Virginie Bettonville

Determination of inhibitory potency of argatroban toward thrombin by electrophoretically mediated microanalysis

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value.

Study of intact virus-like particles of human papillomavirus by capillary electrophoresis.

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil® and Cervarix®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen® fluorescence‐based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short‐end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.

Quantitation and biospecific identification of virus-like particles of human papillomavirus by capillary electrophoresis

Capillary electrophoresis (CE) for HPV-VLP quantitation is a very interesting alternative technique compared to those currently used in viral analysis, such as SDS-PAGE, Western blot or protein assay that are destructive and semi-quantitative or non specific. In this study, the quantitative performance of the CE method was evaluated. A main issue in virus quantitation is the absence of reference material. Therefore, the concentration of a HPV16-VLP sample produced in the laboratory was determined using ELISA with Gardasil®, after adjuvant dissolution, as reference material and conformational H16.V5 antibody. HPV16-VLP concentration was found to influence particles electrophoretic mobility until a plateau was reached for concentrations ≤ 50µgml-1. As zeta potential is directly proportional to the electrophoretic mobility, it was measured at different HPV-VLP concentrations and the results were in complete accordance with the measured electrophoretic mobilities. The concentration dependence of the electrophoretic mobility could be explained by an overlap of the electrical double layers of adjacent particles. The HPV16-VLP peak identity was demonstrated unequivocally by the study of HPV16-VLP/H16.V5 antibody complex formation using affinity CE. Finally, the CE method was successfully validated following the ICH Q2R1 guidelines. To overcome the sample heterogeneity issue, a well-designed sample preparation was used. Considering sample complexity, validation results were satisfactory with maximum repeatability and intermediate precision RSD of 12.2% and a maximum relative bias of 1.4%.