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Exploring the potential of molecular spectroscopy for the detection of post-translational modifications of a stressed biopharmaceutical protein

Author(s):

Marco Pinto Corujo, Vivian Lindo, Nikola Chmel and Alison Rodger*  

Abstract:


Background: Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex structures (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as post-translational modifications (PTMs). PTMs may affect the protein’s functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins.

Materials & Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs.

Conclusion: This work required some novel computational approaches to pre–process Raman and IR spectra and a review of the band assignments for proteins existing in the literature.

Keywords:

Post–Translational Modifications, Biopharmaceuticals, Protein secondary structure, Spectroscopy, Raman, Circular Dichroism, Mass spectrometry, Infrared absorbance

Affiliation:

Department of Chemistry, University of Warwick, Coventry, Astrazeneca, Cambridge, Department of Chemistry, University of Warwick, Coventry, Department of Molecular Sciences, Macquarie University, North Ryde, 2109, New South Wales



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