Characterization and downstream processing of HIV-1 core and virus-like-particles produced in serum free medium

This work aimed at the characterisation and downstream processing of HIV-1 core and virus-like particles (CLPs and VLPs) produced in serum free medium. The produced core and virus-like particles have similar characteristics to those of the native immature HIV-1 virus in terms of protein composition, diameter, density and shape. The molecular mass (determined by gel filtration chromatography) and the diameter (determined by electron microscopy) were similar for CLPs and VLPs, this being correlated with the difference between the particle densities (1.14 g/cm3 and 1.19 g/cm3, respectively, for CLPs and VLPs). After this characterization, several concentration and purification methods were tested to obtain enough material for further tests. For this purpose, a downstream processing strategy was developed, involving clarification by centrifugation and microfiltration, concentration and partial purification by ultrafiltration, and final purification by gel filtration chromatography. Quality analysis using Western immunoblot and gel filtration chromatography was performed to define the best operational conditions for each purification step. The particle recovery obtained was 87% with the main losses occurring in the ultrafiltration step. In addition, by evaluating the effect of operational conditions on product quality, it was possible to obtain a final product with high content of intact high molecular weight particles.