attributed to the effect of cell immobilization on plasmid stability . Because GM-CSF production was growth-associated, as was found in batch fermentation with free cells, this stabilization effect cannot be attributed solely to the reduced cell growth in the immobilized cell environment . Plasmid-carrying cells were preferentially retained in the fibrous matrix, perhaps because their abilities to adhere to the fiber surface and to form cell aggregates were higher than those of plasmid-free cells.

Hokkaido Igaku Zasshi, 1996 Jul, 71(4), 543 - 55
{Metabolic evaluation of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients--basic study on development of a bioreactor in hybrid artificial liver.}; Matsue H; In order to develop a functional bioreactor for hybrid artificial liver, function of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients (group III, n = 5) was investigated . Culture media, Leibovitz L-15 medium (group I, n = 7) and normal human plasma (group II, n = 3), were used as controls . Morphologically, no degeneration of porcine hepatocytes in hepatic failure plasma was observed for 5 days in culture . Levels of ureogenesis showed no significant difference against the controls at day 1, 2, 5 in culture, but the level at day 3 was significantly higher than that of group II . Levels of gluconeogenesis showed a close tendency as those of ureogenesis, but the level at day 3 was significantly lower than that of group I . Levels of intracellular DNA contents, showing between 1.85 +/- 0.39 and 1.35 +/- 0.05 microgram/cm2, were compatible with those of controls during first three days in culture, but level of group III at day 5 was significantly higher than that of group I . After incubation of porcine hepatocyte in hepatic failure plasma, the amount of valine, leucine, isoleucine, glutamine, arginine, and citrulline was significantly decreased . Elevated phenylalanine and tyrosine were also decreased, but Fischer's ratio, the ratio of branched chain amino acid against aromatic amino acids, was not significantly increased . Data obtained by this investigation showed that cultured porcine hepatocytes held proper hepatic function in the hepatic failure plasma . It is concluded that culture porcine hepatocyte monolayers were a promising candidate for a bioreactor of a hybrid artificial liver.

Appl Biochem Biotechnol, 1996 Jul, 60(1), 1 - 17
Immobilization of manganese peroxidase from Lentinula edodes on alkylaminated Emphaze AB 1 polymer for generation of Mn3+ as an oxidizing agent; Grabski AC et al.; Manganese peroxidase (MnP) is secreted by white-rot fungi and participates in the degradation of lignin by these organisms . MnP uses H2O2 as an oxidant to oxidize MnII to MnIII as the manganic ion Mn3+ . The Mn3+ stabilized by chelation, is a highly reactive nonspecific oxidant capable of oxidizing a variety of toxic organic compounds . Previous attempts at immobilization of MnP, purified from Lentinula edodes through reactive amino groups, have been hindered by the protein's low lysing content of only 1% and its instability above pH 6.0 . As an alternative to amine coupling, the enzyme has now been covalently immobilized through its carboxyl groups, using an azlactone-functional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent . The immobilization reaction was performed under acidic (pH 5.25) conditions, and 90% coupling efficiency was achieved within 2h . Net immobilization efficiencies, expressed as the product of protein coupling efficiency and enzyme activity, have been measured at > 95% within 4h . The MnP-NH-polymer and the free soluble protein were characterized and compared for their pH, temperature, and storage stabilities, as well as their H2O2 dependence and kinetics . The tethered MnP, employed in an immobilized enzyme bioreactor for generation of chelated Mn3+ may have industrial applications as a nonspecific oxidant of organopollutants.

J Biotechnol, 1996 Jun 27, 47(2-3), 113 - 27
Cultivation of Saccharomyces cerevisiae in a bioreactor in microgravity; Walther I et al.; Yeast cells were cultured for 8 d in a newly developed bioreactor during the Spacelab IML-2 mission . Two bioreactors, one stirred and one without stirring, were installed in the Biorack facility in space . Two control units were installed in the Biorack module at the Kennedy Space Center . Samples were drawn on mission day 3, 5, 6, 7 and 8 and preserved either by freezing or chemically fixed for post-flight analysis . The values of pH, pH regulation, temperature and redox potential were transmitted on-line to the ground station throughout the mission . The performance of the bioreactor was satisfactory except for a partial failure of the medium micropump . Despite the failure of the pump, the data support the following conclusions: There is a significant difference in the distribution of the bud scars between cells cultured at 0 x g and at 1 x g . The percentage of randomly distributed bud scars was significantly higher in the flight (17%) than in the ground control cells (5%) . No remarkable differences were noted in the cell cycle, ultrastructure, cell proliferation, cell volume, ethanol production and glucose consumption.

Biotechnol Appl Biochem, 1996 Jun, 23 ( Pt 3), 221 - 6
Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: I: Preparation and in vitro characterization; Jean-Francois J et al.; The feasibility of the immobilization of Escherichia coli L-asparaginase into a hydrogel matrix made of poly-(ethylene glycol) (PEG) and BSA was demonstrated . After immobilization a 200-fold increase in the Km value was observed . The use of an L-aspartic acid analogue, carbobenzoxy-L-aspartic acid and surface modification by methoxy-PEG of molecular mass 5 kDa cause a only a slight gain in affinity of the enzyme for its natural substrate . The immobilized L-asparaginase has an optimal activity over a larger range of pH than the native enzyme, owing to the effect of the matrix . At a physiological pH of 7.3, the immobilized enzyme retained 90% of its activity compared with only 43% for the native form . The immobilized enzyme retained a high proportion of its initial activity, more than 90% after 50 days of incubation at 37 degrees C, even in the presence of its substrate . This may be compared with a half-life of 2 days observed for native enzyme incubated under the same conditions . These results suggest that the BSA-PEG matrix can be very useful for enzyme immobilization and, taking into account the good biocompatibility of the matrix, one can expect that this matrix will provide a functional bioreactor for use in vivo.

Biochim Biophys Acta, 1996 May 23, 1294(2), 195 - 203
The pressure-dependence of two beta-glucosidases with respect to their thermostability; Hamon V et al.; A comparative study of temperature and pressure effects were carried out by using two homologous enzymes exhibiting different thermostability and oligomery: almond beta-glucosidase and Sulfolobus solfataricus beta-glucosidase . Both the activity and stability were studied using an in-house built bioreactor allowing injection, stirring, sampling and on-line spectrophometric monitoring with retention of pressure up to 2.5 kbar and temperature control possible up to 150 degrees C . Almond beta-glucosidase, the most pressure sensitive enzyme of the two was continuously affected by pressure up to 1.5 kbar . Activation volume changes revealed that the inactivation of almond beta-glucosidase was due to both catalytic step inactivation and enzyme-substrate binding inactivation . Structural modifications generated by pressure, related to a loss of activity did not affect the global conformation of almond beta-glucosidase, after depressurization . In contrast, Sulfolobus solfataricus beta-glucosidase was a highly barostable enzyme . It maintained a half-life of 91 h at 60 degrees C and 2.5 kbar . Moreover, this enzyme appeared to be activated by pressure between atmospheric pressure and 2.5 kbar with a maximal activity at 1.25 kbar . However, this enzyme still displayed the best catalytic efficiency at atmospheric pressure because of a Km value drastically increased by pressure . Activation volume changes indicated that the hydrolysis reaction catalysed by Sulfolobus solfataricus beta-glucosidase, was alternatively favoured and disfavoured by pressure due to the catalytic step activation or inactivation associated with the enzyme-substrate binding step being continuously inactivated by pressure.

Ann N Y Acad Sci, 1996 May 15, 782, 87 - 96
Rate limitations in posttranslational processing by the mammary gland of transgenic animals; Subramanian A et al.; Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs . Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM . Gamma carboxylation is a vitamin K-dependent posttranslational modification . The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied . We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression . Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes . Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively . Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively . A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC . Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC . Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response . This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Ann N Y Acad Sci, 1996 May 15, 782, 391 - 401
Bioprocessing with genetically modified and other organisms: case studies in processing constraints; Moo-Young M et al.; Whereas the gene stability related considerations are important in bioprocessing with recombinant cultures, bioreactor design and scale-up require attention to the often reduced shear tolerance of the genetically altered biocatalysts relative to the corresponding wild strains . In addition, the peculiarities of expression of the rDNA product impact the downstream recovery methods . As a consequence, a bioprocessing scheme and the process machinery designed for a naturally occurring organism may need significant modifications for use with a genetically modified variety of the same organism . The case studies described highlight some of the processing constraints and consideration of general relevance.

Enzyme Microb Technol, 1996 May 1, 18(6), 392 - 416
Bioreactors with immobilized lipases: state of the art; Balcao VM et al.; This review attempts to provide an updated compilation of studies reported in the literature pertaining to reactors containing lipases in immobilized forms, in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject . Highlights are given to industrial applications of lipases (including control and economic considerations), as well as to methods of immobilization and configurations of reactors in which lipases are used . Features associated with immobilized lipase kinetics such as enzyme activities, adsorption properties, optimum operating conditions, and estimates of the lumped parameters in classical kinetic formulations (Michaelis-Menten model for enzyme action and first-order model for enzyme decay) are presented in the text in a systematic tabular form.

Artif Cells Blood Substit Immobil Biotechnol, 1996 May, 24(3), 201 - 18
Microencapsulated genetically engineered E . coli DH5 cells for plasma urea and ammonia removal based on: 1 . Column bioreactor and 2 . Oral administration in uremic rats; Prakash S et al.; We report a novel approach for plasma urea and ammonia removal using artificial cells microencapsulated genetically engineered bacteria E . coli DH5 cells . This has been evaluated for use in a column bioreactor for removing plasma urea and ammonia . It has also been evaluated in uremic rats for urea removal by oral administration . In 30 minutes, microencapsulated E . coli DH5 in a column bioreactor in-vitro lowered plasma urea to 10.47 +/- 3.45 mg/dl from 45.85 +/- 2.98 mg/dl and lowered plasma ammonia concentration to 46.00 +/- 4.00 microM from 679 +/- 32 microM/1 . The efficiency of this bioreactor for plasma urea and ammonia removal is much higher than any other available methods . Initial plasma urea and ammonia concentration does not affect the plasma urea and ammonia removal efficiency of the column bioreactor . In in-vivo studies of oral administration to uremic rats, both free and encapsulated bacteria both lowered systemic urea from the initial 52.08 (S.D.2.37) mg/dl to 10.58 (S.D.0.85) mg/dl . Unlike free bacteria, microencapsulated bacteria was not retained in the intestine.

Ann Biomed Eng, 1996 May-Jun, 24(3), 373 - 81
Determination of specific oxygen uptake rates in human hematopoietic cultures and implications for bioreactor design; Peng CA et al.; Oxygen plays an important role in the cultivation of primary cells ex vivo . In this study, we used hermetically sealed tissue culture well inserts equipped with oxygen electrodes to measure the oxygen utilization of cultured human bone marrow mononuclear cells (BM MNCs) . The oxygen uptake rate (OUR) of BM MNCs was determined during a 14-day culture in which both adherent and nonadherent cells were present . Early in the culture, the cells exhibited very low OURs . The specific OURs (uptake rate per cell) were at approximately 0.005 mumol/10(6) cells/hr shortly after the initiation of culture . The OUR then increased as the cultures developed . After about 8 to 10 days of cultivation the specific OURs had increased to 0.038 +/- 0.006 and 0.025 +/- 0.005 mumol/10(6) cells/hr for adherent and nonadherent cells, respectively, after which no further increase was observed . Based on these oxygen uptake rate data, a mathematical model of oxygen diffusion was formulated and use to investigate issues associated with hematopoietic bioreactor design, including initial cell density, medium depth, reactor configuration, and oxygen partial pressure . In situ OUR measurements confirmed predicted oxygen limitations based on the mathematical model and the experimentally determined OURs . High-density hematopoietic cultures present design challenges in terms of sufficient and uniform delivery of oxygen to an active hematopoietic culture . These challenges can be met by using parallel-plate bioreactors with thin liquid layers.

Artif Organs, 1996 May, 20(5), 396 - 402
Ex vivo manipulation of cell subsets for cell therapies; Nordon RE et al.; Large-scale cell separation and ex vivo expansion technologies will form the basis for development of new cellular products for the treatment of cancer and fatal viral diseases . The cell subsets that are likely to play a significant role in cellular therapy include hematopoietic stem cells, platelet and granulocyte precursors, cytotoxic lymphocytes, and genetically modified hematopoietic or lymphoid precursors . Cell enrichment techniques are required to eliminate tumor cells from autologous stem cell grafts and to reduce the size of culture systems required for expansion or gene transfection . The consumption of expensive culture components such as cytokines and serum may be reduced by the use of perfusion bioreactor devices . Methods that have been developed for the production of cell subsets for cellular therapy are reviewed.

Biosci Biotechnol Biochem, 1996 May, 60(5), 811 - 7
Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium; Inoue Y et al.; A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells . We found that the ras-rBHK-IgG cell could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite . The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium . True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system . After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

Anticancer Res, 1996 May-Jun, 16(3B), 1393 - 7
Phosphomonoester metabolism as a function of cell proliferative status and exogenous precursors; Aiken NR et al.; Elevations of phosphomonoesters (PMEs) correlate with increased cell growth or increased cell degradation, and have been shown to occur in human tumors as well as animal tumor models and cell lines . Furthermore, decreased PME levels have been observed in tumor patients who respond to therapy . Therefore, understanding the mechanisms underlying the interactions of intrinsic and extrinsic control of PMEs may assist diagnosis and treatment of tumors at the clinical level . In order to probe the underlying mechanisms controlling PME concentrations, we used cells grown in bioreactors and 31P nuclear magnetic resonance spectroscopy to study the effects of proliferative status and exogenous precursor amines on the PMEs phosphorylcholine (PCho) and phosphorylethanolamine (PEtn) . In general, PEtn demonstrated an inverse correlation with cell growth, beginning to rise as the stationary growth phase was approached . PCho, on the other hand, generally decreased during log growth, an effect that was reversed by the addition of exogenous choline . The net effect of these changes was a consistent and dramatically lower PCho/PEtn ratio in stationary cultures compared to actively proliferating cultures.

Biotechnol Prog, 1996 May-Jun, 12(3), 393 - 7
A miniature bioreactor for sensing toxicity using recombinant bioluminescent Escherichia coli cells; Gu MB et al.; A miniature bioreactor was fabricated as a contactor between biosensing cells and toxic materials . This miniature bioreactor (58 mL working volume) showed performance similar to that of a conventional bioreactor, as well as the advantages of easy installation, facile operation, and small medium requirements during long-term continuous operation . A performance evaluation measured the response to ethanol in continuous operation by using a recombinant bioluminescent Escherichia coli strain . Continuous cultures were repeatedly induced by the ethanol challenge . Steady-state cell concentrations (OD) were found to be decreased, the induced specific bioluminescence (SBL) peak value was found to be increased, and the peak response time, which is the time constant of this continuous monitoring system, was found to be decreased with increasing dilution rate . Finally on- and off-line bioluminescence monitoring was shown to be reliable, suggesting that this system is suitable for applications such as monitoring the influent and effluent streams of waste water biotreatment plants.

Biotechnol Prog, 1996 May-Jun, 12(3), 310 - 5
Reductive microbial dechlorination of indigenous polychlorinated biphenyls in soil using a sediment-free inoculum; Klasson KT et al.; In laboratory experiments, unagitated soil slurry bioreactors inoculated with micro-organisms extracted from polychlorinated biphenyl-contaminated (PCBs) sediments from the Hudson River were used to anaerobically dechlorinate PCBs . The onset of dechlorination activity was accelerated by the addition of certain organic acids (pyruvate and maleate) and single congeners (2,3,6-trichlorobiphenyl) . Dechlorination was observed under several working conditions after 19 weeks of incubation with PCB-contaminated soil and nutrient solution . Best results showed a drop in average chlorine content from 4.3 to 3.6 chlorines per biphenyl due to a loss of m-chlorines . Soil used for these experiments was obtained from a PCB-contaminated (weathered Aroclor 1248) site at an electric power substation . Dechlorination was observed with no sediment particles or other matrix being added.

Xenobiotica, 1996 Apr, 26(4), 447 - 57
Study of coumarin metabolism by Chinese hamster lung fibroblasts expressing a human cytochrome P450 using H-nmr; Street JC et al.; 1 . Human cytochrome P450 2A6 is expressed in Chinese hamster lung fibroblasts . The isozyme hydroxylates coumarin at the 7-position . 2 . Metabolism of coumarin in these lung fibroblasts was monitored using 1H-nmr . Media samples were taken from cells grown in flasks and also in a fluidized bed bioreactor . In each case 7-hydroxycoumarin was readily observable by nmr in crude extracts of the medium . 3 . The rate of formation of 7-hydroxycoumarin observed in the acute bioreactor experiments on a per cell basis was found to be much higher than that obtained under chronic monolayer conditions, and correlated with glucose consumption rates.

