The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression...

Area of Science:

• Biotechnology
• Cell Biology
• Genetic Engineering

Background:

• Chinese hamster ovary (CHO) cell production of biologics is limited by challenges in coordinating multiple transgene expressions.
• Existing selection systems, like single-marker or multi-auxotrophic platforms, have limitations including low efficiency and metabolic burdens.

Purpose of the Study:

• To develop a novel, high-threshold cooperative selection system for CHO cells to overcome limitations in multi-gene co-expression.
• To engineer a tyrosine-auxotrophic system for stringent and efficient enrichment of cells expressing multiple transgenes.

Main Methods:

• Reconstruction of an essential tyrosine biosynthesis pathway involving pterin-4α carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR).
• Generation of a triple-knockout CHO host cell line using CRISPR/Cas9 technology.
• Application of the system for monoclonal antibody (mAb) production and evaluation of cell population enrichment and productivity.

Main Results:

• Achieved 97.49% enrichment of triple-positive cell populations, demonstrating high homogeneity and coordinated antibody chain expression.
• Obtained titers of 0.35 g/L in fed-batch and 1.60 g/L in perfusion cultures without tyrosine supplementation.
• Demonstrated pathway reconstitution's impact on central metabolism, reducing byproducts and enhancing biosynthesis.

Conclusions:

• The developed tyrosine-auxotrophic system provides a new paradigm for stringent multi-gene co-expression in CHO cells.
• This antibiotic-free platform significantly advances CHO cell engineering for the production of next-generation biologics.
• The system enables efficient co-enrichment and enhanced productivity for complex biopharmaceutical manufacturing.