Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions.

We have shown previously that recombinant NS/0 myelomas expressing sufficient amounts of E1B-19K were resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. However, no significant increase in monoclonal antibodies (MAb) was observed during the prolonged stationary phase of these batch cultures. Here, we show that E1B-19K can enhance cell survival and improve MAb productivity in high cell density perfusion culture. Typically, lymphoid cells grown under steady state in perfusion exhibit decreasing viabilities with concomitant accumulation of apoptotic cells. By modulating the ability of these cells to resist to induction of apoptosis in low nutrient environment, a 3-fold decrease in specific death rate from 0.22 day-1 for NS/0 control to 0.07 day-1 for E1B-19K cells was achieved, resulting in a significant improvement in cell viability throughout perfusion. E1B-19K cells at the perfusion plateau phase also exhibited a 3-fold reduction in specific growth rate concomitant with a lower percentage of S and higher percentage of G1 phase cells. This was associated with a 40% decrease in specific oxygen consumption rate, likely related to a reduction in the specific consumption rates of limiting nutrient(s). Expression of E1B-19K consequently had a significant impact on the steady-state viable cell density, allowing maintenance of 11.5 x 10(6) E1B-19K cells/mL versus 5.9 x 10(6) control NS/0 cells/mL for the same amount of fresh medium brought into the system (half a volume per day). Whereas MAb concentrations found in perfusion culture of control NS/0 myelomas were almost 3-fold higher than those found in batch culture; in the case of E1B-19K-expressing myelomas, the MAb concentration in perfusion was more than 7-fold higher than in batch. This was attributable to the 2-fold increase in viable cell plateau and to a 40% increase in the perfusion to batch ratio of specific MAb productivity (2.2-fold for E1B-19K myelomas versus 1.6-fold for NS/0 control).     

Enhanced productivity of NS0 cells in fed-batch culture with cholesterol nanoparticle supplementation

NS0 cells are an important industrial cell line for the production of therapeutic monoclonal antibodies. Culturing these cells is challenging because they are cholesterol auxotrophs, and providing cholesterol to the cells is hampered by the low solubility of lipids in aqueous medium. Limited loading capacity, precipitation, instability, and toxicity are associated with traditional delivery methods that involve solvents or carrier molecules. In this work, nanoparticle cholesterol mixtures (NCM) were produced by electrohydrodynamic spraying and added directly to a cholesterol auxotroph NS0 cell line. Compared to a cholesterol-cyclodextrin solution and a commercial proprietary cholesterol solution, SyntheChol NS0 supplement, NCM is significantly less cytotoxic. In the fed batch cell culture process, product titer was increased by 32% when the NCM supplement replaced SyntheChol NS0 supplement. An even greater product titer improvement, 64%, was achieved when both NCM and SyntheChol NS0 supplements were used in the fed-batch process.     

Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1.

We have constructed NS0 myeloma cell lines that inducibly express the p21CIP1 cyclin dependent kinase inhibitor, using the Lacswitch system. Ectopic p21(CIP1) protein expression was rapidly induced within 12 h of addition of IPTG, causing G1-phase arrest and almost complete inhibition of cell proliferation. The production of a chimeric IgG4 antibody, expressed constitutively from an independent promoter, was found to be significantly increased by more than 4-fold in p21CIP1-arrested cells. This study demonstrates for the first time the successful construction of anchorage-independent and proliferation-controlled NS0 cell lines with enhanced secreted chimeric antibody production independent of the inducible promoter activity used to achieve cytostasis.     

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.     

The loss of antibody productivity in continuous culture of hybridoma cells

Two hybridoma lines, HB8178 and AFP-27, were grown in continuous culture. The concentrations of viable cells as well as those of various nutrients and metabolites reached steady-state values. The concentrations of either total IgG or antigen-specific antibody, however, failed to reach steady-state values but rather continuously decreased over the course of the cultures. The fraction of antibody-producing cells in the total cellular population also continuously decreased in the AFP-27 cultures. Comparison of the specific antibody productivity based on either the entire population or the antibody-producing fraction of the population over time suggests that the decrease in productivity was at least partly due to the occurrence of a nonproducing subpopulation of cells.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line

