Nanoscopy

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps towards not only the detection of glycosylation status, but the characterisation of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). Whilst this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms, therefore with stricter orientation control, we believe this has the potential to lead to further glycan characterisation using TERS, which would have use in biopharmaceutical synthesis and formulation research.