Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in
therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide
I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be
seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to
quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance,
and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region.
In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region
to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation
coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein
secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between
changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions,
which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure.
These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and
demonstrate comparability of protein secondary structure during downstream process and formulation development of
Keywords: Amide I, Amide II, chemometrics, formulation development, FTIR, higher order structure, therapeutic proteins,
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