Upstream Processing:Vendor Notes: Generating Stable, High-Expressing Cell Lines for Recombinant Protein Manufacture - - BioPharm International

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Upstream Processing:Vendor Notes: Generating Stable, High-Expressing Cell Lines for Recombinant Protein Manufacture


BioPharm International
Volume 20, Issue 3

CASE STUDIES

Performance of Candidate Cell Lines After Single-Load and Double-Load Processes

This case study focused on comparing the expression of candidate cell lines under non-fed, batch shake-flask conditions generated by both the single and double load processes. The Platform ACE Cell Line was transfected with an ATV containing the sequences for the heavy and light chains of an IgG1 monoclonal antibody flanked by cHS4 insulator sequences and the hygromycin resistance gene. After two transfections, 350 drug resistant colonies were generated at the 96-well stage and screened for antibody expression by ELISA. Selected primary transfectant colonies with titers >1 μg/mL were expanded to 24-well plates and subjected to terminal over growth assays, where titers were measured by ELISA. The top 20 primary transfectants, as determined by the 24-well overgrowth assay, were expanded to shake flask cultures for assessment of protein yield and growth characteristics. This process took six weeks to complete and resulted in primary transfectants with titers of ~450 mg/L under terminal, non-fed, shake-flask culture (unsupplemented CD-CHO medium from Invitrogen). These primary transfectants were used to generate approximately 5 g of research material in a 10-L batch Wave bag bioreactor.


Figure 3
The top primary transfectants were single-cell subcloned by limiting dilution, selected, and expanded to shake-flask cultures, and subjected to performance testing in terminal non-fed, shake-flask cultures. The growth profile and corresponding fluorescent in situ hybridization (FISH) image from the top single-load subcloned cell line are shown in Figure 3A. Cells reached a viable cell density of 7.0 x 106 cells/mL, with titers of 740 mg/L and a specific productivity of 14 pg/cell/day. The corresponding FISH images demonstrated that the ACE was intact and contained the antibody heavy and light chain sequences. Moreover, no integration of the antibody sequences onto the host CHO genome was detected.

The single-load primary transfectants, in addition to being subcloned, were also subjected to a double load with a second ATV containing the heavy and light chain sequences and an alternate secondary drug resistance marker (zeocin). This double-load process resulted in primary transfectants with titers reaching 833 mg/L under terminal, non-fed, shake flask cultures. As with the single-load process, the top double-load primary transfectants were single-cell subcloned, resulting in a number of clonal cell lines reaching yields of approximately 1,000 mg/L in terminal, non-fed shake flask cultures. The growth profile and corresponding FISH image from a top, double-load subcloned cell line are shown in Figure 3B. Cells reached a viable cell density of 7.0 x 106 cells/mL, with titers of 1,140 mg/L and a specific productivity of 45 pg/cell/day. This increase in expression is believed to be from an increase in gene copies resulting from the second round of transfection. Once again, FISH analysis shows an intact ACE loaded with the antibody heavy and light chain sequences with none integrated on the host genome.


Table 1
The top candidate, double-load, single-cell subcloned cell line was subjected to process development and fed-batch by supplementing the basal CD-CHO medium with glucose and plant hydrolysates. Compared with batch conditions, feeding resulted in a threefold improvement in expression (3 g/L) for a fed-batch 250-mL shake flask and a fourfold improvement in expression (>4 g/L) for a fed-batch 1-L bioreactor (Table 1).


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