Yoann Ladner

Ce manuscrit est consacré à l'élaboration de phases stationnaires monolithiques organiques pour lesmicrosystèmes électrochromatographiques plastiques. La partie bibliographique situe d'abord l'intérêt destechniques électrocinétiques en microsystème. Ensuite, l'état de l'art sur l'utilisation des monolithes dans cestechniques séparatives est présenté en portant une attention particulière à la description du processus dephotopolymérisation. La fonctionnalisation de surface du copolymère d'oléfine cyclique (COC) est ensuitedétaillée afin d'envisager l'ancrage du monolithe aux parois du canal.La partie expérimentale a porté tout d'abord sur le développement et la caractérisation de deux monolithesacrylates dédiés à la chromatographie à polarités des phases inversées. Le travail expérimental s'est ensuiteorienté vers le développement d’un nouveau procédé permettant la synthèse et l’ancrage du monolithe dansles microcanaux en COC, ceci en une seule étape et à partir des photoinitiateurs de type I. Ce protocolepermet l'obtention d'efficacités intéressantes qui restent cependant inférieures à celles obtenues en capillaire.Des adaptations des conditions d'irradiation ont alors été apportées pour la synthèse de ces monolithes àl’intérieur des microsystèmes afin d'obtenir des efficacités de l’ordre de 250 000 plateaux/m. Pour terminer,les domaines d'applications et la longévité du dispositif ont été illustrés par la séparation de différentesfamilles de solutés (PAH, mycotoxines, catécholamines, acides aminés, amines biogènes) et l'analysed'échantillons réels tels que les vins (pour déterminer la teneur en certaines amines biogènes : histamine ettyramine).

Phenolic preservatives are critical excipients in biopharmaceutical formulations such as insulin and its analogs. The determination of these analytes in insulin formulations and human plasma is crucial for various applications. This study reports the first use of a method combining salting‐out assisted liquid–liquid extraction (SALLE) with capillary zone electrophoresis (CZE). The CZE‐based separation of phenolic preservatives remains particularly challenging, as the alkaline conditions required for adequate resolution lead to elevated electric current and accelerated capillary deterioration. Analytical parameters, including the nature of the background electrolyte (BGE) and ionic strength, were optimized to ensure reliable separation of phenol, m ‐cresol, and the internal standard. SALLE was performed using acetonitrile (ACN) as the extraction solvent and ammonium acetate as the salting‐out agent. CZE separation was completed in less than 6 min using a carbonate buffer at pH 10.3. The SALLE procedure yielded good recoveries (96%–102%) and repeatability (relative standard deviation [RSD] for concentration determination < 5.5%). The SALLE–CZE–UV methodology showed excellent linearity ( r ² > 0.994) and repeatability (RSD for corrected migration times: 0.4% for phenol and 0.5% for m ‐cresol; RSD for the ratio of corrected peak areas [analyte/internal standard]: 2.6% for phenol and 1.8% for m ‐cresol). Moreover, appropriate limits of detection (LOD) and quantification (LOQ) were obtained for insulin formulations in line with regulatory requirements. In addition, suitable LOD and LOQ (0.014; 0.047 g.L ⁻¹ for phenol and 0.007; 0.022 g.L ⁻¹ for m ‐cresol) were obtained for spiked human plasma.

We explore a bioinspired approach to design tailored functionalized capillary electrophoresis (CE) surfaces based on covalent grafting for biomolecules analysis. First, the approach aims to overcome well‐known common obstacles in CE protein analysis affecting considerably the CE performance (asymmetry, resolution, and repeatability) such as the unspecific adsorption on fused silica surface and the lack of control of electroosmotic flow (EOF). Then, our approach, which relies on new amino‐amide mimic hybrid precursors synthesized by silylation of amino‐amides (Si–AA) derivatives with 3‐isocyanatopropyltriethoxysilane, aims to recapitulate the diversity of protein–protein interactions (π–π stacking, ionic, Van der Waals…) found in physiological condition (bioinspired approach) to improve the performance of CE protein analysis (electrochromatography). As a proof of concept, these silylated Si–AA (tyrosinamide silylation, serinamide silylation, argininamide silylation, leucinamide silylation, and isoglutamine silylation acid) have been covalently grafted in physiological conditions in different amount on bare fused silica capillary giving rise to a biomimetic coating and allowing both the modulation of EOF and protein–surface interactions. The analytical performances of amino‐amide functionalized capillaries were assessed using lysozyme, cytochrome C and ribonuclease A and compared to traditional capillary coatings poly(ethylene oxide), poly(diallyldimethylammonium chloride), and sodium poly(styrenesulfonate). EOF, protein adsorption rate, protein retention factor k, and selectivity were determined for each coating. All results obtained showed this approach allowed to modulate the EOF, reduce unspecific adsorption, and generate specific interactions with proteins by varying the nature and the amount of Si–AA in the functionalization mixture.

Ordered mesoporous materials and their modification with multiple functional groups are of wide scientific interest for many applications involving interaction with biological systems and biomolecules (e.g., catalysis, separation, sensor design, nano-science or drug delivery). In particular, the immobilization of enzymes onto solid supports is highly attractive for industry and synthetic chemistry, as it allows the development of stable and cheap biocatalysts. In this context, we developed novel silylated amino acid derivatives (Si-AA-NH₂) that have been immobilized onto SBA-15 materials in biocompatible conditions avoiding the use of toxic catalyst, solvents or reagents. The resulting amino acid-functionalized materials (SBA-15@AA) were characterized by XRD, TGA, EA, Zeta potential, nitrogen sorption and FT-IR. Differences of the physical properties (e.g., charges) were observed while the structural ones remained unchanged. The adsorption of the enzyme lysozyme (Lyz) onto the resulting functionalized SBA-15@AA materials was evaluated at different pHs. The presence of different functional groups compared with bare SBA-15 showed better adsorption results, for example, 79.6 nmol of Lyz adsorbed per m² of SBA-15@Tyr compared with the 44.9 nmol/m² of the bare SBA-15.

For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom‐up level to generate specific fingerprints that can be used to tackle post‐translational modifications or ensure production consistency between lots. To circumvent time‐consuming and labor‐intensive off‐line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in‐line methodology for the routine quality control of mAbs at the bottom‐up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in‐line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In‐line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS‐compatible BGE was used to obtain high resolution separation of released peptides and in‐line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE‐fingerprints were successfully established for different mAbs and an antibody‐drug conjugate.

Therapeutic monoclonal antibodies (mAbs) are dominating the biopharmaceutical field due to the fact of their high specificity in the treatment of diverse diseases. Nevertheless, mAbs are very complex glycoproteins exhibiting several macro- and microheterogeneities that may affect their safety, quality, and efficacy. This complexity is very challenging for mAbs development, formulation, and quality control. To tackle the quality issue, a combination of multiple analytical approaches is necessary. In this perspective, capillary electrophoresis has gained considerable interest over the last decade due to the fact of its complementary features to chromatographic approaches. This review provides an overview of the strategies of mAbs and derivatives analysis by capillary electrophoresis hyphenated to ultraviolet, fluorescence, and mass spectrometry detection. The main sample preparation approaches used for mAb analytical characterization (i.e., intact, middle-up/down, and bottom-up) are detailed. The different electrophoretic modes used as well as integrated analysis approaches (sample preparation and separation) are critically discussed.