Metabolic profiling or metabolomics is the analysis of a larger number of small metabolic compounds within cells. While this technique has been utilized to study microbial and yeast strains under different physiochemical conditions, very little has been reported regarding its application in mammalian cell culture. Here, the physiological and metabolic changes observed during the proliferation arrest of an antibody producing GS-NS0 mouse myeloma cell line were studied using conventional biochemical analysis and one-dimensional nuclear magnetic resonance (NMR)-based metabolic profiling. Proliferation-arrested cells had increased antibody productivity, enhanced normalized mitochondrial membrane potential, and showed changes in the consumption of several amino acids. Further investigation into these physiological changes was carried out by ¹H NMR profiling followed by principle component analysis (PCA). The resulting data showed a clear separation of the arrested and control spectra that related to the altered metabolic state of the arrested culture. Metabolites associated with phosphatidylcholine homeostasis, lipid and fatty acid metabolism, and ascorbate formation were found to be present in significant amount in these cultures. Taken together, the results suggested that there was a link between the metabolic alterations and the hyper-productive state, possibly relating to vesicle recycling and secretory functions, and mechanism to counteract against the generation of reactive oxygen species. While the use of metabolic profiling is still in its infancy, its potential to enhance the understanding of physiological processes in mammalian cell lines used for antibody production is certain.     

A fully defined,fed‐batch,recombinant NS0 culture process for monoclonal antibody production

To manufacture a glycoprotein, mammalian cells expressing the desired protein are often grown in fed‐batch mode. Feeding an undefined, nonanimal hydrolysate helps the cells receive sufficient nutrition, but makes systems difficult to optimize. Even different lots of the same hydrolysate may have significant variability; furthermore, individual components may actually be detrimental to the cells. Switching to fully defined feeds could eliminate these issues. For monoclonal antibody (mAb) production by fed‐batch NS0 cells, this article describes the replacement of a hydrolysate‐based feed with a fully defined, animal‐component‐free feed system. The defined feed initially had 67 components, but additional experiments allowed a reduction to 25 components. The mAb titer is approximately 20% higher than in the undefined system, and the feed volume is circa 20% lower. The two systems generated antibodies with similar glycosylation profiles. Other benefits of the defined feed system include lower raw material costs, the ability to optimize key nutrient concentrations, greater confidence in raw material quality, and the elimination of potential, hydrolysate‐associated endotoxin issues. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

表达重组抗CD52单克隆抗体CHO细胞灌流培养基的筛选

目的筛选重组抗CD52单克隆抗体CHO细胞株培养和连续灌流表达用培养基,以提高抗体表达量。方法通过调整原有批培养用培养基中谷氨酰胺和植物水解蛋白,获得5种培养基配比。使用模拟灌注方式进行细胞培养,分析细胞密度、活细胞比率和目标蛋白表达,筛选连续灌流细胞培养和表达用培养基。最后在7 L反应器中采用灌注培养方式对筛选获得的培养基进行验证。结果使用50 mL细胞培养管进行模拟灌注培养时,活细胞比率较高,达到90%以上;CHO细胞在添加谷氨酰胺至4.0 mmol/L和植物水解蛋白至5.0 g/L的批培养用培养基中生长速度最快;在基础培养基中抗体表达量比优化前高15%。20 d培养周期内,优化的培养基在7 L反应器中可以维持CHO细胞密度在(2 727±253)万个/mL,活细胞比率在95%以上。结论通过模拟灌注培养,筛选获得了一种在7 L反应器灌流培养中适宜于重组抗CD52单克隆抗体CHO细胞表达的培养基。     

Effect of oxygen on antibody productivity in hybridoma culture

Summary The effect of the inlet gas oxygen concentration on hybridoma growth, viability and monoclonal antibody production was studied. Maximum viable cell cencentrations of 1.2×10⁶ cells/mL were obtained in split-flow bioreactors mixed with a gas containing 10% oxygen. However, maximum antibody concentration was attained at a sub-optimal level of growth with an oxygen concentration of 2.5%.     

Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process

Controlled feeding of nutrient supplements to a cell culture to enhance monoclonal antibody productivity has been practiced widely in high-yield, fed-batch processes. In this study, a similar feeding concept has been applied to a perfused culture and evaluated for the effects on bioreactor productivity and product quality. Our experimental results show that, by using such a "controlled-fed perfusion" approach, the volumetric antibody productivity (antibody per liter per day) was significantly increased by nearly twofold over the perfusion process, and surpassed fed-batch and batch processes by almost tenfold. The substantial boost in the overall productivity is attributable primarily to the combined effects of increased cell density as well as reduced product dilution. Both were achieved through careful nutrient supplementation in conjunction with metabolite minimization. As the manufacturing process evolved from roller bottles to the controlled-fed perfusion bioreactor system, the immunoreactivity and the cDNA sequences of the antibody were well preserved. However, the product glycosylation distribution patterns did alter. The controlled-feed perfusion process demonstrated a unique encompassment of the advantages of fed-batch and perfusion methods; that is, high product concentration with high volume throughput. Therefore, it may be very suitable for large-scale production of monoclonal antibodies.