STABILIZATION OF TETANUS AND DIPHTHERIA TOXOIDS AGAINST MOISTURE-INDUCED AGGREGATION

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release po lymers, such as poly(lactide-co-glycolide). For example, herein we rep ort that lyophilized tetanus toroid aggregates during incubation at 37 degrees C and elevated humidity-i.e., conditions relevant to its rele ase from such systems, The mechanism and extent of this aggregation ar e dependent on the moisture level in the solid protein, with maximum a ggregation observed at intermediate moisture contents, The main aggreg ation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon fo rmalinization (exposure to formaldehyde during vaccine preparation) re act with nucleophiles of a second vaccine molecule to form intermolecu lar cross-links, This process is inhibited by the following: (i) succi nylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbit ol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toroid, is also retarded by succinylation, suggesting the g enerality of this mechanism for formalinized vaccines, Hence, mechanis tic stability studies of the type described herein may be important fo r the development of effective single-dose vaccines.