References of "Servais, Anne-Catherine"
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See detailIdentification and quantitation of intact virus-like particles of human papillomavirus (HPV-VLP) using capillary electrophoresis
Bettonville, Virginie ULiege; Nicol, Jérôme; Furst, Tania et al

Conference (2017, September 19)

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See detailChallenges in the determination of amyloid oligomeric species by two electrophoretic techniques
Napp, Aurore ULiege; Houbart, Virginie ULiege; Demelenne, Alice ULiege et al

Poster (2017, September 18)

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already ... [more ▼]

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already occurred. Knowing that, it is of high interest to have one (or more) reliable biomarker(s) at our disposal to diagnose Parkinson before the first symptoms appear. Alpha-synuclein (aSyn) is a protein physiologically expressed at high level by neuronal cells, under a monomeric form. This protein would play a critical role in the development of the disease because under certain conditions, aSyn is capable of self-assembly to form fibrils like those found in Lewy bodies. Other intermediate soluble forms like dimers and oligomers are also formed. As these forms seems to be the toxic species, they are the center of many attentions. The quantification of each form would be a great help, but for the moment only the total forms (of monomeric or oligomeric) can be quantified. In this study, aSyn oligomers were generated after optimization of incubation conditions (pH, temperature, agitation, …). Then, different approaches were investigated to detect and follow the different species formed during the aggregation. We analyzed the oligomers by capillary gel electrophoresis (CGE) and SDS-PAGE. We found that capillary gel electrophoresis is a promising automated technique to analyze aSyn oligomers, due to the fact that it separates the aggregates according to their size, like the SDS-PAGE, but with more advantages. To gain sensitivity and selectivity by CGE, we used a laser-induced fluorescence detector. As aSyn do not have a native fluorescence, we derivatized it. After careful screening and optimization of various derivatization reagents, we could quantify with high sensitivity aSyn oligomers by CGE-LIF. We realized different calibration curves, and we had promising results that will allow us to quantify the different aSyn oligomeric forms in biological fluids. [less ▲]

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See detailQuality control of insulin formulations: API , protamine and aggregates follow-up
Demelenne, Alice ULiege; Napp, Aurore ULiege; Lamalle, Caroline et al

Poster (2017, June 21)

The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are ... [more ▼]

The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are expensive and require a prescription, they are an important target for counterfeiting in developing countries. Therefore, there is a need for fast and efficient methods for quality control of biopharmaceutical products such as insulin formulations. A fully validated micellar electrokinetic chromatography (MEKC) method was developed for quantification of human insulin and NPH insulin (insulin combined with protamine) in formulations. The BGE used was made of 50 mM ammonium acetate (pH 9.0), 20 mM Sodium dodecyl sulfate and 13 % (v/v) acetonitrile and samples were introduced at the short capillary end allowing a separation of insulin and two major excipients (phenol and m-cresol) within 3 minutes. This method was compared with HPLC method using a mobile phase composed of water with 0.1% formic acid / acetonitrile with 0.1 % formic acid in gradient mode. Secondly a MEKC method was also optimized to analyze protamine peptides in insulin formulations. The major protamine peptides could be separated using a BGE made of 100 mM phosphate buffer (pH 2) with 50 mM Thesit®. This method was used to check conformity of protamine in commercially available formulations. Finally, different approaches were investigated in order to follow insulin aggregation. Indeed, insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolds protein in bloodstream results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. On the other hand, during its production, insulin is often subjected to extreme conditions making aggregation, as well as protein stability, important parameters to be controlled during its quality control. In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method. [less ▲]

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See detailOptimization of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Conference (2017, June 20)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

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See detailOptimiaztion of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Poster (2017, June 19)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

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See detailWhole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry
Nys, Gwenaël ULiege; Gallez, Anne ULiege; Kok, Miranda ULiege et al

in Journal of Pharmaceutical & Biomedical Analysis (2017), 140

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See detailSeparation of FLEC diastereomers by CE vs. LC approaches in the context of neurometabolomics
Moldovan, Radu-Cristian ULiege; Bodoki, Ede; Servais, Anne-Catherine ULiege et al

Poster (2017, June)

Some of the D-amino acids (D-Ser, D-Asp, D-Glu) have gained an increasing attention during the last decades, due to the discovery of their role as neurotransmitters and their implication in different ... [more ▼]

