Ion Mobility–Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates

Abstract

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility−mass spectrometry (IM−MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1β. Our data covering both heat-and pH-based forced degradation conditions, aquired on two different IM−MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1β, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1β monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

Native Mass Spectrometry and Collision-Induced Unfolding of Laser-Ablated Proteins

“…Coupling mass spectrometry with ion mobility for empirical determination of macromolecule cross sections provides a unique opportunity to probe protein structural changes as a function of various experimental parameters and ion internal energies. , For example, dynamic interactions such as allosteric binding effects, structural changes upon drug interactions, and mutant-induced structural aberrations can be studied with ion mobility-mass spectrometry techniques. − Gas-phase relative drift times can be converted to averaged collisional cross sections (CCSs) and be used to elucidate protein interactomics and validate protein identities. − In addition, the effect of stressors on protein stability and structure, such as gas-phase collisional denaturing via collision-induced unfolding (CIU) − interrogation, can provide valuable information about dynamic changes to protein structure − beyond what can be discerned from conventional single-energy collision cross section measurements.…”

Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section

“…342 Using fingerprint maps, they were able to isolate one out of the three antigen−antibody complexes that had different binding topology and stability and proved that CIU can be used to classify antigen−antibody complexes based on their epitope maps. 340 Similar to the work cited above, 337 Yuwein et al also used CIU to study a model antibody−biotin conjugate complex and revealed some structural destabilization upon biotin conjugation. 337 Watanabe et al employed CIU to show how domain exchange of an anti-HIV IgG1 mAb is shut down by an engineered mutant.…”

“…Similar to the work cited above, Yuwein et al also used CIU to study a model antibody–biotin conjugate complex and revealed some structural destabilization upon biotin conjugation . Watanabe et al employed CIU to show how domain exchange of an anti-HIV IgG1 mAb is shut down by an engineered mutant .…”

“…340 Similar to the work cited above, 337 Yuwein et al also used CIU to study a model antibody−biotin conjugate complex and revealed some structural destabilization upon biotin conjugation. 337 Watanabe et al employed CIU to show how domain exchange of an anti-HIV IgG1 mAb is shut down by an engineered mutant. 343 Finally, CIU has been used by many groups to correlate glycosylation and disulfide bonding patterns to the stability of antibody biotherapeutics.…”

Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies

“…Arrival times may be further converted into rotationally averaged collision cross sections (CCS, Ω), which reflect the three-dimensional conformations of ions in the gas phase. Native IM-MS has been used for the study of the NIST mAb, to assess the influence of mAb (de)­glycosylation, to monitor Fab-arm exchange and bispecific antibody formation, for forced degradation studies, or to measure the drug-to-antibody ratio of ADCs. − Native IM-MS thus appears as an attractive technique for the study of biopharmaceuticals, but it is yet to gain popularity in industry …”