Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions

Abstract

Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2-8 and 0-300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions.

Computational investigation of protein surface polarity and pH-dependence: application to antibodies shows that antibody-antigen interfaces tend to be relatively polar

“…Note that these are extreme values and, as we show in a subsequent section, antibody-antigen interfaces are relatively polar. and CH3 domains (Yageta et al, 2015). Measured phase diagram boundaries for these domains, derived at acidic pH (Yageta et al, 2015) has been marked (Figure 3), showing a good match between these calculations and experiment.…”

“…and CH3 domains (Yageta et al, 2015). Measured phase diagram boundaries for these domains, derived at acidic pH (Yageta et al, 2015) has been marked (Figure 3), showing a good match between these calculations and experiment. For example, looking across the pH variation at 0.15 M ionic strength, there is a greater variation in pH-dependence for CH2 than for CH3 domains.…”

“…In order to compare qualitatively with experiment, CH2 and CH3 domains from the IgG1 PDB structure 1HZH are used (Saphire et al, 2001), since pH and ionic strength variations in stability have been reported for IgG1 CH2 and CH3 domains (Yageta et al, 2015). Measured phase diagram boundaries for these domains, derived at acidic pH (Yageta et al, 2015) has been marked (Figure 3), showing a good match between these calculations and experiment. For example, looking across the pH range at 0.15 M ionic strength, there is a greater variation in pH-dependence for CH2 than for CH3…”

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

“…17 Furthermore, Seiki Yageta et al reported C H 3 domain plays the most critical role in driving intact IgG aggregation under acidic conditions. 18 Here, we report nivolumab was prone to aggregation during a purification process conventionally used for mAbs. Aggregate and monomer fractions of nivolumab bulk substance were characterized in-depth by liquid chromatography-mass spectrometry (LC-MS).…”

Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

“…Subsequent work from the same group suggests that before acid treatment, the CH2 exhibits stronger interactions with the CH3 domain, but upon renaturation, the CH2 instead forms stronger interactions with the CH1 domain (Martsev et al, 1995). Lastly, experimental determination of the conformational stability of isolated IgG constant domains using circular dichroism suggests that CH2 and and CH3 domains are less stable than the CH1-CL dimer at pH 2 (Yageta et al, 2015). Our work suggests that of the Fc domains, both structures exhibit a relative sensitivity to acid pH, but as CH2 exhibits a more destabilising energy in the acidic regime we suggest CH2 will be more acid sensitive.…”