title: Identification of peptide sequences as a measure of Anthrax vaccine stability during storage.
abstract: The UK anthrax vaccine is an alum precipitate of a sterile filtrate of Bacillus anthracis Sterne culture (AVP). An increase in shelf life of AVP from 3 to 5 years prompted us to investigate the in vivo potency and the antigen content of 12 batches with a shelf life of 6.4 to 9.9 years and one bulk with a shelf life of 23.8 years. All batches, except for a 9.4-year-old batch, passed the potency test. Mass spectrometry (MS) and in-gel difference 2-dimensional gel electrophoresis (DIGE) were used to examine antigens of the pellet and supernatant of AVP. The pellet contained proteins with a MW in excess of 15 kDa. DIGE of desorbed proteins from the pellet revealed that with aging, 19 spots showed a significant change in size or intensity, a sign of protein degradation. MS identified 21 proteins including protective antigen (PA), enolase, lethal factor (LF), nucleoside diphosphate kinase, edema factor, and S-layer proteins. Fifteen proteins were detected for the first time including metabolic enzymes, iron binding proteins, and manganese dependent superoxide dismutase (MnSOD). The supernatant contained131 peptide sequences. Peptides representing septum formation inhibitor protein and repeat domain protein were most abundant. Five proteins were shared with the pellet: 2,3,4,5-tetrahydropyridine-6-dicarboxylate N-succinyltransferase, enolase, LF, MnSOD, and PA. The number of peptide sequences increased with age. Peptides from PA and LF appeared once batches exceeded their shelf life by 2 and 4 years, respectively. In conclusion, changes in antigen content resulting from decay or desorption only had a limited effect on in vivo potency of AVP. The presence of PA and LF peptides in the supernatant can inform on the age and stability of AVP.
authors.affiliation: Division of Bacteriology; National Institute for Biological Standards and Control; Hertfordshire, UK.
authors.forename: Gail
authors.initials: G
authors.lastname: Whiting
authors.affiliation: Laboratory of Molecular Structure; National Institute for Biological Standards and Control; Hertfordshire, UK.
authors.forename: Jun X
authors.initials: JX
authors.lastname: Wheeler
authors.affiliation: Division of Bacteriology; National Institute for Biological Standards and Control; Hertfordshire, UK.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Rijpkema
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines / Q000737:chemistry* / Q000276:immunology*; D000942:Antigens, Bacterial / Q000032:analysis*; D001408:Bacillus anthracis / Q000276:immunology; D004356:Drug Storage; D015180:Electrophoresis, Gel, Two-Dimensional; D006168:Guinea Pigs; D013058:Mass Spectrometry; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D006113:United Kingdom; D064166:Vaccine Potency
chemical_list: D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D010455:Peptides
keywords: anthrax vaccine; mass spectrometry; peptides; protective antigen; shelf life; stability