Understanding the Role of Polyols and Sugars in Reducing Aggregation in IgG and IgG Monoclonal Antibodies During Low-pH Viral Inactivation Step.

: Low-pH treatment of monoclonal antibodies (mAbs) is commonly employed to inactivate enveloped viruses following Protein A capture chromatography. However, this treatment can sometimes induce conformational changes and aggregation in pH-sensitive proteins. This study investigates the stabilizing effects of four compendial excipients-mannitol, sorbitol, trehalose, and sucrose-on aggregation, as well as on the conformational and colloidal stability of pembrolizumab (IgG) and denosumab (IgG) in the context of low-pH viral inactivation (VI) operations. : Following Protein A chromatography, pembrolizumab and denosumab were subjected to low-pH incubation and neutralization to mimic the conditions encountered in the VI step. Excipients (10% /) were added to the eluate, and aggregation was assessed post-neutralization. Effects on conformational stability (measured by thermal unfolding) and colloidal stability (via diffusion interaction parameter K) were investigated. Additionally, the impact of excipients on the dynamic binding capacity of the cation-exchange (CIEX) column was evaluated. : All tested excipients significantly reduced aggregation of both mAbs during low-pH incubation. For instance, mannitol reduced aggregation by sixfold in pembrolizumab and by threefold in denosumab. These additives enhanced conformational stability, as evidenced by the increased melting temperatures (Tm), with a pronounced stabilizing effect on the CH2 domain at low pH. Furthermore, the presence of sugars and polyols was associated with higher K values, indicating improved colloidal stability under acidic conditions. Importantly, adding these sugars or polyols did not impact the dynamic binding capacity of the CIEX column used in subsequent processing. : This study offers valuable insights and supports the rational use of polyols and sugars for stabilizing mAbs during the low-pH VI step.