Effects of Different Lengths of a Nucleic Acid Binding Region and Bound Nucleic Acids on the Phase Behavior and Purification Process of HBcAg Virus-Like Particles

Abstract

Virus-like particles (VLPs) are macromolecular structures with great potential as vehicles for the targeted administration of functional molecules. Loaded with nucleic acids, VLPs are a promising approach for nanocarriers needed for gene therapy. There is broad knowledge of the manufacturing of the truncated wild-type lacking a nucleic acid binding region, which is mainly being investigated for vaccine applications. Whereas for their potential application as a nanocarrier for gene therapy, hepatitis B core antigen (HBcAg) VLPs with a nucleic acid binding region for efficient cargo-loading are being investigated. VLP structure, loading, and phase behavior are of central importance to their therapeutic efficacy and thereby considerably affecting the production process. Therefore, HBcAg VLPs with different lengths of the nucleic acid binding region were produced in E. coli. VLP attributes such as size, zeta potential, and loading with host cell-derived nucleic acids were evaluated. Capsid's size and zeta potential of the VLP constructs did not differ remarkably, whereas the analysis of the loading with host cell-derived nucleic acids revealed strong differences in the binding of host cell-derived nucleic acids dependent on the length of the binding region of the constructs, with a non-linear correlation but a two-zone behavior. Moreover, the phase behavior and purification process of the HBcAg VLPs as a function of the liquid phase conditions and the presence of host cell-derived nucleic acids were investigated. Selective VLP precipitation using ammonium sulfate was scarcely affected by the encapsulated nucleic acids. However, the disassembly reaction, which is crucial for structure homogeneity, separation of encapsulated impurities, and effective loading of the VLPs with therapeutic nucleic acids, was affected both by the studied liquid phase conditions, varying pH and concentration of reducing agents, and the different VLP constructs and amount of bound nucleic acids, respectively. Thereby, capsid-stabilizing effects of the bound nucleic acids and capsid-destabilizing effects of the nucleic acid binding region were observed, following the two-zone behavior of the construct's loading, and a resulting correlation between the capsid stability and disassembly yields could be derived.

Keywords: capsid stability; disassembly; downstream processing; gene therapy; nanocarrier (nanoparticle); nucleic acid binding; phase behavior; virus-like particles.

FIGURE 1

Typical HBcAg VLP purification process (Hillebrandt et al., 2021) with additional process steps for application as a transport vector for nucleic acids highlighted in blue. The central purification steps captured by selective precipitation and disassembly, investigated in this study, are highlighted in grey. HBcAg VLP construct characterization was conducted after purification by CaptoCore 400 chromatography and dialysis (marked with an asterisk *). HBcAg, hepatitis B core antigen; VLP, virus-like particle.

FIGURE 2

Reducing SDS-PAGE scan of HBcAg VLP construct monomers after re-dissolution in lanes two to seven, with Invitrogen Mark 12 Unstained Standard in lane 1. Molecular weights of the proteins in the standard are displayed on the left. Protein staining by Coomassie blue; HBcAg, hepatitis B core antigen; VLP, virus-like particle.

FIGURE 3

Hydrodynamic radius in black and zeta potential in grey of all VLP construct capsids. Measurements were conducted directly after CaptoCore 400 chromatography purification step. DLS measurements were conducted in six replicates and zeta potential measurements in five replicates; VLP, virus-like particle.

FIGURE 4

Loading of the VLP capsids with host cell-derived nucleic acids for all constructs. SEC chromatography was used to evaluate the loading of the purified VLP capsids by A260/A280 coefficient after scatter correction (Porterfield and Zlotnick, 2010). For every construct, samples were analyzed by SEC in triplicates, respectively. Error bars are negligible and therefore omitted. Details on capsid peak identification can be found in Supplementary Material S2; VLP, virus-like particle; SEC, size-exclusion chromatography.

FIGURE 5

Remaining VLP constructs in precipitation supernatant (A) and re-dissolved VLP constructs after precipitation and re-dissolution overnight for different tested ammonium sulfate concentrations. After precipitation with different ammonium sulfate concentrations, the supernatant (A) and re-dissolution solution (B) were analyzed by SDS-PAGE, and gel scans were evaluated by the intensity of the VLP band on the gel lane with respect to the highest determined intensity for the respective construct. Corresponding gel images can be found in Supplementary Material S4; VLP, virus-like particle.

FIGURE 6

Dimer yields after disassembly reaction for different liquid phase conditions and for all constructs. In subfigures (A) and (B), results for a urea concentration of 4 M and varying pH values are displayed. In subfigures (C) and (D), results for a pH of eight and varying urea concentrations can be found. According to the two-zone behavior, subfigures (A) and (C) show results for Cp149, Cp154, and Cp157 and subfigures (B) and (D) for Cp164, Cp167, and Cp183, respectively. Results for 3 M urea, 3.5 M urea, pH 7, and pH 7.5 can be found in Supplementary Material S5. SEC was used to evaluate the dimer yield after the disassembly reaction. Details on capsid peak identification can be found in Supplementary Material S2; SEC, size-exclusion chromatography.