title: Coupling purification and on-column PEGylation of tumor necrosis factor alpha analogue.
abstract: Trends in preparation of PEGylated protein drugs strive for simple, fast, and cheap processes, resulting in well-defined homogeneous products. We investigated the on-column PEGylation of tumor necrosis factor alpha (TNF-α), where purification and conjugation were performed in one step by using immobilized metal affinity chromatography (IMAC). The same quality of the PEGylated product was obtained by the on-column approach starting from either the crude Escherichia coli protein extract or the purified protein. In comparison with the PEGylation in solution, the on-column approach resulted in more homogeneous PEGylated product. The on-column PEGylation reduces the number of production steps, costs, and preparation time.
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia. tanja.milunovic@ki.si
authors.forename: Tatjana
authors.initials: T
authors.lastname: Milunović
authors.forename: Menči
authors.initials: M
authors.lastname: Kunstelj
authors.forename: Katarina
authors.initials: K
authors.lastname: Fidler
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Vladka
authors.initials: V
authors.lastname: Gaberc Porekar
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols