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TL;DR
Acid treatment at pH 3 weakened intermolecular interactions, increased thermal transition temperature, and induced partially irreversible conformational changes, with implications for antibody formulation and aggregation resistance.
Pharmaceutical Research · 4 authors, 5 centres
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Acid treatment at pH 3 weakened intermolecular interactions, increased thermal transition temperature, and induced partially irreversible conformational changes, with implications for antibody formulation and aggregation resistance.
Raman spectroscopy was employed to investigate pH-induced conformational changes in human serum IgG and recombinant rituximab at a high concentration of 50 mg/ml across pH 3 to 7, complemented by thermal transition analysis, dynamic light scattering (DLS), particle tracking analysis (PTA), and surface plasmon resonance imaging (SPRi). At pH 3, Tyr band intensity ratios (I850/I830) and Trp band widths at 1555 cm⁻¹ indicated weakened intermolecular interactions, and the thermal transition temperature (Tm) was highest at pH 3. Acid-treated samples neutralized back to pH 7 retained higher Tm values than pH 7 controls, suggesting partially irreversible conformational changes, which were more pronounced for hIgG than rituximab. DLS and PTA confirmed reduced aggregate formation at pH 3, consistent with suppressed intermolecular interactions attributed to charge-charge repulsions. SPRi showed decreased antigen-binding activity in acid-treated rituximab.