title: Continuous protein purification using functionalized magnetic nanoparticles in aqueous micellar two-phase systems.
abstract: A novel technique for technical-scale continuous purification of proteins is presented. It is based on the combined use of functionalized magnetic nano-particles and an Aqueous Micellar Two-Phase System featuring the non-ionic surfactant, Eumulgin ES, which undergoes temperature induced phase separation at ∼25°C. In the first step, conducted below the transition temperature (i.e. 15°C), the magnetic sorbent particles are added into the single dispersed phase and bind the protein of interest. Next, on raising the temperature to 30°C the protein-laden magnetic particles partition strongly into the micelle-rich top phase of the micellar two-phase system that's formed. The magnetically susceptible top phase is then continuously separated from the micelle-poor phase in a flowthrough tailor-made magnetic extractor featuring a permanent magnet providing an upwardly acting magnetic force. This separation device was shown to be effective for continuous separation of a wide range of differently sized magnetic particle sorbents (i.e. from 2μm diameter to as small as 25nm) from a 10% (w/w) Eumulgin ES system; high separation efficiencies were recorded for the phase-forming surfactant (87 to >98%), and all magnetic sorbent particles tested (95-99.9%). Finally, protein purification by continuous magnetic extraction was demonstrated at 15L scale for the recovery of an antibody fragment, A33 Fab', from a crude extract of Escherichia coli periplasm. Nearly 70% of the A33 Fab' initially present in the extract at 15.6% of the total protein content was recovered in a 2-fold concentrated and highly purified (>98%) state. Further, the amounts of magnetic sorbent and phase-forming surfactant lost in the process were very small; thus recycling of both components into subsequent rounds of continuous magnetic extraction is highly feasible.
authors.affiliation: Institute for Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Fischer
authors.forename: Chia-Chang
authors.initials: CC
authors.lastname: Hsu
authors.forename: Markus
authors.initials: M
authors.lastname: Gärtner
authors.forename: Christine
authors.initials: C
authors.lastname: Müller
authors.forename: Tim W
authors.initials: TW
authors.lastname: Overton
authors.forename: Owen R T
authors.initials: OR
authors.lastname: Thomas
authors.forename: Matthias
authors.initials: M
authors.lastname: Franzreb
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D008280:Magnetics; D008823:Micelles; D053758:Nanoparticles; D011506:Proteins / Q000302:isolation & purification*; D014867:Water
chemical_list: D008823:Micelles; D011506:Proteins; D014867:Water
keywords: Antibody fragments; Aqueous two-phase extraction systems; E. coli periplasm; Ion exchange adsorption; Magnetic sorbents; Non-ionic surfactants