Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation

Abstract

Inserting foreign epitopes to hepatitis B core (HBc) virus‐like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost‐effective purification process was developed for two chimeric HBc VLPs displaying Epstein–Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core‐HBc was found to be less stable in water environment compared with EBNA1‐HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α‐helix of HCV core‐HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost‐effective purification approach.

Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids

“…The standard deviation of nucleic acid concentrations significantly exceeded those determined with a prior extraction. This might also explain the conflicting results in the literature between UV-spectroscopy-based analysis (A260/A280) and a dye-based fluorescence assay without a prior separation of the HBcAg VLPs and bound NA hc [ 27 ]. However, the analysis of DNA impurities by the PicoGreen assay in HBcAg VLP purification [ 14 ] does not seem to be affected by a lack of nucleic acid extraction, as the DNA presumably was not bound to the VLPs and binding of the dye to the DNA was not sterically impeded.…”

“…This might also be useful when evaluating the removal of specific NA hc from HBcAg VLPs, as in RNA-specific lithium chloride precipitation [ 8 ] or enzymatic treatments [ 18 , 39 ]. For the evaluation of general nucleic acid depletion [ 15 , 16 , 27 ]), it may be sufficient and advantageous in terms of time and costs to perform the silica-SC-based extraction procedure in combination with a dye-based fluorescence assay such as PicoGreen or RiboGreen, but without an enzymatic treatment. Depending on the objective of the specific experiment and the expected nucleic acid species, the complexity of the sample treatment between the silica-SC-based extraction proposed here and the absolute quantification of nucleic acids by dye-based fluorescence assays can be varied and adapted to the specific purpose.…”

“…Both methodologies overcome the limitations of existing techniques, and effective absolute quantification of HBcAg proteins and bound nucleic acids is added to the analytical toolbox for HBcAg VLP research. Today, only qualitative analytical techniques such as SDS-PAGE for proteins and NAGE for nucleic acids and proteins, in combination with microvolume UV/Vis spectroscopy measurements for approximations of protein and nucleic acid concentrations, are used for HBcAg VLP process development for gene delivery [ 8 , 10 , 15 , 16 , 27 ]. Size exclusion HPLC is also frequently used to separate and quantify different HBcAg VLP protein species (capsids and dimers) and impurities such as proteins and free nucleic acids [ 19 , 20 , 21 ].…”

“…Relative analysis of the A260/A280 ratio of HBcAg proteins and bound NA hc has been used to correlate HBcAg VLP constructs with different lengths of the nucleic acid binding region to particle loading with NA hc [ 26 ]. It can also be used to evaluate the purity of the VLPs after the depletion of NA hc [ 8 , 15 , 27 ]. For the loading of HBcAg VLPs with NA ther of known nucleotide sequences, a method to approximate the absolute amounts of protein and nucleic acid was proposed in the literature [ 28 ] and used to estimate protein and nucleic acid composition [ 10 , 16 ].…”

“…However, for HBcAg VLPs with a nucleic acid binding region for gene delivery and thus bound nucleic acids, appropriate nucleic acid extraction seems to be necessary to quantify the nucleic acids precisely. This can be concluded from the conflicting results of the relative analysis of the A260/A280 ratio with UV spectroscopy and a dye-based fluorescence assay without prior extraction of the HBcAg VLPs and bound NA hc [ 27 ].…”