Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Abstract

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 V_H domain, the constant domain (C_H2), and the hinge region (C_H1-C_H2 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.

Keywords:

• reversible self-association
• protein-protein interactions
• monoclonal antibody
• immunoglobulin G1
• hydrogen exchange
• mass spectrometry
• flexibility
• stability
• aggregation
• high protein concentration

Disclosure of Potential Conflicts of Interest

MedImmune authors are employees of MedImmune. JA and RM are graduate students, JMH is senior research associate, and CRM, DDW and DBV are professors at The University of Kansas. DDW is a consultant for Agilent Technologies.

Acknowledgments

We would like to thank Dr. Hasige A. Sathish from MedImmune for his critical review of this manuscript.

Funding

This study was financially supported by MedImmune and the Kansas Biosciences Authority.

Supplemental data

Supplemental data for this article can be accessed on the publisher's website.