Heparin-affinity chromatography is a generic purification platform for chimeric gag VLPs displaying different viral surface antigens

Chimeric virus-like particles formed by the co-expression of HIV-1 gag capsid protein and surface proteins from other viruses can be used as a platform for production of a wide range of viral vaccines. These virus-like particles can be rapidly produced in an insect cell culture using the baculovirus expression vector system. However, a variety of different process-related impurities such as host cell proteins, double stranded DNA, chromatin, and structurally similar bionanoparticles like extracellular vesicles and baculovirus particles are present in the harvested supernatant and complicate the chromatographic purification steps. Heparan sulfate is the first entry point of a lot of enveloped viruses into the cell. Taking advantage of this functional property, its sub-variant, the glycosaminoglycan heparin immobilized on a chromatography media is a universal tool for the separation of enveloped virus-like particles. A purification process was developed consisting of clarification of the cell culture supernatant by membrane filtration, a pre-purification step using core–shell bead flow-through chromatography with Capto™ Core 700, followed by heparin affinity chromatography with Capto™ Heparin. The combination of Capto™ Core 700 and Capto™ Heparin allows for direct load of the flow-through material from the first into the second step without buffer exchange in between. In addition, lengthy buffer screening, as required for ion exchange and multimodal chromatography, can be omitted. Linear salt gradient elution in heparin affinity chromatography enabled separation of virus-like particles and baculovirus. The platformability of the process was demonstrated by purification of virus-like particles expressing influenza A virus hemagglutinin or neuraminidase, or SARS-CoV-2 spike protein as surface antigens and the virus-like particle without co-expressed surface protein. Western blotting, host cell protein and DNA assays, TCID50 (for residual baculovirus), mass spectrometry, multi-angle light scattering and nanoparticle tracking analysis were used to characterize the different fractions obtained during the purification. A similar elution profile was obtained for all tested virus-like particles and deviations could be explained by batch-to-batch variations and differences in productivity for the different constructs. Double stranded DNA was reduced to 7 – 36 ng/109 particles and host cell protein content was below the limit of quantification; in the main product fraction an average seven-fold reduction of baculovirus and a yield of 9 % − 14 % of bound particles with a concentration of 1.4 – 1.8 × 1010 particles/mL in the eluate were achieved meeting requirements for a viral vaccine. Accordingly, this process is suitable as a downstream process platform for chimeric HIV-1 gag-based VLP products.