CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.

Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations.

Decreases in polysorbate (PS80) content were observed while evaluating the long-term storage stability of Chinese hamster ovary-derived, purified monoclonal antibodies. It was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. Polysorbate degrading activity was enriched from Chinese hamster ovary media leading to the identification of group XV lysosomal phospholipase A2 isomer X1 (LPLA2) by shotgun proteomics. Recombinant LPLA2 was over expressed, purified, and functional integrity confirmed against a diheptanoyl phosphatidylcholine substrate. Incubation of recombinantly produced LPLA2 with PS20 and PS80 resulted in hydrolysis of PS20 and PS80 monoester but a much slower rate was observed for higher-order PS80. Endogenous LPLA2 was detected and quantitated at less than 1 ppm in 3 formulated antibodies while LPLA2 was not detected (or less than 0.1 ppm) in a fourth formulated antibody. Furthermore, antibodies with detectable quantities of endogenous LPLA2 demonstrated polysorbate hydrolysis while in contrast the antibody without detectable LPLA2 did not show polysorbate hydrolysis. Comparison of polysorbate degradation products generated from the formulated antibody and samples of polysorbate incubated with recombinant LPLA2 resulted in similar elution profiles by liquid chromatography-mass spectrometry. These results suggest that LPLA2 may play a key role in polysorbate degradation in some antibody preparations.