Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293.

Area of Science:

• Biotechnology
• Pharmaceutical Science
• Cell Biology

Background:

• Therapeutic protein production relies on mammalian cell cultures, but current transient transfection methods face challenges.
• Optimizing transfection is key for efficient industrial-scale production of biopharmaceuticals.

Purpose of the Study:

• To identify a reliable, high-throughput, microliter-scale transfection assay for predicting protein expression in large-scale bioproduction.
• To compare the efficacy of various transfection reagents, including lipid, polymeric, and cell-penetrating peptides (CPPs).

Main Methods:

• Evaluated multiple transfection reagents (lipid, polymeric, CPPs) using high-throughput assays.
• Correlated microliter-scale assay results with protein (antibody) expression in CHO and HEK293 producer cell lines at milliliter and liter scales.
• Assessed predictive power of EGFP(+) population in CHO suspension cultures and SEAP quantitation, compared to adhesion assays and pLuc transfection.

Main Results:

• Microliter-scale assays using adhesion cultures or pLuc transfection did not reliably predict large-scale protein production.
• CHO suspension culture EGFP(+) population and SEAP quantitation showed strong correlation with protein yield.
• Cell-penetrating peptide (CPP)-based transfection significantly enhanced protein yields in both CHO and HEK293 cells compared to PEI and lipoplex methods.

Conclusions:

• A predictive, high-throughput assay for transient protein expression can be established using CHO suspension culture metrics (EGFP(+) population, SEAP).
• Cell-penetrating peptide (CPP) transfection represents a superior alternative to current industry standards like PEI for large-scale therapeutic protein production.
• This CPP-based approach offers a significant advancement for efficient and scalable biopharmaceutical manufacturing.