Developement of serum-free media in CHO-DG44 cells using a central composite statistical design.

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.

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor.

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.

Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A.

Production of recombinant antibodies against botulinum neurotoxin is necessary for the development of a post-exposure treatment. CHO-DG44 cells were transfected with a plasmid encoding the light and heavy chains of a chimeric monoclonal antibody (S25) against botulism neurotoxin serotype A. Stable cell lines were obtained by dilution cloning and clones were shown to produce nearly equivalent levels of light and heavy chain antibody by an enzyme-linked immunosorbent assay (ELISA). In suspension culture, cells produced 35 microg/ml of chimeric antibody after 6 days, corresponding to a specific antibody productivity of 3.1 pg/cell/day. A method for the harvest and recovery of an antibody against botulism neurotoxin serotype A was investigated utilizing ethylenediamine-N,N'-tetra(methylphosphonic) acid (EDTPA) modified zirconia and MEP-hypercel, a hydrophobic charge interaction chromatography resin. Purification of the S25 antibody was compared to that achieved using rProtein A-Sepharose Fast Flow resin. After the direct load of culture supernatant, analysis by ELISA and gel electrophoresis showed that S25 antibody could be recovered at purities of 41 and 44%, from the EDTPA modified zirconia and MEP-hypercel columns, respectively. Although the purity obtained from each of these columns was low, the ability to withstand high column pressures and nearly 90% recovery of the antibody makes EDTPA modified zirconia well suited as an initial capture step. Combining the EDTPA modified zirconia and HCIC columns in series resulted in both purity and final product yield of 72%.

Acute physiologic and chronic histologic changes in rats and mice exposed to the unique hallucinogen salvinorin A.

Salvinorin A is a unique hallucinogen that is seeing increased use in humans. It is not currently a controlled substance and is used as a legal alternative to controlled substances. Usually smoked or buccally absorbed by chewing, doses of approximately 200 mcg can produce profound hallucinogenic effects of short duration. The mechanism of action of salvinorin A is at the kappa-opioid receptor. Little data is available on the medical effects of this substance so animal studies were undertaken to explore the acute toxic effects of this substance in rats and the chronic effects in mice. Rats were anesthetized and administered salvinorin A at 1600 mcg/kg or vehicle. Recordings were made of galvanic skin response, EKG, temperature, and pulse pressure for 100 minutes. Mice were chronically exposed to vehicle or 400, 800, 1600, 3200, or 6400 mcg/kg of salvinorin A for two weeks. After exposure the animals were sacrificed and brain, heart, kidney, bone marrow, blood and spleen were removed, fixed, sectioned, stained and examined by light microscopy. No effects were seen on cardiac conduction, temperature, or galvanic skin response. A nonsignificant rise was seen in pulse pressure. Histologic studies of spleen, blood, brain, liver, kidney, and bone marrow did not find any significant histologic changes at any of the doses examined. These data suggests that the toxicity of salvinorin A is relatively low, even at doses many times greater than what humans are exposed to. However, further studies should be done on blood pressure effects. The psychological impact of this potent hallucinogen should also be investigated.

Increased production of a secreted glycoprotein in engineered CHO cells through amplification of a transcription factor.

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.