Purpose: The aim of the study is to evaluate Fourier-transform infrared spectroscopy (FTIR) as an analytical tool for high-concentrated protein formulations.
Methods: FTIR is used to determine the melting temperature (T(m (FTIR))) of various proteins, such as bovine serum albumin (BSA), immunoglobulin (IgG1), beta-lactoglobulin (beta-LG), and lysozyme (HEWL), at different protein concentrations (5-100 mg/mL), where four data interpretation methods are discussed. The obtained T(m (FTIR)) values are further compared to the T(m) measured by the nanodifferential scanning calorimetry (nDSC) technique.
Results: The T(m (FTIR)) values of IgG1 and beta-LG showed strong consistency and corresponded to the nDSC results irrespective of the method of data interpretation and the protein concentration applied. In contrast, the T(m (FTIR)) of BSA and HEWL is characterized by significant deviations. Only the midpoint of the second-derivative intensity-temperature curve of the intermolecular beta-sheet mode measured at a concentration of 100 mg/mL is consistent with the nDSC results.
Conclusions: Determination of a T(m (FTIR)) is feasible by the midpoint of the intensity-temperature plot of the arising intermolecular beta-sheet band. More significant results are obtained for proteins, which are predominantly composed of intramolecular beta-sheet elements as well as at higher protein concentrations. A further study was started to assess the predictability of long-term protein stability by T(m (FTIR)).