{"doi":"10.1016/j.xphs.2020.11.033","title":"Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine","abstract":"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.","journal":"Journal of Pharmaceutical Sciences","year":2020,"id":87844,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9467,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":445959,"name":"Jian Xiong","orcid":"0000-0002-8485-7428","position":1,"is_corresponding":false},{"id":446587,"name":"Nishant Sawant","orcid":null,"position":2,"is_corresponding":false},{"id":446588,"name":"Sanjeev Agarwal","orcid":null,"position":3,"is_corresponding":false},{"id":446589,"name":"John M. Hickey","orcid":null,"position":4,"is_corresponding":false},{"id":445960,"name":"David A. Holland","orcid":"0000-0003-2702-7038","position":5,"is_corresponding":false},{"id":445961,"name":"Tarit Mukhopadhyay","orcid":"0000-0003-2362-0635","position":6,"is_corresponding":false},{"id":445962,"name":"Joseph R. Brady","orcid":"0000-0002-2284-3872","position":7,"is_corresponding":false},{"id":445963,"name":"Neil C. Dalvie","orcid":"0000-0002-7912-0309","position":8,"is_corresponding":false},{"id":446590,"name":"Mary Kate Tracey","orcid":null,"position":9,"is_corresponding":false},{"id":445964,"name":"Kerry R. Love","orcid":"0000-0003-1840-3820","position":10,"is_corresponding":false},{"id":63891,"name":"John Christopher Love","orcid":"0000-0003-0921-3144","position":11,"is_corresponding":false},{"id":445965,"name":"David D. Weis","orcid":"0000-0003-3032-1211","position":12,"is_corresponding":false},{"id":446591,"name":"Sangeeta B. Joshi","orcid":null,"position":13,"is_corresponding":false},{"id":445966,"name":"David B. Volkin","orcid":"0000-0002-1448-1998","position":14,"is_corresponding":false},{"id":445958,"name":"Kawaljit Kaur","orcid":"0000-0003-2624-9755","position":0,"is_corresponding":true}],"reference_count":59,"raw_metadata":null,"created_at":"2026-07-18T22:00:29.831859Z","pmid":"33278412","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}