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This study reports findings of an unusual cluster of mutations spanning 22 bp (base pairs) in a monoclonal antibody expression vector. It was identified by two orthogonal methods: mass spectrometry on expressed protein and next-generation sequencing (NGS) on the plasmid DNA. While the initial NGS analysis confirmed the designed sequence modification, intact mass analysis detected an additional mass of the antibody molecule expressed in CHO cells. The extra mass was eventually found to be associated with unmatched nucleotides in a distal region by checking full-length sequence alignment plots. Interestingly, the complementary sequence of the mutated sequence was a reverse sequence of the original sequence and flanked by two 10-bp reverse-complementary sequences, leading to an undesirable DNA recombination. The finding highlights the necessity of rigorous examination of expression vector design and early monitoring of molecule integrity at both DNA and protein levels to prevent clones from having sequence variants during cell line development.

Suspension cultivation is the preferred mode of operation for the large-scale production of many biologics. Chinese Hamster Ovary (CHO) cells are anchorage-dependent in origin, but they have been widely adapted to suspension culture. In suspension culture, formation of CHO cell aggregates is a common phenomenon and compromises cell culture performance in multiple ways. To better understand the underlying mechanisms that regulate cell aggregation, we utilized CHO-specific transcriptome profiling as a screening tool and demonstrated that many genes encoding extracellular matrix (ECM) proteins were upregulated in the cultures with increased cell aggregation. Significantly, hypoxia was identified to be a cause for promoting CHO cell aggregation, and transforming growth factor beta1 (TGFβ1) pathway activation served as an intermediate step mediating this biological cascade. These transcriptomics findings were confirmed by cell culture experiments, and it was further demonstrated that adding recombinant TGFβ1 to the culture significantly increased ECM protein fibronectin expression and cell aggregation. The results of this study emphasize the importance of adequate mixing and oxygen supply for suspension cultures from a new angle, and regulating the TGFβ1 pathway is proposed as a new strategy for mitigating cell aggregation to improve cell culture performance.

The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half-life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4-Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases alpha2,3-sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU-486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5-L controlled bioreactors. Addition of 1 microM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N-linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4-Ig fusion protein expressed in CHO cells.