Comparative analysis of pH shift-induced aggregation in IgG subclasses: Unveiling distinct pathways, mechanisms, and kinetics

Abstract

Therapeutic proteins are inherently challenging to stabilize due to their structure, physicochemical properties, and pronounced sensitivity to environmental factors such as pH and ionic strength. This research focuses on understanding the aggregation mechanisms and kinetics of three IgG subclasses during acidic pH exposure and subsequent neutralization, mimicking pH shift encountered in downstream processing conditions. Using orthogonal analytical methods, including SEC, AUC, and DLS, we tracked the formation of initial dimers and their progression to high-molecular-weight species (HMWs). Conformational changes leading to aggregation were analyzed with DSC, CD, ATR-FTIR and fluorescence spectroscopy. The Lumry-Eyring model, which accounts for the reversibility of each step in the aggregation process, was employed. The kinetic rate constants of each step were optimized using MATLAB. This model enabled detailed mapping of transitions from monomers to intermediates, and further to dimers or higher oligomers. We have analyzed the product formation during neutralization of acid-stressed samples and observed distinct behavior among the IgG subclasses. IgG1 dimers, not only dissociated into monomers but also formed trimers or HMWs, whereas IgG2 and IgG4 predominantly formed tetramers or HMWs with limited reversion to monomers. Such fundamental investigations are crucial for optimizing monoclonal antibody development, ensuring therapeutic efficacy, and mitigating safety risks associated with aggregation.