Artificial induction of bottlenecks in the endoplasmic reticulum for rational improvement of antibody secretion in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used as hosts for therapeutic antibody production. Bottlenecks in the secretion process constrain the productivity of specific antibodies, although these bottlenecks occur in different and specific processes in the secretion depending on the product and host. Among secretion processes, those associated with the endoplasmic reticulum (ER)-including folding, assembly, cargo selection, and vesicle transport-have been reported as common bottlenecks. Here, bottlenecks in ER-associated secretion processes were artificially induced by knocking down relevant proteins, and their impact on antibody production and secretion was analyzed. In assays using translational and vesicle transport inhibitors, cycloheximide and brefeldin A, intracellular accumulation and secretion of antibodies and their intermediates differed among the knockdown cells having bottlenecks in certain secretion-related processes. Intracellular IgG accumulation at 0.968pg cell⁻¹ was observed in cells with the vesicle transport bottleneck when adding cycloheximide. Meanwhile, heavy-chain homodimers characteristically accumulated to 0.118pg cell⁻¹ in cells with the assembly bottleneck 8h after adding brefeldin A. Moreover, secretion profiles of cells with artificially induced bottlenecks were used to characterize secretion bottlenecks in three distinct antibody-producing CHO cells, followed by validation by overexpressing the corresponding proteins. CHO cells producing bevacizumab exhibited characteristics highly similar to those of cells with folding bottlenecks, suggesting that they also have a folding bottleneck. This was confirmed by overexpressing folding-related proteins, which improved the specific antibody productivity in batch culture by 18.9%. These findings should help to simplify the screening of secretion bottlenecks in antibody-producing CHO cells.