Effects of Salts from the Hofmeister Series on the Conformational Stability, Aggregation Propensity, and Local Flexibility of an IgG1 Monoclonal Antibody

Abstract

This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAb's backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (Tm) for three observed conformational transitions within the mAb by 6-9 °C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 °C over 12 months, as monitored by size exclusion chromatography. Backbone flexibility, as measured via H/D exchange mass spectrometry, increased in two segments in the CH2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the Tm values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the VL, CH1, and CH3 domains. In contrast, 0.5 M sulfate increased Tm values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the CH1 and VL domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the CH2 domain (especially residues 241-251 in the heavy chain), the stabilizing anion sulfate did not affect these CH2 regions.

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…37,38 Formic acid (0.1% v/v) was used for digestion and desalting. A mobile phase composed of water and acetonitrile, both containing 0.1% (v/v) formic acid, was use to elute the peptides.…”

“…[34][35][36] For example, HX-MS has been recently applied to examine the effect of various solution factors and physicochemical structural changes on the local flexibility of mAbs, including salts from the Hofmeister series, 37 pharmaceutical excipients, 38 freeze-thaw cycles, 39 methionine oxidation, 40 asparagine deamidation, 41 antibody-drug conjugation, 42 deglycosylation and glycan modifications, 43 and engineered point mutations. 44 In this study, antibody clusters were characterized by a combination of DLS and chemical cross-linking experiments to determine the effect of solution conditions on the extent of RSA for an IgG1 mAb (referred to as "mAb-C") as a function of mAb-C protein concentration (1-10 mg/mL).…”

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity

“…33 Mass spectrometry coupled to HX (HX-MS), extends the HX technique to complex, multi-domain macromolecules like mAbs. [37][38][39][40][41][42][43] Recently, we described a novel HX-MS method to map protein interfaces formed between mAbs undergoing reversible protein-protein interactions directly at up to 60 g/L. 39 Here, we applied this technique to investigate the molecular mechanism by which an IgG1 mAb (mAb-J) undergoes reversible self-association, and further probed this mechanism by a variety of other biophysical techniques.…”

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

“…Although the interrelationships between dynamics and stability of proteins are complex, 32 growing evidence suggests that physical destabilization of mAbs can, in at least some cases, be mediated by changes in local dynamics of specific sequences. 33,34 Hydrogen/deuterium exchange coupled to mass spectrometry (H/D-MS) is now a well-established technique to explore the local dynamics of the amide backbone of mAbs with peptidelevel resolution. 35,36 H/D-MS has been used to study changes in mAb conformational dynamics imparted by stabilizing and destabilizing excipients 34 and salts, 33 mutation in the IgG1 C H 3 domain, 37 deglycosylation, 35 chemical and post-translational modifications, 38,39 thermal and freeze-thaw stress, 40 and cytotoxic drug conjugation 41 in IgG1 mAbs.…”