Without a trace: multiple knockout of CHO host cell hydrolases to prevent polysorbate degradation in biologics

Enzymatic degradation of polysorbates (PS) in biologic drug formulations is often caused by hydrolytic host cell proteins (HCPs) and can lead to particle formation and reduced shelf-life. Here, we present a host cell line-engineering approach by removing nine Chinese hamster ovary (CHO) host cell hydrolases, which have previously been confirmed to degrade PS. Strikingly, the sequential genomic knockout (KO) of these hydrolases, including the genetic removal of two entire gene clusters of unprecedented size, yielded viable CHO host cell line variants. This novel host cell line was further optimized using the additional KO of the two key proapoptotic genes, Bax and Bak1. Thus, we generated a competitive multi-hydrolase KO CHO host cell line that was further shown to be highly suitable for the generation of recombinant therapeutic glycoproteins. Most importantly, PS degradation and hydrolytic activity were drastically reduced, providing an avenue toward future PS degradation-free biologics-manufacturing processes.