Impact of buffer concentration on the thermal stability of immunoglobulin G

초록

Purpose: This study determined the effect of buffer concentration on the unfolding and aggregation propensity of immunoglobulin G (IgG) in acidic buffers and evaluated the viscosity of highly concentrated IgG solutions. Methods: Nano differential scanning fluorimetry (nanoDSF) was used to determine the thermal stability of IgG as a function of pH and buffer concentration. Protein–protein interaction (PPI) properties were measured using dynamic light scattering (DLS). A stability study was performed at 40 °C for 8 weeks, and the monomers and multimers of IgG were determined using size-exclusion chromatography. The viscosities of the IgG formulations were measured using m-VROC viscometer. Results: In sodium acetate buffer at pH 4.6, the midpoint temperature of thermal unfolding (Tm) and the onset temperature of protein aggregation (Tagg) determined using nanoDSF tended to increase as the buffer concentration decreased from 50 to 10 mM. The PPI study using DLS suggested that the intermolecular interactions became repulsive as the buffer concentration decreased. In a stability study with IgG samples formulated in sodium acetate or sodium citrate buffers, the best stability was observed in 10 mM sodium acetate buffer. At IgG concentrations above 194 mg/mL in 10 mM sodium acetate buffer, the viscosity was 9.6 ± 0.1 cP. Conclusion: The aggregation propensity of IgG in sodium acetate buffer depended on the buffer concentration, attributed to the charge shielding effect by binding the buffer anions to the positively charged region of IgG. This study showed that IgG could be concentrated to 200 mg/mL with a viscosity of approximately 10 cP or less in 10 mM sodium acetate buffer.

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