Poly(Beta-Amino Ester)s as High-Yield Transfection Reagents for Recombinant Protein Production

Abstract

Purpose Transient transfection is an essential tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and the resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of a class of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection in comparison to linear 25 kDa PEI, a commonly used transfection reagent. Methods Transfection efficiency and protein production were assessed in human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions using PBAE-based nanoparticles in comparison to linear 25 kDa PEI. Production of both a cytosolic reporter and secreted antibodies was investigated. Results In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and enhanced production of a cytosolic reporter compared to linear 25 kDa PEI. This result extended to secreted proteins, as a model PBAE increased the production of 3 different secreted antibodies compared to linear 25 kDa PEI at culture scales ranging from 20 to 2000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticle formulation led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types. Conclusion These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.

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“…Unmodified and CBD-fused ICs were recombinantly expressed in HEK 293-F cells via transient co-transfection of plasmids encoding the corresponding heavy chain (HC) and light chain (LC) using the high-yield transfection reagent poly(beta-amino ester) 4-4-6. 82 HEK 293-F cells were grown to 1.2×10 6 cells/mL and diluted to 1.0×10 6 cells/mL in a fresh culture medium on the day of transfection. A total of 1 mg of maxiprepped (Qiagen), endotoxin-free plasmid DNA (1:2 ratio HC:LC) and 63.15 mg of poly(beta-amino ester), 4-4-6, per L of cells were independently diluted to 0.05 and 3 mg/mL in 25 mM magnesium acetate buffer (Sigma-Aldrich) pH 5.2 respectively, combined by vortexing and incubated at room temperature for 15 min.…”

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“…The most effective polymer had greater-than twofold higher transfection efficiency than PEI in HEK-293 cells and approximately eight times higher than PEI in CHO cells. 416 Targeted delivery can be achieved by modifying PBAE composition. For example, Deng et al 417 demonstrated that fluorinating PBAE results in lung endothelium-targeted delivery after intravenous injection in vivo.…”