Understanding the discrimination and quantification of monoclonal antibodies preparations using Raman spectroscopy.

The use of Raman spectroscopy for analytical quality control of anticancer drug preparations in clinical pharmaceutical dispensing units is increasing in popularity, notably supported by commercially available, purpose designed instruments. Although not legislatively compulsory, analytical methods are frequently used post-preparation to verify the accuracy of a preparation in terms of identity and quantity of the drug in solution. However, while the rapid, cost effective and label free analysis achieved with Raman spectroscopy is appealing, it is important to understand the molecular origin of the spectral contributions collected from the solution of actives and excipients, to evaluate the strength and limitation for the technique, which can be used to identify and quantify either the prescribed commercial formulation, and/or the active drug itself, in personalised solutions. In the current study, four commercial formulations, Erbitux®, Truxima®, Ontruzant® and Avastin® of monoclonal antibodies (mAbs), corresponding respectively to cetuximab, rituximab, trastuzumab and bevacizumab have been used to highlight the key role of excipients in discrimination and quantification of the formulations. It is demonstrated that protein based anticancer drugs such as mAbs have a relatively weak Raman response, while excipients such as glycine, trehalose or histidine contribute significantly to the spectra. Multivariate analysis (partial least square regression and partial least square discriminant analysis) further demonstrates that the signatures of the mAbs themselves are not prominent in mathematical models and that those of the excipients are solely responsible for the differentiation of formulation and accurate determination of concentrations. While Raman spectroscopy can successfully validate the conformity of mAbs intravenous infusion solutions, the basis for the analysis should be considered, and special caution should be given to excipient compositions in commercial formulations to ensure reliability and reproducibility of the analysis.