C. Mark Smales

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.