An Arrayed CRISPR Screen Identifies Knockout Combinations Improving Antibody Productivity in HEK293 Cells.

Area of Science:

• Biotechnology
• Molecular Biology
• Bioprocessing

Background:

• Mammalian cells, like human embryonic kidney 293 (HEK293) cells, are crucial for producing complex biologics such as multispecific antibodies.
• High expression titers and purity of these biologics can be challenging to achieve during production.

Purpose of the Study:

• To investigate cellular responses to antibody and multispecific expression.
• To identify genetic targets for host-cell engineering to improve biologic production.

Main Methods:

• Bulk RNA sequencing (RNA-seq) was performed on HEK293 cells expressing five different molecules.
• An arrayed CRISPR knockout screen was conducted targeting differentially expressed genes and literature-motivated candidates.
• Knockout targets impacting antibody productivity were identified and validated.

Main Results:

• A differential expression signature of secretory and protein folding stresses was observed, correlating with lower titers.
• Eight novel knockout targets were identified, increasing expression titers by 20-80%.
• HIST2H3C and INHBE knockouts demonstrated significant titer increases and additivity, even up to 100% for difficult-to-express antibodies, without compromising purity.

Conclusions:

• Disruption of HIST2H3C and INHBE represents a novel host-cell engineering strategy to enhance antibody and multispecific productivity.
• These findings are generalizable to different HEK293 hosts and multispecific production systems.
• This approach holds promise for improving biopharmaceutical manufacturing efficiency.