Volume 19, Issue 9, Pages 1274-1282 (September 2011) Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry  Rebecca J. Rose, Aran F. Labrijn, Ewald T.J. van den Bremer, Stefan Loverix, Ignace Lasters, Patrick H.C. van Berkel, Jan G.J. van de Winkel, Janine Schuurman, Paul W.H.I. Parren, Albert J.R. Heck  Structure  Volume 19, Issue 9, Pages 1274-1282 (September 2011) DOI: 10.1016/j.str.2011.06.016 Copyright © 2011 Elsevier Ltd Terms and Conditions

Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Structure of Human IgG (A) Modular schematic of an IgG antibody. Heavy-chain variable (VH), first constant (CH1), second constant (CH2), and third constant (CH3) domains, and light-chain variable (VL) and constant (CL) domains are indicated. The hinge region, joining the Fc region to the Fab arms, and where disulfide bonds link the heavy chains, is indicated. (B) Model of an IgG4 CH2CH3 dimer based on crystal structure 1ADQ (Corper et al., 1997). The four antiparallel β strands that make intermolecular contacts are indicated in one CH3 domain (red β strands). Colors of CH2CH3 domains correspond to (A). (C) Schematic structure of IgG4Δhinge in equilibrium between HL and (HL)2. See also Figure S2. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Relative Distribution of HL and (HL)2 Populations Monitored by Native MS (A, B, and D–F) Mass spectra of 1μM (HL equivalent) (A) IgG4Δhinge, (B) IgG4, (D) IgG4-CAM, (E) IgG4-R409KΔhinge, and (F) IgG4-F405QΔhinge. Lower m/z peaks relate to HL molecules (left), and higher m/z peaks relate to (HL)2 molecules (right). (C) Mass spectra were determined at different IgG4Δhinge concentrations; the proportion of IgG4Δhinge populating the HL state is plotted as a function of overall protein concentration (HL equivalents, see also Figure S1). Combined data points from five separate experiments are shown. All spectra were obtained in 100 mM NH4Ac (pH 7). Cartoons depicting HL and (HL)2 species are shown. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Relative KD Values of IgG4ΔHinge Variants Nine amino acids at the IgG4Δhinge CH3-CH3 interface were mutated individually as indicated. Residues subjected to mutation are shown in red stick representation on the IgG4 CH3 domain based on crystal structure 1ADQ (Corper et al., 1997). Relative KD values, obtained by dividing the apparent KD for each IgG4Δhinge variant, as derived from the native MS analysis (Table S1), by the apparent KD of wild-type IgG4Δhinge, are plotted as bars. Error bars relate to standard error from KD fit. See also Figure S3. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Confirmation of Native MS Data by Other Techniques (A) Native PAGE analysis of IgG4Δhinge, IgG4-R409KΔhinge, and IgG4-F405QΔhinge. Molecular sizes of (HL)2 and HL are indicated. (B) Size-exclusion chromatography retention times (of main peaks) and (C) sedimentation coefficients determined by SV-AUC were plotted against MS-derived apparent KD values for 20 representative IgG4Δhinge variants (Table S1). Wild-type IgG4Δhinge is shown as solid black circles. See also Table S2. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Kinetics of HL Exchange in IgG4ΔHinge Enzymatically deglycosylated IgG4Δhinge (black circles), IgG4-L368AΔhinge (white circles), and IgG4-R409KΔhinge (gray triangles) were mixed with equimolar amounts of their nontreated (glycosylated) equivalents (2 μM total antibody). The generation of mixed (HL)2 molecules (1 glycosylated HL, 1 deglycosylated HL) from glycosylated (HL)2 and deglycosylated (HL)2 was plotted as a function of time. Cartoons representing the different (HL)2 molecules are indicated. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 Dissociation of Half Molecules in Full-Length IgG4 Antibodies Proportion of (A) IgG4Δhinge, (B) IgG4 in presence of reductant, and (C) IgG4 in absence of reductant populating the HL state as a function of overall concentration for wild-type (black circles) and F405L (gray circles) constructs. In (A), wild-type IgG4Δhinge in the presence of reductant is additionally shown as gray crosses. See also Figure S4. Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 7 Model for FAE Reaction Two different IgG4 molecules are depicted. Four reaction steps are identified: (1) reduction of hinge disulfide bridges, (2) dissociation into HL molecules, (3) reassociation into (HL)2 molecules, and (4) oxidation of hinge disulfide bridges. Because the kinetics of reduction or oxidation for interchain disulfides and intrachain disulfides may differ, these are indicated separately (A and B, respectively). Structure 2011 19, 1274-1282DOI: (10.1016/j.str.2011.06.016) Copyright © 2011 Elsevier Ltd Terms and Conditions