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Gupta, Shivani

University of British Columbia

Since 2015 more than 39 biosimilars have been approved by the FDA, with over 20 launched, providing alternatives for widely used protein therapeutics. A major challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture these biologics. Many biologics approved before 2011 were expressed in murine cell lines, whereas Chinese Hamster Ovary (CHO) cells have become the preferred hosts for production. Differences between glycosylation have been identified in biologics produced using murine CHO cell line. In the case of monoclonal antibodies (mAbs), differences in glycans can significantly impact critical antibody effector function, binding activity, efficacy, and in vivo half-life. To more closely match the reference biologic (RB) glycosylation, a mAb expressing CHO cell line was genetically engineered to express mAb with murine-like glycans. This was done by selectively co-expressing two key murine enzymes, α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-Acetylneuraminic acid hydroxylase (CMAH). These CHO cells were then analyzed using a spectrum of analytical methods to demonstrate biosimilarity, including high-resolution mass spectrometry and biochemical and cell-based assays. In fed-batch cultures, two CHO cell clones were identified that maintained stable production while matching the glycosylation profile and function of the reference product. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans, to reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development. In parallel, a high-throughput CHO-specific assay was designed and evaluated to quantify N-glycosylation-related enzyme mRNA expression levels. This assay is referred to as the N-glycan enzyme mRNA expression profiler. Two different mAbs produced by two CHO cell hosts were investigated to assess the relationships between glycan mRNA levels and mAb glycan profiles. Genetic understanding of the N-glycosylation pathway in the CHO cells helped identify gene expression patterns that correlated to the N-glycan profile of the produced mAb. The results indicated that this assay could identify clones with desirable genetic makeups early in cell line development and aid in designing cell line engineering strategies to obtain the desired glycan profiles for both novel biologic and biosimilar development.

Text

2023-01-02

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83532

Chemical and Biological Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/