Spotlight on Formulation & Delivery
Researchers assess the impact of buffer pH on antibody structure to identify conditions that maintain therapeutic stability and minimize conformational degradation. Using HRPF, this study revealed that Adalimumab undergoes significant unfolding at low pH and early structural changes in the Fc region at high pH. All critical insights for optimizing formulation conditions that preserve efficacy, reduce aggregation risk, and ensure safe delivery.
Figure Legend: Adalimumab Volcano Plot comparing the Average Peptide Oxidation (APO) at 1000V for pH 3, 5, or 11 against pH 7. The dotted line represents a p-value threshold of 0.01. Peptides with a p-value < 0.01 and a fold change > 50% are labeled. A positive fold change indicates increased exposure compared to pH 7, while a negative fold change indicates increased protection.
Figure Legend: Structure of Adalimumab Heavy Chain, modeled using AlphaFold, highlighting regions affected by changes in pH. The structures compare the changes in solvent accessibility of the protein at pH 3, pH 5, and pH 11 against a reference condition at pH 7. Regions detected with oxidation under all conditions are colored in green. Regions that become more exposed are highlighted in red, indicating unfolding or increased flexibility, while regions that become more protected are highlightd inblue, suggesting a change in structure or potential interaction site. (A) pH 3 vs. pH 7: Several peptides (12) in both the Fc and Fab regions become more exposed (red), suggesting significant unfolding of the protein structure at the lower pH. (B) pH 5 vs. pH 7: Minimal changes in solvent accessibility are observed, except for a single peptide (260-278) becoming more exposed. This indicates that Adalimumab remains stable under slight pH variations around neutral pH. (C) pH 11 vs. pH 7: One peptide becomes more exposed, and two peptides become more protected, all within the Fc region, indicating initial signs of instability and unfolding starting in this region as pH becomes more basic.
