Enhancement of foreign protein productivity in recombinant CHO cells by inhibition of cellular apoptosis under various stress conditions

Abstract

Sodium butyrate (NaBu) can enhance the expression of genes from some of the mammalian promoters including the CMV and SV40 while it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, a survival protein, human Bcl-2, was overexpressed in the recombinant CHO cells (SH2-0.32) producing a humanized antibody directed against the S surface antigen of hepatitis B virus. When batch cultures of both control cells transfected with bcl-2 deficient plasmid (SH2-0.32-Δbcl-2) and cells transfected with bcl-2 expression plasmid (14C6-bcl-2) were performed in the absence of NaBu, both cells showed similar profiles of cell viability and antibody production. Compared with SH2-0.32-Δbcl-2 culture, under the condition of NaBu addition at the exponential growth phase, overexpression of bcl-2 gene considerably suppressed the NaBu-induced apoptosis of 14C6-bcl-2 by inhibiting caspase-3 activity and extending the culture longevity by more than 2 days. As a result, the final antibody concentration of 14C6-bcl-2 culture was 2 times higher than that of SH2-0.32-Δbcl-2 culture in the presence of NaBu and 3 times higher than that of SH2-0.32-Δbcl-2 and 14C6-bcl-2 cultures in the absence of NaBu. Overexpression of human Bcl-2 protein in SH2-0.32 considerably suppressed NaBu-induced apoptosis during batch culture using a commercially available serum-free medium, extending the culture longevity. Due to the enhanced transcription efficiency and the extended culture longevity, the final antibody concentration of 14C6-bcl-2 culture (23㎍/mL) was 2 times higher than that of SH2-0.32-Δbcl-2 culture (10.5㎍/mL) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and SH2-0.32-Δbcl-2 culture...