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).