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

Abstract

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 °C. The temperature perturbation induces a conformational change from a native α-helical to an intermolecular β-sheet secondary structure in BSA. Systematic investigation of the concentration dependence of the α-β 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.

Figure 1

(a) IR absorbance spectra and (b) inverted second-derivative spectra of BSA at a concentration of 60 mg mL^–1. Panels (a) and (b) show the formation of intermolecular β-sheets and the depletion of the α-helical structure of BSA due to temperature-induced aggregation.

Figure 2

(a) Normalized second-derivative band heights at 1617 cm^–1 attributed to parallel β-sheets plotted against temperature for BSA concentrations ranging from 30 to 90 mg mL^–1. The transition temperatures of sigmoidal fits (dashed lines) are indicated by arrows. (b) Transition temperatures of individual secondary structure elements plotted for different concentrations.

Figure 3

(a) Concentration profiles of the four secondary structure components resolved through MCR-ALS versus temperature. (b) Spectral profiles of the four components in the thermal denaturation of BSA.

Figure 4

Dependence of thermal denaturation temperatures of BSA on the concentration of sugar type and concentration present in the solution.