Qualification of FTIR spectroscopic method for protein secondary structural analysis.

Fourier transform infrared (FTIR) spectroscopy is widely used to study protein secondary structure both in solution and in the solid state. The FTIR spectroscopic method has also been employed as a characterization method by the biopharmaceutical industry to determine the higher order structure of protein therapeutics, and to determine if any changes in protein conformation have occurred as a result of changes to process, formulation, manufacture, and storage conditions. The results of these studies are often included in regulatory filings; when comparability is assessed, the comparison is often qualitative. To demonstrate that the method can be quantitative, and is suitable for these intended purposes, the precision and sensitivity of the FTIR method were evaluated. The results show that FTIR spectroscopic analysis is reproducible with suitable method precision, that is, spectral similarity of replicate measurements is greater than 90%. The method can detect secondary structural changes caused by pH and denaturant. The sensitivity of the method in detecting structural changes depends on the extent of the changes and their impact on the resulting spectral similarity and characteristic FTIR bands. The results of these assessments are described in this paper.