title: 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 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.
authors: a Department of Pharmaceutical Chemistry; Macromolecule and Vaccine Stabilization Center; University of Kansas ; Lawrence , KS , USA.
authors: Jayant
authors: J
authors: Arora
authors: John M
authors: JM
authors: Hickey
authors: Ranajoy
authors: R
authors: Majumdar
authors: Reza
authors: R
authors: Esfandiary
authors: Steven M
authors: SM
authors: Bishop
authors: Hardeep S
authors: HS
authors: Samra
authors: C Russell
authors: CR
authors: Middaugh
authors: David D
authors: DD
authors: Weis
authors: David B
authors: DB
authors: Volkin
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology; D041961:Deuterium Exchange Measurement; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D013058:Mass Spectrometry; D051379:Mice; D056004:Molecular Dynamics Simulation
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G
keywords: ADCs, antibody-drug conjugates; BS2G, bis (sulfosuccinimidyl) 2,2,4,4 glutarate; BsAbs, bispecific antibodies; CD, circular dichroism; CDR, complementarity-determining regions; CH1-CH3, constant domains 1–3 respectively of the heavy chain; DLS, dynamic light scattering; Fab, antigen binding fragment; Fc, crystallizable fragment; HC, heavy chain; HPLC, high-performance liquid chromatography; HX-MS, hydrogen exchange mass spectrometry; IgG1, immunoglobulin G1; LC, light chain; RSA, reversible self-association; SC, subcutaneous; SEC, size-exclusion chromatography; VH/VL, variable domain of the heavy/light chain; aggregation; flexibility; high protein concentration; hydrogen exchange; immunoglobulin G1; mAb, monoclonal antibody; mass spectrometry; monoclonal antibody; protein-protein interactions; reversible self-association; stability