Effect of CHO cell line constructed with CMAH gene-directed integration on the recombinant protein expression.

Chinese hamster ovary (CHO) cells are the most widely used platform for recombinant therapeutic protein (RTP) production. Traditionally, the development of CHO cell lines has mainly depended on random integration of transgenes into the genome, which is not conducive to stable long-term expression. Cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) is expressed in CHO cells and produces N-hydroxyacetylneuraminic acid, which may cause a human immune response. However, the effects of transgene integration at the CMAH site on RTP expression in CHO cells remain unclear. In this study, we selected CMAH gene, which is lacking in humans, as the target site to construct recombinant CHO cell line using the CRISPR/Cas9 technique. Erythropoietin (EPO) and EGFP integration at the CMAH site resulted in more stable expression levels and lower heterogeneity than random integration. In addition, the proportion of N-glycosylation levels in the EPO glycoside of CMAH integration site also changed. In conclusion, CMAH site integration improved the stability of RTP expression in CHO cells.