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During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post-transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence-activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three-fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin-opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines., (© 2010 American Institute of Chemical Engineers)

To date, the FDA has approved 18 monoclonal antibody (MAb) therapeutic drugs with targets ranging from asthma and rheumatoid arthritis to leukemia. Many of these approved products are produced in Chinese hamster ovary cells (CHO) making CHO a significant and relevant host system. We studied the applicability of CHOK1SV cells as a potential host cell line for MAb production in terms of timelines, achievable titers, transfectant stability, and reproducibility. CHOK1SV, developed by Lonza Biologics, is a suspension, protein-free-adapted CHOK1-derivative utilizing the glutamine synthetase (GS) gene expression system. CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants were obtained under the dual selection of methionine sulfoximine (MSX) and glutamine-free media. We examined outgrowth efficiencies, specific productivities, and achievable batch titers of three different IgG MAbs transfected into CHOK1SV. Reducing the MSX concentration in the initial selection medium resulted in a decreased incubation time required for transfectant colonies to appear. Specific productivities of "high-producers" ranged between 11 and 49 pg/c/d with batch titers ranging from 105 to 519 mg/L. Transfectant stability and the effects of MSX also were investigated, which indicated that the addition of MSX was necessary to maintain stable MAb production. Cell growth was stable regardless of MSX concentration.