Laser-Based Mid-Infrared Spectroscopy for Monitoring Temperature-Induced Denaturation of Bovine Serum Albumin and De-/Stabilization Effects of Sugars

Stability of high-concentration protein formulations is considered a major challenge in current biopharmaceutical development. In this work, we introduce laser-based mid-infrared (IR) spectroscopy as a versatile technique to study the effect of protein concentration and presence of sugars on the thermal denaturation of the model protein bovine serum albumin (BSA). Many analytical techniques struggle to characterize the complex structural transition that occurs during protein denaturation. To this end, a commercially available laser-based mid-IR spectrometer equipped with a customized flow cell was employed to record IR spectra of BSA in the temperature range of 25-85 degrees C. The temperature perturbation induces a conformational change from a native alpha-helical to an intermolecular beta-sheet secondary structure in BSA. Systematic investigation of the concentration dependence of the alpha-beta transition temperature between 30 and 90 mg mL(-1) shows a trend of decreasing denaturation temperatures at higher BSA concentrations. In-depth chemometric analysis by a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of the spectra, suggested the formation of not one but two intermediates in the denaturation of BSA. Subsequently, the impact of sugars on denaturation temperatures was investigated, revealing both stabilizing (trehalose, sucrose, and mannose) and destabilizing (sucralose) effects, illustrating the applicability of this method as an investigative tool for stabilizers. These results highlight the potential and versatility of laser-based IR spectroscopy for analysis of protein stability at high concentrations and varying conditions.

Automated summary

The stability of high-concentration protein formulations is a significant challenge in biopharmaceutical development. This study used laser-based mid-infrared spectroscopy to investigate the effect of protein concentration and the presence of sugars on the thermal denaturation of bovine serum albumin (BSA). The results showed that high BSA concentrations led to lower denaturation temperatures, while different sugars had both stabilizing and destabilizing effects.