A single anion exchange chromatography step enables scalable, high-yield purification of hepatitis B virus capsid virus-like particles
5 affiliations
- 1Institute of Virology, School of Medicine, Technical University of Munich and Helmholtz Munich
- 2Program in Emerging Infectious Diseases, Duke-NUS Medical School
- 3Electron Microscopy Faculty, Chemistry Department, Technical University of Munich
- 4Hemholtz Munich, Institute of Structural Biology
- 5German Center for Infection Research (DZIF), Munich Partner Site
Abstract
Introduction: Hepatitis B Virus (HBV) causes a significant global health burden. The HBV core (HBc) protein forms the HBV capsid and has a crucial role in the virus replication cycle. When expressed across different platforms, HBc can self-assemble into HBV-capsid-based virus-like particles (VLPs), with a wide range of applications in both fundamental and pharmaceutical research. Due to their ability to display or crosslink foreign antigens on the surface, HBc VLPs are used as vaccine antigens. However, conventional VLP purification methods face challenges in scalability and achieving high yields. Therefore, investigating the generation and purification process of these VLPs is of high interest.
Methods: HBc VLPs composed of three different forms of HBc protein were expressed in E. coli – D (full-length, 183 aa), ΔD ₁₆₃ (C-terminal partially truncated, 163 aa), and ΔD ₁₄₉ (C-terminal fully truncated, 149 aa). Polyethyleneimine precipitation (PEI) was performed for excess nucleic acid reduction, followed by two stages of ammonium sulfate precipitation (AS). For a final purification anion-exchange chromatography (AEX) was performed. Purified HBc VPLs were analyzed for purity and stability by SDS-PAGE, native agarose gel electrophoresis, ELISA, Dynamic light scattering, electron microscopy and host cell protein and DNA and endotoxin assays.
Results: The single AEX step method resulted in high purity (>85%) and reduced host-cell residues comparable to those detected in a sample produced under good manufacturing practice (GMP) conditions. The HBc VLP variants yielded 5.2 mg, 25.9 mg, and 27.8 mg purified capsids per liter E. coli culture, respectively. Our data showed that all three VLP forms preserved their correct assembly and exhibited minimal aggregation.
Discussion: We successfully designed an HBc-VLP purification technique for the purification of different HBV core antigen particles. The purification resulted in highly pure and little aggregated VLP. This purification method is therefore well-positioned to accelerate studies of HBc VLPs.
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BibTeX
@article{seidler2026single,
title = {{A single anion exchange chromatography step enables scalable, high-yield purification of hepatitis B virus capsid virus-like particles}},
author = {A. Seidler and S. Ambike and C. Peters and R. Bester and M. Sattler and A. Geerlof and U. Protzer},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1904236},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1904236/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A single anion exchange chromatography step enables scalable, high-yield purification of hepatitis B virus capsid virus-like particles
AU - A. Seidler
AU - S. Ambike
AU - C. Peters
AU - R. Bester
AU - M. Sattler
AU - A. Geerlof
AU - U. Protzer
PY - 2026
DA - 2026/10/09
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1904236
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1904236/full
AB - Introduction: Hepatitis B Virus (HBV) causes a significant global health burden. The HBV core (HBc) protein forms the HBV capsid and has a crucial role in the virus replication cycle. When expressed across different platforms, HBc can self-assemble into HBV-capsid-based virus-like particles (VLPs), with a wide range of applications in both fundamental and pharmaceutical research. Due to their ability to display or crosslink foreign antigens on the surface, HBc VLPs are used as vaccine antigens. However, conventional VLP purification methods face challenges in scalability and achieving high yields. Therefore, investigating the generation and purification process of these VLPs is of high interest. Methods: HBc VLPs composed of three different forms of HBc protein were expressed in E. coli – D (full-length, 183 aa), ΔD ₁₆₃ (C-terminal partially truncated, 163 aa), and ΔD ₁₄₉ (C-terminal fully truncated, 149 aa). Polyethyleneimine precipitation (PEI) was performed for excess nucleic acid reduction, followed by two stages of ammonium sulfate precipitation (AS). For a final purification anion-exchange chromatography (AEX) was performed. Purified HBc VPLs were analyzed for purity and stability by SDS-PAGE, native agarose gel electrophoresis, ELISA, Dynamic light scattering, electron microscopy and host cell protein and DNA and endotoxin assays. Results: The single AEX step method resulted in high purity (>85%) and reduced host-cell residues comparable to those detected in a sample produced under good manufacturing practice (GMP) conditions. The HBc VLP variants yielded 5.2 mg, 25.9 mg, and 27.8 mg purified capsids per liter E. coli culture, respectively. Our data showed that all three VLP forms preserved their correct assembly and exhibited minimal aggregation. Discussion: We successfully designed an HBc-VLP purification technique for the purification of different HBV core antigen particles. The purification resulted in highly pure and little aggregated VLP. This purification method is therefore well-positioned to accelerate studies of HBc VLPs.
SN - 1664-302X
ER -