Purpose: Protein therapeutics are large complex molecules that are easily susceptible to structural perturbations due to physical stress. Several studies reported that structural perturbations to protein molecules can cause alteration to physiologically active conformations and the individual molecules can oligomerize and aggregate. Emerging scientific opinion is gravitating towards the idea that aggregation in protein drugs acts as a trigger for the generation of antidrug antibodies, which in turn decreases efficacy of a drug over long term administration in patients. Although proteins are inherently susceptible to structural perturbations, at what level such structural deviations from active-state conformations are going to be significant for occurrence of dimers and oligomers leading to aggregation needs to be critically assessed. Novel methods of analysis that delineate minor structural perturbations are therefore important to understand how small changes affect stability of proteins. We hypothesize that subtle changes in physicochemical properties of the proteins are reflected in intra-molecular vibrations and structure collectively, measurable by Fourier Transform Infrared (FTIR) spectroscopy or circular dichroism (CD) spectroscopy and multivariate statistical analysis.
Methods: We have analyzed eight diverse proteins with a wide range of isoelectric points by attenuated total reflectance (ATR)-FTIR (using Tensor 27, Bruker) and CD spectroscopy (using Jasco J815, Jasco) to find out how they can be differentiated by spectral characteristics. We have also analyzed changes caused by temperature (15°C- 50°C) at less than denaturing or unfolding temperature points in specific proteins by both ATR-FTIR and CD spectroscopy. The spectral data obtained from both spectroscopic techniques have been analyzed by ANOVA and multivariate analysis (principal component analysis, PCA). Furthermore, occurrence of dimers and oligomers in the proteins under the experimental temperature conditions were analyzed by dynamic laser light scatter (DLS) using the DynaPro II (Wyatt) particle analyzer.
Results: The ATR-FTIR spectral data analyzed by ANOVA and comparison of means did not separate all studied proteins although the FTIR absorbance spectra appeared to have variation, whereas multivariate analysis of the same spectral data separated the variations. To differentiate the effect of temperature we analyzed lysozyme (LYS). PCA was applied to the ATR-FTIR data and the principal component (PC) score values were compared by Student’s t-test (Fig. 1a and 1b). CD spectroscopy followed by traditional analysis of determining secondary structural components did not help to distinguish the temperature effects at many different temperature points in a range of 15-50 °C. However, by PCA of CD spectral data and comparison of PC scores by Student’s t-test it was possible to separate out the varying effects of different temperature points on LYS (Fig. 1c and 1d). Analysis by DLS showed varying polydispersity at different temperature points that corroborated that the proteins indeed suffered structural perturbation which led to deviations from native states.
Conclusion: Multivariate analysis of ATR-FTIR and CD data can be applied to the study of minor structural perturbations of protein drugs that may occur at much less than the denaturing or aggregating temperature points. This analytical approach can be used in a control strategy for maintaining protein drug quality throughout the product life cycle and may also be useful in release and stability testing and in biosimilarity assessments.
Disclaimer: The conclusions reflect the views of the authors and should not be construed to represent FDA’s views or policies.