Effects of buffer type and pH on biophysical properties and stability of IgG4 monoclonal antibodies: Uncovering dynamic shifts in stability predictors.

Rapid development of biopharmaceuticals requires reliable predictive tools to assess the stability of therapeutic proteins. This study presents a comprehensive biophysical characterization of an IgG4 monoclonal antibody formulated in three buffers, sodium acetate, sodium citrate, and histidine, across a pH range of 5.0-6.0, aiming to identify effective stability predictors. We employed the following orthogonal analytical techniques: dynamic light scattering (DLS) for colloidal stability, Fourier-transform infrared spectroscopy (FTIR) for secondary structure, and nano differential scanning fluorimetry (nanoDSF) for conformational stability. These biophysical parameters were correlated with the stability outcomes under accelerated storage (25 °C, 8 weeks) and high-temperature stress (50 °C, 24 h), which were quantified using size exclusion chromatography (SEC). The results indicate that both the zeta potential and diffusion interaction parameter (kD) can serve as predictors of stability under accelerated storage conditions. A key finding was that the predictive power of zeta potential and kD diminished under high-temperature stress, whereas the correlation between the thermal unfolding temperature (Tm) and SEC-derived stability (monomer content) increased (r = 0.74). Our findings validated this dynamic predictor model for IgG4 and provided key mechanistic insights into its modulation. We demonstrated that the transition between predictive indicators is governed by buffer-specific effects, such as citrate-induced charge reversal in IgG4, a phenomenon distinct from other subclasses. This understanding establishes a rational framework for matching formulation strategies to the specific storage and processing requirements.