Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody

Abstract

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-C2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability

“…Future directions should therefore focus on the characterization of mAb dimers, which can be very different in terms of both intermolecular linkages (i.e., covalent vs. noncovalent) and IgG domains involved in the connections. [57][58][59][60] In this context, it is important to combine modeling activities with advances in analytical methods. The characterization of dimers and other aggregate end products involves a variety of orthogonal techniques [61][62][63] and can benefit from emerging approaches based on microfluidic technology.…”

Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis

“… 43 − 45 Recent HDX-MS studies of IgG2, for example, showed significant structural changes in two regions of Fc-CH 2 when proteins were thermally stressed. 46 In that study, it was observed that dimeric species obtained under conditions that would most likely promote the formation of LT dimers were associated with weak non-covalent bonds, whereas species corresponding to HT dimers exhibited rearranged disulfide bonds. These results are in general agreement with our observations ,suggesting that HT aggregation is accompanied by partial mAb unfolding.…”

Next generation Fc scaffold for multispecific antibodies

“…HDX-MS experiments were performed to pinpoint differences in protein structure between CPMv1 and CPMv103, as previously described ( Zhang et al., 2010 , 2018 ). The H/D exchange reaction was initiated by diluting protein samples with 10 mM acetate in D2O (pD 5.2) as indicated for a predetermined time at 37°C.…”