Use of Raman spectroscopy and PLS for the quantification of critical quality attributes in biopharmaceutical products

The pharmaceutical industry continually seeks innovative ways to streamline process development and ensure that the products meet stringent quality standards and regulatory requirements. In this context, a cutting-edge approach that is increasingly being used is Process Analytical Technology (PAT), a systematic methodology which provides real-time insights into the production process by timely in-line, online or at-line measurements of critical quality and performance attributes of raw and in-process materials and processes. In this framework, this paper deals with the application of Raman spectroscopy, a rapid and non-destructive analytical tool, and chemometric techniques, namely Partial Least Squares (PLS), for the quantification of multiple Critical Quality Attributes (CQAs) of biopharmaceutical products. Specifically, the protein/excipient quantification, protein oxidation, protein aggregation, and protein fragmentation were evaluated. In fact, the monitoring of these product quality attributes at different process development stages and process steps allows a better understanding of the impact of the process parameters on the product quality. As an example, the quantification of protein/excipients composition is fundamental during the compounding and filtration step. Instead, the HMW (High Molecular Weight) and LMW (Low Molecular Weight) species and the oxidation level can be affected during manual or automated visual inspection step as the product is exposed to the light source as well as to the ambient temperature for some time. A monoclonal antibody (mAb) was used as model protein, with different excipients such as Methionine and Polysorbate 20. Dilutions were performed to get samples with different concentrations. Thermal stress was applied to investigate changes in protein aggregation (HMW) and protein fragmentation (LMW), while an oxidative stress was performed to obtain samples characterized by different oxidation levels. The present study shows that the combination of Raman spectroscopy and PLS turned out in excellent performances as far as it concerns the quantification of HMW, LMW and the product composition, when compared to the conventional analytical techniques. However, Raman spectroscopy was not able to discern the oxidation levels in the samples, probably due to the low sensitivity of the tool or due to the intrinsic difficulty of sample preparations for model calibration.