Effects of culture conditions on the quality of fc-fusion protein produced in CHO cells

Abstract

Quality of recombinant therapeutic proteins, such as glycosylation, has effects on biological activities, in vivo half-life, solubility, and so on. Due to the importance of quality, Chinese hamster ovary (CHO) cells are the predominant host cell lines for therapeutic proteins because they can produce recombinant proteins having a similar quality with those of human. Recombinant protein quality, expressed in CHO-based production system, can be determined at the stages of genetic sequences, cell lines, culture modes and key parameters of cultivation. In this study, we analyze product quality determined at the stage of cell lines and cultivations and investigate what causes differences of quality. Understanding potential steps and effectors in each step causing varieties of product quality will improve our abilities to control critical product quality in the CHO cell based production system. DUKX-B11 and DG44 are the widely used CHO host cells for therapeutic protein production. To examine the effect of cell line on therapeutic protein quality, therapeutic Fc fusion protein producing CHO cell lines, originated from DUKX-B11 and DG44 host, were cultivated in batch culture with three temperature conditions. Product qualtity was evaludaed as sialylation quality and isoelctric foucusing (IEF) gel analysis was used for evaluation. By IEF analysis for protein pI isoform distribution, we observed IEF profiles of Fc fusion proteins, expressed by twelve rCHO subclones and they were categorized into two groups based on the location of major pI isoforms. To identify possible causes make the differences of pI isoform, we investigated the effects of cultivation temperature, cell growth profile (cell counts and viability), culture duration, sialidase activity and metabolites affecting product quality. Based on the analysis data, rCHO cells producing acidic pI isoforms as major isoforms had a low sialidase acitivity and they were originated from DUKX-B11 host cells. To identify whether the difference of sialidase acitivity was originated from cell line level, we compared sialidase and sialyltransferase gene expression levels between CHO host cells. DUKX-B11 had about 34.6% of sialidase mRNA expression but, there was no difference in sialyltrnsferase mRNA expression level. Taken together, differences of Fc fusion protein sialylation quality between rCHO cells, were originated from the difference in sialdisade gene expression of CHO host cells. Therapeutic proteins quality is also determined depending on cell culture modes and key culture process parameters. Fed-batch is a culture mode to feed nutrients selectively based on the predicted requirements of the cell for growth and production. It has been a dominant mammalian cell culture method because it can be possible to get a large amount of therapeutic protein, simple operations and reliabilities. There have been numerous researches for the effects of culture processes and conditions on the resulting glycosylation profile. Despite these efforts to control over product quality like glycosylation, many studies were perfomed in simple culture mode in small scale model and success studies with a particular mammalian cell lines expressing a particular protein do not carry over to others. For understanding these differences, we investigate the effects of culture moed, temperature, DO and pH on product quality during fed-batch cultures using stirred bioreactor. Fc fusion protein with relatively low sialylation was produced in batch culture, as compared to fed-batch culture, and it might be caused by deficiency of essential nutrient involved in glycosylation. Temperature shift down had a dominant effect to maintain sialic acid contents but there were no differences in sialic acid contents between bi-phasic and tri-phasic temperature shift down. DO 30 condition was preferable culture conditions in terms of high sialylation of Fc fusion protein but, pH shows no significant effect on product quality. Although pH and DO are also critical process parameters, their effect on product quality might be minor than temperature. Therapeutic protein quality like glycosylation has a complex metabolic pathway. It is characterized by step-wise addition and removal of individual monosaccharides and variety of enzymes related to glycosylation are involved. For depth understanding glycosylation pathways, numerous comparative transcriptomic analyses on rCHO cells have been conducted with a targeted quantitative polymerase chain reaction (PCR) but, its application is limited due to its low scalability in terms of the number of genes analyzed. So, we investigated fifty-two N-glycosylation-related gene expressions changes in rCHO cell producing Fc fusion protein using an emerging technology, NanoString nCounter system. The addition of NaBu to the production process reduced the relative proportion of acidic pI isoforms and sialic acid content of the glycoprotein. Fifty-two N-glycosylation-related gene expressions were also assessed by the NanoString nCounter system which can measure a direct digital readout using custom-designed color-coded probes. Among them, ten genes (ugp, slc35a2, ganc, man1a, man1c, mgat5a, st3gal5, glb1, neu1, and neu3) were up-regulated and three genes (b4galt2, st3gal3, and neu2) were down-regulated significantly. Altered expression patterns in st3gal3, neu1, and neu3, which have the roles in the sialic acid biosynthesis pathway, correlated with the reduced sialic acid content of the glycoprotein by NaBu. In conclusion, product quality is influence by host cell line and culture condition in fed-batch and the measurable phenotypic quality changes correlate with the gene expression profile changes. Taken together, the results obtained in this study provides a better understanding to produce therapeutic proteins with a cocnsistent and preferable quality profile.