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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 detailStudy of insulin aggregation by SEC and CGE
Demelenne, Alice ULiege; Napp, Aurore ULiege; Servais, Anne-Catherine ULiege et al

Conference (2016, October)

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient ... [more ▼]

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient but only the monomeric form is active once dissociated in the bloodstream. Insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolded proteins 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”. Moreover, during its production, insulin is often subjected to extreme conditions making lack of aggregates an important parameter to be controlled during its quality control. United States and European Pharmacopoeias use both size exclusion chromatography (SEC) to assess the level of covalent high molecular weight species. This technique is reproducible, and easy to use but shows many drawbacks including possible changes in the aggregates composition by dilution into the HPLC system or adsorption of sample onto the stationary phase. For those reasons other techniques have been considered in the literature for studying aggregation of insulin. Optical microscopy, electron microscopy, dynamic light scattering, turbidimetry, Fourier Transform infrared spectroscopy, Raman spectroscopy, thioflavin T fluorescence and circular dichroism spectroscopy are some of them. In any cases, the use of orthogonal techniques is essential to assess the relevance of the results. Results: In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by SEC and capillary electrophoresis (CE). CE shows many advantages in terms of sample and solvent consumption and enables analysis of samples under their native form. 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 detailAlpha-synuclein as biomarker in Parkinson’s disease: strategies for detection in CGE-LIF
Houbart, Virginie ULiege; Napp, Aurore ULiege; Rudaz, Serge et al

Conference (2016, April)

Detailed reference viewed: 29 (2 ULiège)
See detailAlpha-synuclein as biomarker in Parkinson’s disease: strategies for detection in CGE-LIF
Houbart, Virginie ULiege; Napp, Aurore ULiege; Rudaz, Serge et al

Poster (2016, April)

Detailed reference viewed: 41 (21 ULiège)
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See detailHepcidin determination in dried blood by microfluidic LC-MS/MS: comparison of DBS and volumetric absorptive microsampling for matrix effect and recovery.
Houbart, Virginie ULiege; COBRAIVILLE, Gaël ULiege; Servais, Anne-Catherine ULiege et al

in Bioanalysis (2015), 7(21), 2789-99

BACKGROUND: Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new ... [more ▼]

BACKGROUND: Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new alternatives are commercialized as volumetric absorptive microsampling (VAMS) that are expected to overcome hematocrit influence. RESULTS: The feasibility of hepcidin (a peptide hormone) extraction and determination from DBS and VAMS blood sampling was investigated. Experimental design was used to determine the optimal extraction conditions. Matrix effect and extraction recovery were studied and a special attention was paid to phospholipid removal. CONCLUSION: The data suggest that the combination of VAMS and phospholipid removal plates provides low matrix effect and high sensitivity, and constitutes an easy and promising protocol for hepcidin analysis. [less ▲]

Detailed reference viewed: 57 (17 ULiège)
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See detailEVALUATION OF α-SYNUCLEIN AS BIOMARKER OF PARKINSON'S DISEASE
Napp, Aurore ULiege; Garraux, Gaëtan ULiege; Servais, Anne-Catherine ULiege et al

Conference (2013, October 17)

Detailed reference viewed: 49 (23 ULiège)
See detailLes biomarqueurs de la maladie de Parkinson
Napp, Aurore ULiege; Fillet, Marianne ULiege

Scientific conference (2013, May 23)

Detailed reference viewed: 55 (16 ULiège)