A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM's F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM's F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 x 10(5) cells to maximum cell density of 1.04 x 10(6) cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2x) EAA, 1.4 mM (0.5x) NEAA, 1x ITS supplement, 0.7x Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 x 10(6) cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 +/- 3.4 mug/mL, a 50% improvement.

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.

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Expression levels of reporter protein driven by Mouse Mammary Tumor Virus Promoter system were improved by expressing its specific transcription factor (glucocorticoid receptor) from a different expression vector. The vector that expresses glucocorticoid receptor (GR) also contained dihydrofolate reductase (dhfr) gene as a selection marker. In the presentstudy we amplified the glucocorticoid receptor gene (gr)along with the dhfr gene by adapting the cell lines to increasing concentrations of methotrexate, an antifolate analog. Stepwise increases in the volumetric titers of a secreted reporter glycoprotein, Secreted Alkaline Phosphatase (SEAP), were observed in recombinant Chinese hamster ovary (CHO) cellsgrowing in increased concentrations of methotrexate. Western andRT-PCR analysis showed that this increase in volumetric titers is associated with higher levels of GR expressed in CHO cellsgrowing in increased concentration of methotrexate. A stablytransfected cell line growing in 10(-6) M methotrexate wasgrown in suspension culture and induced with 10(-7) Mdexamethasone. The SEAP volumetric titers reached a peak of approximately 23 mug ml(-1) on the 5th day after induction.Inducing these cells with increasing concentrations of dexamethasone resulted in increased specific productivity. These high volumetric productivities were further increased in fed-batch bioreactors.

Analysis of BCL-2 content by flow cytometry 51 Determination of antibody, substrate and metabolite concentrations 52 Results and Discussion 52 Effect of bcl-2 on cell density and viability 52 Determining the level of BCL-2 55 Analysis of high expressing clones 59 Effect of bcl-2 on the extent of apoptosis 64 Effect of bcl-2 on cell metabolism and antibody productivity 66 Cell cycle analysis 72 Conclusions 72 References 74