Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry

Abstract

Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.

Brain Bioavailability of Human Intravenous Immunoglobulin and its Transport through the Murine Blood–Brain Barrier

“…The interaction between monoclonal antibodies and antigens display high specificity and affinity, with equilibrium dissociation ranging from 10 À 11 to 10 À 4 mol/L. 26,27 Therefore, if we assume similar affinity of IVIg for CNS targets, the brain concentrations measured here after systemic injections are therapeutically relevant.…”

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

“…Electrospray ionization mass spectrometry (ESI-MS) represents a generic method to discriminate between nonexchanged parental mAbs and the resulting bsAb product with intermediate mass (20,24). Therefore, ESI-MS was used to visualize the protein species in the samples used to generate the ELISA data in Fig.…”

“…Because breaking the covalent linkage between half-molecules is a prerequisite for FAE, residue S228 (opposed to P228) is thought to act by making the interheavy chain disulfide bonds more susceptible to reducing conditions (22). Dissociation of the noncovalently associated CH3 domains has been shown to be the rate-limiting step in the FAE reaction (23) and heavily dependent on the composition of the CH3-CH3 interface residues (24). Indeed, residue R409 decreased the CH3-CH3 interaction strength (compared with K409 in IgG1), thus enabling human IgG4 to engage in FAE (21).…”

Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3–CH3 Interaction Strength

“…Apparent dissociation constant (K D ) values were determined by nanoelectrospray ionization mass spectrometry, as described elsewhere (17). In brief, purified proteins (at 50 mM stock concentrations) were buffer exchanged into 100 mM NH 4 Ac (pH 6.9), serial diluted to concentrations ranging from 25 nM to 20 mM (monomer equivalent), and analyzed by nano-electrospray ionization mass spectrometry.…”