Optimizing Nuclease Treatment to Enhance Anion Exchange Chromatography of HIV-Derived Virus-Like Particles

Residual host cell chromatin imposes numerous challenges on purifying HIV-derived enveloped virus-like particles (VLPs) using anion-exchange chromatography (AEX). According to FDA guidelines, DNA must be reduced to less than 10 ng per dose at a fragment size of less than 200 bp. To prove the fulfillment of these quality criteria, methods for the qualitative and quantitative analysis of DNA fragments must be applied and adapted to chromatin. DNA and chromatin impede the purification of HIV VLPs with AEX, co-eluting in the same fractions as the VLPs. Although nuclease treatments can be employed, the chromatin structure can shield DNA from nuclease activity. To address these challenges, we adjusted our analytical focus on characterizing the chromatin in our clarified HIV VLP supernatant. We identified two DNA subpopulations: a main large fragment population and a minor population consisting of short fragments below 200 bp. Our findings demonstrated that the larger DNA fragments are the primary issue in our process, as they co-elute with the desired VLPs. To remove the long DNA fragment population, we optimized the nuclease treatment using a Design of Experiment approach to digest the DNA despite the tight chromatin structure. The nucleases Benzonase, Denarase, and M-SAN efficiently digested the DNA removing over 90 % of the DNA. By shredding the long DNA fragments before the AEX step, we successfully separated the HIV VLPs from the remaining short DNA fragments. Combined with nuclease treatment, AEX membrane chromatography offers an efficient single-step purification platform for HIV VLP-based vaccines and other therapeutics.