title: High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography.
abstract: The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany.
authors: Christopher
authors: C
authors: Ladd Effio
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany; Boehringer Ingelheim Pharma GmbH & Co. KG, Germany.
authors: Stefan A
authors: SA
authors: Oelmeier
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors: Jürgen
authors: J
authors: Hubbuch
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D029821:Enterovirus A, Human / Q000276:immunology; D006512:Hepatitis B Core Antigens / Q000276:immunology; D057166:High-Throughput Screening Assays; D016732:Parvovirus B19, Human / Q000302:isolation & purification; D011120:Polyomavirus / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology; D058425:Vaccines, Virus-Like Particle / Q000302:isolation & purification*
chemical_list: D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins; D058425:Vaccines, Virus-Like Particle
keywords: Aggregates; High-throughput analytics; Papillomavirus; Process analytical technology; Size-exclusion HPLC; Virus-like particle vaccines