title: Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.
abstract: In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 105U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Gesa
authors.initials: G
authors.lastname: Gieseler
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany. pepelanova@iftc.uni-hannover.de.
authors.forename: Iliyana
authors.initials: I
authors.lastname: Pepelanova
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Stuckenberg
authors.affiliation: Sartorius Stedim Biotech GmbH, August-Spindler-Straße 11, 37079, Göttingen, Germany.
authors.forename: Louis
authors.initials: L
authors.lastname: Villain
authors.affiliation: Miltenyi Biotec GmbH, Friedrich-Ebert-Straße 68, 51429, Bergisch Gladbach, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Nölle
authors.affiliation: Miltenyi Biotec GmbH, Friedrich-Ebert-Straße 68, 51429, Bergisch Gladbach, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Odenthal
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Beutel
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
authors.affiliation: Institute of Technical Chemistry, Leibniz University of Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
mesh_terms: D055396:Bone Morphogenetic Protein 2 / Q000302:isolation & purification* / Q000378:metabolism*; D002845:Chromatography / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D055396:Bone Morphogenetic Protein 2; D011994:Recombinant Proteins
keywords: Animal-component-free; BMP-2; Membrane adsorbers; Mixed-mode chromatography