Targeted CHO cell engineering approaches can reduce HCP-related enzymatic degradation and improve mAb product quality.

Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification.

Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells.

IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.