FTIR and nDSC as analytical tools for high-concentration protein formulations

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 (IgG(1)), 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 IgG(1) 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).