Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines

Abstract

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS−/− CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10–19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500‐L and 1000‐L bioreactors replicated laboratory results using 5‐L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

“…Copy number analysis was performed by real time qPCR analysis [34]. Cell pellets taken from specified age seed cultures were used to extract genomic DNA.…”

“…Genomic DNA isolated from the seed culture cell pellets was digested with the restriction enzymes specific for the target gene expression plasmids. The digested DNA was subjected to agarose gel electrophoresis and hybridization with the gene-specific hybridization probes [34].…”

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

“…Our data compares favorably with Turan et al 57 MSX inhibits the activity of the enzyme GCL, which catalyzes the synthesis of reduced glutathione (GSH) from its precursor amino acids: glutamic acid, cysteine, and glycine. 59 Alteration in the amount of glutathione (either GSH or GSGG forms) has been shown to have dramatic consequences on productivity 39,60,61 and oxidative status 62 in RI derived cell lines.…”

Integrating plant molecular farming and materials research for next-generation vaccines

“…Since then, biologics have been increasingly used as pharmaceutical agents, outpacing the market of small-molecule drugs 1 with an estimated market size of around US$400 billion by 2025 (ref. 2 ). Biologics are a class of medications that are derived from a biological source and typically fall into one of four major categories: monoclonal antibodies (mAbs), receptor modulators, enzyme modulators and vaccines 3 .…”