The Chinese hamster ovary (CHO) cell line is the most widely used host cell for therapeutic antibody production. Although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (DTE) antibodies remain at low secretion levels. To improve the production of various therapeutic antibodies, it is necessary to determine possible ratelimiting steps in DTE antibody secretion in comparison with other high IgG producers. Here, we analyzed the protein secretion process in CHO cells producing the DTE immunoglobulin G (IgG) infliximab. The results from chase assays using a translation inhibitor revealed that infliximab secretion could be nearly completed within 2 h, at which time the cells still retained about 40% of heavy chains and 65% of light chains. Using fluorescent microscopy, we observed that these IgG chains remained in the endoplasmic reticulum and Golgi apparatus. The cells inefficiently form fully assembled heterodimer IgG by making LC aggregates, which may be the most serious bottleneck in the production of DTE infliximab compared with other IgG high producers. Our study could contribute to establish the common strategy for constructing DTE high-producer cells on the basis of rate-limiting step analysis.
GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.
Chinese hamster (Cricetulus griseus) ovary-derived Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the industrial production of recombinant therapeutics because of their ability to fold, assemble, and perform post-translational modifications, such as glycosylation, on proteins. They are also valuable for their ability to grow in serum-free suspension cultures. In this study, we established a cell line derived from lung tissue of Chinese hamsters, named Chinese hamster lung (CHL)-YN cells. The biosafety of CHL-YN cells was confirmed by in vitro sterility testing, mycoplasma detection, and reverse transcriptase assays. One of the key characteristics of CHL-YN cells was their doubling time of 8.1 h in chemically defined culture medium; thus, they proliferate much faster than conventional CHO cells and general mammalian cells. Transgenes could be introduced into CHL-YN cells with high efficiency. Finally, between 50% to > 100% of the amount of glycosylated immunoglobulin G (IgG)1 produced by CHO-K1 cells was produced by CHL-YN cells over a shorter period of time. In summary, fast-growing CHL-YN cells are a unique cell line for producing recombinant proteins.
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