Continuous protein purification using functionalized magnetic nanoparticles in aqueous micellar two-phase systems

• Published in: Journal of Chromatography A Vol. 1305; pp. 7 - 16
• Main Authors: Fischer, Ingo, Hsu, Chia-Chang, Gärtner, Markus, Müller, Christine, Overton, Tim W., Thomas, Owen R.T., Franzreb, Matthias
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 30.08.2013; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Antibody fragments; Aqueous two-phase extraction systems; Biological and medical sciences; Biotechnology; E. coli periplasm; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Ion exchange adsorption; Magnetic sorbents; Magnetics; Methods. Procedures. Technologies; Micelles; Nanoparticles; Non-ionic surfactants; Others; Proteins; Proteins - isolation & purification; Various methods and equipments; Water; Magnetic sorbents; Aqueous two-phase extraction systems; Antibody fragments; Non-ionic surfactants; E. coli periplasm; Ion exchange adsorption; Nanoparticle; Escherichia coli; Solvent extraction; Separation method; Membrane-oligosaccharide glycerophosphotransferase; Sample preparation; Aqueous medium; Bacteria; Fab'-Fragment; Enterobacteriaceae; Purification; Enzyme; Antibody; Liquid liquid extraction; Transferases; Non ionic surfactant; Protein; Pilot plant scale; Magnetic particles; Biphasic system; Adsorption; Magnetic separation; Micellar solution
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2013.06.011

•A novel large-scale protein separation process is described.•It is based on magnetic sorbents and aqueous micellar two-phase systems.•The process equipment is described and characterized.•The process is applied to a Fab α33 fermentation broth in a 15L scale.•Recycling of the phase forming surfactants and magnetic particles is possible. 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.