Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody
Autor: | C. Russell Middaugh, David D. Weis, Jayant Arora, David B. Volkin, Ranajoy Majumdar, Steven M. Bishop, Hardeep S. Samra, John M. Hickey, Reza Esfandiary |
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Rok vydání: | 2015 |
Předmět: |
Work (thermodynamics)
medicine.drug_class Immunology Analytical chemistry protein-protein interactions Molecular Dynamics Simulation Mass spectrometry Monoclonal antibody Mass Spectrometry Protein–protein interaction Viscosity Antibodies Monoclonal Murine-Derived Mice immunoglobulin G1 Dynamic light scattering medicine Immunology and Allergy Animals reversible self-association Hydrogen exchange Chemistry aggregation Deuterium Exchange Measurement high protein concentration stability hydrogen exchange Complementarity Determining Regions flexibility Deuterium monoclonal antibody Immunoglobulin G Biophysics Reports |
Zdroj: | mAbs |
ISSN: | 1942-0870 |
Popis: | There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VH domain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA. |
Databáze: | OpenAIRE |
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