A Highly Stringent Split Intein-Mediated DHFR Selectable Marker Enables Efficient Development of High-Producing CHO...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Line Development

Background:

• Traditional dihydrofolate reductase (DHFR)-based selection systems for Chinese hamster ovary (CHO) cell line development have limitations, including low stringency and inefficient multigene selection.
• These limitations hinder the efficient production of therapeutic proteins, necessitating improved selection strategies.

Purpose of the Study:

• To develop a highly stringent split DHFR selectable marker for enhanced CHO cell line development.
• To improve the efficiency of therapeutic protein production and multigene selection in CHO cells.

Main Methods:

• Integrated a computationally guided split-site design with split intein-mediated protein ligation (SiMPl) to create a synthetic SiMPl-DHFR selection system in CHO cells.
• Evaluated the system's stringency, protein productivity, and efficiency in multigene selection for antibody production.

Main Results:

• The SiMPl-DHFR system achieved 98.5% Fc-fusion glycoprotein-producing cells in stable pools, a 22.5-fold increase in specific productivity compared to wild-type DHFR (WT-DHFR).
• SiMPl-DHFR demonstrated superior performance, with non-amplified pools showing higher expression than WT-DHFR amplified pools.
• Efficient generation of stable CHO cell pools for monoclonal and bispecific antibody production requiring coordinated expression of multiple genes was achieved.

Conclusions:

• The synthetic SiMPl-DHFR selection system offers a highly stringent and extensible platform for mammalian cell line development.
• This novel approach rewires DHFR selection logic, overcoming limitations of traditional methods and enhancing therapeutic protein production.