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Pharm Res ; 36(1): 24, 2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30536043

RESUMO

PURPOSE: Tryptophan's (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. METHODS: Selective Trp oxidation was induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. RESULTS: Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. CONCLUSIONS: Trp oxidation in CDRs can adversely impact mAbs' conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.


Assuntos
Anticorpos Monoclonais/química , Regiões Determinantes de Complementaridade/química , Deutério/química , Hidrogênio/química , Triptofano/química , Antígenos/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Oxirredução , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície/métodos
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