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.