Quantification of Linear Polyethylenimines in Recombinant Adeno-Associated Virus by High-Performance Liquid Chromatography with Charged Aerosol Detection

Abstract

Polyethylenimine (PEI) is widely employed as a transfection reagent in recombinant adeno-associated virus (rAAV) manufacturing, but it must be removed from the final product due to its potential toxicity. Accurate quantification of PEI in complex biological matrices such as rAAVs is challenging, largely because the strong electrostatic attraction between PEI and nucleic acids can hinder the accuracy of its quantification. Here, we report a robust high-performance liquid chromatography method with charged aerosol detection for the quantification of residual linear PEI in purified AAV samples. Sample preparation includes treatment with trifluoroacetic acid and hydrochloric acid at 60°C to denature capsid protein, disrupt PEI–DNA polyplexes, and hydrolyze nucleic acids. The method achieves a limit of detection of 5 µg/mL and a limit of quantitation of 10 µg/mL in spike-and-recovery studies, with quantification confirmed via visual peak identification. This approach enables sensitive, specific, and reproducible PEI measurement and provides a valuable tool for process monitoring and quality control in gene therapy manufacturing.