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Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine
Name
1-s2.0-S0022354920307632-main.pdf
Description
Published version
Size
2.64 MB
Format
Adobe PDF
Checksum (MD5)
026eb8acf23882c22924f25df21ea0e1
Author(s)
Kaur, Kawaljit
Xiong, Jian
Sawant, Nishant
Agarwal, Sanjeev
Hickey, John M
Holland, David A
Mukhopadhyay, Tarit K
Brady, Joseph R
Dalvie, Neil C
Tracey, Mary Kate
Date Issued
2021
Journal
Journal of Pharmaceutical Sciences
Publisher
Elsevier BV
Version
Final published version
Abstract
© 2020 The Authors In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.
Terms of Use
Creative Commons Attribution 4.0 International license
Persistent DSpace Link
DOI of Published Version
10.1016/j.xphs.2020.11.033