Biotechniques, 1996 Apr, 20(4), 702 - 7
Viable AC-2, a new adult bovine serum- and colostrum-based supplement for the culture of mammalian cells; Viander B et al.; In this study we have shown that Viable AC-2, a medium based on the ultrafiltrate fraction of bovine colostrum and adult bovine serum, can be used successfully as a fetal bovine serum (FBS) substitute in the culture of several anchorage-dependent and independent cell lines . Of the 15 cell lines cultured in 8% Viable AC-2 in microplates, 10 reached the maximum cell density of 65%-123% of that in 10% FBS, 4 cell lines reached maximum cell density of 35%-65% of that in 10% FBS and only one cell line, a human osteosarcoma G-292, grew slowly in Viable AC-2 . In a small-scale suspension culture, 8%-15% Viable AC-2 supports the growth of Chinese hamster ovary cells (CHO-K1) on microcarriers in spinner flasks significantly better than 10% FBS . Shionogi mouse mammary tumour cell line (S115) transfected with human alpha 2-adrenergic receptor subtype C2 was used as a model to study recombinant protein production in Viable AC-2-supplemented medium . The results showed that in cell culture flasks and in an ACUSYST-R bioreactor, the alpha 2-C2 receptor expression level per mg of total protein was similar in both Viable AC-2 and FBS.

Curr Opin Biotechnol, 1996 Apr, 7(2), 223 - 30
Ex vivo culture systems for hematopoietic cells; Collins PC et al.; During the past few years, hematopoietic cell culture technologies for transplantation therapies have progressed significantly on several fronts . Advances include the discoveries of the growth factors thrombopoietin and Flt-3 ligand, the development of a variety of bioreactor systems, and results from preliminary clinical trials that demonstrate the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.

Am J Clin Nutr, 1996 Apr, 63(4), 627S - 32S
The mammary gland as a bioreactor: factors regulating the efficient expression of milk protein-based transgenes; Rosen JM et al.; Specific regulatory regions required for hormonal regulation and tissue-specific expression of rat beta-casein and why acidic protein (WAP) genes in the mammary gland have been defined . Composite response elements with multiple binding sites for several transcription factors have been identified . Mammary gland-specific gene expression appears not to be mediated by a single transcription factor, but instead requires cooperative interactions among several factors . Signal transduction pathways regulated by lactogenic hormones result in transcription factor binding and interaction within these elements, chromatin-structure changes, and milk-protein gene expression . Intragenic sequences in the 5' and 3' untranslated regions of the beta-casein and WAP mRNAs, respectively, also appear crucial for the efficient expression of these genes . Vectors to target the expression of heterologous genes, such as insulin-like growth factor I, to the mammary gland can be designed . This technology can be used to manipulate milk composition in transgenic animals, one result being improved infant formulas.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 20 - 5
Azidothymidine homodinucleotide-loaded erythrocytes and bioreactors for slow delivery of the antiretroviral drug azidothymidine; Benatti U et al.; A new Azidothymidine derivative, di-(thymidine-3'- azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphate (AZTp2AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing . Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZTp2AZT to 3'-Azido-3'-deoxythymidine which was then released linearly in plasma . The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZTp2AZT to yield two 3'-Azido-3'- deoxythymidine-5'-phosphate molecules which were then dephosphorylated to 3'-Azido-3'-deoxythymidine . At late times of incubation, also a limited asymmetrical hydrolysis of AZTp2AZT became apparent in the intact erythrocytes, yielding 3'-Azido-3'-deoxythymidine-5'-diphosphate that was then converted to the triphosphorylated derivative . Therefore, erythrocytes loaded with AZTp2AZT act "in vitro" as bioreactors ensuring sustained and potentially useful release of 3'-Azido-3'-deoxythymidine.

Artif Cells Blood Substit Immobil Biotechnol, 1996 Mar, 24(2), 91 - 106
Oxygen diffusivity in calcium alginate gel beads containing Gluconobacter suboxydans; Mehmetoglu U et al.; Gaining insight into the mass transfer characteristics in immobilized enzyme and microorganism systems bears much importance because of their widely increased use in industrial scale production as well as for analysis purposes . In this study, the effective diffusion coefficient of oxygen in calcium alginate gel with and without cells has been determined by the Moment Analysis Method for the first time in literature . When the gel concentration was increased from 1 to 3 %, De values of oxygen varied between 2.54 x 10(-5) cm2/s and 2.58 x 10(-5) cm2/s indicating almost no dependency on gel concentration . However, a decrease in effective diffusion coefficient (from 2.55 cm2/s to 2.47 cm2/s) was observed with increased immobilized Gluconobacter suboxydans concentration in the range of 0 to 6 % . Experimental results on liquid-particle mass transfer coefficient revealed a negligible external mass transfer resistance . Effectiveness factor for the bioreactor system was also calculated and found to be 0.39 and 0.21 for gel beads of 0.1 and 0.2 cm radius respectively . It is concluded therefore that the use of smaller gel beads could substantially improve the production efficiency in similar bioreactors.

Biotechnol Prog, 1996 Mar-Apr, 12(2), 257 - 65
Influence of aeration on cytoplasmic pH of yeast in an NMR airlift bioreactor; Melvin BK et al.; Nuclear magnetic resonance spectroscopy has been increasingly pursued as a tool for noninvasive, real-time studies of metabolic processes of cell suspension in bioreactors . One acute challenge in NMR bioreactor design has been supplying enough oxygen for cell respiration in a suspension that contains sufficient cells for NMR signal detection . The use of cytoplasmic pH as an intracellular marker of adequate oxygenation was evaluated from 31P NMR spectra of the yeast Saccharomyces cerevisiae at several cell densities, ranging from low (0.9% (v/v)) to very high (45% (v/v)) cell densities, in an airlift bioreactor . 31P NMR spectra were obtained for derepressed yeast cells prior to, and during, glycolysis under nongrowth conditions . During endogenous respiration, pHcyt can be used as an intracellular marker for aeration for cell densities up to 18% (v/v) based on two criteria: a value of pHcyt at least 0.2 pH units higher under aerobic than anaerobic conditions and an absolute pHcyt value of 7.1-7.2 . These results were more conservative than values of the maximum cell density obtained from calculations using kLa and respiration rate estimates and highlight the utility of intracellular measurements in conjunction with engineering design calculations . During glycolysis, pHcyt values were similar under aerobic and anaerobic conditions and hence pHcyt cannot be used as a marker under these conditions . Carbon dioxide in the influent gas was observed to aid cells in maintaining physiological pHcyt at high cell densities.

J Immunol Methods, 1996 Feb 5, 189(2), 217 - 31
Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production; Jackson LR et al.; The objective of this study was to compare monoclonal antibody production in hollow fiber bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice . Three hybridoma cell lines were grown in each of three different hollow fiber bioreactor systems and in groups of 20 mice . Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10(6) hybridoma cells . Each mouse was tapped a maximum of three times for collection of ascites . Ascites volumes and daily clinical observations were recorded . Bioreactors were harvested three times weekly for 65 day and were monitored by cell counts, cell viability and media glucose consumption . Time and materials logs were maintained . The total quantity of monoclonal antibody produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 455 mg vs . 168 mg; cell line 3C9, 446 mg vs . 565 mg; and cell line RMK, 997 mg vs . 1023 mg . Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, and from 0.71 to 11.10 mg/ml in hollow fiber bioreactor system . Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber bioreactor system merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

J Biol Chem, 1996 Feb 2, 271(5), 2514 - 22
Expression in high yield of pig alpha 1 beta 1 Na,K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae; Pedersen PA et al.; Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity . In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae . The alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-microns-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage . The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 +/- 3,000 {3H}ouabain sites/cell . In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 micrograms of Na,K-pump protein/g of cells . Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein . The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min-1) were close to those of native pig kidney Na,K-ATPase . Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same alpha-subunit concentration and {3H}ouabain binding capacity as the wild type Na,K-ATPase . The high yield and absence of endogenous activity allowed assay of {3H}ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).

Appl Microbiol Biotechnol, 1996 Feb, 44(6), 774 - 7
Overexpression of the gene for N-acylamino acid racemase from Amycolatopsis sp . TS-1-60 in Escherichia coli and continuous produciton of optically active methionine by a bioreactor; Tokuyama S et al.; A DNA sequence encoding N-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c . The recombinant plasmid was introduced into Escherichia coli MM 294 lysogenized with a bacteriophage lambda having a T7 RNA polymerase gene . The amount of AAR produced by the E . coli transformant was 1100-fold more than that produced by Amycolatopsis sp . TS-1-60, the DNA donor strain . The AAR was purified to homogeneity from the crude extract of the E . coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography . Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR and D- or L-aminoacylase . D- or L-methionine was continuously produced with a high yield from N-acetyl-DL-methionine by the bioreactor.

Appl Microbiol Biotechnol, 1996 Feb, 44(6), 724 - 30
Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: experiments and computer simulations; Gron S et al.; Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor . Membrane filtration was used to separate cells from the broth . Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations . Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling . Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling . Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.

Curr Opin Biotechnol, 1996 Feb, 7(1), 46 - 9
Non-invasive glucose monitoring; Arnold MA; Several recent reports claim success in measuring blood glucose non-invasively in human subjects with near-infrared spectroscopy . A critical examination of these published results suggests more fundamental research is needed to verify the validity of these claims . In addition, progress continues in assessing the utility of near-infrared spectroscopy as a non-invasive probe for continuous bioreactor monitoring during fermentation processes . Recent work demonstrates that five critical fermentation components, including glucose, may be measured simultaneously.

Artif Organs, 1996 Feb, 20(2), 186 - 92
Function of culturing monolayer hepatocytes by collagen gel coating and coculture with nonparenchymal cells; Koike M et al.; Since 1987, we have been developing a bioartificial liver (BAL) using multiplated cultured hepatocyte monolayers . With the goal of promoting hepatic functions of cultured hepatocyte monolayers, we combined the use of a collagen gel layer over the monolayers of hepatocytes and/or cocultured hepatocytes with nonparenchymal cells (NPCs) . The study was divided into four groups according to culture configurations: Group 1: hepatocyte monolayer culture (control); Group 2; coculture of hepatocytes and NPCs; Group 3: hepatocyte monolayer with a overlaid collagen gel layer; and Group 4: coculture with a overlaid collagen gel layer . The culture continued for 14 days . Morphological changes and hepatic functions were evaluated by urea and albumin syntheses . The morphological status of the hepatocytes remained for 2 weeks in Groups 3 and 4 . Deterioration and detachment of hepatocytes and/or NPCs started in Group 1 and 2 on the third day in culture . Significantly high urea synthesis was noted in Group 4 (p < 0.001 compared with Group 1 and 2: p = 0.0014 compared with Group 3) . Although there was no significant difference in albumin synthesis among the four groups, those hepatocytes covered by the collagen gel (Groups 3 and 4) tended to secrete albumin throughout the observation period . These results indicted that the environment, although artificial (but close to the in vivo state), supplied with collagen gel and the coculture, enhanced the activities of the cultured hepatocyte monolayers . We suggest that use of cocultured hepatocytes under a collagen gel is a promising candidate for a bioreactor of multiplated BAL.

Acta Microbiol Immunol Hung, 1996, 43(4), 359 - 70
Effect of medium composition on hybridoma growth and antibody production; Hencsey Z et al.; Two murine hybridoma cell lines named 1D12 and 3B1 secreting antibody against human blood group B were developed . Effect of medium composition and hyperosmotic stress on cell growth, viability and monoclonal antibody (MAb) production was examined during down-stream process in suspension culture . Both of the cell lines secreted MAb of higher titre if the basal medium was supplemented with 2.5 g/l L-glucose and 419 mg/l L-glutamine . Additional nutrients like transferrine, amino acids, ethanolamine, selenium were added to promote cell growth and viability . In case of cell line 1D12 enhancement of MAb production was observed using hyper-osmotic medium as well . Having data of these preliminary experiments we optimized the large scale production of MAbs in bioreactor . These MAbs serve as a base of the anti-B blood group reagent registered in Hungary.

Acta Microbiol Immunol Hung, 1996, 43(4), 345 - 57
Large scale cultivation of an anti-D (IgM) antibody producing human/mouse heterohybridoma; Fizil A et al.; A human/mouse heterohybridoma cell line secreting anti-Rh(D) antibody was established by the fusion of Epstein-Barr Virus (EBV) transformed human B lymphocytes and human/mouse heteromyelomas . During scaling-up the cell line was adapted to suspension culture, then medium composition was gradually altered into a more economical one . The adapted cell line has kept its multiplication and antibody secreting characteristics in the new medium . Parameters of large scale batch cultivation in bioreactors were optimized in working volume of 3.6 litre and production was carried out under these parameters in working volume of 24 litre . Optimized parameters were the follows: (i) the cell line is highly sensitive to dissolved oxygen (DO) level, its optimal DO is 10%; (ii) optimal inoculum cell count is 3 x 10(5) cell/ml; (iii) Marine-blade impeller was chosen for supporting the requested oxygen transfer . Using the optimized parameters HUMAN Co . Ltd . produces the Monoclonal Anti-D (IgM) reagent, which has been registered in 1995 in Hungary and now it is on the market.

Chin J Biotechnol, 1996, 12(2), 119 - 29
Studies on high-density cultivation of Vero cells with biosilon solid microcarrier; Wang D et al.; From eight kinds of microcarriers, Biosilon has been chosen as the most suitable microcarrier for high-density cultivation of African green monkey kidney (Vero) cells . Conditions affecting high-density cultivation were studied with a 500 ml modified Wheaton spinner flask . When cultivation with a 1.5 L CelliGen bioreactor at inoculum density of 4.9 x 10(6)/ml and perfusion volume increased from 0.20 L to 3.65 L gradually, the cell density could reach 2.05 x 10(7)/ml after 22 days of cultivation . When Vesicular Stomatitis Virus (VSV) was inoculated into the cultures of 1.21 x 10(6) ml at the some MOI (multiplicity of infection), the virus titers reached 7 log TCID50/ml and 8 log TCID50/ml, respectively.

Adv Exp Med Biol, 1996, 389, 261 - 9
Endosome-lysosomes, ubiquitin and neurodegeneration; Mayer RJ et al.; Before the advent of ubiquitin immunochemistry and immunogold electron microscopy, there was no known intracellular molecular commonality between neurodegenerative diseases . The application of antibodies which primarily detect ubiquitin protein conjugates has shown that all of the human and animal idiopathic and transmissible chronic neurodegenerative diseases, (including Alzheimer's disease (AD), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD) and scrapie) are related by some form of intraneuronal inclusion which contains ubiquitin protein conjugates . In addition, disorders such as Alzheimer's disease, CJD and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins which may be associated with cytoskeletal reorganisation . Although our knowledge about these diseases is increasing, they remain largely untreatable . Recently, attention has focused on the mechanisms of production of different types of amyloid and the likely involvement within cells of the endosome-lysosome system, organelles which are immuno-positive for ubiquitin protein conjugates . These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials or their precursors which subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities . Such common features of the disease processes give new direction to therapeutic intervention.

Crit Rev Biotechnol, 1996, 16(3), 185 - 215
Scleroglucan; Wang Y et al.; Cultures of filamentous fungi that secrete significant amounts of exopolysaccharides are among the most difficult of fermentation fluids, presenting difficulties in the areas of aeration, agitation, mixing, and control that may in turn impact the physiology of the microorganism in an undesirable manner . The fungus Sclerotium glucanicum, which produces a potentially useful exopolysaccharide known as scleroglucan, illustrates many such difficulties . This review discusses in detail the range of physiological studies on the producing microorganism itself, including those concerning formation of "undesirable" byproducts, principally oxalate, but also, under certain conditions, other TCA cycle acids . In addition, the bioreactor technology in use for production of this type of biopolymer is discussed in relation to the difficulties such fluid types present . The potential of pneumatically agitated reactors for such production is evaluated, and the lack of fundamental studies on such reactors and on the hydrodynamics and mixing behavior of such complex fluids is pointed out.

