Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter

Area of Science:

• Biotechnology
• Cell Line Development
• Protein Expression

Background:

• Chinese hamster ovary (CHO) cells are vital for therapeutic protein manufacturing.
• Efficient cell line development is key to accelerating biological drug discovery.
• The glutamine synthetase (GS)-CHO system uses GS expression and MSX inhibition for selection.

Purpose of the Study:

• To enhance the efficiency and stringency of cell line selection in CHO systems.
• To improve the identification of high-producing cell lines for recombinant protein manufacture.
• To optimize the balance between GS expression and MSX inhibitor levels.

Main Methods:

• Utilized GS-knockout CHO cells to eliminate endogenous GS background expression.
• Engineered weakened SV40E promoters to modulate plasmid-driven GS expression.
• Confirmed promoter activity reduction using TaqMan RT-PCR and GFP expression profiling.

Main Results:

• Achieved significant productivity gains in bulk culture and clonal cell lines.
• Demonstrated increased selection stringency, evidenced by a shift in producing-cell populations.
• Showcased effective selection even without MSX, indicating enhanced control.

Conclusions:

• Combined GS-knockout CHO cells with weakened SV40E promoters significantly improve cell line development.
• This strategy enhances selection stringency and protein productivity.
• The approach holds potential for targeted integration and transient CHO expression systems.