Effect of pH on stability of recombinant botulinum serotype A vaccine in aqueous solution and during storage of freeze-dried formulations.

The purpose of this study was to evaluate the importance of prelyophilization solution pH on the stability of botulinum neurotoxin, serotype A (rBoNTA(H(c))). This understanding is of significant importance for proteins such as rBoNTA(H(c)), a potential constituent of a multivalent vaccine product. For multivalent vaccines it may be difficult to identify a liquid formulation satisfying the stability requirements for all constituent protein antigens. Consequently, a lyophilized multivalent vaccine formulation may be a more viable alternative. Therefore evaluating the effect of prelyophilization pH (may be suboptimal) on the stability of antigens such as rBoNTA(H(c)) during lyophilization/storage becomes important. We hypothesize that when rBoNTA(H(c)) is lyophilized from a suboptimal pH, using the appropriate stabilizers can provide adequate physicochemical stability during lyophilization and long-term storage. We identified pH 5 and 8 in which the protein was stable and unstable against aggregation. Excipients were identified that could stabilize rBoNTA(H(c)) during lyophilization and storage in a stable solution of pH 5. These excipients were 7.5% (w/v) trehalose and 2.5% (w/v) trehalose with 2.5% (w/v) HES, with and without 0.01% (w/v) polysorbate 20. In support of our hypothesis, these excipients were found to provide adequate physicochemical stability to rBoNTA(H(c)) during lyophilization/storage, when freeze-dried from a prelyophilized solution of pH 8.