Biotechnol Prog, 1996 Jan-Feb, 12(1), 57 - 64
Practical considerations in operation and scale-up of spin-filter based bioreactors for monoclonal antibody production; Deo YM et al.; Minimum spin-filter fouling and optimum cell retention at high specific perfusion rates are important for efficient operation of a spin-filter based continuous perfusion bioreactor . We examined the effect of operation conditions and spin-filter configuration on the performance of continuous perfusion bioreactors using a perfusion recycle scheme . This study showed that single cell suspensions foul a spin-filter screen, partially but irreversibly, in the early stages of the bioreactor run . A high perfusion rate and cell density contribute to screen fouling, while an increase in rotational velocity and screen surface area per reactor volume reduce screen fouling . An empirical model was developed to describe the effects of these parameters on the perfusion capacity of a spin-filter . Examination of screen samples by scanning electron microscopy confirmed that partial screen fouling occurs at relatively early stages of fermentation . Once the initial partial screen fouling has occurred, further fouling continues slowly due to cell growth on the screen surface, and this gradually leads to the overflow state . The rate of this gradual fouling phase probably depends upon the number of cells deposited on the screen surface during the initial partial fouling . The usefulness of this model was confirmed by successful scale-up of high-cell-density (> 10 x 10(6)/mL) long-term (> 30 days) continuous perfusion process for commercial scale production of monoclonal antibodies.

Adv Space Biol Med, 1996, 5, 341 - 56
Bioregeneration in space; Wolf L; ESA has been studying a small-scale bioregenerative system to support long-term biological experiments on-board spacecraft with oxygen, water, and food . Core component of this system is a special photo-bioreactor in which a maltose-producing strain of the green alga Chlorella is cultivated . In initial experiments this bioreactor has been tested, and the physiology of Chlorella has been studied . The optimal conditions for CO2 to O2 conversion and maltose production have been determined, and the possibility of controlling the culture so as to match the needs of the consumer has been established . A microgravity-compatible photo-bioreactor, and a maltose separator have been developed and are functioning on the ground according to the design specifications . Tests in weightlessness will have to be performed in the future . The components are to be integrated to a complete bioregenerative life support system, which will then be subjected to extensive testing . The EXEMSI project afforded an opportunity to study the mutual influence of a Chlorella culture and real biological oxygen consumers, the four crew members in the laboratory module of the isolation facility . Chlorella 241.80 was batch cultured in an airlift bioreactor by the crew for 25 days with air aspirated from the module . The crew members determined pH and cell density in samples withdrawn from the culture . Microscopic observations showed no evidence of contamination of the culture by other organisms . Growth rates were smaller than those observed in laboratory conditions . This is attributed to the relatively low average CO2 concentration in the module atmosphere: 0.1% against 0.5% in the air supply during the laboratory experiments . The data show no evidence of trace contaminant accumulation in the Chlorella culture . The results are encouraging and suggest the value of further simulated operational testing of the system.

Adv Exp Med Biol, 1996, 397, 141 - 51
Production of influenza virus in cell cultures for vaccine preparation; Merten OW et al.; Influenza virus strains of different types for use as an inactivated vaccine have been successfully grown in different cell lines . Increasing titres were obtained with BHK-21/BRS, VERO and MDCK cells . Cultures in stationary flasks, in spinner cultures or in large bioreactor systems were tested and the optimal conditions were studied . MDCK cells grown in serum-free medium before and during the virus production phase were found to yield high titres in the presence of trypsin . Satisfactory results were obtained with egg-adapted strains of human and equine origin as well as with strains just isolated from human patients without any further passages in eggs or cell culture.

Appl Microbiol Biotechnol, 1996 Jan, 44(5), 568 - 75
Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus; Zhang J et al.; Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease . Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation . A fermentation process that used chemically defined medium was therefore developed for its production . By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale . Further medium optimization resulted in a yield of 790 mg/l in a 23-1 bioreactor using a batch process . A titer of 880 mg/l was attained during scale-up in a 800-1 fermentor by employing a nutrient-feeding strategy . This production represents a 44-fold increase over the yield from the initial process in shake-flasks . The defined-medium fermentation broth was very amenable to downstream processing.

Int J Artif Organs, 1996 Jan, 19(1), 72 - 8
Matrix-induced liver cell aggregates (MILCA) for bioartificial liver use; Kong LB et al.; Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use . We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates . Porcine hepatocytes were obtained by EDTA/collagenase digestion . Cell viability was assessed by trypan blue exclusion and LDH release . Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC) . At 4 hours, the viability of MILCA was 92 +/- 2%, LDH release was 100 +/- 22 U/L and 7-HC formation was 140 +/- 34 nM/g cells . At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194 +/- 43 vs 481 +/- 78 nM/g cells; p < 0.002) . On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules) . MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n = 6; 5 x 10(9) cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min) . In these studies, lidocaine (20 micrograms/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71 +/- 8 nM/g cells . Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.

Berl Munch Tierarztl Wochenschr, 1996 Jan, 109(1), 8 - 9
Production of veterinary vaccines in the Sudan; Babiker SH; At present the number of domestic animals in the Sudan is approx . 89 millions . Animals disease control depends mainly on mass vaccination . The production of veterinary vaccines in the Sudan started as early as 1924 using the classical and conventional methods of production . The introduction of the Goettingen-IBT bioreactor technology for vaccine production to the Sudan in 1985 resulted in significant improvements in bacterial vaccine production both quantitatively and qualitatively . In addition to other benefits, the running cost of production was reduced by at least 80% . Plans are made to get the use of the Goettingen-IBT bioreactor technology, to produce other vaccines in the future.

Transgenic Res, 1996 Jan, 5(1), 67 - 72
Synthesis and secretion of the mouse whey acidic protein in transgenic sheep; Wall RJ et al.; The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk . Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs . To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes . Two lines of transgenic sheep were produced . The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre . Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe . WAP RNA was not detected in skeletal muscle and intestine . These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences . Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.

J Immunol Methods, 1995 Dec 15, 188(1), 73 - 8
Production and characterization of a monoclonal antibody against Schistosoma japonicum glutathione S-transferase; Campbell MJ et al.; Schistosoma japonicum glutathione S-transferase (GST), expressed from a pGEX plasmid, was isolated from Escherichia coli cells and used to immunize mice in order to generate specific anti-GST monoclonal antibodies . Using a modified immunization and fusion procedure, one stable hybridoma clone secreting an anti-GST antibody (alpha GST-1) was obtained . Milligram quantities of this antibody were produced in vitro in a miniPERM bioreactor and subsequently purified by protein G affinity chromatography . The characteristics of this antibody were investigated by enzyme-linked immunosorbent assays and immunoblotting experiments . The alpha GST-1 antibody was found to react specifically with GST and GST fusion proteins and demonstrated no reactivity with normal E . coli proteins . This monoclonal antibody should be a valuable reagent for tracing the production of GST fusion proteins and possibly for affinity purification of GST fusion proteins.

Gene Ther, 1995 Dec, 2(10), 723 - 30
Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production; Forestell SP et al.; Efforts to improve gene transfer (transduction) efficiency achieved with retroviral vectors often focus on increasing the end-point titer . In this study, we assayed more than 70 retroviral vector supernatants for end-point titer and for the ability to transfer reporter genes into cell populations (referred to as transduction efficiency) . We found no correlation between end-point titer and transduction efficiency . We also show that increasing end-point titer by ultrafiltration does not necessarily increase transduction efficiency . Evidence presented shows that nontransducing retroviral particles interfere with transducing virions and reduce transduction efficiency without reducing end-point titer . We have investigated production parameters and stability of retroviral vector particles using transduction efficiency as a measure of supernatant potency . Analysis of the production kinetics showed that the rate of virus production was marginally higher at 37 degrees C than at 32 degrees C . However, recombinant amphotropic retrovirus particles are significantly more stable at 32 degrees C than at 37 degrees C . In addition, we show that short incubation periods are sufficient to yield supernatants with high transduction efficiencies . We have implemented improved culture conditions, including short incubation periods, by continually perfusing medium over producer cells in a packed-bed bioreactor incubated at 32 degrees C . By operating the packed-bed bioreactor in perfusion mode, retroviral vector supernatants with a high transduction efficiency can be routinely produced in large quantities.

Protein Expr Purif, 1995 Dec, 6(6), 789 - 98
Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture; Cosgrove L et al.; Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene . Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine . The expression levels of ectodomain were subsequently increased by gene amplification . Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS) . By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days . Harvests were concentrated 20-fold by anion-exchange chromatography . Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography . Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500 . Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR . The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M) . These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Xenobiotica, 1995 Dec, 25(12), 1283 - 91
S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride by rat hepatic flavin-containing monooxygenase 1 expressed in yeast; Nunoya K et al.; 1 . Rat hepatic flavin-containing monooxygenase 1 (FMO1) expressed in yeast catalyzed the S-oxidation of (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) in vitro . 2 . S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide, known inhibitors of FMO, but was not enhanced by n-octylamine, a known enhancer of FMO . 3 . The rate of S-oxide formation from SM-12502 was about four-fold lower than that from (+/-)-trans-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-9979) and enantioselectivity and diastereoselectivity of the S-oxidation reaction were observed . 4 . The ability of the recombinant yeast to produce the S-oxide from SM-12502 was maintained for long periods and exemplifies the recombinant yeast as a bioreactor to produce a large amount of the S-oxide.

Berl Munch Tierarztl Wochenschr, 1995 Dec, 108(12), 471 - 5
{Protective effect in mice of the live rabies vaccine produced using the Göttingen bioreactor}; Roth F et al.; For the control of urban rabies in developing countries, the SAD-virusstrain was used in the Gottingen two-step Bioreactorsystem as a model-virus for oral vaccine production . The quality of the antigen had been tested by challenge . In the Bioreactorsystem, controlled by computer, a high standard of cell quality and cell density up to 6 x 10(6) cells/ml had been maintained . The high cell quality is correlated to high proliferation of rabies virus . Virus titres up to 10(8.5) pfu/ml were reached . The protectivity of the antigen had been tested by challenge . In comparison to a commercial vaccine with a PD50 of 10(-1.92) the SAD-Antigen of the Gottingen Bioreactorsystem reached a PD50 of 10(-1.63) by a challenge doses of 70-75 MLD50.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 519 - 25
Growth inhibition by ammonia and use of a pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum; Wittmann C et al.; The inhibitory effect of ammonia on the growth of the polychlorinated xenobiotic-degrading bacterium Mycobacterium chlorophenolicum was examined . The strain is inhibited by both the ionized and nonionized forms of ammonia, At pH 6.9, 50% reduction of the growth rate was observed at 6.8 g/l total ammonium . For 23 experiments performed in shake-flask culture at different pH values, and ammonium concentrations a growth model based on the extended Monod kinetic fits the data with a deviation of 5.3% . To overcome growth inhibition in bioreactors, a pH- controlled feeding strategy was developed for effective cultivation of M . chlorophenolicum at an ammonium level below 0.3 g/l . The ammonium addition was controlled online by the stoichiometric interdependence of ammonium consumption and pH decline . With this online control strategy, a biomass concentration as high as 26.2 g/l can be achieved within less than a week of cultivation . The yield is also increased from 0.32 g to 0.43 g biomass (per gram glucose) . The strategy developed provides an effective method for the production of biomass o M . chlorophenolicum serving as the inoculum in remediation technologies.

Toxicol Lett, 1995 Dec, 82-83, 807 - 13
Genetically engineered bacterial cells and applications; Waterman MR et al.; Heterologous expression systems have proven to be very important in P450 research, permitting investigation of many P450s identified only by recombinant (cloning) technologies . They also make it possible to study human P450s since tissue sources for naturally occurring enzymes are difficult to obtain . A variety of different heterologous systems have been applied to the study of P450s and each one has its own unique advantages . For the study of biophysical properties and structure/function relationships, E . coli has proven to be a particularly good system . Both microsomal and mitochondrial P450s can be expressed to high levels in E . coli and subsequently purified to homogeneity for detailed analysis . Techniques of both site directed mutagenesis and random chimeragenesis are very facile in bacteria providing excellent opportunity to analyze specific aspects of structure/function relationships of P450s . Furthermore microsomal P450s are active in intact E . coli, activities being supported by flavodoxin and flavodoxin reductase, providing the opportunity to develop bioreactors expressing designer P450s . The salient features of P450 expression in bacteria are summarized herein.

Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 53 - 8
Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection; Tom RL et al.; The extracellular domain of the epidermal growth factor receptor (EGFR) was expressed using the baculovirus expression vector system . The maximum level of the EGFR extracellular domain secreted into the medium in Sf-9 (Spodoptera frugiperda or fall army-worm) cell batch culture was approximately 2.5 micrograms ml-1 . In order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant virus, and replacement of spent medium . By using this process, the specific yield of recombinant protein, which in batch culture drops when infection is carried out at densities greater than 3 x 10(6) cells ml-1, can be maintained at a maximum in cultures infected at densities of 10(7) cells ml-1 or greater . The process, when applied to 3-1 and 11-1 bioreactor cultures, allowed a maximum volumetric yield of triple the maximum value attainable in batch culture . Spent-medium analysis indicates that medium replacement provides certain nutrients that could otherwise be limiting for recombinant protein production.

J Biotechnol, 1995 Nov 21, 43(1), 33 - 40
Effects of the hydrodynamic environment and shear protectants on survival of erythrocytes in suspension; Zhang Z et al.; Survival of media-suspended porcine erythrocytes exposed to various hydrodynamic environments was investigated with and without such shear protectant additives as bovine serum albumin, dextran and the non-ionic surfactant Pluronic F68 . Erythrocytes provided a model cell population with cells of a uniform size, metabolic state and shear tolerance . Because the cells were non-growing, any shear adaptation effects were avoided . Cell lysis was followed by microscopic counts or release of haemoglobin . The cells were susceptible to agitation damage in unaerated shake flasks agitated at 100 rpm or greater . Relative to additives-free operation, the presence of 0.1% (w/v) dextran or albumin prolonged cell survival, but Pluronic F68 actually enhanced cell lysis in flasks agitated at 100 rpm . The protective effect of the additives depended on the hydrodynamic conditions . The protective effect of albumin was demonstrated also in aerated conditions in a split-cylinder airlift bioreactor (aspect ratio of 8.8; riser-to-downcomer cross-sectional area ratio of 1.0; specific power input of 0.34 W m-3) . Comparison of the cell lysis characteristics in the airlift device and the best case performance of the shake flask showed longer survival in the flask (100 rpm); however, the length of survival in the reactor (approx . 70 h) was sufficient for practical purposes . In all cases, the cell lysis pattern conformed initially to zero-order dependence in cell concentration, becoming first-order after varying degrees of exposure to hydrodynamic forces . Fatigue failure of cells was inferred.

J Biotechnol, 1995 Nov 21, 43(1), 21 - 32
Adaptive predictive control of dissolved oxygen concentration in a laboratory-scale bioreactor; Diaz C et al.; We present an algorithm for the adaptive control of dissolved oxygen concentration in a bioreactor, based on the agitation rate . The dynamics are represented by an incremental first-order model with variable dead-time and parameters . These are estimated on-line by a recursive least-squares identification method with a forgetting factor and a constant sensitivity . The model is employed to predict the behaviour of the dissolved oxygen concentration over a finite horizon, using an original method which requires little computation . Then, a Generalized Predictive Control optimisation strategy computes the agitation rate from the predictions and the desired set point, while gradually updating the controller smoothness . This algorithm, which requires little preliminary knowledge, has been implemented on a laboratory-scale fed-batch bioreactor for which the use of conventional controllers showed limited performance, due to the unpredictable and evolutive nature of the dynamics . The new controller proved to be robust and effective over a wide range of operating conditions, while requiring no operator adjustments.

Blood, 1995 Nov 15, 86(10), 3754 - 62
Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels; Eipers PG et al.; Hematopoietic stem cell gene therapy holds the promise of being able to treat a variety of inherited and acquired diseases of the hematopoietic stem cell . However, to date, genetic modification of the human hematopoietic stem cell has been relatively inefficient . Here, we report the results of using a bioreactor system to expand hematopoietic cells after a brief retrovirus infection using a high titer, replication defective virus encoding for murine CD18 . The retrovirus transduced culture continued to produce genetically modified hematopoietic progenitors for up to 6 weeks, the duration of the culture period . Up to one-third of the long-term culture initiating cell (LTC-IC) are genetically modified by the culture conditions . Murine CD18 can be expressed on the cell surface of up to 20% of the mature cells generated by the culture system, suggesting that clinically significant levels of gene transfer may be occurring . These results demonstrate the feasibility of using continuous perfusion bioreactors as a method of efficiently modifying human hematopoietic stem cells.

Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10462 - 6
Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin; Drews R et al.; Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains . This processing was inefficient in the mammary gland of transgenic mice and pigs . We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes . We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland . In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites . These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 297 - 308
Characterization of microbial communities in anaerobic bioreactors using molecular probes; Raskin L et al.; The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria . These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays . It was determined that methanogens in "healthy" mesophilic, single-phase sewage sludge digesters accounted for approximately 8-12% of the total community and that Methanosarcinales and Methanomicrobiales constituted the majority of the total methanogen population . Methanobacteriales and Methanococcales played a relatively minor role in the digesters . Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels: Desulfovibrio and Desulfobulbus spp . were the dominant sulfate-reducing populations, Desulfobacter and Desulfobacterium spp . were present at lower levels, and Desulfosarcina, Desulfococcus, and Desulfobotulus spp . were absent . Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study . In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates . The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase . It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.

Appl Microbiol Biotechnol, 1995 Nov, 43(6), 978 - 84
Optimization of glucose oxidase production by Aspergillus niger using genetic- and process-engineering techniques; Hellmuth K et al.; Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god) . The gene was placed under the control of the gpdA promoter of A . nidulans . For more efficient secretion the alpha-amylase signal peptide from A . oryzae was inserted in front of god . Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions . Addition of yeast extract (2 gl-1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively . With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.

Appl Microbiol Biotechnol, 1995 Nov, 43(6), 1028 - 33
Oxygenation of intensive cell-culture system; Emery AN et al.; The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor . Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration) . Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10(7) ml-1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh . Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter . A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth . However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation.

Antimicrob Agents Chemother, 1995 Nov, 39(11), 2523 - 7
Efficacy of constant infusion of A-77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, in limiting acute HIV-1 infection in vitro; Bilello JA et al.; A-77003, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is effective for both acute and chronic infection in vitro and was evaluated clinically by continuous intravenous infusion administration . The minimum effective dose (the concentration required to completely inhibit viral replication) was determined in vitro in a population of uninfected (99%) and HIV-infected (1%) cells exposed to A-77003 by continuous infusion in hollow-fiber bioreactors . The production of infectious HIV and release of p24 antigen from infected cells were completely inhibited in cultures exposed to A-77003 at or above a concentration of 0.5 microM . Measurement of unintegrated HIV-1 DNA synthesis and flow cytometric analysis for cells expressing HIV p24 antigen demonstrated that the spread of HIV to uninfected cells was also blocked at 0.5 microM A-77003 . Dose deescalation to 0.25 microM or removal of A-77003 resulted in the limited spread of the virus throughout the culture, the resumption of viral DNA synthesis, and release of p24 . HIV produced after exposure to 0.5 microM A-77003 was noninfectious for a period of 72 h after the removal of the drug . Addition of 1 mg of alpha 1-acid glycoprotein per ml to this in vitro system completely ablated the anti-HIV effect of 0.5 microM A-77003 . These data suggest that determination of the minimum effective dose under conditions which simulate human pharmacodynamic patterns may be useful in determining the initial dose and schedule for clinical trials . However, other factors, such as serum protein binding, may influence the selection of a therapeutic regimen.

Biotechnol Prog, 1995 Nov-Dec, 11(6), 664 - 76
An energetically structured model of mammalian cell metabolism . 1 . Model development and application to steady-state hybridoma cell growth in continuous culture; DiMasi D et al.; Incomplete understanding of mammalian cell culture kinetics hinders the ability of the biochemical engineer or biologist to design and control mammalian cell culture systems and to develop operating strategies . To address this problem, a mechanistic, structured mathematical model has been developed to simulate mammalian cell culture kinetics under a variety of bioreactor operating conditions . An important feature of in vitro mammalian cell metabolism in conventional cell culture media is the partial substitutability of the substrates glucose and glutamine for provision of energy in the cell . The utilization of glucose and glutamine by cells can therefore vary substantially, and these changes can profoundly affect the culture behavior . The model developed here specifically addresses the dynamics of substrate consumption and energy metabolism in mammalian cell culture . This energetically structured (ES) model is also distinguished by the consideration of changes in the specific cell mass with specific growth rate and the consideration of essential amino acids as potentially growth rate limiting . The model is applied here to simulate literature data on the growth and metabolism of a murine hybridoma in continuous culture.

Sheng Li Ke Xue Jin Zhan, 1995 Oct, 26(4), 299 - 304
{Expression and regulation of milk protein gene in mammalia}; Yue JM et al.; On the basis of introducing the structures and relations of evolution in milk protein genes, the factors involved in gene expression including cis-acting elements, trans-acting factors as well as induction of hormones were discussed . Finally the applicable prospect of mammary gland as a bioreactor was estimated.

Hybridoma, 1995 Oct, 14(5), 495 - 500
Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody; Roggenbuck D et al.; Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced . A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system . The process of fermentation has been carried out continuously over a period of 4 months . In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased . Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody . During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable . Furthermore, we detected formation of cell clusters in the fermentor system . These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies . Clusters were found to represent fusions of hybridoma cells using electron microscopy . Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas . We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 826 - 37
Optimal dynamic experiments for bioreactor model discrimination; Cooney MJ et al.; This paper describes a general approach for dynamic model discrimination for continuous cultures and presents dynamic models for pure cultures of E . coli and C . utilis obtained using the method . For each pure culture system, four candidate models representing various levels of structure were considered . All models reduce to Monod growth kinetics at steady state . An optimized set of multivariable step inputs in selected manipulative variables was used to discriminate between candidate models . The models that best predicted the dynamic behavior were selected by comparison of model predictions with experimental data . Two discrimination functions were compared in terms of their ability to determine the optimal set of multivariable step inputs to discriminate between candidate models . Results indicate that model discrimination based on maximizing the minimum absolute difference between any two models for a given set of inputs possessed good potential for discrimination between candidate models . Models selected for E . coli and C . utilis from the model discrimination work are presented and compared with experimental data.

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 822 - 5
Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells; Compagno C et al.; Among the main agro-industrial wastes, whey and starch are of prime importance . In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose . Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation) . A yeast strain for the simultaneous utilization of lactose and starch has also been developed . Biotechnological perspectives are discussed.

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 772 - 80
Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line; Bohmann A et al.; A bioreactor system for the continuous cultivation of animal cells with a high potential for scale-up is presented . This reactor system consists of radial-flow fixed-bed units coupled with a dialysis module The dialysis membrane enables the supply of low-molecular-weight nutrients and removal of toxic metabolites, while high-molecular-weight nutrients and products (e.g., monoclonal antibodies) are retained and accumulated . This concept was investigated on the laboratory scale in a bioreactor with an integrated dialysis membrane . The efficiency of the reactor system and the reproducibility of the cell activity (hybridoma cells) under certain process conditions could be demonstrated in fermentations up to 77 days . Based on model calculations, an optimized fermentation strategy was formulated and experimentally confirmed . Compared to chemostat cultures with suspended cells, a ten-times higher mAb concentration (383 mg1(-1)) could be obtained . The highest volumetric specific mAb production rate determined was 6.1 mg mAb (1 fixed bed)-1h-1.

Biotechnol Appl Biochem, 1995 Oct, 22 ( Pt 2), 233 - 46
Effect of aerobic pretreatment with Aspergillus terreus on the anaerobic digestion of olive-mill wastewater; Borja R et al.; A kinetic study was carried out on the anaerobic digestion of olive-mill wastewater (OMW) and OMW that was previously fermented with Aspergillus terreus . The bioreactors used were batch fed and contained saponite as support for the mediating bacteria . The anaerobic digestion process followed first-order kinetics, from which the kinetic constant A was calculated using a non-linear regression . This kinetic parameter was influenced by the pretreatment carried out, and was 3.7 times higher for pretreated OMW than for untreated OMW . The anaerobic processing of pretreated OMW seemingly involved no inhibition phenomena as the biotoxicity and the total phenolic compound content (analysed by HPLC) were reduced by 71.2% and 77.9% respectively as a result of the pretreatment . Finally, the yield coefficient of methane production was 0.345 litres of methane (at standard temperature and pressure)/g of chemical oxygen demand, that is, 23% higher than that provided by untreated wastewater.

J Biotechnol, 1995 Sep 29, 42(2), 117 - 31
Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions; Gawlitzek M et al.; The N-glycosylation patterns of a genetically engineered human interleukin-2 variant glycoprotein (IL-Mu6), produced by BHK-21 cells from long-term suspension and microcarrier cultures in the presence and absence of fetal calf serum were compared . IL-Mu6 was used as a model protein in studying the effect of different controlled cell culture conditions on the expression of N-glycans in recombinant glycoproteins . IL-Mu6 contains a single amino acid substitution (Glu100<==>Asn) generating a potential N-glycosylation recognition site (Asn100-Xxx-Thr/Ser) in addition to the natural O-glycosylation at position Thr3 . Parallel cell cultivations were carried out in two continuously perfused 2.5-liter stirred bioreactors under defined culture conditions . Major differences were found in the glycoprotein products obtained during these different cultivation conditions . Serum-free cultures resulted in a higher level of terminal sialylation and proximal alpha 1-6 fucosylation . The ratio of O- to N-glycans as well as the amount of nonglycosylated product and the antennarity of N-linked carbohydrates in the model protein exhibited major differences depending on the presence or absence of serum, the condition of growth and the cultivation procedure.

Artif Organs, 1995 Sep, 19(9), 941 - 50
Reconstruction of liver tissue in vitro: geometry of characteristic flat bed, hollow fiber, and spouted bed bioreactors with reference to the in vivo liver; Bader A et al.; Bioreactors currently being developed for hybrid artificial livers vary greatly with respect to their microenvironment . The specific architecture modifies the relationship parenchymal and nonparenchymal cells have with the exchange surfaces of the bioreactor . Most designs are either based on hollow fiber, spouted bed, or flat bed devices . This diversity is contrasted by the uniform and unique organization of the in vivo liver . The liver cells are arranged as plates and both sinusoidal surfaces of the hepatocytes are enclosed within the matrix of the space of Disse . In this study we intended to define the in vivo liver tissue characteristics in a manner useful for an organotypical approach to hepatic tissue engineering . Transmission electron microscopy of an in vivo liver was utilized to describe these ratios . The ratios defined in this study are based on the constant hepatocellular expression of two sinusoidal surfaces . A relationship is established between the expression of the sinusoidal surfaces and their use as attachment and exchange surfaces inside a bioreactor . The presence of biliary surfaces and nonparenchymal cell surfaces is compared . The functional relevance of an in vivo like extracellular matrix geometry for oxidative biotransformation of primary hepatocytes in vitro was studied using the two model drugs cyclosporin and rapamycin . The generation of the hydroxylated cyclosporin metabolites AM 9 and AM 1 and four rapamycin metabolites was analyzed by high performance liquid chromatography (HPLC) . It is shown that the cell-specific biotransformation rates at 1 week in culture in matrix overlayed hepatocytes was 5-10 times that of hepatocytes without matrix overlay . Bilaminar membrane (BLM) bioreactors were used to reconstruct extracellular matrix geometry, three-dimensional cell plates, and sinusoidal analogs in between cell plates.

Vaccine, 1995 Sep, 13(13), 1244 - 50
An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process: use of serum-free medium and perfusion-reactor system; Perrin P et al.; An experimental rabies vaccine was prepared from the BHK-21 cell line adapted to culture in suspension using bioreactors . A new serum-free medium (MDSS2) (Merten et al., Cytotechnology, 1994, 14, 47) developed for the culture of various cell lines and for the production of several biologicals, was used for cell culture and virus production . The PV-Paris/BHK-21 rabies virus strain (adapted to the BHK-21 grown in monolayer) was adapted to BHK-21 cells cultivated in suspension and in the serum-free medium . High titres of rabies virus were obtained with bioreactors equipped with a perfusion system using BHK-21 cells grown in suspension in MDSS2 . Experimental vaccines were prepared and had satisfactory protective activity when tested in mice . This new and low cost technology for rabies vaccine production could be suitable for developing countries where rabies is an important health problem.

Biotechnol Prog, 1995 Sep-Oct, 11(5), 584 - 8
Partial and total cell retention in a filtration-based homogeneous perfusion reactor; Banik GG et al.; Suspended mammalian cells can be cultivated in a variety of operational modes (pure chemostat, total cell retention, or partial cell retention) in a homogeneous perfusion bioreactor by varying the cell bleed rate . Hybridomas were grown in the reactor at a perfusion rate of 2.0 day-1 for over 10 weeks at different specific growth rates and viable cell densities achieved by varying the extent of cell retention . Cell metabolism in the reactor was found to vary with the extent of cell retention, which determined both cell density and specific growth rate . With partial cell retention, the nutrient consumption and metabolite production rates decreased with both increasing growth rate and increasing cell density . The specific and volumetric antibody production rates, however, increased dramatically with cell density (and to a lesser extent with decreasing growth rate) . The specific MAb production rate was lower with total cell retention than with partial retention at the same growth rate . Since the reactor can be operated over a range of perfusion rates and extents of cell retention, the system can be used to culture cell lines with widely different productivity patterns.

Biotechnol Prog, 1995 Sep-Oct, 11(5), 518 - 24
Cell growth and monoclonal antibody production in the presence of antigen and serum; Dandulakis G et al.; The impact that the continuous presence in the fermentation broth of the cognate antigen has on the serum-supplemented hybridoma cell cultures was investigated . Both soluble and immobilized antigen at various concentrations was applied . The cell line (ATCC TIB191) was cultured in a serum-supplemented PFHM-II medium in T-flasks . Sepharose gel beads provided the immobilization matrix, and bovine gamma-globulin was the carrier protein upon which the antigen, picric acid, was conjugated . Produced antibody, after elution from the beads by displacement with free picric acid, was measured with an ELISA . Soluble antigen--carrier protein conjugates showed no effect on the cultures, but the immobilized antigen had a strong influence on them . Cell growth rate and total antibody production decreased as the amount of immobilized antigen increased from 16:10 (units are in (mol of Ag/mol of carrier):(mg of carrier/mL of beads) to 36:40 . Most importantly, the specific antibody production rate switched from growth phase-independent behavior in the antigen-free cultures to growth phase-dependent behavior in the immobilized antigen cultures . Furthermore, during the early stationary phase of the cultures with immobilized antigen, the specific antibody production was 20-100% higher relative to the production rate in antigen-free cultures . This increased specific antibody production rate would be particularly useful in increasing the volumetric productivity of perfusion-type bioreactors for hybridoma cell cultures.

Microbiologia, 1995 Sep, 11(3), 337 - 42
Effect of cultivation conditions on glycerophosphate oxidase production by a mutant strain of Aerococcus viridans; Mackova-Suchova M et al.; The effect of different concentrations of carbon source (lactose) and inducer (glycerol) on biomass and glycerophosphate oxidase (GPO) production by mutant strain of Aerococcus viridans 1509 was tested . The best combination of lactose and glycerol concentrations for good enzyme productivity was 0.5% lactose and 2.5% glycerol . Further improvement of GPO levels was achieved after scaling-up of the bioprocess and cultivation of the cells in a 3 liter laboratory bioreactor . Using 4.5 x 10(-5) m3 s-1 air flow rate during growth, GPO activity increased 20-times in comparison with cultivation in flasks.

Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7705 - 9
Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1; Faulkner KM et al.; The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein . In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase) . The present paper shows that NADPH can be replaced by an electrochemically generated reductant {cobalt(II) sepulchrate trichloride} for the electrocatalytically driven omega-hydroxylation of lauric acid . Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 {mRat4A1/mRatOR}L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase . Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor . These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

Anal Biochem, 1995 Jul 20, 229(1), 133 - 8
A chemiluminescence-flow injection analysis of serum 3-hydroxybutyrate using a bioreactor consisting of 3-hydroxybutyrate dehydrogenase and NADH oxidase; Tabata M et al.; We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase . The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide . The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction . Among three immobilized enzyme columns, a coimmobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction . The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries . The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods . The coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyses.

Appl Biochem Biotechnol, 1995 Jul-Sep, 54(1-3), 259 - 70
A simple method for quantifying activity and survival of microorganisms involved in bioremediation processes; Schmidt SK et al.; We have developed a substrate-induced growth response (SIGR) method for quantifying activity and population dynamics of microorganisms involved in bioremediation processes in soil and bioreactors . The biomass of organisms that can mineralize a given chemical can be estimated based on the concentration of that chemical needed to induce the growth of the standing population . Estimates of population size are obtained by using nonlinear regression techniques to fit a simple model of microbial population dynamics to biodegradation curves . Using this approach we obtain estimates of values for parameters such as initial population size and growth rate of organisms carrying out biodegradative processes . Our approach was validated by comparing model parameter estimates with independent estimates of the same parameters from the same bioremediation systems . Examples studied include pentachlorophenol degraders introduced into soil and 2,4-dinitrophenol degrading organisms in a bioreactor.

