Study On Gigaporous Chromatographic Process Of Virus-like Particles Purification

The virus like particles (VLPs) represent a class of important modern vaccines that are designed specifically for prevention of virus infections. It is necessary to purify the VLPs to ensure its efficacy and safety. Chromatography is one of the most powerful techniques for separation and purification of biomacromolecules. Unfortunately, common chromatographic packing beads designed for protein purification usually have pore sizes smaller than 30 nm, which close or even smaller than the size of VLPs that are self-assembled from unusually more than 100 subunits and have size ranging from twenty up to hundreds of nanometers. Therefore, when the beads are applied to VLPs purification, they will encounter several of severe limitations such as slow diffusion of VLPs and even blockage of the pores, long separation time, and loss in bioactivity of the target product. To solve these problems, this work studies the application of gigaporous media with pore size larger than 100 nm in purification of hepatitis B virus surface antigen VLPs (HB-VLPs). By comparing with several other types of media, the chromatographic process of gigaporous media for VLPs was systematically studied.(1) Two agarose media (DEAE-FF and DEAE-Capto), four gigaporous media (DEAE-AP-120nm, DEAE-AP-280nm, POROS D and POROS HQ) and one monolithic anion exchange media were performed to investigate the effect of pore size on dynamic binding capacity (DBC) of proteins of different size protein. The agarose beads with small pore size were found more suitable for the binding of small proteins, while the gigaporous media were more suitable for HB-VLPs adsorption and benefit to improve the antigen recovery.(2) The average pore radius of agarose and gigaporous media was precisely estimated by using single and double pore size distribution model, respectively. The effect of pore size on the static adsorption capacity, dynamic binding capacity and intraparticle diffusion of proteins of different size was investigated in depth. For the protein smaller than 5.4 nm, the accessible surface area of the media is the most important factors affecting the static and dynamic adsorption capacity. For protein larger than 5.4 nm, the binding capacity is mainly influenced by diffusivity. Larger pores provide more accessible surface area and larger diffusivity.(3) Combining with confocal laser scanning microscopy, high performance liquid chromatography (HPLC) and transmission electron microscope analyses, the mechanism of the pore size affecting on the VLPs’assembly and antigen activity during purification process was studied and discussed. The adsorption of the VLPs in DEAE-FF and DEAE-Capto was mostly confined to a thin shell on the outer surface of the beads, while the large pores in gigaporous media enabled the VLPs to access to the interior pore spaces by diffusion transport efficiently. Compared to the DEAE-FF, DEAE-AP-280nm gained about 12.9 times increase in static adsorption capacity,8.0 times increase in DBC, and 11.4 times increase in effective pore diffusivity. The gigaporous structure also significantly improved the VLPs stability by lowering the multi-point interaction between the VLPs and binding sites in the pores. At 2.0 mg/mL media loading quantity, about 85.5% VLPs were correctly self-assembled after the chromatography with DEAE-AP-280nm media; oppositely about 85.2% VLPs lost their normal assembly with DEAE-FF due to irreversible disassembly. In actual VLPs purification, DEAE-AP-280nm media were demonstrated the best results showing the highest recovery of 68.33% and purification fold of 3.47, at 2.98 mg protein/mL-media loading quantity and a flow rate of 240 cm/h.(4) To elucidate the thermodynamic mechanism for the effect of pore size or ligand density of two giga-porous IEC media on the adsorption and conformational change of VLPs, a thermodynamic model was proposed to obtain the intrinsic molar enthalpy changes related to the binding of VLPs and the accompanying conformational change at the liquid-solid interface during its adsorption. Combining with isothermal titration calorimetry and chromatography experiments, the thermodynamic parameters of adsorption and conformational change were acquired. The intrinsic binding of intact HB-VLPs on media were all positive values, indicting an entropically driven process; while the negative ΔconfHdis values suggested a spontaneous enthalpy-driven process for the formation of HB-VLPs disassembly. As pore size decrease or ligand density increase, ΔconfHdis became more negative, which was in agreement with the findings from chromatography experiments that smaller pore size or higher ligand density led to more serious disassembling of HB-VLPs.In summary, the current study on gigaporous chromatographic process of HB-VLPs purification will provide a useful guidance to establish efficient separation/purification process for a variety of VLPs. Application of gigaporous media to VLPs purification not only provided high binding capacity and diffusivity, but also stabilized the assembly of VLPs thermodynamically on the liquid-solid interface. Those well demonstrated superiorities will enable the gigaporous media a wide application prospect for VLPs purification.