Monoclonal antibody expressed from PLBD2-knockout CHO cell line showed no significant reduction in co-purifying lipase activity
Polysorbate degradation was measured for monoclonal antibody drug substances produced from either control CHO cell lines or from PLBD2-knockout CHO cell lines. Drug substances were purified using Protein A and ion exchange chromatography.
The lack of PLBD2 expression in the PLBD2 knockout cell line was confirmed by Western blot analysis. The results clearly showed the clearance of PLBD2 in the knockout cell line (Figure 4-III). The representative degradation profiles of PS20 and PS80 when incubated with mAb-2 (generated by PLBD2 knockout cell line) are shown in Figure 4-I. The percentage of polysorbate degradation is calculated by summing changes in peak areas of monoesters (Figure 4-I). Surprisingly, the lipase activities in antibody samples expressed from the PLBD2 knockout cell line for both PS20 and PS80 were slightly higher than that from the control cell line (Figure 4-II), suggesting that PLBD2 does not play a significant role in degrading PS in drug substances. It is theoretically possible that knocking out PLBD2 in CHO cells resulted in increased expression of a different esterase, which in turn is able to degrade polysorbates. To address that possibility we performed proteomics analysis on the mAb-2 sample that was produced by PLBD2 knockout cell line. No new active lipase was found (data not shown).