title: Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine.
abstract: To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography-mass spectrometry (LC-MS). The RP-HPLC method has a linear range of 0.51-12 μg protein, an accuracy of 96-106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development.
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States.
authors.forename: Anastasija
authors.initials: A
authors.lastname: Shytuhina
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States.
authors.forename: Pavlo
authors.initials: P
authors.lastname: Pristatsky
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States.
authors.forename: Jian
authors.initials: J
authors.lastname: He
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States.
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.affiliation: Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Gaithersburg, MD 20878, United States.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Schwartz
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States.
authors.forename: Van M
authors.initials: VM
authors.lastname: Hoang
authors.affiliation: Vaccine Bioprocess Research & Development, Merck Research Laboratories, West Point, PA, 19486, United States. Electronic address: sha_ha@merck.com.
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
mesh_terms: D002646:Chikungunya virus / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D058425:Vaccines, Virus-Like Particle / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis; D014765:Viral Vaccines / Q000032:analysis*
chemical_list: D006023:Glycoproteins; D058425:Vaccines, Virus-Like Particle; D014764:Viral Proteins; D014765:Viral Vaccines
keywords: Analytical characterization; CHIKV; Chikungunya; RP-HPLC; VLP; Vaccine