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

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 2 μm diameter to as small as 25 nm) 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 15 L 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.