The demand for biopharmaceutical products continues to grow significantly, driven by the increasing availability of highly effective and potent medicines. This is coupled with the patient’s and physician’s desire for improved therapeutic outcomes.
Besides microbial and insect host cell lines, the use of mammalian cells to produce high-quality recombinant proteins at cost-effective yields continues to grow dramatically. Mammalian cell lines such as the widely used CHO enable production of complex, multisubunit proteins such as full-length therapeutic monoclonal antibodies.
While a well-characterized, clonal, stable mammalian cell line remains the de facto standard for the production of high-quality bulk drug substances, transient expression systems for generating sufficient protein to support early-stage preclinical and clinical development may become a valuable option for companies that do not wish to make a major investment in the production of stable cell lines prior to proof of concept clinical studies.
Compared to the time and cost of generating stable cell lines, the speed, flexibility, and cost effectiveness of transient gene expression (TGE) has enabled its broad adoption for early discovery, applications in research, and for supporting preclinical studies.
TGE enables the rapid expression of several proteins in parallel and can be applied to a wide variety of mammalian cell lines; it also provides flexibility in testing different constructions of expression vectors in a short period of time.
TGE with PEI
In the last decade, advances in the development of transfection reagents have emerged from different sources—all with the aim of overcoming some of the key limitations of TGE systems (such as low protein yield and quality and batch-to-batch inconsistency). Polyethylenimine (PEI) is the most popular transfection reagent for TGE due to its high DNA delivery efficiency and affordability.
Until recently PEIs available in the market have not been specifically designed for transfection. Often, these products are poorly characterized in terms of chemical structure and impurities. They are not optimized for transfection, and most significantly they are not formulated, tested, or qualified specifically for use in biomanufacturing applications. Such unqualified PEI for transfection applications may lead to inconsistency in the outcomes of TGE experiments.
Polyplus Transfection has developed and manufactured a PEI formulation for transfection and, in particular, for TGE in suspension-adapted cells for the production of recombinant proteins. PEIpro™ is the latest PEI product from Polyplus-transfection.
Positively charged reagent/DNA complexes are needed to achieve high transfection efficiency. The global charge of the complexes is determined by the PEI/DNA ratio [N/P ratio, i.e., number of nitrogen residues (N) in the PEI per phosphate (P) of DNA]. To obtain positively charged complexes, an N/P ratio of greater than three is needed.
The number of nitrogen residues available in the PEI depends on the molecular weight of the polymer, its structure (branched or linear), the deprotection of the protonable residues (deacylation), and the distribution of the fragment length (polydispersity) following hydrolysis.
The branched PEI results in a lower availability of protonable amines. Further, although mainly based on the pH conditions, the availability of protonable amines can also vary with the chemical properties of the complexation medium, which also influence the particles stability in solution.
Linear PEIs ranging from 19 to 28 kDa are most suitable for the efficient transfection of DNA into mammalian cells. Indeed high molecular weight fragments (>200 kDa) are involved in cell toxicity probably due to damage to the cell membrane during the release of DNA into the cell cytoplasm, whereas low molecular weight fragments are not effective in the formation of complexes with DNA.
In addition, although some linear PEIs appear to be of interest for transfection, the partial acidic hydrolysis of the intermediate form (deacylation) during synthesis leads to the presence of propionate side chains in the final product that are then responsible for poor transfection efficiency due to the reduction in free protonable amines.
Finally, even in the case of what is claimed to be fully deacylated linear PEI, inconsistencies in the manufacturing process often result in the presence of multilength PEI fragments with a high polydispersity in the final product causing uncontrollable variations in protein yields batch to batch manufacturing.
PEIpro is manufactured from a well-qualified monomer unit that is then converted into PEOX intermediate by cationic ring-opening polymerization. The PEOX intermediate is purified and then fully hydrolyzed into a linear PEI.
The molecular weight of PEIpro has been optimized to maximize transfection efficiency and to make certain that lot-to-lot molecular weights are reproducible. The result is the production of high recombinant protein levels using TGE systems (Figure 1).
The quality of PEIpro is continuously assessed during the manufacturing process with the appropriate control testing (Figure 2).
Further, systematic lot management and release testing is performed for each lot produced. To assess activity a standardized transfection assay is carried out with a suspension-adapted HEK-293 cell line in animal-component-free media to ensure the reliability and reproducibility of the transfection efficiency under conditions that are suitable for biomanufacturing.
Figure 3 demonstrates PEIpro’s ability to deliver reproducible protein production from lot-to-lot.
In addition to ensuring reliable production of recombinant proteins during the entire development process, any PEI transfection reagent selected should have the potential to meet cGMP guidelines from FDA, EMA, or ICH when the transfection reagent will be used to produce therapeutic recombinant proteins that are in late-stage clinical development (i.e., post-Phase I).
PEIpro is tested for complete sterility, the absence of mycoplasma, and the presence of low levels of bacterial endotoxins (following pharmacopeia standards).
Combined with appropriate and advanced quality controls, as well as lot release testing, PEIpro enables biopharmaceutical companies to develop consistent, reliable, and secure TGE systems for the reproducible production of recombinant proteins.