Quantification of clinical mAb solutions using Raman spectroscopy: Macroscopic vs microscopic analysis.

Raman Spectroscopy is well emerged in the field of Analytical Quality Control (AQC) as a rapid and cost-effective technique useful in many applications. The advantage of Raman spectroscopy is the non-invasiveness of measurements that enablesto analyse samples directly in its container. In this study, the potential of Raman spectroscopy was investigated for analysis of clinical preparations of mAbs. Three commercial formulations of monoclonal antibodies (mAbs) Avastin®, Ontruzant® and Tecentriq® corresponding to Bevacizumab (BVC), Trastuzumab (TRS) and Atezolizumab (ATZ) respectively, were analysed in quartz cuvette in macroscopic analysis and through the wall of perfusion bags in microscopic analysis. The spectra have been compared to those of excipients (trehalose and sucrose) and of γ-Globulin, in order to investigate the origin of Raman bands. As expected, Raman spectra were a combination of bands from monoclonal antibodies and correspoding excipients found in formulas. For quantitative analysis of the solutions, models have been constructed using Partial Least Square Regression (PLSR) with Leave K-Out Cross Validation (LKOCV). The quantification performance was comparable for both macroscopic and microscopic analysis, in terms of error and linearity. The results are thus promising for future AQC in situ, in perfusion bags.

In situ Analytical Quality Control of chemotherapeutic solutions in infusion bags by Raman spectroscopy.

Analytical Quality Control (AQC) in centralised preparation units of oncology centers is a common procedure relying on the identification and quantification of the prepared chemotherapeutic solutions for safe intravenous administration to patients. Although the use of Raman spectroscopy for AQC has gained much interest, in most applications it remains coupled to a flow injection analyser (FIA) requiring withdrawal of the solution for analysis. In addition to current needs for more rapid and cost-effective analysis, the risk of exposure of clinical staff to the toxic molecules during daily handling is a serious concern to address. Raman spectroscopic analysis, for instance by Confocal Raman Microscopy (CRM), could enable direct analysis (non-invasive) for AQC directly in infusion bags. In this study, 3 anticancer drugs, methotrexate (MTX), 5-fluorouracil (5-FU) and gemcitabine (GEM) have been selected to highlight the potential of CRM for withdrawal free analysis. Solutions corresponding to the clinical range of each drug were prepared in 5% glucose and data was collected from infusion bags placed under the Raman microscope. Firstly, 100% discrimination has been obtained by Partial Least Squares Discriminant Analysis (PLS-DA) confirming that the identification of drugs can be performed. Secondly, using Partial Least Squares Regression (PLSR), quantitative analysis was performed with mean % error of predicted concentrations of respectively 3.31%, 5.54% and 8.60% for MTX, 5-FU and GEM. These results are in accordance with the 15% acceptance criteria used for the current clinical standard technique, FIA, and the Limits of Detection for all drugs were determined to be substantially lower than the administered range, thus highlighting the potential of confocal Raman spectroscopy for direct analysis of chemotherapeutic solutions.

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.