Some of the D-amino acids (D-Ser, D-Asp, D-Glu) have gained an increasing attention during the last decades, due to the discovery of their role as neurotransmitters and their implication in different neurological pathologies (Alzheimer’s disease, schizophrenia etc.). Nevertheless, their use as biomarkers is particularly relevant when correlated with the levels of other neurotransmitters. In order to develop a fast and efficient separation method widely accessible for the quantitation of these molecules, we used only common separation tools such as RP-18 stationary phases for reversed phase liquid chromatography (RP-LC) or bare fused capillaries for capillary zone electrophoresis (CZE). For achieving chiral resolution, a derivatization procedure was implemented. (-)-FLEC was the chiral derivatization agent of choice due to its fast and quantitative reaction with primary and secondary amines and the ability of performing in-capillary derivatization. Moreover, the derivatization process implies only a simple mix of the sample and reagent, at room temperature. The separation of the FLEC derivatives of several biologically relevant D- and L- amino acids (Asp, Glu, Ser, Tyr, Trp, Phe, His) together with certain neurotransmitter molecules have been optimized using CZE or RP-LC, chiral resolution being achievable for all amino acids of interest. By the CZE approach the running buffer’s pH turned out to be critical in achieving baseline separation of the targeted analytes. The derivatives of most amino acids could be separated using 60mM acetate buffer at pH 5, while for Asp derivatives the separation could be achieved only at pH 4. Being stronger bases, a third run at a more alkaline pH was needed for the separation of the remainder neurotransmitters. Moreover, the implemented in-capillary derivatization allows a fast and fully automated separation procedure. As for the RP-LC approach 50 mM acetate buffer in combination with an organic modifier (methanol, acetonitrile or tetrahydrofuran (THF)) was tested as mobile phase using gradient elution. Once again, the strong influence of pH on the resolution was observed. The organic modifier nature was of critical importance, where only THF enabled baseline resolution for all amino acid derivatives. [less ▲]

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See detailLc-chip versus UHPLC-tandem mass spectrometry for the quantitation of estetrol and estradiol without derivatization after whole blood microsampling
Nys, Gwenaël ULiege; Servais, Anne-Catherine ULiege; Pequeux, Christel ULiege et al

Conference (2017, March 29)

the aim of this work was to conduct a PK study on mice to select the most appropriate administration route for E2 and E4 formulations. To achieve this goal, a reference method for the quantitation of both ... [more ▼]

the aim of this work was to conduct a PK study on mice to select the most appropriate administration route for E2 and E4 formulations. To achieve this goal, a reference method for the quantitation of both estrogens after whole blood microsampling was developed and validated on a UHPLC-MS/MS system. This reference method was later transferred on LC-chip device and both methods were compared in terms of analytical parameters such as response function, accuracy, precision, trueness and limit of quantification. [less ▲]

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See detailPartial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.
Farcas, E.; Bouckaert, C.; Servais, Anne-Catherine ULiege et al

in Analytica Chimica Acta (2017), 984

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD ... [more ▼]

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the muM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD. [less ▲]

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See detailQuantitation and biospecific identification of virus-like particles of human papillomavirus by capillary electrophoresis.
Bettonville, Virginie ULiege; Nicol, Jerome T. J.; Furst, Tania et al

in Talanta (2017), 175

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 ... [more ▼]

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(R), 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 </= 50microgml-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%. [less ▲]

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See detail(+) or (-)-1-(9-fluorenyl)ethyl chloroformate as chiral derivatizing agent: A review.
Moldovan, Radu-Cristian ULiege; Bodoki, Ede; Servais, Anne-Catherine ULiege et al

in Journal of Chromatography. A (2017), 1513

Over the last 30years, (+/-)-1-(9-fluorenyl)ethyl chloroformate ((+/-)-FLEC) was used as a chiral derivatizing agent in various analytical applications involving a wide range of endogenous, pharmaceutical ... [more ▼]

Over the last 30years, (+/-)-1-(9-fluorenyl)ethyl chloroformate ((+/-)-FLEC) was used as a chiral derivatizing agent in various analytical applications involving a wide range of endogenous, pharmaceutical and environmentally relevant molecules. This comprehensive review aims to present all the significant aspects related to the state of the art in FLEC labeling and subsequent chiral separation of the resulting diastereomers using LC, SFC and CE techniques. [less ▲]

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See detailCapillary electrophoresis in the context of drug discovery.
Farcas, Elena ULiege; Pochet, Lionel; Crommen, Jacques ULiege et al

in Journal of Pharmaceutical & Biomedical Analysis (2017)

Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be ... [more ▼]

Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be explained by the relatively low number of experienced CE practitioners, the maturity of HPLC in the pharmaceutical industry and some intrinsic limitations of the technique. The objective of this review is to focus our attention on recent developments of this technique in three different drug discovery areas: bioassays, drug-plasma interactions and drug metabolism studies. These developments were based on two important abilities of CE: the capacity to measure non-covalent interactions in solution and the ability to use a portion of the capillary as a reactor while the rest of the capillary is used for the separation of the product of the reaction. [less ▲]

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See detailSingle and dual cyclodextrins systems for the enantiomeric and diastereoisomeric separations of structurally related dihydropyridone analogues.
Delplanques, Thibaut; Boulahjar, Rajaa; Charton, Julie et al

in Electrophoresis (2017)

Cyclodextrin capillary electrophoresis methods (CD-CZE) were developed for complete enantiomeric and diastereoisomeric separations of a series of ten dihydropyridone analogues, of which eight were neutral ... [more ▼]

Cyclodextrin capillary electrophoresis methods (CD-CZE) were developed for complete enantiomeric and diastereoisomeric separations of a series of ten dihydropyridone analogues, of which eight were neutral, one was anionic and one was cationic. Ten different systems comprising one or two cyclodextrins were found to successfully separate the isomers thanks to a screening approach. Among the tested cyclodextrins, HS-gamma-CD, either in a single or in a dual system, in a phosphate buffer using capillaries dynamically coated with polyethylene oxide (PEO), and SBE-beta-CD, either in a single or in a dual system, in a borate buffer using uncoated capillaries, were the most selective selectors. The effects of different parameters such as the nature and concentration of the cyclodextrins, nature and concentration of the buffer, and voltage were examined. The precision and limits of detection and quantification were evaluated for the optimized methods. This article is protected by copyright. All rights reserved. [less ▲]

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See detailStudy of virus-like particles of human papillomavirus in capillary electrophoresis
Bettonville, Virginie ULiege; Nicol, Jérôme; Furst, Tania et al

Poster (2017)

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See detailPharmacokinetic study of estetrol in murin model developing a whole blood microsampling, extraction procedure and an UHPLC-MS/MS method
Nys, Gwenaël ULiege; Servais, Anne-Catherine ULiege; Pequeux, Christel ULiege et al

Conference (2016, October 17)

We developed a sensitive UHPLC-MS/MS method with a simple yet efficient sample collection and preparation protocol to accurately quantify E4 in whole blood. This method was then successfully applied for ... [more ▼]

We developed a sensitive UHPLC-MS/MS method with a simple yet efficient sample collection and preparation protocol to accurately quantify E4 in whole blood. This method was then successfully applied for the pharmacokinetic study of E4 in a murin model providing the best administration route for E4. The innovative sampling allowed decreasing the number of animals used for the study as VAMS only draws few microliters of blood thus leaving the mice alive for the whole PK study reducing the inter-individual variability. [less ▲]

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See detailOPTIMIZATION OF A FULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline et al

Conference (2016, October 01)

Objective of the study Since CYP1A1, a member of cytochrome P450 superfamily, has been described to be over expressed in various types of cancer, our study was focused on the optimization of a fully ... [more ▼]

Objective of the study Since CYP1A1, a member of cytochrome P450 superfamily, has been described to be over expressed in various types of cancer, our study was focused on the optimization of a fully automated system for the monitoring of this particularly interesting enzyme. Moreover, the potentiality of this approach to screen CYP1A1 inhibitors was investigated. Materials and methods The experiments were carried out on a HP3DCE system using an on-column DAD. The EMMA procedure was performed by injecting a plug containing the co-factor(NADPH) and the substrate(7-ethoxycoumarin) between two plugs of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed. Results Satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. A DoE was performed to find the best mixing conditions. The amount of metabolite obtained was comparable to the one detected after conventional off-line metabolization. The ability of our system to monitor CYP1A1 inhibition was then proven with apigenin, a well-known CYP1A1 inhibitor. Conclusions The present study describes the development of a fully automatized in-capillary method for CYP1A1 activity monitoring and proves the potentialy of our system to be used for the screening of CYP1A1 inhibitors. The advantages of performing inline metabolization assays are mainly the miniaturization and the automatization of the process. Besides, the reagents consumption is drastically reduced due to the injection of few tens of nanoliters. [less ▲]

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