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

Area of Science:

• Biophysical Chemistry
• Protein Analytics
• Spectroscopy

Background:

• High-concentration protein formulations are crucial in biopharmaceuticals.
• Accurate characterization of protein stability is essential for drug development.
• Traditional methods may face limitations with concentrated protein solutions.

Purpose of the Study:

• To evaluate Fourier-transform infrared spectroscopy (FTIR) as an analytical tool for high-concentration protein formulations.
• To determine the melting temperature (Tm) of various proteins using FTIR.
• To compare FTIR-derived Tm values with those obtained from nanodifferential scanning calorimetry (nDSC).

Main Methods:

• Proteins (BSA, IgG1, beta-LG, HEWL) were analyzed at concentrations ranging from 5-100 mg/mL.
• Four different data interpretation methods were applied to FTIR spectra.
• Melting temperatures (Tm (FTIR)) were determined and compared to Tm values measured by nDSC.

Main Results:

• FTIR accurately determined Tm for IgG1 and beta-LG, consistent with nDSC across concentrations and interpretation methods.
• Significant deviations were observed for BSA and HEWL.
• Only the second-derivative midpoint of the intermolecular beta-sheet mode at 100 mg/mL aligned with nDSC for BSA and HEWL.

Conclusions:

• Melting temperature determination using FTIR is feasible, particularly using the midpoint of the intensity-temperature plot of the intermolecular beta-sheet band.
• Higher protein concentrations and proteins rich in intramolecular beta-sheet structures yield more reliable FTIR results.
• Further research is warranted to assess FTIR's predictive capability for long-term protein stability.