title: A capillary zone electrophoresis method to detect conformers and dimers of antithrombin in therapeutic preparations.
abstract: Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Marie
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.
authors.forename: Nguyet Thuy
authors.initials: NT
authors.lastname: Tran
authors.affiliation: Université Paris Sud, UMR-S1176, Le Kremlin-Bicêtre, France.|INSERM, U1176, Le Kremlin-Bicêtre, France.
authors.forename: François
authors.initials: F
authors.lastname: Saller
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.
authors.forename: Youmna Mohamed
authors.initials: YM
authors.lastname: Abdou
authors.affiliation: LFB, Les Ulis, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Zeau
authors.affiliation: LFB, Les Ulis, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Plantier
authors.affiliation: ECDYSIS Pharma, Loos, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Urbain
authors.affiliation: Université Paris Sud, UMR-S1176, Le Kremlin-Bicêtre, France.|INSERM, U1176, Le Kremlin-Bicêtre, France.|AP-HP, Hôpital Necker, Service d'Hématologie Biologique, Paris, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Borgel
authors.affiliation: Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.
authors.forename: Myriam
authors.initials: M
authors.lastname: Taverna
mesh_terms: D000990:Antithrombin III / Q000302:isolation & purification*; D002021:Buffers; D019281:Dimerization; D004339:Drug Compounding; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D011092:Polyethylene Glycols; D011487:Protein Conformation; D020033:Protein Isoforms / Q000302:isolation & purification
chemical_list: D002021:Buffers; D020033:Protein Isoforms; D011092:Polyethylene Glycols; D000990:Antithrombin III
keywords: Capillary zone electrophoresis; Conformers; Heterodimer; Latent form; Therapeutic antithrombin