Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry
✍ Scribed by Tia Estey; Christina Vessely; Theodore W. Randolph; Ian Henderson; LaToya Jones Braun; Rajiv Nayar; John F. Carpenter
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 462 KB
- Volume
- 98
- Category
- Article
- ISSN
- 0022-3549
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✦ Synopsis
Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short-and long-term stability of the protein remain poorly defined. In our companion paper (Vessely et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(H c ), rBoNTB(H c ), and rBoNTE(H c ). In the current study, we evaluated the effect of binding to adjuvant (Alhydrogel TM , aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(H c ) as well as a trivalent rBoNT(H c ) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(H c ) protein antigens. We found that all three of the rBoNT(H c ) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls.
Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens. ß 2008