Capture and purification of Human Immunodeficiency Virus-1 virus-like particles: Convective media vs porous beads.

Downstream processing (DSP) of large bionanoparticles is still a challenge. The present study aims to systematically compare some of the most commonly used DSP strategies for capture and purification of enveloped viruses and virus-like particles (eVLPs) by using the same staring material and analytical tools. As a model, Human Immunodeficiency Virus-1 (HIV-1) gag VLPs produced in CHO cells were used. Four different DSP strategies were tested. An anion-exchange monolith and a membrane adsorber, for direct capture and purification of eVLPs, and a polymer-grafted anion-exchange resin and a heparin-affinity resin for eVLP purification after a first flow-through step to remove small impurities. All tested strategies were suitable for capture and purification of eVLPs. The performance of the different strategies was evaluated regarding its binding capacity, ability to separate different particle populations and product purity. The highest binding capacity regarding total particles was obtained using the anion exchange membrane adsorber (5.3 × 1012 part/mL membrane), however this method did not allow the separation of different particle populations. Despite having a lower binding capacity (1.5 × 1011 part/mL column) and requiring a pre-processing step with flow-through chromatography, Heparin-affinity chromatography showed the best performance regarding separation of different particle populations, allowing not only the separation of HIV-1 gag VLPs from host cell derived bionanoparticles but also from chromatin. This work additionally shows the importance of thorough sample characterization combining several biochemical and biophysical methods in eVLP DSP.

Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP.

Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100-200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.

Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography.

Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed. In the first step, virus-like particles and extracellular vesicles were collected and separated from smaller impurities in a flow-through mode. Benzonase® treated HEK 293 cell culture supernatant was directly loaded onto a column packed with core-shell beads. The collected flow-through was further purified using heparin affinity chromatography. In heparin affinity chromatography 54% of the total particle load were found in the flow-through, and 15% of the particles were eluted during the salt linear gradient. The particle characterization, especially particle size distribution and mass spectrometry data, suggests that extracellular vesicles dominate the flow-through fraction and HIV-1 gag VLPs are enriched in the elution peak. This is in part in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography. The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day.