Raman spectroscopy for in situ, real time monitoring of protein aggregation in lyophilized biotherapeutic products.

Quality of biotherapeutic products is of paramount importance for ensuring patient safety. Analytical tools that can facilitate rapid quality assessment of the therapeutic product at the point of care are very much in demand. In this article, we apply chemometrics based analysis of Raman spectra towards quantitative prediction of protein aggregation in lyophilized biotherapeutic products. Two commercially available therapeutic proteins, erythropoietin (EPO) and human growth hormone (HGH), have been used to demonstrate the applicability of the proposed approach. Thermally induced protein aggregation was monitored by size exclusion chromatography as well as Raman spectroscopy with a 785 nm wavelength laser. Partial least square (PLS) regression was used to analyse the Raman spectra and create a model for quantitative determination of aggregate. Satisfactory performance was observed with both EPO and HGH with R of 0.91 and 0.94, cross-validation correlation coefficient of 0.85 and 0.89, and Root Mean Square Error computed from cross calibration (RMSEcv) of 5.25 and 1.92, respectively. The developed approach can enable rapid and accurate assessment of aggregation in lyophilized samples of biotherapeutic products. The study also demonstrates novel use of Raman spectroscopy for protein quantification through a vial.