C6 Cells for the Manufacture of Recombinant Proteins

The first step in the manufacturing train is the generation and selection of a high-producing cell line. This has been performed 

The first step in the production of monoclonal antibodies is generation of the expression construct. For antibody generation, the antibody construct depicted in Fig. 3.4 has mostly been used. Here, expression of both the tight chain and the heavy chain genes is driven by a cytomegalovirus (CMV) promoter that has been modified to 

A total of 300-400 clones is isolated and transferred to 96-well plates and cultured in DMEM supplemented with 10% EBS. After 5-10 days, culture supernatants are sampled and screened for the presence of IgG either by Protein A HPLC or by ELISA. Production titers from two independent screening rounds are used to rank the transfectants, and the top 20-30 are selected and expanded for cryopreservation and further evaluation. Selection pressure is maintained until cryopreservation, after 

The highest ranked cell lines selected from the primary screens are then screened in 6-well plates using DM EM plus 10% FBS. Cells are seeded at 0.5 X 10 cells per well in duplicate and incubated for 4 days at 37 °C and 10% CO2. Culture supernatants are then harvested and the IgC concentration determined by Protein A HPLC or ELISA. Final cell concentrations are measured and the data used to calculate cell-specific production rates. The cell lines with the 

Selected PER.C6 cell lines possess low copy numbers, typically below 10 copies per cell as measured by Southern analysis (Fig. 3.6). 

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