HIV-Gag VLP-based vaccine purification at manufacturing scale: Combination of counterflow centrifugation and nuclease treatment as a key to DNA depletion.

Virus-like particles (VLPs) hold promising potential as vaccines, necessitating their production in large quantities at high purity. The FDA requires DNA depletion to be less than 10 ng per dose, a challenge exacerbated by the release of DNA from dead cells during both the upstream processes and the clarification. Our study identified the clarification step using conventional filtration cascades as the main source of DNA contamination, exceeding DNA release during cultivation significantly. Since standard nuclease treatment before clarification showed only limited potential for process improvement, we focused our efforts on an alternative clarification approach. By substituting the primary 20 µm filter in the filtration cascade with low shear force counterflow centrifugation, we successfully minimized cell disruption and DNA release during VLP clarification, while also greatly improving the overall clarification filter capacity many times over. Coupled with nuclease pre-treatment, DNA was efficiently digested, reducing the DNA amount after clarification below the FDA criteria, reaching 3.2 ng/dose in a batch of approximately 6 million doses. These results were consistently reproduced, achieving an overall VLP recovery near 100% during clarification across lab- and production-scale. Our findings demonstrate that counterflow centrifugation is a highly effective clarification method for enveloped VLPs, paving the way for its application in extracellular particles and enhancing the purification of gene therapeutics like viral vector particles, where high purity with regard to DNA removal as a key performance indicator during purification is a common challenge.