Studies on enhancing therapeutic protein production and quality in recombinant CHO cells

Abstract

Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the production of therapeutic proteins because of the ability of human like glycosylated protein production. Considering productivity and quality is most important to produce therapeutic proteins by recombinant CHO (rCHO) cell culture. Addition of sodium butyrate (NaBu) or lithium chloride (LiCl) incuced cell cycle arrest with a concurrent increase in the specific productivity for a number of therapeutic proteins. However, these chemical reagents are also cytotoxic and induce apoptotic cell death, and may negatively affect the quality of glycoproteins produced in CHO cells. Dex-tran sulfate (DS) is probably the most widely applied anti-aggregation agent in suspension cultures of CHO cells. However, the effet of different molecular weights (MWs) and concentration of DS on the cell growth, protein productivity, and quality attributes of rCHO cells has not been fully substantiated yet. Glycosylation structure is the most critical factor to determine the product quality of therapeutic proteins. However, some proteases and glycosidases that accumulated in culture supernatant negatively affect the quality of therapeutic proteins in rCHO cell cultures. Recently, identification and quantification of intracellular and extracellular proteome were performed using mass spectrometry based on omics technology. In this study, to find a less harmful and more effective chemical reagent, eight chemical reagents were evaluated for cell cycle arrest, producitivity, and quality of monoclonal antibody (mAb). And to examine the effect of DS on cell growth and mAb production of rCHO cells, three different MWs of DS at various concentrations were evaluated in two different rCHO cell lines producing mAb. Finally, in an effort to maintain good quality of mAb and Fc-fusion protein during the cultures, host cell proteins (HCPs) accumulated culture medium were characterized based on omics study. To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL-sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to re-combinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate ($\mu$), but increased culture longevity and specific mAb productivity ($q_mAb$ in a dose-dependent manner. The beneficial effect of valeric acid on culture longevity and $q_mAb$ outweighed its detrimental effect on $\mu$, resulting in 2.9-fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galacto-sylation. Unexpectedly, galactosylation of the mAb increased by the addition of 1.5 mM valeric acid. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells. To investigate the effect of dextran sulfate (DS), a widely used anti-aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM-0.025 and CS13-1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viabil-ity in the decline phase of growth. However, it increased mAb production only in the CS13- 1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13-1.00 cells, and showed the highest maximum mAb concentration. For SM-0.025 cells, it significantly de-creased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not neg-atively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its effi-cacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addi-tion of DS did not affect N-glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13-1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3？1.0 g/L. Taken together, to maxim-ize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good quality of mAb and Fc-fusion proteins during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb producing rCHO cell line (SM-0.025) and Fc-fusion protein producing rCHO cell lines (DG-Fc and DUKX-Fc) were identified and quantified by nanoflow liquid chromatography-tandem mass spec-trometry, followed by their gene ontology and functional analysis. Clustering analysis of HCPs, classified into four clusters according to their concentration profiles during the cultures, showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, Neu1, and B4galt1) correlated with changes in quality at-tributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the da-taset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be re-moved during both the cultures and purification steps for ensuring good mAb quality.