Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells

Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 degrees C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.     

Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.

     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Production of xylitol from D-xylose by recombinant Lactococcus lactis

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.     

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.     

Comparative Metabolic Analysis of CHO Cell Clones Obtained through Cell Engineering,for IgG Productivity,Growth and Cell Longevity

Cell engineering has been used to improve animal cells’ central carbon metabolism. Due to the central carbon metabolism’s inefficiency and limiting input of carbons into the TCA cycle, key reactions belonging to these pathways have been targeted to improve cultures’ performance. Previous works have shown the positive effects of overexpressing PYC2, MDH II and fructose transporter. Since each of these modifications was performed in different cell lines and culture conditions, no comparisons between these modifications can be made. In this work we aim at contrasting the effect of each of the modifications by comparing pools of transfected IgG producing CHO cells cultivated in batch cultures. Results of the culture performance of engineered clones indicate that even though all studied clones had a more efficient metabolism, not all of them showed the expected improvement on cell proliferation and/or specific productivity. CHO cells overexpressing PYC2 were able to improve their exponential growth rate but IgG synthesis was decreased, MDH II overexpression lead to a reduction in cell growth and protein production, and cells transfected with the fructose transporter gene were able to increase cell density and reach the same volumetric protein production as parental CHO cells in glucose. We propose that a redox unbalance caused by the new metabolic flux distribution could affect IgG assembly and protein secretion. In addition to reaction dynamics, thermodynamic aspects of metabolism are also discussed to further understand the effect of these modifications over central carbon metabolism.     

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14?%, respectively), while the control cells produced predominantly bi-antennary branching (70?%). About 53?% of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77?%, whereas only 61?% of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6?% after the addition of 0.2?mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.     

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.     

Characterization of D-ribose biosynthesis in Bacillus subtilis JY200 deficient in transketolase gene

D-Ribose is a functional five-carbon sugar, which has been used for the commercial production of riboflavin. Mechanisms of d-ribose biosynthesis from xylose were investigated in the genetically engineered Bacillus subtilis JY200 with a deficiency in transketolase. A transketolase gene (tkt) disruption cassette in plasmid pUNKC was introduced into the chromosomal tkt gene in the wild type B. subtilis 168. Analysis of culture broth by thin layer chromatography confirmed that the disruption of tkt allowed B. subtilis JY200 to produce d-ribose. In a batch culture of B. subtilis JY200, a loss of cell viability was observed after glucose depletion. Fed-batch cultivation by feeding 400 gl(-1) glucose solution as a co-substrate was carried out to supply energy to xylose metabolism and to maintain cell viability throughout cultivation. Fed-batch cultivation of B. subtilis JY200 in a complex medium containing 11 gl(-1) xylose and 5 gl(-1) glucose initially gave the best result of 10.1 gl(-1)D-ribose concentration, 0.24 gg(-1)D-ribose yield and 0.29 gl(-1)h(-1) productivity, corresponding to 40-, 5- and 12-fold increases compared with those in the batch culture. A kinetic study of D-ribose production in fed-batch cultivations of B. subtilis JY200 suggested that xylose uptake might be critical to maximize D-ribose biosynthesis from xylose.     

Enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase

As commonly recognized, the excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. Accordingly, this may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle was made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase (aspartase). As a result, in the glucose minimal medium containing aspartate, the production of two recombinant proteins, beta-galactosidase and green fluorescent protein, in the aspartase-producing strain was substantially increased by 5-fold in association with 30-40% more biomass production. This preliminary study illustrates the great promise of this approach used to enhance the production of these two recombinant proteins.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.     

风疹病毒包膜糖蛋白E1中二硫键对其细胞融合活性的影响

为了探讨风疹病毒包膜糖蛋白E1中二硫键对风疹病毒细胞融合活性的影响,在构建重组载体pBSK-SPE2E1的基础上,利用PCR定点突变与体内同源重组相结合的方法,构建了11个突变体,分别将E1外功能区的11个半胱氨酸残基突变为其它氨基酸残基,从而去除一个二硫键,利用Giemsa染色法定性检测由此引起的细胞融合情况,流式细胞术检测导入的外源DNA在细胞表面的表达效率,血吸附检测重组表达的突变体蛋白的受体识别活性。结果表明E1外功能区的10个二硫键对RV的细胞融合活性都有重要影响,任何一个二硫键的去除均导致E1的细胞融合活性丧失;其中第5和第8个半胱氨酸残基所形成的二硫键与E2和E1的相互作用有关,第3、第4和第13个半胱氨酸残基所形成的二硫键可能直接影响E1的细胞融合功能。     

Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes

To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated ‘primed’ ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO₂ concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO₂ condition, only the slight effect can be seen. However when cultured in 8% CO₂ condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 μg/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of glucose on the production of recombinant protein C in mammalian cell culture

Effects of glucose on a cultured Chinese hamster ovary cell line producing recombinant human protein C were investigated. After the recombinant cells reached confluency, they were maintained in the medium containing 10% serum and different levels of glucose in either batch or daily-exchange mode. High concentrations of glucose to the cultures yielded higher cell densities. Daily exchanges of media produced higher cell densities than the corresponding batch culture. Total protein C production per cell decreased with time in batch culture, in accordance with the declined glucose metabolism. Supplementation of the media with high levels of glucose diminished both the expression and gamma-carboxylation activities of the recombinant cells. Production of protein C persisted in daily-exchange culture, resulting in a constant production rate of protein C. In this case again, glucose reduced the specific productivity of recombinant protein C. An apparent glucose inhibition constant was determined to be 0.11 mg/mL by Dixon plots. The ability to gamma-carboxylate recombinant protein C was also impaired at the highest level of glucose. From these results, a strategy to maximize recombinant protein C productivity is discussed.     

Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21(host)) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21(Hsp70)) due to an inhibition of apoptotic cell death and an increased cellular expression of rFVIII [Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. Biotech. Bioeng. 97, 144-155]. In the present study we investigated the difference in adherence of rFVIII to the cell membrane surface by comparing changes in cell viability and extent of phosphatidylersine (PS) exposure in apoptosis between rBHK-21(host), rBHK-21(Hsp70), and parental BHK-21 cells devoid of rFVIII expression (BHK-21(native)) during batch cell culture experiments. The Zenon technique was used to double stain for cell surface and intracellular rFVIII using flow cytometric Guava PCA analysis. By this quantitative analysis intracellular rFVIII was shown to decrease in rBHK-21(host) cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21(Hsp70) cell cultures displayed higher cell viability and intracellular rFVIII with less cell surface rFVIII staining. Time dependent increases of rFVIII adherence to the surface of rBHK-21(host) cells and its reduction on the surface of rBHK-21(Hsp70) cells was also confirmed by fluorescence microscopy. Furthermore, greater rFVIII cell surface staining correlated with an increase in detectable PS exposure on the surface of BHK-21(native) batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21(host) cells despite a similar decline in cell viability between rBHK-21(host) and BHK-21(native) batch cultures. Any exposed PS on rBHK-21(host) cells was most likely masked by secreted rFVIII, mimicking the effect on activated platelets where the externalization of PS also occurs, and serves as a ligand for FVIII activation in the blood coagulation cascade. Taken together we have identified that rFVIII sequestration on the membrane surface is another potential limitation to rFVIII productivity and one which can also be alleviated by reduction of apoptosis in a clone expressing human HSP70.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".