Cue to Acid-Induced Long-Range Conformational Changes in an Antibody Preceding Aggregation: The Structural Origins of the Subpeaks in Kratky Plots of Small-Angle X-ray Scattering.

Antibody aggregation, followed by acid denaturation and neutralization of pH, is one of the reasons why the production of therapeutic monoclonal antibodies (mAbs) is expensive. Determining the structural details of acid-denatured antibodies is important for understanding their aggregation mechanism and for antibody engineering. Recent research has shown that monoclonal antibodies of human/humanized immunoglobulin G1 (IgG1) become smaller globules at pH 2 compared to their native structure at pH 7. This acid-denatured species is unstable at pH 7 and prone to aggregation by neutralization of pH. Small-angle X-ray scattering (SAXS) data have revealed an acid-induced reduction in the subpeaks in Kratky plot, indicating conformational changes that can lead to aggregation. The subpeaks are well resolved at pH > 3 but less pronounced at pH ≤ 2. One of the weakened subpeaks indicates loosely organized inter-region (Fab-Fab and Fab-Fc) correlations due to acid denaturation. However, the structural origin of the other subpeak (called peak in this study) has not been established because its region could represent the various inter-region, inter-domain, and intra-domain correlations in IgG1. In this study, we aimed to untangle the effects of domain-domain correlations on Kratky's peak based on the computed SAXS of the crystal structure of IgG1. The peak appeared in the static structure and was more prominent in the Fc region than in the Fab or isolated domains. Further brute-force analysis indicated that longer domain-domain correlations, including the inter-region, also positively contribute to Kratky's peak. Thus, the distortion of the Fc region and a longer inter-region correlation initiate acid denaturation and aggregation.