Interest of flow injection spectrophotometry as an orthogonal method for analyzing biomolecule aggregates: Application to stressed monoclonal antibody study.

This study aimed to explore the suitability of flow injection spectrophotometry (FIS) to analyze three degraded therapeutic monoclonal antibodies (bevacizumab, nivolumab, and rituximab). For this purpose, aggregates were generated with stirring, freeze-thaw, and heat stresses. The intact and stressed mab samples were filtered with 0.22 µm hydrophilic filters and analyzed by size exclusion chromatography (SEC), cation-exchange chromatography (CEX), and FIS. In terms of quantitative and qualitative analysis, protein loss and structural changes were assessed. Various aggregates profiles were obtained according to the mabs and the stresses. FIS allowed performing very satisfactory quantifications for each mab with intermediate precision RSD < 3.0 % and recovery between 97.9 and 102.0 %. From the protein loss measurements, it appears that SEC underestimates the mab aggregate proportions up to two times less as compared with FIS since the latter avoids any non-specific interactions (electrostatic or hydrophobic interactions). Using second derivative spectroscopy and multivariate data analysis, we noticed apparent structural differences, located in the regions 245-265 nm for rituximab and nivolumab and 280-300 nm for bevacizumab, depending on the stress. The FIS complementarity with the other techniques used in this study allowed us to demonstrate that the three mabs behave differently for a given stress condition. While extreme mechanical stress formed large aggregates irrespective of the mabs, rituximab showed to be less stable and more sensitive than the two other mabs under freeze-thaw and heat stresses, generating large aggregates (>200 nm) and partial unfolding. Nivolumab tends to form small aggregates less than 50 nm when heated and freeze-thawed. Moreover, freeze-thaw seems to generate native IgG-1 aggregates with rituximab. Similarly, bevacizumab showed to form these IgG-1 aggregates and was resistant to freeze-thaw, likely thanks to trehalose cryoprotectant from its formulation. Finally, FIS associated with multivariate analysis can provide rich information in one single run and appears to be a fast, simple, and reliable method to set complementary and orthogonal approaches for protein aggregates monitoring.

UV spectroscopy and least square matching for high throughput discrimination of taxanes in commercial formulations and compounded bags.

The need for high-throughput quality control of pharmaceuticals after compounding is often required before the treatment of the patients. Ultra-fast analysis using flow injection analysis coupled to UV spectroscopy and least square matching was assessed for the simultaneous quantification and identification of three therapeutic taxanes after dilution in physiological saline (cabazitaxel, docetaxel, and paclitaxel). In-depth preliminary analysis of the zero and first order UV spectra of the taxanes using principal component analysis (PCA) allowed us focusing on relevant spectral range with very low formulation influence. Least square-matching algorithm available on basic HPLC software was applied to these spectra yielding very high match scores (>999) with significant difference (P < 0.0001). The approach was qualitatively assessed through specificity and sensitivity which were excellent for the three taxanes (100%, n = 378), irrespective of their formulation. In terms of quantification, satisfactory linearity and accuracy were achieved for each of the taxanes according to their therapeutic range (0.05 to 1.02 mg·mL-1). The RSD (%) of the precision was satisfactory (<3%). Finally, the suitability of the approach for the taxanes QC has been demonstrated under routine application.

Simple and ultra-fast recognition and quantitation of compounded monoclonal antibodies: Application to flow injection analysis combined to UV spectroscopy and matching method.

Compounding of monoclonal antibody (mAbs) constantly increases in hospital. Quality control (QC) of the compounded mAbs based on quantification and identification is required to prevent potential errors and fast method is needed to manage outpatient chemotherapy administration. A simple and ultra-fast (less than 30 s) method using flow injection analysis associated to least square matching method issued from the analyzer software was performed and evaluated for the routine hospital QC of three compounded mAbs: bevacizumab, infliximab and rituximab. The method was evaluated through qualitative and quantitative parameters. Preliminary analysis of the UV absorption and second derivative spectra of the mAbs allowed us to adapt analytical conditions according to the therapeutic range of the mAbs. In terms of quantitative QC, linearity, accuracy and precision were assessed as specified in ICH guidelines. Very satisfactory recovery was achieved and the RSD (%) of the intermediate precision were less than 1.1%. Qualitative analytical parameters were also evaluated in terms of specificity, sensitivity and global precision through a matrix of confusion. Results showed to be concentration and mAbs dependant and excellent (100%) specificity and sensitivity were reached within specific concentration range. Finally, routine application on "real life" samples (n = 209) from different batch of the three mAbs complied with the specifications of the quality control i.e. excellent identification (100%) and ±â€¯15% of targeting concentration belonging to the calibration range. The successful use of the combination of second derivative spectroscopy and partial least square matching method demonstrated the interest of FIA for the ultra-fast QC of mAbs after compounding using matching method.