C7 Protein Purification with Functionalized Magnetic Nanoparticles
One of the greatest challenges in biotechnology remains the downstream processing and the selective and efficient purification of the desired products. A possible solution, as an alternative to chromatographic methods, can be magnetic separation, in which the products are extracted directly from the culture medium using functionalised magnetic nanoparticles.
The SFB project C7 is particularly concerned with purification of the recombinant proteins produced in the associated sub-projects, namely antibodies and glycosyltransferases. We seek to develop a highly efficient, selective, reusable and cost-effective system based upon the principle of magnetic separation. Here at the Institute for Particle Technology we have developed a non-aqueous method for the synthesis of magnetic iron oxide nanoparticles (MNPs). This allows the controlled synthesis of particles with specified properties, in particular, uniform magnetic properties.
Iron oxide nanoparticles display a number of properties not observed in their bulk solid counterpart. The most interesting of these properties is room temperature superparamagnetism for nanoparticles of less than 30 nm: due to the presence of only a single magnetic domain fluctuating at room temperature, the particles appear paramagnetic, but the domains are oriented by an external field and thus the particles can be manipulated.
We plan to exploit this behaviour in the purification of biological macromolecules, as in the absence of a magnetic field the particles form stable dispersions, giving an extremely high surface area for product binding. Upon the application of an external magnetic field, the particles are then held in place, allowing unbound impurities to be washed away. Removal of the magnetic field and un-binding of the product complex then affords the purified macromolecule and the magnetic particles ready for reuse.
This strategy requires magnetic particles of small size (10-20 nm) that are appropriately functionalised to provide specific binding of target substances and allow dispersions in water to form. We aim to realize a modular system of functionalization in order to utilize highly specific interactions for optimum purification.
At the Institute of Electrical Measurement and Fundamental Electrical Engineering a magnetic column is being designed and built to effect the efficient separation of particles from biological impurities. The gradient strength, magnetic field strength, length of column and solvent flow rate must be optimised. Thereby the purification selectivity and regeneration efficiency can be further enhanced for optimum results.