Raman Spectroscopy to Monitor Post-Translational Modifications and Degradation in Monoclonal Antibody Therapeutics

Bethan Mcavan, Leo A Bowsher, Thomas Powell, John F O'Hara, Mariangela Spitali, Royston Goodacre, Andrew Doig

Research output: Contribution to journalArticlepeer-review


Monoclonal antibodies (mAbs) represent a rapidly expanding market for biotherapeutics. Structural changes in the mAb can lead to unwanted immunogenicity, reduced efficacy and loss of material during production. The pharmaceutical sector requires new protein characterisation tools that are fast, applicable in situ and to the manufacturing process. Raman has been highlighted as a technique to suit this application as it is information rich, minimally invasive, insensitive to water background and requires little to no sample preparation. This study investigates the applicability of Raman to detect Post-Translational Modifications (PTMs) and degradation seen in mAbs. IgG4 molecules have been incubated under a range of conditions known to result in degradation of the therapeutic including varied pH, temperature, agitation, photo and chemical stresses. Aggregation was measured using size-exclusion chromatography and PTM levels were calculated using peptide mapping. By combining principal component analysis (PCA) with Raman spectroscopy and circular dichroism (CD) spectroscopy structural analysis we were able to separate proteins based on PTMs and degradation. Furthermore, by identifying key bands that lead to the PCA separation we could correlate spectral peaks to specific PTMs. In particular, we have identified a peak which exhibits a shift in samples with higher levels of Trp oxidation. Through separation of IgG4 aggregates, by size, we have shown a linear correlation between peak wavenumbers of specific functional groups and amount of aggregate present. We therefore demonstrate the capability for Raman spectroscopy to be used as an analytical tool to measure degradation and PTMs in-line with therapeutic production.
Original languageEnglish
JournalAnalytical Chemistry
Publication statusAccepted/In press - 1 Jul 2020


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