Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.
Keywords: HIV-1 Gag-eGFP VLP; chromatography; clarification; downstream processing; nanoparticle quantification.
© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.