Environ Health Perspect, 1995 Jun, 103 Suppl 5, 75 - 8
Coupling transport and biodegradation of VOCs in surface and subsurface soils; Hunt JR et al.; Volatile organic chemicals present at Superfund sites preferentially partition into the soil gas and may be available for microbial degradation . A simple mass transfer model for biodegradation for volatile substrates has been developed for the aerobic decomposition of aromatic and aliphatic hydrocarbons . The mass transfer analysis calculates diffusive fluxes from soil gas through water and membrane films and into the cell . This model predicts an extreme sensitivity of potential biodegradation rates to the air-water partition coefficients of the compounds . Aromatic hydrocarbons are removed rapidly while the aliphatic hydrocarbons are much slower by orders of magnitude . Furthermore, oxygen transfer is likely to limit aromatic hydrocarbon degradation rates . The model presents results that cast doubt on the practicality of using methane or propane for the co-metabolic destruction of trichloroethylene in a gas phase bioreactor . Toluene as a primary substrate has better mass transfer characteristics to achieve more efficient trichloroethylene degradation . Hence, in sites where these contaminants coexist, bioremediation could be improved.

Appl Environ Microbiol, 1995 Jun, 61(6), 2203 - 10
Genetic diversity of Desulfovibrio spp . in environmental samples analyzed by denaturing gradient gel electrophoresis of {NiFe} hydrogenase gene fragments; Wawer C et al.; The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified {NiFe} hydrogenase gene fragments . Five different PCR primers were designed after comparative analysis of {NiFe} hydrogenase gene sequences from three Desulfovibrio species . These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria . One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also . By using this specific primer pair, we were able to amplify the {NiFe} hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples . However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined . DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample . Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.

Appl Environ Microbiol, 1995 Jun, 61(6), 2145 - 50
Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid; Mohn WW; Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill . The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized . In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33 . The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination . Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849) . Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids . For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively . Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture . Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture . DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile . Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Immunol, 1995 Jun, 32(8), 595 - 602
Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma; Leibiger H et al.; Glycosylation of the human monoclonal IgG1 lambda antibody (mAb) CBGA1 was analysed by lectin blotting . The CBGA1 antibody binds to several antigens including donor self antigens, as detected by ELISA immunoblotting techniques and an erythrocyte binding assay . The mAb producing cell line was obtained by EBV transformation of peripheral blood lymphocytes of a healthy donor followed by fusion to the heteromyeloma cell line, CB-F7 . The resulting heterohybridoma was cultivated in a hollow fibre bioreactor system . A bulk pool of 0.9 g antibody was produced . Fab and Fc fragments of the purified mAb were prepared and analysed . A noteworthy heterogeneity of CBGA1 and its fragments in SDS-PAGE and IEF was detected . We found glycosylation in the Fab fragment of CBGA1 in addition to the conserved glycosylation site in the Fc fragment at Asn 297 . Fab glycosylation was detected in both the Fd region and the lambda-chain . The glycosylation pattern of the gamma-chain differs from that of the lambda-chain . Sequence analysis of the VH gene shows a potential N-glycosylation site located in framework III at position Asn 75.

J Hematother, 1995 Jun, 4(3), 159 - 69
Bioreactor expansion of human bone marrow: comparison of unprocessed, density-separated, and CD34-enriched cells; Koller MR et al.; Scale-up of human hematopoietic cultures was previously described in continuously perfused systems with bone marrow mononuclear cells (BMMNC), yielding expansion of both progenitors and long-term culture-initiating cells (LTC-IC) . We report here on the use of these systems for expansion of unprocessed whole BM cells (WBMC) and CD34-enriched cells . Density separation recovered 84% of CFU-GM and 65% of LTC-IC from WBMC . Subsequent CD34 selection recovered 17% of CFU-GM and 48% of LTC-IC from the MNC fraction . The unabsorbed (CD34-depleted) fraction contained 37% of CFU-GM and 38% of LTC-IC, accounting for most of the lost cells . WBMC, BMMNC, and CD34-depleted cells were each placed directly in bioreactors, whereas CD34-enriched cells were placed in bioreactors containing preformed irradiated stroma . After 14 days, an average of 3.82 x 10(7) (12.7-fold expansion), 3.54 x 10(7) (11.8-fold), 2.85 x 10(7) (9.5-fold), and 3.65 x 10(7) (1298-fold) total cells were obtained from bioreactors inoculated with WBMC, BMMNC, CD34-depleted, and CD34-enriched cells on stroma, respectively . These cultures yielded 1.64 x 10(5) (27.9-fold expansion), 1.69 x 10(5) (14.3-fold), 8.36 x 10(4) (13.0-fold), and 1.91 x 10(5) (41.4-fold) CFU-GM each, respectively . Cell recovery and expansion data were combined to determine the number of expanded CFU-GM obtained per ml of BM aspirate, allowing direct comparison of performance between the four culture inocula . WBMC generated 3.76 x 10(6) CFU-GM per ml BM aspirate, whereas MNC resulted in 1.42 x 10(6) CFU-GM . CD34-enriched cells (on irradiated stroma) gave 7.00 x 10(5) CFU-GM per ml BM aspirate, whereas CD34-depleted cells generated 4.97 x 10(5) CFU-GM . The high productivity from WBMC cultures was studied further and was found to be reproducible at different inoculum densities . WBMC cultures had elevated levels of endogenous EGF and PDGF production, which may have been responsible for the more extensive stromal development observed . Flow cytometric analysis showed that the final culture composition, with respect to T and B lymphocytes, monocytes, granulocytes, and erythrocytes, was not significantly affected by the inoculum composition and in all cases was comprised of multiple lineages . Therefore, each step in cell purification resulted in the loss of primitive and accessory cells, which in turn resulted in a net decrease in the number of expanded cells obtained per ml BM aspirate.

Curr Opin Genet Dev, 1995 Jun, 5(3), 342 - 8
YAC transgenics and the study of genetics and human disease; Lamb BT et al.; Advances in yeast artificial chromosome (YAC) technologies over the past decade have enabled the precise identification and manipulation of large genomic regions (>100 kb) of DNA . Introduction of YACs into the mouse germline has now been accomplished through transfection of mouse embryonic stem cells as well as through pronuclear microinjection, allowing the efficient transfer defined genomic loci into mice . YAC transgenics will have a profound impact on the development of transgenic mice as bioreactors and as models of human disease, and on the functional analysis of higher order genomic structure.

J Biotechnol, 1995 May 15, 40(1), 49 - 58
Transgenic rabbits as bioreactors for the production of human growth hormone; Limonta JM et al.; Gene farming is one of the most promising areas in modern biotechnology . To assay the potential usefulness of transgenic rabbits as bioreactors, one call embryos were microinjected with a chimeric gene comprising 5' sequences from mouse whey acidic protein gene (mWAP) linked to the human growth hormone (hGH) gene . Transgenic animals were obtained and the presence of the foreign protein was detected in the milk and serum of these animals at levels of up to 50 micrograms ml-1 and 0.6 ng ml-1, respectively . Founder transgenics were able to transmit the microinjected gene to the first filial generation in a Mendelian fashion . These results showed that transgenic rabbits could constitute a suitable system for the rapid production of recombinant proteins in the milk of lactating females.

Biochem Biophys Res Commun, 1995 May 5, 210(1), 14 - 20
Scaled-up expression of human alpha 2,6(N)sialyltransferase in Saccharomyces cerevisiae; Borsig L et al.; Expression of recombinant full length human alpha 2,6(N)sialyltransferase has been scaled-up in S . cerevisiae in a 150-l bioreactor yielding 47 U at a concentration of 0.31 U/l . The protein specific activity as measured in reconstituted yeast lyophilisate was 0.8 mU/mg protein . The recombinant enzyme exhibited similar Michaelis constants as previously determined for the native rat enzyme . By immunoblotting the enzyme was shown to be heterogeneous by size (44-48 kD) and N-glycosylated . We conclude that recombinant alpha 2,6(N)sialyltransferase expressed in S . cerevisiae is retained in the endoplasmic reticulum as a fully active enzyme.

Mikrobiol Z, 1995 May-Jun, 57(3), 71 - 7
{The effect of hydrodynamic conditions on the interferon-synthesizing activity of swine splenocytes in the production of alpha-interferon}; Dumanskii VD et al.; Parameters of physical constituents of the agitation process and mass-exchange characteristics of the suggested bioreactor for suspension cultivation of pig splenocytes are determined . The effect of hydrodynamic conditions on viability and interferon-synthesizing activity of cells in production of alpha-interferon of pigs has been studied . The relation existing between interferonogenesis processes proceeding under suspension and under stationary biosynthesis has been analyzed by the pair correlation method.

J Biotechnol, 1995 Apr 15, 39(2), 137 - 48
Evaluation of the E . coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study; Tedin K et al.; A concept study devised for the development of a biological containment system has been conducted . We show that the lysis genes of different phage origin function in a variety of bacteria . They may therefore be suited for conditional suicide cassettes . Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E . coli production strains from a bioreactor . Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off . These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions . A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.

J Biotechnol, 1995 Apr 15, 39(2), 129 - 36
Large-scale preparation and biological activity of recombinant human parathyroid hormone; Paulsen J et al.; Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-beta-galactosidase-hPTH fusion protein . We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure . We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.

Biosci Biotechnol Biochem, 1995 Apr, 59(4), 753 - 5
Application of direct current to protect bioreactor against contamination; Tokuda H et al.; Bacteria cell suspensions were sterilized by direct electric current and the cell death rate was proportional to the current . When repeated hydrolysis of casein by immobilized mycelia was done under 0.06 A of direct electric current, contaminants were inhibited while the hydrolysis was stable for more than 10 batches (250 h).

Trends Biotechnol, 1995 Apr, 13(4), 150 - 5
Cell death (apoptosis) in cell culture systems; Cotter TG et al.; In this review we describe the process of cell death known as apoptosis, outlining the key morphological and biochemical features . We also identify the stress elements in bioreactors that drive cells to their death . Finally, we outline methods that might be used to enhance the life span and increase the robustness of cells in culture.

Curr Opin Biotechnol, 1995 Apr, 6(2), 192 - 7
Nuclear magnetic resonance analysis of cell metabolism; Zupke C et al.; Nuclear magnetic resonance (NMR) continues to be a useful tool for the study of cellular metabolism . A variety of NMR techniques have been developed or newly applied to the analysis of cell systems . Many of these techniques are particularly useful for the analysis of immobilized cell bioreactors . The use of several NMR techniques has been an integral part of recent comprehensive metabolic studies . Novel computer-based models and methods have been developed which may make NMR study of metabolism more accessible and powerful.

Artif Organs, 1995 Apr, 19(4), 368 - 74
A novel bioreactor design for in vitro reconstruction of in vivo liver characteristics; Bader A et al.; We have constructed a bioreactor aimed at imitating the three-dimensional micro- and macroenvironment of the liver . In vivo hepatocytes are arranged in plates of cell monolayers and are specifically attached with both sinusoidal surfaces to the space of Disse which contains extracellular matrix . Nonparenchymal cells are located on the other side of the space of Disse toward the sinusoid . For supporting monolayer hepatocytes with bipolar attachment to the extracellular matrix, we used a double gel culture technique that sandwiches hepatocytes between two layers of collagen . In double gel cultures, albumin production increases during an adaptive period to the in vitro environment . In contrast to conventional single gel hepatocytes, double gel hepatocytes maintain expression of sinusoidal microvilli and a polyhedric cell shape in culture as seen by transmission electron microscopy . Albumin production in the bioreactor was stable . The organotypical bioreactor concept is an example of organ mimicry and may provide the basis for the organ-otypical development of a full-sized hybrid artificial liver.

Trends Biotechnol, 1995 Mar, 13(3), 106 - 15
Potential applications of lifetime-based, phase-modulation fluorimetry in bioprocess and clinical monitoring; Bambot SB et al.; The measurement of analyte concentration is a critical part of successful bioreactor and clinical monitoring . Although strategies exist for measuring the majority of relevant analytes, industrial on-line bioreactor control is carried out primarily by measurement and control of pH, pO2 and, in some cases, cell density . This is because the available technology cannot be easily and inexpensively adapted to (a) measure the analyte in an aseptic manner and/or allow for remote sensing, and (b) measure in real time so that on-line control is possible . Similar issues need to be addressed for biosensors for clinical applications . A rapidly emerging technology that has the potential of meeting these challenges is liftime-based phase-modulation fluorimetry, an optical technique that uses the measurement of fluorescence lifetime rather than intensity for determining the concentration of an analyte.

Biotechnol Prog, 1995 Mar-Apr, 11(2), 133 - 9
Computer simulation of low-density lipoprotein removal in the presence of a bioreactor containing phospholipase A2; Shefer SD et al.; High concentrations of low-density lipoproteins (LDL) in the blood can lead to coronary heart disease, the primary cause of death in the Western hemisphere . A new treatment to reduce LDL levels is now being tested on rabbits, which are model animals for hypercholesteremia . The treatment involves using an immobilized enzyme within a bioreactor that is incorporated in an extracorporeal circuit . The enzyme modifies LDL to a form that is much more rapidly removed from the circulation . A mathematical model to describe LDL metabolism in the presence of the bioreactor was developed to give a better understanding of the biodistribution of modified LDL during and following treatment . A four-compartment model was developed on the basis of previous studies on human lipid metabolism, with the specific values of the constants taken from the experimental data on rabbits . A Macintosh II computer with a Stella II modeling program was used to simulate the treatment and to predict LDL levels over time given different values for initial enzyme activity, length of treatment, rate of enzyme denaturation, and other relevant parameters . The model provided a close fit with the experimental results for the change in total cholesterol . It confirmed the observed delay in the plasma cholesterol rebound level after the end of the extracorporeal treatment . One conclusion derived from both the experimental data and the model is that during the first 1.5 h, the limiting step for LDL removal is the rate at which modified LDL is taken up by the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Microbiol Biotechnol, 1995 Mar, 42(6), 813 - 7
On-line monitoring of ethanol, acetaldehyde and glycerol during industrial fermentations with Saccharomyces cerevisiae; Rank M et al.; Industrial fermentations carried out in a 500-l bioreactor were monitored on-line by a prototype of a split-flow modified thermal biosensor . Acetaldehyde and glycerol in the extracellular broth were monitored over the first 48 h of fed-batch fermentations . The aim was to determine the usefulness of these secondary metabolites for on-line monitoring and control . When fermentation of the 13-16 g/l batch sugar was monitored, using immobilised aldehyde dehydrogenase, the acetaldehyde reached a peak value of 0.3 g/l . With immobilised alcohol oxidase a much larger peak of 3.5 g/l ethanol was seen immediately after the acetaldehyde peak . When glycerokinase was used a delayed peak of 1 g/l glycerol was monitored . Of the three metabolites monitored, the ethanol proved the most valuable indicator of suitable timing for the start of the feeding phase and later for controlling and preventing overfeed using the on-line biosensor system.

Magn Reson Med, 1995 Mar, 33(3), 422 - 6
Increase of GPC levels in cultured mammalian cells during acidosis . A 31P MR spectroscopy study using a continuous bioreactor system; Galons JP et al.; The purpose of this study was to study the metabolic events during a slow acidosis in three different cell lines by combining 31P magnetic resonance spectroscopy and hollow fiber bioreactor technology . The rate of change in intracellular pH, glycerophosphorylcholine (GPC), phophorylcholine (PCho), and nucleoside-triphosphate (NTP) levels were measured during 8 h of acidosis and 16 h of recovery in EPO, EAT, and RN1a cells, three cultured mammalians cell lines . Our results show a significant increase in GPC levels to 330 +/- 21.540 +/- 25, and 220 +/- 21% of their initial value correlated to a decrease of PCho levels to 57 +/- 14.58 +/- 17 and 45 +/- 15% of their initial value in EAT, RN1a, and EPO cells, respectively . These changes are discussed in terms of perturbation of energetic metabolism in cells undergoing a slow acidosis.

Protein Eng, 1995 Mar, 8(3), 301 - 14
Engineering disulfide-linked single-chain Fv dimers {(sFv')2} with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides; McCartney JE et al.; Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies . As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors . Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields . Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione . Disulfide-bonded (sFv')2 homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group . Both 26-10 (sFv')2 and 741F8 (sFv')2 existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-association . The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas . The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification . Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l . An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.

