Control of injectable preparations in a centralized preparation unit: identification of an assay interference between monoclonal antibodies and 5-Fluorouracil by UV/Raman spectrophotometry

QCRx (Icônes Services) is a UV-visible/Raman spectrophotometry method adapted to cytotoxics and monoclonal antibodies (mAbs) dosage in sterile drug preparation units. Small series of samples (1 to 15) can be analyzed as the activity progresses. In our center, around sixty preparations are daily assayed by this method and an analysis of non-conformities (NCs) is carried out each month. The aim of this study was to analyze more precisely the mAbs dosage results, investigate the NCs observed and implement corrective measures to reduce their rate.

All mAbs assays carried out between november 2023 and march 2024 were analyzed (n=2300) revealing an interference related to the mAbs assay after dosing a sample containing 5-Fluorouracil (5FU). In order to confirm the NC, quality controls (QC) of known concentration of Bevacizumab (5 mg/mL) were analyzed (determination of the concentration, analysis of UV and Raman spectra), during the same series of dosages, before and after 5FU (n=6). Series with NaCl (0.9%) solutions following 5FU samples were also analyzed with QCRx, then by HPLC-UV (Dionex Ultimate 3000®-Chromeleon^®) to detect cross-contamination between 5FU and the following samples (Limit of Detection=0.041 mg/mL). Data analysis was carried out using Python^® and Excel^®.

Over the study period, the overall NC rate for mAbs was 5.1% with a significant difference (pval_chi2<0.001) when they were analyzed after a 5FU sample (NC=20.7%, n=365) or not (NC=2.2%, n=1935). The average recovery rate was also significantly higher (113.9% versus 99.6%, pval_ttest<0.001). The QC assay confirmed this result (average recovery: 117.9% after 5FU and 102.7% before). Raman and UV spectra revealed an upward shift in intensities, explaining the observed higher recovery rates. Nevertheless, no peak attributable to a contamination was observed on the spectra. NC were observed only during the same series of dosages and not after circuit washing between a sample of 5FU and mAbs. Therefore, a deposition or crystallization of 5FU in the fluidic circuit of the QCRx was suspected. HPLC-UV analysis did not reveal the presence of cross-contamination with 5FU. Further investigations will therefore be necessary to determine the cause of the NCs. In the meantime, a simple corrective measure was applied and consisted of the systematic passage of mAbs at the start of the dosage series. This reduced the NC rate of mAbs from 5.1% (Nov 2023-Mar 2024) to 1.7% (April 2024) (pval_chi2=0.003).

The regular and in-depth analysis of the QCRx analytical NCs revealed an interference between 5FU and mAbs linked to the order in which samples were run. The corrective measure has enabled to divide these NCs by 3, demonstrating the value of this type of monitoring to highlight NCs that are undetectable on a daily basis.

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