title: Performance of protein-A-based affinity membranes for antibody purification.
abstract: The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5-10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL-protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL-protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL-protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption-elution cycles.
authors.affiliation: a Department of Chemistry, Biochemistry Division, Hacettepe University, 06800 Ankara, Turkey.
authors.forename: Lokman
authors.initials: L
authors.lastname: Uzun
authors.forename: Deniz
authors.initials: D
authors.lastname: Türkmen
authors.forename: Veyis
authors.initials: V
authors.lastname: Karakoç
authors.forename: Handan
authors.initials: H
authors.lastname: Yavuz
authors.forename: Adil
authors.initials: A
authors.lastname: Denizli
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification*; D001780:Blood Coagulation Tests; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008422:Materials Testing; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D015394:Molecular Structure; D055668:Photochemical Processes; D010949:Plasma / Q000737:chemistry / Q000276:immunology; D058105:Polymerization; D016062:Porosity; D013205:Staphylococcal Protein A / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D000906:Antibodies; D008567:Membranes, Artificial; D013205:Staphylococcal Protein A; D014867:Water; D003488:Cyanogen Bromide