J Biotechnol, 1995 Feb 21, 39(1), 59 - 65
Simple fed-batch technique for high cell density cultivation of Escherichia coli; Korz DJ et al.; A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed . A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium . Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (mu < mu crit) . Cell concentrations of 128 and 148 g per 1 dry cell weight (g l-1 DCW) were obtained using glucose or glycerol as carbon source, respectively.

Pharmazie, 1995 Feb, 50(2), 128 - 9
Development of biologically-controlled insulin pumps with glucose-dependent release; Groning R et al.; An experimental insulin pump has been developed in which living microorganisms are used to measure glucose concentration and to produce the energy required to control the release of insulin . The insulin pump consists of a miniaturised bioreactor containing 50 mg freeze-dried yeast . 300 microL of glucose-containing liquid are injected into the bioreactor through a septum . When solutions containing 100 mg or 400 mg glucose in 100 mL are used, the amounts of insulin released differ significantly even within the first measuring interval of 15 min (t-test, p < 0.05) . Within 120 min, 4 I.U . and 17 I.U . insulin respectively are released from the two glucose solutions.

Acta Cient Venez, 1995, 46(2), 129 - 34
{Optimization of the pertussis vaccine production process}; German Santiago J et al.; The production of Pertussis Vaccine was reevaluated at the Instituto Nacional de Higiene "Rafael Rangel" in order to optimise it in terms of vaccine yield, potency, specific toxicity and efficiency (cost per doses) . Four different processes, using two culture media (Cohen-Wheeler and Fermentacion Glutamato Prolina-1) and two types of bioreactors (25 L Fermentador Caracas and a 450 L industrial fermentor) were compared . Runs were started from freeze-dried strains (134 or 509) and continued until the obtention of the maximal yield . It was found that the combination Fermentacion Glutamato Prolina-1/industrial fermentor, shortened the process to 40 hours while consistently yielding a vaccine of higher potency (7.91 +/- 2.56 IU/human dose) and lower specific toxicity in a mice bioassay . In addition, the physical aspect of the preparation was rather homogeneous and free of dark aggregates . Most importantly, the biomass yield more than doubled those of the Fermentador Caracas using the two different media and that in the industrial fermentor with the Cohen-Wheeler medium . Therefore, the cost per doses was substantially decreased.

Aviakosm Ekolog Med, 1995, 29(6), 61 - 4
{Specificity of mass transfer processes in some photobioreactors}; Zhavoronkov VA et al.; In compact photo-bioreactors with high concentration of micro organisms in cultural medium the zone volume of photoabsorption is essentially smaller then general working volume of the apparatus . This discrepancy is overcome by providing an intensive exchange between the dark and light zones . A quantitative characteristic of the intensity of migration exchange is introduced . It is the specific coefficient of mass transfer determined on the basis of solving the problem of Brown convective diffusion near the photoabsorption surface . The structural dependence of the quantity on the problem parameters is found . The qualitative and quantitative difference of this dependence near solid and free surface is discussed.

Chin J Biotechnol, 1995, 11(2), 101 - 7
Studies of protective properties of pluronic and other agents on the hybridoma cell culture; Xu D et al.; The potential toxicity and optimal concentrations of different protective agents such as pluronic F-68, methylcellulose (MC), CMC, and GPE on the in vitro growth of murine hybridoma 2F7 cells that secrete monoclonal antibody against small-cell lung cancer were studied . The effect on the rate of glucose utilization of adding protective agents was investigated . The protective effects of different concentrations of the protective agents at high agitation speed were also observed . It showed that 0.05% to 0.10% (w/v) of pluronic F-68 and 0.10% to 0.20% (w/v) of MC could protect hybridoma cells from shear stress at high agitation speed . Adding pluronic F-68 could increase glucose utilization rate, but increased the ammonia production rate . Although CMC did not affect 2F7 cell growth at a concentration less than 0.10% (w/v), it exhibited no protective property . GPE could lyze hybridoma cells . In a 1.5-L CelliGen bioreactor, when the pluronic F-68 concentration was 0.10% (w/v) in the medium and agitation speed was 70 r/min, the hybridoma cells could grow normally.

Artif Cells Blood Substit Immobil Biotechnol, 1995, 23(5), 587 - 95
Poly(ethylene glycol)-serum albumin hydrogel as matrix for enzyme immobilization: biomedical applications; D'Urso EM et al.; Poly(ethylene glycol)-albumin hydrogels were implanted in mice in subcutaneous position to study their biocompatibility . After one month of implantation, the fibrous capsule formed around the implant was thin and the inflammatory tissue was limited . Acid phosphatase (AP) was selected to evaluate the hydrogel as matrix for enzyme immobilization . AP-hydrogels were prepared using activated PEG (PEGa) of different molecular weights (M.W . 4,600 to 20,000) to evaluate the effect of the matrix composition on the activity of AP . The apparent Km of the immobilized AP was 16 to 20 times higher than the Km of the soluble enzyme . The apparent Km value decreases with the increase of the chain length of the PEGa used . This can be correlated to an increase in the hydrogel porosity . The operational stability of the AP was markedly improved after immobilization by 110 to 160 times according to the PEGa molecular weight involved . Also, asparaginase (ASNase) was immobilized in PEGa (M.W . 10,000)-albumin-hydrogel as a model for in vivo bioreactor . ASNase hydrogels were implanted in the peritoneal cavity of rats; 7 days later, 75% of the initial enzyme activity were retrieved.

Scand J Gastroenterol Suppl, 1995, 208, 101 - 10
Transplantation of isolated hepatocytes and their role in extrahepatic life support systems; Dixit V; Transplantation of isolated hepatocytes for the replacement of liver function and the use of isolated hepatocytes as a bridge-to-transplantation in extrahepatic bioartificial liver support devices offer important therapeutic advances for treating severe liver disease . Progress in cell biology, tissue culture techniques and biotechnology have led the way for the potential therapeutic use of isolated hepatocytes in a wide array of liver disorders . Transplanted hepatocytes show considerable promise of performing the full range of liver functions in several animal models of liver disease, ranging from fulminant hepatic failure to congenital metabolic liver disease . Recently, several interesting designs for extrahepatic liver support systems have been proposed . Although there is no current consensus on its eventual design configuration, the hollow fiber hepatocyte bioreactor design has the greatest potential for therapeutic benefit.

J Ind Microbiol, 1995 Jan, 14(1), 10 - 6
Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors; Breen A et al.; The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated . Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification . The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors . One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions . The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology . DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor . This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes . Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions . DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.

Appl Microbiol Biotechnol, 1995 Jan, 42(5), 671 - 4
Preparation of enantiomerically pure (R)-(1-hydroxyethyl)dimethyl(phenyl)silane using resting cells of Saccharomyces cerevisiae (DHW-S-3) as biocatalyst; Fischer L et al.; The prochiral sila-ketone acetyldimethyl(phenyl)silane (1) was reduced enantioselectively into (R)-(1-hydroxyethyl)dimethyl(phenyl)silane {(R)-2} using resting cells of the commercially available yeast Saccharomyces cerevisiae (DHW S-3) as the biocatalyst . The bioconversion was performed on a 2.0-g scale in a 5-1 bioreactor . Starting with a substrate (1) concentration of 0.4 g.l-1, the highest production rate measured for this bioconversion was about 45-55 mumol (R)-2.l-1.min-1 . After an incubation time of 1 h, all substrate in the medium had been converted, either biocatalytically reduced to (R)-2 or (probably chemically) converted into dimethyl(phenyl)silanol (Me2PhSiOH) . After extraction of the cell-free medium with ethyl acetate/dichloromethane and subsequent purification of the extract by Kugelrohr distillation and chromatography on silica gel (medium-pressure liquid chromatography), 800 mg (yield 40%) of the bioconversion product (R)-2 was isolated . As shown by HPLC studies (cellulose triacetate as the chiral stationary phase) and 1H-nuclear magnetic resonance experiments (after derivatization of the bioconversion product with a chiral auxiliary agent), compound (R)-2 was almost enantiomerically pure (> 99% enantiomeric excess).

Bioprocess Technol, 1995, 20, 61 - 110
Cell adhesion in animal cell culture: physiological and fluid-mechanical implications; Koller MR et al.; We have reviewed the general forces through which cells interact with substrata in their first nonspecific contact . The complex, fast-emerging biology of specific cell adhesion and the structure of the extracellular matrix were reviewed in substantial detail, and the most updated conceptual model of biological cell adhesion was assembled from past efforts and new literature data . The chemistries of the various possible substrata for cell adhesion have been reviewed extensively in the past, and here only a brief summary was presented, with particular emphasis on the materials for traditional and porous microcarriers . The fascinating molecular and cellular implications of cell adhesion were reviewed in detail to establish that cell adhesion and the extracellular matrix provide more than structural support for the cells and their assemblies, and that in fact they constitute fundamental regulators of cell function, metabolism, and differentiation . We reviewed the fluid-mechanical mechanisms of cell damage in microcarrier systems and provided experimental evidence for the importance of the cell-adhesion quality in the ability of cells to withstand fluid forces in bioreactors . We provided evidence that the interplay of cell adhesion and fluid forces is likely to produce cell responses more complex than that of simple life and death, and we suggested that such responses are awaiting investigation and exploration for new applications and culturing possibilities . We also reviewed the experimental evidence on the importance of cell adhesion in cell and microcarrier aggregation and discussed the implications of such aggregation on the culturing environment and the operation of bioreactors . Finally, we discussed the possible implications of cell adhesion as it relates to the developing field of tissue engineering, using the example of bone marrow culture, which involves a large variety of cells and constitutes one of the most complex cell culture systems.

Nucl Med Biol, 1995 Jan, 22(1), 105 - 9
Preparation of {11C}formaldehyde using a hollow fiber membrane bioreactor; Hughes JA et al.; A bioreactor consisting of the enzymes alcohol oxidase and catalase immobilized onto a hollow fiber membrane was used to convert {11C}methanol to {11C}formaldehyde . Using an alcohol oxidase:catalase ratio of 1:500 U, conversion yields of 90-95% were obtained allowing the production of up to 7400 MBq (200 mCi) of {11C}formaldehyde in 5 min . The hollow fiber bioreactor allowed for a convenient, rapid synthesis with yields significantly higher than the standard chemical procedures, has demonstrable advantages over glass bead immobilized systems (primarily due to convective flow), and was amenable to hot cell conditions.

Arch Med Res, 1995 Spring, 26(1), 59 - 63
Rabies veterinary virus vaccine produced in BHK-21 cells grown on microcarriers in a bioreactor; Gallegos Gallegos RM et al.; BHK-21 cells were grown in microcarriers in the CELLIGEN CL 50 bioreactor to produce a stock of rabies veterinary virus vaccine PV (Pasteur virus) strain . Perfusion mode operation of this bioreactor produced between two- and fourfold larger yields (cells/ml) than traditional stationary cell culture systems (i.e., Blake, and Roller bottles or cell factory multitrays) . The method employed harvested 281 of rabies virus in 200 h (infectivity titer 0.6 +/- 1.4 x 10(7) LD50 per ml) in a single operation . The risk of contamination is thus reduced when compared with traditional stationary methods which, in order to obtain the same amount of virus, would require the operation of 285 Blake bottles, or 143 Roller bottles, or 15 Cell Factory multitrays (10 trays) . By perfusion mode operation of the bioreactor, 89% of the cell culture medium was recovered as vaccinal virus, which contrasts with the yield of only 50-59% using traditional cell culture systems . On the other hand, only 925 ml of fetal serum was required to obtain the 281 of rabies virus harvest as compared to the 3420 ml required by traditional methods.

Appl Biochem Biotechnol, 1995 Spring, 51-52, 179 - 95
Comparative energetics of glucose and xylose metabolism in ethanologenic recombinant Escherichia coli B; Lawford HG et al.; This study compared the anaerobic catabolism of glucose and xylose by a patented, recombinant ethanologenic Escherichia coli B 11303:pLOI297 in terms of overall yields of cell mass (growth), energy (ATP), and end product (ethanol) . Batch cultivations were conducted with pH-controlled stirred-tank bioreactors using both a nutritionally rich, complex medium (Luria broth) and a defined salts minimal medium and growth-limiting concentrations of glucose or xylose . The value of gamma ATP was determined to be 9.28 and 8.19 g dry wt cells/mol ATP in complex and minimal media, respectively . Assuming that the nongrowth-associated energy demand is similar for glucose and xylose, the mass-based growth yield (Yx/s, g dry wt cells/g sugar) should be proportional to the net energy yield from sugar metabolism . The value of Yx/s was reduced, on average, by about 50% (from 0.096 g/g glu to 0.51 g/g xyl) when xylose replaced glucose as the growth-limiting carbon and energy source . It was concluded that this observation is consistent with the theoretical difference in net energy (ATP) yield associated with anaerobic catabolism of glucose and xylose when differences in the mechanisms of energy-coupled transport of each sugar are taken into account . In a defined salts medium, the net ATP yield was determined to be 2.0 and 0.92 for glucose and xylose, respectively.

Boll Soc Ital Biol Sper, 1995 Jan-Feb, 71(1-2), 13 - 20
Enzymatic hydrolysis of DNA using a membrane bioreactor; Donato L et al.; Aim of this work was a kinetic study of the deoxyribonuclease I (E.C . 3.1.21.1.) reaction in several conditions . Optimal reaction conditions with enzyme free in solution were determined . DNase activity was studied as function of several parameters such as pH, enzyme concentration, substrate concentration, cofactor concentration . The Km value of the free enzyme was also determined . Subsequently, the possibility to carry out the reaction in systems in which the enzyme was immobilized on the inner surface of the polymeric membranes was evaluated and realized . Experimental procedures, in these new conditions, permitted to determine the mass of immobilized enzyme and the percentage of the reaction product recovery . Results showed that the immobilization procedure determines a decay of the initial activity of enzyme, but there is an important increase of enzyme stability.

Int J Artif Organs, 1995 Jan, 18(1), 27 - 33
Influence of warm ischemia on isolation and primary culture of hepatocytes from rat liver for a hybrid artificial liver; Kon H et al.; To assess the possibility of using hepatocytes from ischemic liver, as a bioreactor of a hybrid artificial liver, we investigated the influence of warm ischemia on the isolation and culture of hepatocytes in rats . Warm ischemia was induced by clamping the liver hilus and the animals were divided into 3 groups according to the duration of ischemia: group A (no ischemia), group B (10 minutes) and group C (20 minutes) . Hepatocytes were isolated by the collagenase perfusion method and cultured for 5 days . The yield and viability of the isolated hepatocytes were lower in group C . Rate of attachment was decreased as the duration of ischemia increased . There was no significant difference observed in functions in culture . Sufficient hepatocytes, as a bioreactor, can be isolated and cultured from warm ischemic liver within 10 minutes . Though the number of available hepatocytes were diminished, hepatocytes procured from longer warm ischemic liver could be utilized as a bioreactor.

J Mol Recognit, 1995 Jan-Apr, 8(1-2), 146 - 50
Photostructurized electrochemical biosensors for bioreactor control and measurement in body fluids; Lobmaier C et al.; This article details a new type of biosensor for the simultaneous analysis of glucose, glutamate and glutamine in complex biological fluids like fermentation broths and blood . Simultaneous analysis was made possible by the application of different enzyme layers onto different electrodes of one photostructurized sensor . Photostructuring was done by means of a new developed photopolymer . Preparation of the photopolymer and the enzyme layers as well as the characterization of the sensors thus constructed with respect to linearity, response time and sensitivity are described.

Zentralbl Chir, 1995, 120(8), 614 - 23
{An artificial liver on its way to clinical reality?}; Thasler WE et al.; Temporary extracorporeal liver assist devices based on liver cell cultures represent a promising approach for the treatment of liver failure as well as for perioperative care in liver surgery and transplantation . Primary hepatocytes from donor animals as well as human cell lines have been discussed as cell sources . A viable cell mass of 110-220 g (= 2.5-5 x 10(10) liver cells) has to be functionally active in bioreactors over a time period of several days . This cell number correlates to 10-20% of liver cells in an adult human liver . Neither in animal experiments, nor in clinical trials a complete liver replacement could yet be shown for cell-based bioreactors, however, beneficial effects have been demonstrated: partial detoxification and specific synthetic functions have been reported in animal experiments . Two authors have shown first preliminary clinical data on different cell-based liver assist devices with influences on blood parameters and the neurological status of treated patients . Nevertheless, clinical improvements also have been achieved by using non-biological support, like charcoal-hemoperfusion and modified hemodialysis . In order to assess the influence of liver support devices on better results in therapy of liver failure, randomized clinical trials have to be performed . Prior to this, artificial liver support systems have to demonstrate their biocompatibility and biosafety for clinical use as well as their efficacy and availability.

