PEI-mediated Transient gene expression for protein production

Transient protein expression in mammalian cell lines has gained increasing relevance as it enables fast and flexible production of high-quality eukaryotic proteins. Milligram amounts of protein can be produced within several days, meaning a significant shortening of process timeline in comparison to protein production from stable clones. While substantial efforts have been made in fine-tuning plasmid vector design to improve protein yields and transgene expression, one of the current bottlenecks that needs to be addressed is the transition from small scale development towards a large-scale manufacturing process that can support potential commercial requirements

Advantages of PEI for transient protein production

Considerable efforts have been made to overcome existing limiting aspects of transient gene expression systems, in terms of cell lines, cell culture-based systems, and protein production in cost-effective manner. In most cases, transfection reagents that are used in small to large scale protein production include calcium phosphate, cationic lipids and polyethylenimine (PEI). Calcium phosphate appears to be the economical option; it is in fact a cumbersome transfection method that requires rigorous control and large quantities of DNA. While the use of cationic lipids leads to less inter-experimental variability, the imputed cost and cellular toxicity are detrimental to their use in large scale protein production. Conversely, PEI is widely used due to its affordability, high DNA delivery efficiency, and reproducibility during upscaling in both adherent and suspension cells, unlike calcium phosphate that is inefficient for transfection of cells grown in suspension. Several PEIs of different molecular weights and chemical properties that were not specifically developed for transfection are commercially available. These PEIs are poorly characterized in terms of chemical structure and purity, and cannot guarantee high reproducibility in protein production.

PEIpro® and PEIpro®-HQ, provided and manufactured by Polyplus-transfection®, are the unique PEIs developed for transfection and suitable for large-scale protein production from process development up to use in GMP processes.

PEI optimization for improved protein yields

PEIpro® benefits from extensive research development in polymer chemistry and formulation, in terms of polymer structure, deacetylation degree, polydispersity index and molecular weight. The polymer branching structure is directly correlated with the availability of protonable amines, which affects the global charge of the polymer. Formation of positively charged reagent/DNA complexes is essential to obtain a higher transfection efficiency, rendering linear PEIs preferable to branched PEI. The polydispersity index should be close to 1, as different lengths of PEI in the final product lead to heterogenous complex size formation, and to unwanted batch-to-batch titer variations. In addition, the molecular weight of PEI should be carefully optimized for high transfection efficiency with minimal cellular toxicity. PEIpro® and PEIpro®-HQ are the unique commercially available PEIs that fulfill the aforementioned characteristics.

Enhanced protein production yields in HEK-293 and CHO cells

PEIpro - comparison PEI

PEIpro - comparison PEI 2
PEIpro® requires less reagent and similar to lower DNA amount compared to other PEIs. Suspension HEK-293 (A) and CHO (B) cells were seeded at 1×106 cells/ml in serum free medium and transfected with PEIpro®, PEI “Max” and L-PEI 25 kDa (Polysciences, Warrington, PA) resuspended at 1 mg/ml. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay.

 

Easily scalable for large scale protein production

PEIpro - Complex size
DNA-PEIpro® complex size is identical, independently from the volume of transfection mix preparation. Complexes were prepared with a DNA concentration of 0.01 mg per mL of complex volume at a DNA:Reagent ratio of 1:4, either in 10 mL or 1 L. The size of the complexes was then measured every ten minutes using the Zetasizer Nanometer ZS (Malvern Instrument, Malvern, UK).
PEIpro - Scale-up protein production
Scale-up protein production results with PEIpro® are highly predictable. Suspension CHO cells were seeded at 1×106 cells/mL in 100 mL serum-free medium. DNA-PEIpro® complexes were prepared with a DNA concentration of 0.01 mg per mL of complex volume at a DNA:Reagent ratio of 1:4, either in 10 mL or 1 L. For the transfection, only 10 mL of transfection mix were added to the 100 mL culture. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay.

 

Compatible with various synthetic medium for HEK-293 and CHO cells

 

PEIpro - Compatible media HEK PEIpro - Compatible media HEK

 

PEIpro® is compatible with several HEK-293 synthetic culture media.  Suspension HEK-293 cells were seeded following the recommended protocol in serum-free media and transfected with PEIpro® using the standard conditions. IgG3-Fc production was assayed 48 h after transfection using protein G affinity quantification (HPLC).

 

PEIpro - Compatible media CHO PEIpro - Table compatible media CHO

 

PEIpro® is compatible with several CHO synthetic culture media.  Suspension CHO cells were seeded following the recommended protocol in serum-free media and transfected with PEIpro® using the standard conditions. IgG3-Fc production was assayed 48 h after transfection using protein G affinity quantification (HPLC).