title: The development of a weak anion micro-capillary film for protein chromatography.
abstract: In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12.4mg BSA/mL adsorbent and 8.2mg BSA/mL adsorbent under static and flow conditions, respectively. The ion exchange capacity, determined by Mohr's titration of chlorine atoms displaced from the functionalised surface, was found to be 195±21μmol Cl-/mL of adsorber, comparable to commercial ion exchangers. BSA adsorption onto the ion exchanger was strongly pH-dependant, with an observed reduction in binding above pH 8.2. Frontal experiments of a BSA (5mg/mL) and lysozyme (5mg/mL) mixture demonstrated successful separation of BSA from lysozyme at more than 97% purity as verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separation between similarly charged anionic molecules was also achieved using BSA (5mg/mL) and herring sperm DNA (0.25mg/mL). BSA was extracted at 100% purity, demonstrating the ability of MMCF-DEAE to remove significant DNA contamination from a protein solution. These experiments highlight the potential for MMCFs to be used for fast protein purification in preparative chromatography application.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: A J M
authors.initials: AJ
authors.lastname: Kouyoumdjian
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: R A
authors.initials: RA
authors.lastname: Lazar
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom. Electronic address: nkhs2@cam.ac.uk.
authors.forename: N K H
authors.initials: NK
authors.lastname: Slater
mesh_terms: D000327:Adsorption; D000838:Anions / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D009113:Muramidase / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000838:Anions; D011506:Proteins; D012996:Solutions; D012710:Serum Albumin, Bovine; D009113:Muramidase
keywords: Chromatography; DEAE; Membranes; Micro-capillary films; Weak anion-exchange