Chin J Biotechnol, 1995, 11(1), 69 - 77
Studies on kinetics of substrate utilization of hydrogen production from wastewater with immobilized cells of photosynthetic bacteria; Xu X et al.; The kinetic characteristics of substrate utilization by immobilized cells of Rhodopseudomonas capsulata 386 and Rhodopseudomonas sp . D for H2 production was investigated . The results showed that substrate utilization did not proceed simultaneously with H2 evolution, H2 producing capacity of immobilized cells with agar was higher than that of alginate immobilized cells, but the appearance of maximum H2 producing activity was later than the last one . The kinetics of substrate utilization (glucose) by immobilized cells of strain D followed the first-order reaction, rate constant k value was 1.2 x 10(-2) L/h, analysis of macrokinetics indicated that substrate utilization by immobilized cells in H2 evolution process was governed by biochemical reaction other than diffusion transfer limitation, because of the Thiele modulus were 0.125 and 0.154 for immobilized cells with D = 3.6 mm and 4.4 mm particles, the corresponding effective factor were determined as 0.998 and 0.988, respectively . An immobilized cell bioreactor, fed by glucose and lactate, was employed for continuous H2 evolving system . It was found that hydrogen production were 0.659 L/d and 0.477 L/d in immobilized cells system of strain 386 and D when lactate used as H2 evolving substrate, while the volumetric H2 rate were all up to 1.0 L/L.d.

Appl Microbiol Biotechnol, 1994 Dec, 42(4), 531 - 5
Growth kinetics of animal cells immobilized within porous support particles in a perfusion culture; Yamaji H et al.; The growth kinetics of anchorage-independent animal cells {mouse myeloma MPC-11 (ATCC CCL 167)} immobilized within porous polyvinyl formal resin biomass support particles (BSPs; 3 x 3 x 3 mm cubes; mean pore diameter, 60 microns) was analyzed by measuring intracellular and extracellular lactate dehydrogenase (LDH) activities in a perfusion culture . First, the intracellular LDH content of cells immobilized within the BSPs was assayed in a shake-flask culture and found to remain almost comparable to that of non-immobilized cells in the exponential growth phase under static culture . Then, cells inoculated in the BSPs were grown in a continuous stirred-tank bioreactor . Using the LDH content of non-immobilized cells, the density of immobilized cells and the numbers of leaked and dead cells were evaluated, respectively, by the intracellular LDH activity of immobilized and leaked cells and the LDH activity in cell-free culture supernatant . In the initial period, immobilized cells exhibited exponential growth at a constant apparent specific growth rate; however, the actual specific growth rate, which takes into consideration cell death and cell leakage, decreased significantly . In the stationary phase, the actual specific growth rate maintained a constant but markedly lower value than during exponential growth.

J Chem Technol Biotechnol, 1994 Dec, 61(4), 337 - 42
Micromixing and the steady-state performance of bioreactors using recombinant bacteria--analysis through a reversed two-environment model; Patnaik PR; A reversed two-environment model has been used to study micromixing in continuous fermentation for growth and product formation by recombinant bacteria . As an example, an Escherichia coli M72 strain harbouring the plasmid pPLc23trpA1 and producing tryptophan synthetase is considered . With excess substrate, 10% plasmid-free bacteria in the initial broth do not affect the steady-state concentrations of plasmid-containing bacteria but their mass fractions decrease significantly . With a smaller substrate concentration, the mass fractions decrease sharply as the dilution rate increases if micromixing is good; but concentrations still remain high . There is also a clear demarcation between regions of good and poor micromixing . These results are explained and an optimal combination of micromixing and dilution rate is suggested to maximise productivity.

Int J Artif Organs, 1994 Dec, 17(12), 663 - 9
Amino acid metabolism by hepatocytes in a hybrid liver support bioreactor; Fuchs M et al.; The amino acid patterns of medium perfusate in a liver cell bioreactor developed for a hybrid liver support system have been measured . There were considerable changes in the concentrations of glutamic acid, glutamine, alanine, arginine, ornithine and branched chain amino acids during the first 10 days which is indicative of dynamic cellular metabolism . From day 15, steady state conditions of nitrogen metabolism are reflected by stable amino acid turnover . Monitoring of urea, K+, and P-450 activity suggests that hepatocytes have switched to a stable protein synthesis with a general amino acid uptake and keto acid release following cell volume increase.

Ann N Y Acad Sci, 1994 Nov 30, 745, 455 - 61
Bioreactor engineering as an enabling technology to tap biodiversity . The case of taxol; Shuler ML; One barrier to exploiting the chemical and genetic diversity in nature is the difficulty of cultivating many organisms in a controlled manner . In some cases it is difficult to achieve growth . In many others, good growth is achieved, but the expression of the organism's genetic potential to make a desired product is not realized . The thesis of this paper is that a coupling of an understanding of reactor engineering principles with the basic knowledge of the biology is often necessary to circumvent these barriers . In many cases the construction of appropriate cultivation systems is a necessary step to better understanding of cellular physiology . In some cases the chemical of interest is of high social utility and comes from a natural source that is uncommon and difficult to secure . In these cases a method of controlled cultivation becomes a prerequisite for commercial exploitation . These points were illustrated using a taxol . Taxol is an important new anticancer drug whose development has been greatly impeded by supply problems . Taxol has been derived from the park of the pacific yew tree, a process that kills the tree . The pacific yew is a relatively uncommon tree and very slow growing . One alternative to the natural source is plant cell culture . Such cultures can produce significant levels of taxol with substantial release into the medium . Taxane products not observed in typical extracts from field-grown plants can be found in cell cultures, indicating the potential unmasking of pathways . These cultures are quite responsive to changes in their environments as illustrated by the summary of initial observations . With regard to natural compounds, biochemical engineers can play a major role in the capture and preservation of producing systems, in the discovery of useful compounds, and in providing the basis for commercial production of natural compounds.

Ann N Y Acad Sci, 1994 Nov 30, 745, 21 - 34
Alteration of the biochemical valves in the central metabolism of Escherichia coli; Liao JC et al.; Although E . coli central metabolism has been studied for several decades, many regulatory features are still unknown . To achieve the goal of rational manipulation of cellular metabolism, it is important to understand how E . coli responds to overexpressed enzymes . By studying the biochemical control of fluxes between PEP, pyruvate, and OAA, we have addressed some fundamental questions that may prove to be essential for applications in metabolic engineering . First, we found that simultaneous overexpression of Pck and Ppc, or Pps alone in the presence of glucose leads to phenotypes consistent with futile cycline . In contrast to our expectation, futile cycling per se does not affect the growth rate significantly . However, excessive futile cycling may cause competitive disadvantage in the natural environment . Overexpression of Pck caused growth inhibition but no futile cycling . Therefore, E . coli controls the expression of gluconeogenic enzymes not only to avoid excessive futile cycling, but also to prevent toxicity effects . In metabolic engineering, futile cycling may be used as a strategy to stimulate metabolism for either production of metabolites or digestion of toxic wastes . Second, we found that the expression levels of Pps and Pck in E . coli are not optimal for growth on pyruvate and succinate, respectively . Overexpression of these enzymes increases the growth rate on pyruvate and on succinate, respectively, indicating that the slow growth rates on these substrates are at least partially caused by the insufficient supply of PEP and its derivatives . Moreover, E . coli also has not optimized the Ppc level for optimal growth yield on glucose in uncontrolled batch cultures . These results demonstrate that the central metabolism is not optimized for growth under defined laboratory conditions . Thus, the possibility exists that adjustment of native enzyme levels in the central metabolism can improve bioreactor performance . Third, we found that overexpression of Pck affects the transcriptional levels of unrelated genes . This example indicates that physiological responses to enzyme (over)expression should be interpreted cautiously, as changing the expression level of a specific enzyme may affect many unlinked genes . Similar results have also been obtained by use of two-dimensional electrophoresis of proteins from E . coli . Although more questions remain to be answered, fast progress in the area of metabolic engineering can be expected in the near future.

J Biotechnol, 1994 Nov 30, 38(1), 81 - 8
A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity; Alvarez P et al.; A system has been developed for the release of heterologous proteins from Saccharomyces cerevisiae, based on the use of thermosensitive osmotic-remedial mutants, deficient in cell integrity, that lyse at the non-permissive temperature, thus releasing the bulk of intracellular proteins and leaving behind cell ghosts and debris . The strains developed combine the lyt2 mutation (which is allelic to gene SLT2/MPK1 coding for a MAP kinase homolog), with the disruption of genes PEP4 and PRB1 known to produce a protease-deficient background . Cells transformed with the appropriate bacterial gene, released about 70% of the heterologous protein chloramphenicol acetyl transferase (CAT) in bioreactor cultivation upon switching growth temperature to 37 degrees C, or by osmotic shock of the cells preincubated at 37 degrees C in the presence of 1 M sorbitol . It is suggested that our release system could be advantageous for obtaining large-scale protein preparations for downstream processing without any mechanical breakage of the cells, enzymatic treatment or chemical extraction.

J Biotechnol, 1994 Nov 30, 38(1), 21 - 32
Development of a miniature bioreactor for continuous culture in a space laboratory; Walther I et al.; A new type of miniature bioreactor for continuous culture of yeast cells in space laboratories has been developed . Silicon microtechnology has permitted the integration of numerous functions and systems in a volume of 87 x 63 x 63 mm3 and a weight of 610 g . The 100 ml of fresh medium can be delivered at variable flow rates to the cultivation chamber (volume 3 ml) by means of a micropump . The culture is agitated by a magnetic stirrer . Microsensors monitor pH, temperature and redox potential . The decrease of pH occurring during the cultivation of Saccharomyces cerevisiae is compensated electrochemically . A window allows the inspection of the culture status . Samples of up to 1 ml can be drawn through a silicone rubber septum . The data measured by the sensors are transmitted on-line to the ground station during operations in space . The bioreactor had to fulfil several requirements related to the safety regulation of the space agencies . In particular, new materials had to be selected and tested for their biocompatibility . The instrument has now passed all space and biological qualification tests and will be used in an experiment selected by ESA for the International Microgravity Laboratory-2 Mission in Spacelab in July 1994 . This paper gives the results of the functional and biological tests and a detailed description of the instrument.

Transplantation, 1994 Nov 15, 58(9), 984 - 8
Bioreactor for a larger scale hepatocyte in vitro perfusion; Gerlach JC et al.; A bioreactor construction for hepatocytes and liver sinusoidal endothelial cells is described . The reactor is based on capillaries for hepatocyte immobilization . Four discrete capillary membrane systems, each serving different purposes, are woven to create a three-dimensional framework for decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2 removal . The biochemical performance of reactors initially seeded with 2.5 x 10(9) hepatocytes were evaluated over 3 weeks . On day 21, pig albumin synthesis was 4.7 mg/day, lidocaine metabolism was 813.7 +/- 23 micrograms/hr, galactose elimination was 210.1 +/- 3 mg/hr, and midazolam metabolism was 37.1 +/- 2 micrograms/hr . The specific construction of the reactor enables scale-up to hybrid liver support systems as extracorporeal bridging devices for liver transplantation.

J Immunol Methods, 1994 Nov 10, 176(1), 67 - 77
Multiparametric analyses of hybridoma growth on glass cylinders in a packed-bed bioreactor system with internal aeration . Serum-supplemented and serum-free media comparison for MAb production; Moro AM et al.; Monoclonal antibodies are one of the most important products of biotechnology and laboratories and companies all over the world are pursuing their large-scale production . Herein we report a protocol for hybridoma cell cultivation over small glass cylinders inside a 3 liter bioreactor vessel which leads to the production and purification--in order of grams--of one MAb intended for human therapeutic use . This protocol proved to be simple, reproducible and cost effective . Three trials are reported: the first two using conventionally serum-supplemented medium culture and producing 3.15 and 2.1 g of purified MAb in 30 and 21 days respectively, and the third one using serum-free medium culture and producing 6 g of purified MAb in 36 days . We have ascertained the stability of the hybridoma by its cloning directly in serum-free medium . The downstream processing of the serum-free trial was done in a single step, concentrating large volumes of supernatant while simultaneously purifying the antibody.

J Cell Biochem, 1994 Nov, 56(3), 340 - 7
Underlying mechanisms at the bone-biomaterial interface; Schwartz Z et al.; In order to understand how biomaterials influence bone formation in vivo, it is necessary to examine cellular response to materials in the context of wound healing . Four interrelated properties of biomaterials (chemical composition, surface energy, surface roughness, and surface topography) affect mesenchymal cells in vitro . Attachment, proliferation, metabolism, matrix synthesis, and differentiation of osteoblast-like cell lines and primary chondrocytes are sensitive to one or more of these properties . The nature of the response depends on cell maturation state . Rarely do differentiated osteoblasts or chondrocytes see a material prior to its modification by biological fluids, immune cells and less differentiated mesenchymal cells in vivo . Studies using the rat marrow ablation model of endosteal wound healing indicate that ability of osteoblasts to synthesize and calcify their extracellular matrix is affected by the local presence of the material . Changes in the morphology and biochemistry of matrix vesicles, extracellular organelles associated with matrix maturation and calcification, seen in normal endosteal healing, are altered by implants . Moreover, the material exerts a systemic effect on endosteal healing as well . This may be due to local effects on growth factor production and secretion into the circulation, as well as to the fact that the implant may serve as a bioreactor.

Bioconjug Chem, 1994 Nov-Dec, 5(6), 577 - 82
Temperature-responsive bioconjugates . 3 . Antibody-poly (N-isopropylacrylamide) conjugates for temperature-modulated precipitations and affinity bioseparations; Takei YG et al.; Immunoglobulin G (IgG) has been modified by poly(N-isopropylacrylamide) (PIPAAm) to create a novel bioconjugate which exhibits reversible phase transition behavior at 32 degrees C in aqueous media . A terminal carboxyl group introduced into PIPAAm molecule by polymerization of IPAAm with 3-mercaptopropionic acid was used for conjugation to IgG via coupling reaction of activated ester with protein amino group . These conjugates exhibited rapid response to changes in solution temperature and significant phase separation above a critical solution temperature corresponding to that for the original PIPAAm . These conjugates bound to antigen quantitatively in aqueous system, and antigen-bound complex also demonstrated phase separation and precipitation above a critical temperature . Precipitate was reversibly redissolved in cold buffer . Though particular conjugate which includes 12 molecules of PIPAAm with 6,100 molecular weight suppressed more than 95% of Fc-dependent binding with protein A, it retained approximately 60% of original specific antigen binding activity . It was manifested that polymer content of conjugate was 20-30 wt% for the case of 6,100 molecular weight of PIPAAm to demonstrate specific antigen binding activity most effectively and to reduce Fc-dependent binding with protein A . IgG-PIPAAm conjugates were soluble in water and formed antigen-bound complex in homogeneous solution system below a critical temperature . These conjugates were separated from solution and other solutes corresponding to PIPAAm nature and scarcely bound to antigen above a critical temperature . It is revealed that temperature-responsive PIPAAm conjugated to biomolecule operated as a switching molecule . These phenomena are attractive for not only reversible bioreactors and protein separations but also carrier substrate to localize biomolecules such as drugs, peptides and hormones in a living body.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 396 - 402
Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen; Kong IC et al.; The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H2 were assessed in serum-bottle microcosms . Experiments were performed over a range of metal concentrations (20-200 mg/l) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum . In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration . However, the extent of inhibition varied for the different biotransformations and for the different metals tested . For example, the concentration of CuCl2 effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/l, respectively . Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic . The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 mg/l respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect . The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.

Trends Biotechnol, 1994 Nov, 12(11), 450 - 5
Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator; Blight MA et al.; Heterologous proteins synthesized in the Gram-negative bacterium Escherichia coli in bioreactor culture may accumulate in one of three 'compartments':the cytoplasm, the periplasm, or the extracellular medium . Many overexpressed proteins from various origins have been purified from each of these locations . However, to date, each system has required specific tailoring to meet the stringent requirements for each protein product to ensure correct folding, activity and appropriate yield . The E . coli haemolysin secretion system appears to provide a flexible mechanism with which to secrete a wide variety of heterologous fusion proteins into the extracellular medium.

