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

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 degrees C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (T-m) for three observed conformational transitions within the mAb by 6-9 degrees C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 degrees 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 C(H)2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the T-m values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the V-L, C(H)1, and C(H)3 domains. In contrast, 0.5 M sulfate increased T-m values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the C(H)1 and V-L domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the C(H)2 domain (especially residues 241-251 in the heavy chain), the stabilizing anion sulfate did not affect these C(H)2 regions.