title: Purification of a PEGylated single chain Fv.
abstract: In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30 kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by (125)I based cell tests. Proteins modified with 5 kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification.
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany. an-na-mo@gmx.de
authors.forename: Anna
authors.initials: A
authors.lastname: Moosmann
authors.forename: Elke
authors.initials: E
authors.lastname: Gerlach
authors.forename: Robert
authors.initials: R
authors.lastname: Lindner
authors.forename: Heiner
authors.initials: H
authors.lastname: Böttinger
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004553:Electric Conductivity; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002412:Cations; D057127:Single-Chain Antibodies; D011092:Polyethylene Glycols; D001120:Arginine; D066246:ErbB Receptors