title: Development of a dose assay for a Clostridium difficile vaccine using a tandem ion exchange high performance liquid chromatography method.
abstract: Clostridium difficile is a gram-positive intestine bacterium that causes a severe diarrhea and could eventually be lethal. The main virulence factor is related to the release of two major exotoxins, toxin A (TcdA) and toxin B (TcdB). Recent C. difficile-associated disease (CDAD) outbreaks have been caused by hypervirulent strains which secrete an additional binary toxin (CDTa/CDTb). Vaccination against these toxins is considered the best way to combat the CDAD. Recently, a novel tetravalent C. difficile vaccine candidate containing all four toxins produced from a baculovirus expression system has been developed. A dose assay to release this tetravalent C. difficile vaccine was developed using tandem ion-exchange HPLC chromatography. A sequential weak cation exchange (carboxyl group) and weak anion exchange (tertiary amine group) columns were employed. The four C. difficile vaccine antigen pIs range from 4.4 to 8.6. The final optimized separation employs salt gradient elution at two different pHs. The standard analytical parameters such as LOD, LOQ, linearity, accuracy, precision and repeatability were evaluated for this method and it was deemed acceptable as a quantitative assay for vaccine release. Furthermore, the developed method was utilized for monitoring the stability of the tetravalent C. difficile vaccine in final container.
authors.affiliation: Vaccine Analytical Development, MRL Merck Co, West Point, PA 19486, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: Vaccine Analytical Development, MRL Merck Co, West Point, PA 19486, United States.
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
authors.affiliation: Vaccine Analytical Development, MRL Merck Co, West Point, PA 19486, United States. Electronic address: richard_rustandi@merck.com.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001428:Bacterial Vaccines / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D016360:Clostridioides difficile / Q000378:metabolism; D004761:Enterocolitis, Pseudomembranous / Q000517:prevention & control; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium
keywords: Binary toxin; Clostridium difficile infection; Dose assay; Ion-exchange HPLC; TcdA/TcdB; Vaccine