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Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance
Hageman, Tyler Wei, Hui Kuehne, Patrick Fu, Jinmei Ludwig, Richard Tao, Li Leone, Anthony Zocher, Marcel Das, Tapan K.
Hageman, Tyler
Wei, Hui
Kuehne, Patrick
Fu, Jinmei
Ludwig, Richard
Tao, Li
Leone, Anthony
Zocher, Marcel
Das, Tapan K.
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Abstract
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.
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This work is licensed under a Creative Commons Attribution 4.0 International License.
Date
2018-12-10
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Springer
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Hageman_2018.pdf
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Keywords
Tryptophan oxidation, Complementarity-determining region, Monoclonal antibody, Hydrogen-deuterium exchange mass spectrometry, Surface plasmon resonance
Citation
Hageman, T., Wei, H., Kuehne, P., Fu, J., Ludwig, R., Tao, L., Leone, A., Zocher, M., & Das, T. K. (2018). Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance. Pharmaceutical research, 36(1), 24. https://doi.org/10.1007/s11095-018-2545-8