Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions

Antibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties at low pH under high concentration conditions is important.

Raman spectroscopy was used to investigate pH-induced conformational changes of antibodies at 50 mg/ml. Experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab.

We detected the evident changes at pH 3 in Tyr and Trp bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the thermal transition temperature (T_m) was highest at pH 3. Acid-treated and neutralized one showed higher T_m than that of pH 7, indicating that their extent of intermolecular interactions correlated with the T_m values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal analyses confirmed the findings of the Raman analysis.

Our studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method for evaluating antibody conformation associated with aggregation propensities.