Bacterial Glutamine Synthetases as a Novel Metabolic Selection Marker to Improve CHO Cell Culture Performance Through...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Metabolic selection markers are crucial for stable cell line generation and productivity in biopharmaceutical manufacturing.
• Current methods often rely on antibiotic resistance genes, posing environmental and regulatory concerns.
• There is a need for novel, efficient, and antibiotic-free selection systems.

Purpose of the Study:

• To investigate the potential of bacterially derived glutamine synthetases (GS) as novel metabolic selection markers.
• To enhance Chinese Hamster Ovary (CHO) cell culture performance and antibody production.
• To evaluate the bioprocess performance and stability of cell lines using bacterial GS markers.

Main Methods:

• GS-I proteins were identified and extracted from enterobacterial and actinomycetes genomes.
• Functional assessment of bacterial GS enzymes in GS-deficient CHO cells via stable transfection.
• Analysis of antibody titer, bioprocess performance, and cell line stability.
• Transcriptome analysis to understand cellular regulatory mechanisms.

Main Results:

• Three bacterial GS enzymes significantly increased antibody titer (3- to 4-fold) in CHO cells compared to endogenous GS.
• Enzyme functionality correlated with solvent accessibility and catalytic pocket geometry.
• Bacterial GS markers demonstrated superior stable pool and clone performance.
• Modulating selection stringency enhanced expression without compromising cell line stability.

Conclusions:

• Bacterially derived GS enzymes are effective metabolic selection markers for mammalian cell lines.
• These markers enhance productivity and bioprocess performance in CHO cell cultures.
• Repurposing bacterial enzymes offers a promising antibiotic-free strategy for cell line development.