LAUSR

Despite evidence that they are not functional or infective, retrovirus‐like particles (RVLPs), originating from endogenous proviral sequences in Chinese hamster ovary (CHO) cells, present a safety risk for biotherapeutics manufactured using this cell line due to their resemblance to other mammalian leukemia viruses. Here, we demonstrate that CRISPR‐ and shRNA‐based cell engineering strategies can be used to disrupt RVLP production by targeting the RVLP nucleotide sequences. Additionally, specific antibodies were generated to monitor RVLP protein expression, including RVLP envelope (Env) protein localized on the surface of CHO cells, greatly facilitating selection of RVLP‐deficient clones. These modified CHO cells showed reduced RVLP production while maintaining or enhancing the ability to produce recombinant virus‐like particles (VLPs), highlighting their potential application in biomanufacturing, especially for complex biologics that are incompatible with standard RVLP mitigation procedures, namely viral inactivation and nanofiltration.