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See detailInsulin aggregation assessment by capillary gel electrophoresis without sodium dodecyl sulfate: Comparison with size-exclusion chromatography
Demelenne, Alice ULiege; NAPP, Aurore ULiege; Bouillenne, Fabrice ULiege et al

in Talanta (2019), 199

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic ... [more ▼]

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic pH mobile phase to assess the content of aggregates in insulin formulations. In this article, we analyzed aggregated human insulin samples and demonstrated that both methods under neutral conditions, namely neutral pH SEC (nSEC) and capillary gel electrophoresis (CGE), yield to similar aggregate content contrary to SEC under acidic conditions (aSEC). aSEC showed polymeric complexes that were not observed in nSEC and CGE. During method development, the effect on SEC profiles of arginine and acetonitrile were highlighted. In CGE, the effect of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition in sieving gel was discussed. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions. In view of the obtained results one may question the relevance of the current pharmacopoeia method to study insulin aggregates by emphasizing the importance of the mobile phase composition and pH in SEC. The new CGE method developed is an easy method for studying non-covalent aggregates of insulin, which could be applied to other proteins. © 2019 Elsevier B.V. [less ▲]

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See detailQuality control and aggregation follow-up of insulin formulations
Demelenne, Alice ULiege; Napp, Aurore ULiege; Radermecker, Régis ULiege et al

Conference (2018, December 19)

The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus ... [more ▼]

The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus required, more particularly methods to assess the content of API and potential aggregates. We started our study on insulin aggregation using the European Pharmacopeia method. Since methods to assess aggregate content are often contradictory, we also developed original and orthogonal methods using size-exclusion chromatography (SEC) and capillary gel electrophoresis (CGE). It was demonstrated that methods under neutral conditions (SEC and CGE) yield to similar aggregate content contrary to pharmacopeia SEC method that works under acidic conditions. Ion-mobility Q-TOF mass spectrometer was also used to confirm the presence and the identity of insulin dimers. Then, we applied the three methods to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. We also used the CGE method to study the stability of human insulin under different storage conditions. Finally, we used UHPLC and mass spectrometry to quantify insulin formulation from different supply chains. We demonstrated that all the analyzed formulations had a potency between 95.0 % and 105.0 % of the potency stated on the label. This was useful to dispel doubts regarding issues in insulin cold chain supply recently described in the litterature. [less ▲]

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See detailInsulin aggregation assessment by size-exclusion chromatography and capillary gel electrophoresis
Demelenne, Alice ULiege; Napp, Aurore ULiege; Servais, Anne-Catherine ULiege et al

Poster (2018, September 10)

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may ... [more ▼]

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may lead to complications in insulin therapy. For that reason, they are important parameters to investigate for insulin quality control. The United States and European Pharmacopoeias currently use a SEC method with acidic mobile phase for the quantification of aggregates in insulin formulations. However, changes in aggregates assessment have been reported when the mobile phase composition differs from the sample dissolution medium. [1] To investigate the impact of mobile phase on human insulin aggregates analysis, the aggregated human insulin samples were analyzed by SEC using neutral (nSEC) or acidic mobile phases (aSEC). The aggregated samples were obtained by dissolving human insulin in acidic media, followed by agitation for 8, 16, 24, 32, 40 and 48 hours respectively. During SEC method development, the impact of arginine and acetonitrile addition to the mobile phase was pointed out. Additionally, an orthogonal capillary-gel electrophoresis method (CGE) for the assessment of insulin aggregates was developed. The optimal pH 8.1 CGE buffer was formulated without SDS in order to preserve the non-covalent aggregates. After the optimization of the methods, human insulin and aggregated samples were analyzed using nSEC, aSEC and CGE. A similar increase of dimers percentage with incubation time was noticed by both nSEC and CGE, while no significant increase of dimers content was observed by aSEC. However, an insulin polymeric complex was detectable for some samples with aSEC. The three methods were used to analyze an insulin formulation and a similar tendency was observed. The results obtained emphasize the importance of mobile phase choice in SEC. The good correlation between dimers percentage obtained by nSEC and CGE suggests that these technics most probably enable the detection of the species initially present in the sample and do not change the composition of the sample during analysis. The developed CGE method is a fast and reliable tool for the study of the complex process of insulin aggregation. Furthermore, the CGE method could be easily applied to other proteins since it proved to be highly reproducible and has the advantages of low sample consumption, and no expensive column or organic solvents are required. [less ▲]

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See detailSeparation and determination of alpha-synuclein monomeric and oligomeric species using two electrophoretic approaches
Napp, Aurore ULiege; Houbart, V.; Demelenne, Alice ULiege et al

in Electrophoresis (2018), 39(23), 3022-3031

Parkinson's disease (PD) is a frequent degenerative disorder that is diagnosed based on clinical symptoms. When the first symptoms appear, more than 70% of the dopaminergic cells are already lost ... [more ▼]

Parkinson's disease (PD) is a frequent degenerative disorder that is diagnosed based on clinical symptoms. When the first symptoms appear, more than 70% of the dopaminergic cells are already lost. Therefore, it is of utmost importance to have reliable biomarkers to diagnose much earlier PD. In this context, alpha-synuclein (aSyn) is a protein of high interest because of its tendency to form oligomers and amyloid fibrils. The oligomeric forms seem to play a critical pathological role in PD. To date, most of studies aiming at detecting and quantifying aSyn oligomers were performed by immunoassays, mainly by ELISA using specific antibodies. In this study a capillary gel electrophoresis (CGE) coupled with fluorescence detection method was developed to detect and quantify the oligomeric forms of aSyn formed in vitro. All the results obtained were supported by SDS–PAGE analysis, a widely used and well-known technique but exhibiting a main drawback since it is not an automated technique. The repeatability and the intermediate precision of the method were evaluated, as well as the stability of the labeled and non-labeled aSyn samples. After careful screening and optimization of various labeling reagents, 4-fluoro-7-nitrobenzofurazan (NBD-F) was selected and used to establish a calibration curve with monomeric fluorescently-labeled aSyn. Finally, the method was used to study the effect of doxycycline on the oligomerization process. Altogether, our results show that CGE is a very promising automated technique to analyze aSyn monomers, as well as small oligomers. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [less ▲]

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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 ▲]

Detailed reference viewed: 98 (24 ULiège)
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 ▲]

Detailed reference viewed: 47 (7 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

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: 61 (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: 57 (16 ULiège)