Abstract
Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined.
Highlights
The development of next-generation sequencing (NGS) technologies has greatly improved the efficiency of sequencing and contributed to the understanding of dynamic changes in gene expression [1]
We explored the use of r6">6]. Massively parallel cDNA sequencing (RNA-seq) technology for the characterization of the mutation rate in a stably transfected Chinese hamster ovary (CHO) cell line expressing a recombinant monoclonal
Feasibility Study. cDNAs from two clones expressing light chain with closely related but slightly differing sequences were mixed in different ratios to assess the ability of NGS to quantitatively detect the fraction of mutant bases in a mixed population
Summary
The development of next-generation sequencing (NGS) technologies has greatly improved the efficiency of sequencing and contributed to the understanding of dynamic changes in gene expression [1]. RNA-seq allows both qualitative and quantitative analysis of the expressed gene product at messenger RNA (mRNA) level with wide dynamic ranges and superior sensitivity [8]. Mammalian cell lines such as the Chinese hamster ovary (CHO) cells have been widely used in the production of recombinant therapeutic product including monoclonal antibodies [9, 10]. Production cell lines are generated by transfecting the host cells with a plasmid vector expressing the gene of interest (GOI) and a selection marker, followed by drug treatment and clone selection
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