Understanding the Role of Polyols and Sugars in Reducing Aggregation in IgG2 and IgG4 Monoclonal Antibodies During Low-pH Viral Inactivation Step

<b>Background/Objectives</b>: 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 (IgG₄) and denosumab (IgG₂) in the context of low-pH viral inactivation (VI) operations. <b>Methods</b>: 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% <i>w</i>/<i>v</i>) 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_D) were investigated. Additionally, the impact of excipients on the dynamic binding capacity of the cation-exchange (CIEX) column was evaluated. <b>Results</b>: 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_D 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. <b>Conclusions</b>: This study offers valuable insights and supports the rational use of polyols and sugars for stabilizing mAbs during the low-pH VI step.