Knight, Tanya J., Povey, Jane F., Vito, Davide, Mohindra, Atul, Jaques, Colin M., Smales, C. Mark (2022) Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc‐fusion protein produced in CHO cells. Biotechnology and Bioengineering, 119 (12). pp. 3408-3420. ISSN 0006-3592. E-ISSN 1097-0290. (doi:10.1002/bit.28230) (KAR id:98242)
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Official URL: https://doi.org/10.1002/bit.28230 |
Abstract
Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.
Item Type: | Article |
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DOI/Identification number: | 10.1002/bit.28230 |
Additional information: | For the purpose of open access, the author(s) has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising. |
Uncontrolled keywords: | Chinese hamster ovary (CHO) cells; clipping; Fc-fusion protein; fragmentation, mRNA translation elongation |
Subjects: |
Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH581.2 Cell Biology |
Divisions: | Divisions > Division of Natural Sciences > Biosciences |
Funders: | Biotechnology and Biological Sciences Research Council (https://ror.org/00cwqg982) |
SWORD Depositor: | JISC Publications Router |
Depositing User: | JISC Publications Router |
Date Deposited: | 28 Nov 2022 10:36 UTC |
Last Modified: | 23 Aug 2024 14:15 UTC |
Resource URI: | https://kar.kent.ac.uk/id/eprint/98242 (The current URI for this page, for reference purposes) |
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