A variety of mechanisms including transcriptional silencing, gene copy loss, and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilized single‐cell RNA‐seq (scRNA‐seq) to study a clonally derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA‐seq data, we identified subclusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality.
The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time-and cost-efficient production platform for recombinant Adenoassociated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.
RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb‐producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post‐transcriptional regulation in CHO cells during biopharmaceutical production.
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