title: Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid.
abstract: The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.
authors.affiliation: Canvax, 3561 Gallager Dr., Mississauga, ON, L5C 2N2, Canada. craiglaferriere@gmail.com
authors.forename: Craig
authors.initials: C
authors.lastname: Laferriere
authors.forename: Neil
authors.initials: N
authors.lastname: Ravenscroft
authors.forename: Seanette
authors.initials: S
authors.lastname: Wilson
authors.forename: Jill
authors.initials: J
authors.lastname: Combrink
authors.forename: Lizelle
authors.initials: L
authors.lastname: Gordon
authors.forename: Jean
authors.initials: J
authors.lastname: Petre
mesh_terms: D000465:Algorithms; D000588:Amines / Q000737:chemistry / Q000276:immunology; D016667:Bacterial Capsules / Q000276:immunology; D003906:Developing Countries; D006192:Haemophilus Infections / Q000276:immunology / Q000517:prevention & control*; D018073:Haemophilus Vaccines / Q000138:chemical synthesis* / Q000276:immunology; D019741:Haemophilus influenzae type b / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D006834:Hydrazines / Q000737:chemistry* / Q000276:immunology; D018796:Immunoconjugates / Q000737:chemistry; D008239:Lysine / Q000737:chemistry / Q000276:immunology; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000276:immunology; D012107:Research Design; D013745:Tetanus Toxoid / Q000737:chemistry* / Q000276:immunology; D014611:Vaccination; D018074:Vaccines, Conjugate / Q000737:chemistry* / Q000276:immunology
chemical_list: D000588:Amines; D018073:Haemophilus Vaccines; C044237:Haemophilus influenzae type b polysaccharide vaccine; D006834:Hydrazines; D018796:Immunoconjugates; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D013745:Tetanus Toxoid; D018074:Vaccines, Conjugate; C029424:hydrazine; C008984:polyribitol phosphate; D008239:Lysine