Trends Biotechnol, 1994 Nov, 12(11), 439 - 43
Can immobilization be exploited to modify enzyme activity?
Clark DS.
Immobilization has long been recognized as a useful tool for retaining enzymes in bioreactors and enabling the continuous operation of enzymic processes . However, the potential of immobilization for modifying enzyme activity in an advantageous, if not predictable, manner is less-well appreciated . This review summarizes selected studies that have used immobilization to tailor the catalytic properties of enzymes, and highlights the application of immobilization to the rational design of biocatalysts.

Vopr Virusol, 1994 Nov-Dec, 39(6), 274 - 6
{Pseudosuspended cultured cells for obtaining influenza A and B viral antigens}; Podcherniaeva RIa et al.; Conditions for pseudosuspension culturing of continuous cells of the kidneys of dogs (MDCK), green marmosets (CV-1), and swine (SPEV) on two types of microcarriers (Cytogel-3 and cytolar-2) under semiproduction conditions (50 liter bioreactor) were developed on a model of reassortants of influenza viruses A and B . To standardize the conditions, native sera were replaced by growth-stimulating proteins isolated from the sera of various animals (cattle, northern deer, swine) . A better adhesive capacity and more active cell proliferation on porous microcarrier cytogel-3 were observed . The highest proliferative activity of cells was observed when porcine growth-stimulating proteins were used . Influenza A virus reassortant (H3N1) was actively reproduced in MDCK cells . The reproduction of influenza B virus reassortant was similarly higher in MDCK cells, in comparison with SPEV cells; at the same time, no differences in hemagglutinin titers in the two cell cultures were observed . The pseudosuspension method of cell culturing is recommended for the preparation of influenza antigens.

J Antibiot (Tokyo), 1994 Oct, 47(10), 1098 - 103
Production of cladospirone bisepoxide, a new fungal metabolite; Petersen F et al.; Cladospirone bisepoxide (1), a novel metabolite, was isolated from cultures of a fungus which was characterized as a coelomycete by the formation of pycnidia . By optimization of media and fermentation conditions, a titer of up to 1.5 g/liter on shake level and 1.16 g/liter on bioreactor scale could be achieved . The isolation of the compound was performed by solvent extraction of the culture broth and subsequent crystallization . Cladospirone bisepoxide displays selective antibiotic activity against several bacteria and fungi and inhibits germinations of Lepidium sativum at low concentrations.

Clin Exp Immunol, 1994 Oct, 98(1), 17 - 20
Plasma half-lives and bioavailability of human monoclonal Rh D antibodies BRAD-3 and BRAD-5 following intramuscular injection into Rh D-negative volunteers; Goodrick J et al.; Two human MoAbs, BRAD-3 (an IgG3 anti-D) and BRAD-5 (an IgG1 anti-D), were produced from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines grown in hollow fibre bioreactors . Six Rh D-negative male volunteers were injected intramuscularly with anti-D; two received BRAD-3 (approx . 1500 micrograms, 2600 IU), two were given BRAD-5 (300 micrograms, 2000 IU), and two had polyclonal anti-D immunoglobulin (500 IU, approx . 100 micrograms anti-D) . Levels of anti-D in plasma samples taken up to 42 days later were measured by a sensitive AutoAnalyser method . The half life of BRAD-5 (mean 22.2 days) was greater, and that of BRAD-3 (mean 10.2 days) less than that of polyclonal anti-D (mean 15.6 days) . The bioavailability (plasma uptake) of the MoAbs (mean 33.9%) was less than that of the polyclonal anti-D (mean 60.3%) . BRAD-3 and BRAD-5 may be suitable for use in antenatal and post-natal prophylaxis against Rh D haemolytic disease of the newborn.

Int J Artif Organs, 1994 Oct, 17(10), 554 - 8
Alpha-keto acid metabolism by hepatocytes cultured in a hybrid liver support bioreactor; Fuchs M et al.; Isolated pig liver cells cultured using a perfusion technique were analyzed over 39 days to test their ability to change the perfusate alpha-keto acid profile . While the pyruvate concentration in the culture medium decreased as of the first day, the alpha-ketoglutarate (KG), alpha-ketoisocaproate (KIC), alpha-ketoisovalerate (KIV) and alpha-keto-beta-methyl-n-valerate (KMV) were synthesized immediately and released by the liver cells . The metabolic capacity of the cell culture system increased up to day 10, decreased during the following 5 days and reached a steady state beyond day 15, which was maintained for at least 30 days . The branched chain alpha-keto acid release, in particular alpha-ketoisocaproate, reflects an effective transamination capacity of the newly developed culture system and shows an intact protein biosynthesis for at least 30 days in vitro.

Int J Artif Organs, 1994 Oct, 17(10), 549 - 53
Hybrid liver support system in a short term application on hepatectomized pigs; Gerlach J et al.; A short term application of a hybrid liver support system in circuits with continuous plasma-separation was investigated in a model of hepatectomized pigs under general anesthesia . Primary pig hepatocytes were immobilized in a bioreactor with three independent capillary systems . An immune barrier is achieved by avoiding the direct contact of blood cells with the hepatocytes by a plasmaseparation step and by an outflow filtration within the reactor . In three groups (hepatectomized pigs and system with- or without hepatocytes as well as untreated pigs with system without hepatocytes), the short term metabolism of the reactors was positively demonstrated by investigating ammonia detoxification, phenylalanine- and lactate metabolism . Limitations of the presented model are discussed.

J Immunother Emphasis Tumor Immunol, 1994 Oct, 16(3), 198 - 210
Intraperitoneal adoptive immunotherapy of ovarian carcinoma with tumor-infiltrating lymphocytes and low-dose recombinant interleukin-2: a pilot trial; Freedman RS et al.; A pilot study was conducted in patients who had advanced epithelial ovarian carcinoma, and who were refractory to platinum-based chemotherapy, to determine the feasibility and clinical effects of a schedule of intraperitoneal (IP) tumor-infiltrating lymphocytes (TIL) expanded in recombinant interleukin-2 (rIL-2), and low-dose rIL-2 IP . TIL were expanded from solid metastases or malignant effusions in serum-free AIM V medium supplemented with low concentrations (600 IU/ml) or rIL-2 using a four-step method of expansion that included a hollow fiber bioreactor (artificial capillary culture system) . Patients received IP TIL suspended in dextrose 5% in sodium chloride 0.2% containing 0.1% human albumin and 6 x 10(5) IU rIL-2 on day 1, followed by 6 x 10(5) IU rIL-2/m2 body surface area, administered daily by bolus IP injection, on days 2-4, 8-11, and 15-18 . In the absence of disease progression, two additional 4-day cycles of IP rIL-2 were administered . Patients (n = 3) whose TIL failed to grow in vitro received IP IL-2 alone . Eight patients received rIL-2 expanded TIL (10(10)-10(11) range) plus rIL-2 followed by several cycles of rIL-2 alone . One of these patients was treated twice with TIL plus rIL-2 . Expanded TIL were primarily CD3+CD4+TCR alpha beta+ (eight TIL-derived T-cell lines) . One TIL-derived T-cell line was comprised mostly of CD3+CD8+TCR alpha beta+ cells . Eleven patients (eight treated with TIL plus rIL-2 and three patients treated with rIL-2 alone) received a total of 38 cycles of rIL-2 without TIL . Grade 3 clinical toxicity (peritonitis) occurred in 1 of 9 cycles of TIL plus rIL-2 and 1 of 38 cycles of rIL-2 alone . Each cycle was 4 days long . Grade 3 anemia occurred in 1 of 9 TIL plus rIL-2 cycles and 3 of 38 cycles of rIL-2 alone . There were no measurable responses; however, four of eight patients treated with IP TIL plus rIL-2 had some indication of clinical activity: ascites regression (two patients), tumor and CA-125 reduction (one patient), and surgically confirmed stable tumor and CA-125 values (one patient) . The schedule of IP TIL plus low-dose rIL-2 shows manageable toxicity and is worthy of further evaluation in patients with epithelial ovarian cancer who have less tumor burden.

Math Biosci, 1994 Oct, 123(2), 147 - 65
Steady-state coexistence of three pure and simple competitors in a four-membered reactor network; Baltzis BC et al.; In this study we investigate the dynamics of pure and simple competition among three microbial populations in a spatially heterogeneous environment . The environment is modeled as a network of four interconnected bioreactors . Growth has been assumed to be noninhibitory, while maintenance requirements have been neglected . Results of numerical studies indicate that the three competitors may coexist in a stable steady state in a domain of the operating parameters space . Results are presented in the form of two-dimensional operating diagrams . No domain was found in the operating parameter space where more than one steady state is meaningful and stable . Since earlier theoretical studies have shown that two pure and simple microbial competitors may coexist in two interconnected bioreactors while numerical studies have shown that three pure and simple competitors cannot coexist in three interconnected bioreactors, the results of the present study may lead one to speculate the N pure and simple competitors may coexist in a network made of 2N-1 bioreactors.

J Chem Technol Biotechnol, 1994 Oct, 61(2), 123 - 38
Mathematical modelling of industrial pilot-plant penicillin-G fed-batch fermentations; Menezes JC et al.; Penicillin-G fermentation with industrial media in 1 m3 stirred tank bioreactors was studied . A model based on the Bajpai-Reuss model structure was developed . Under typical production conditions catabolite repression is nonidentifiable and extensive mycelium differentiation occurs . Thus, the original model was reformulated, neglecting glucose repression of penicillin production and including biomass autolysis . The multi-substrate nature of industrial media was critically analysed . By combining the two most important carbon substrates present, a simple and applicable model was obtained . Model predictions agreed well with experimental data and reproduced the general characteristics observed in the fermentations . The predictive power of the model was tested for fermentations with different sugar feed rate profiles and raw materials (corn-steep liquor and sugar syrup) . Several aspects of parameter estimation and model development are discussed on the basis of direct experimental data inspection and a sensitivity analysis of model parameters.

J Biotechnol, 1994 Sep 30, 37(2), 179 - 84
Use of a hollow fiber bioreactor for large-scale production of alpha 2-adrenoceptors in mammalian cells; Ala-Uotila S et al.; Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct . One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members . Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail . In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production . We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors . These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette . We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.

Biochem Pharmacol, 1994 Sep 15, 48(6), 1121 - 8
A novel dimeric fluoropyrimidine molecule behaves as a remote precursor of 5-fluoro-2'-deoxyuridine in human erythrocytes; Gasparini A et al.; A new dimeric fluoropyrimidine molecule (5-fluoro-2'-deoxyuridilyl-(5'-->3')-5-fluoro-2'-deoxy-5'-uridylic acid, Compound 1) was chemically synthesized from two separately deblocked 5-fluoro-2'-deoxyuridine mononucleotide moieties . Other structurally related nucleotides, 5-fluoro-2'-deoxyuridine-5'-diphosphate (FdUDP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine-3',5'-bisphosphate were also synthesized . The structures of all synthesized molecules were verified by mass spectrometric analyses and were consistent with expected molecular mass values . The metabolic patterns of conversion of Compound 1 were investigated both in human erythrocyte lysates and in intact erythrocytes previously loaded with this molecule according to a highly conservative encapsulation procedure . In hemolysates, Compound 1 was transformed to 5-fluoro-2'-deoxyuridine (FUdR) and to 5-fluorouracil (FU) through the intermediate formation of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) . In intact red cells, Compound 1 still generated FUdR (and to a lesser extent FU), that was then released outside . The conversion pathway involves a phosphodiesterase-catalysed hydrolysis of Compound 1 into two FdUMP molecules, followed by further dephosphorylation to FUdR and by partial conversion to FU . Unlike hemolysates, Compound 1-loaded intact erythrocytes featured transient formation of FdUDP and FdUTP, both metabolites representing storage compounds for the final and sustained production of FUdR and FU . Therefore, human erythrocytes can behave as bioreactors ensuring the time-controlled production and delivery of the two powerful antitumor drugs FUdR and FU from encapsulated Compound 1 . This new molecule and other compounds as well (e.g . FdUDP and FdUTP) can be viewed as useful pre-prodrugs, exploitable for intraerythrocytic bioconversion reactions.

PDA J Pharm Sci Technol, 1994 Sep-Oct, 48(5), 236 - 40
Role of quality control in validation of biopharmaceutical processes: case example of clean-in-place (CIP) procedure for a bioreactor; Geigert J et al.; If ever clear instruction and close teamwork is needed, it is in the validation of manufacturing processes . All members of the Validation Team need to understand how the Quality Control testing fits into the overall validation work plan . This affords the team members the opportunity to understand how data will be used and avoids a situation where the test results either invalidate or inadequately support the validation plan . A case example is presented for an approach used to validate Clean-in-Place (CIP) procedures for 1600 L bioreactors which are operated on a campaign basis for multi-biopharmaceutical synthesis.

Magn Reson Med, 1994 Sep, 32(3), 310 - 8
In vitro and in vivo 13C and 31P NMR analyses of phosphocholine metabolism in rat glioma cells; Gillies RJ et al.; In vivo magnetic resonance spectroscopy (MRS) has revealed that phosphomonoesters (PME) such as phosphocholine (PCho) and phosphoethanolamine (PEth) are elevated in tumors and rapidly proliferating tissues . The regulation of PME levels and their relationship to proliferation are not well known . In the present study, we investigated the regulation of PCho and PEth levels in rat glioma cells grown in vivo and in vitro using 31P and 13C MRS . However, the ability of cells to produce choline endogenously is variable . To fully understand regulation of PCho levels, it is necessary to characterize the activity of the endogenous pathway, if it exists . This was first investigated by following the metabolic fate of 13C-labeled methionine of 9L glioma tumors in vivo . Our results indicate that there is a significant amount of de novo choline synthesis in vivo . However, similar experiments performed in vitro using cells cultured in bioreactors indicated that glioma cells themselves are unable to synthesize choline de novo, suggesting that the in vivo results were due to the involvement of extra-tumoral organs, e.g., liver . Further in vitro experiments demonstrated that the uptake and phosphorylation of physiologically relevant concentrations of exogenous choline is very active in these systems . Thus, it appears that the exogenous pathway for PCho biosynthesis predominates and regulates PCho levels in glioma cells . Our results also demonstrate that PCho levels are lowest, and PEth levels are highest, in non-proliferating cells . These observations indicate that there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth . The source of the increased PEth is, as yet, undefined.

J Biochem (Tokyo), 1994 Sep, 116(3), 682 - 6
Temperature modulated solubility-activity alterations for poly(N-isopropylacrylamide)-lipase conjugates; Matsukata M et al.; Chemical modification of proteins by use of functional polymers is expected to endow them with new properties without destroying their native functions, thus providing useful materials for application in different fields . We have synthesized poly(N-isopropylacrylamide) {poly(IPAAm)} co-oligomer with N,N-dimethylacrylamide (DMAAm) and reactive end groups by telomerization of IPAAm . This co-oligomer exhibits a lower critical solution temperature (LCST) at 37 degrees C . Using this temperature-responsive semitelechelic co-oligomer, we prepared polymer-enzyme conjugates of lipase by covalent coupling via carboxyl end-groups . This bioconjugate exhibits a LCST at 37 degrees C, having rapid, reversible hydration-dehydration changes due to highly mobile free polymer end groups . The conjugate retained its native enzymatic activity below this critical temperature, above which it precipitated and its catalytic function was shut off . This conjugate can be readily separated from reaction mixtures as a precipitate by simple temperature changes after reaction and reused in cycles without denaturation . Such a modulated system is attractive for application as a novel bioreactor system.

Trends Biotechnol, 1994 Sep, 12(9), 365 - 71
Designing microbial systems for gene expression in the field; de Lorenzo V; Unlike bacteria grown in the laboratory, genetically modified microorganisms destined for deliberate release as agents of bioremediation, or as live vaccines must be able to express their phenotype under the control of external signals that are present in the environment into which they are released . This is a major difference from other biotechnological processes (for example, in a bioreactor) in which the working conditions can be fixed at the will of the operator . In the field, operating conditions are determined by the external environment . The main problem is, therefore, how to programme bacteria physiologically and genetically to express the desired phenotype at the correct level and the right time, under physicochemical circumstances over which we have little or no control . This challenge has encouraged the development of new broad-host-range expression systems specifically tailored for bacteria, particularly Pseudomonas, but also various other Gram-negative organisms, for use in the field.

Biotechnology (N Y), 1994 Sep, 12(9), 909 - 14
Expansion of hematopoietic progenitor cell populations in stirred suspension bioreactors of normal human bone marrow cells; Zandstra PW et al.; We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells . Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors, detected as long-term culture-initiating cells (LTC-IC), could be achieved as well in stirred suspension cultures as in static cultures . Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment . Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC, CFC and total cell numbers in stirred cultures . Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold, respectively, above input values . Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer . Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations . In addition, stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms.

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