title: Physico-chemical and immunological examination of the thermal stability of tetanus toxoid conjugate vaccines.
abstract: The thermal stability of meningococcal C (MenC)- and Haemophilus influenzae b (Hib)-tetanus toxoid (TT) conjugate vaccines was investigated using spectroscopic and chromatographic techniques and immunogenicity assays in animal models. In this stability study, both the bulk concentrate and final fills were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to cycles of freeze-thawing. The structural stability, hydrodynamic size and molecular integrity of the treated vaccines were monitored by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopic techniques, size exclusion chromatography (FPLC-SEC), and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Only storage at 55 degrees C for 5 weeks caused some slight unfolding and modification in the tertiary structure of the carrier protein in the MenC-TT conjugate. Substantial loss of saccharide content from the MenC conjugates was observed at 37 and 55 degrees C. Unexpectedly, the experimental immunogenicity of MenC-TT vaccine adsorbed to Alhydrogel was significantly reduced only by repeated freeze-thawing, but not significantly decreased by thermal denaturation. Neither the molecular integrity nor the immunogenicity of the lyophilised Hib-TT vaccines was significantly affected by freeze-thawing or by storage at high temperature. In conclusion, the MenC- and Hib-TT conjugate vaccines were relatively stable when stored at higher temperatures, though when MenC-TT vaccine was adsorbed to Alhydrogel, it was more vulnerable to repeated freeze-thawing. When compared with CRM(197) conjugate vaccines studied previously using similar techniques, the tetanus toxoid conjugates were found to have higher relative thermal stability in that they retained immunogenicity following storage at elevated temperatures.
authors.affiliation: Bacteriology Division, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts, EN6 3QG, Potters Bar, UK. mho@nibsc.ac.uk
authors.forename: Mei M
authors.initials: MM
authors.lastname: Ho
authors.forename: Fatme
authors.initials: F
authors.lastname: Mawas
authors.forename: Barbara
authors.initials: B
authors.lastname: Bolgiano
authors.forename: Xavier
authors.initials: X
authors.lastname: Lemercinier
authors.forename: Dennis T
authors.initials: DT
authors.lastname: Crane
authors.forename: Rachel
authors.initials: R
authors.lastname: Huskisson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Corbel
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D005260:Female; D018073:Haemophilus Vaccines / Q000737:chemistry* / Q000276:immunology; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D022401:Meningococcal Vaccines / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D013745:Tetanus Toxoid / Q000737:chemistry* / Q000276:immunology
chemical_list: D018073:Haemophilus Vaccines; C055753:Haemophilus influenza type b polysaccharide vaccine-tetanus toxin conjugate; D022401:Meningococcal Vaccines; D013745:Tetanus Toxoid; C410218:serogroup C meningococcal conjugate vaccine