High-gradient magnetic separation for technical scale protein recovery using low cost magnetic nanoparticles

Downstream processing still lacks efficient and integrated separation techniques. We present a high-gradient magnetic separation (HGMS) process for the successful purification of recombinant histidine-tagged Green Fluorescent Protein (His-GFP) from an E. coli cell lysate by means of superparamagnetic iron oxide nanoparticles functionalized with a pentadentate chelate ligand. The separator is an improved rotor stator prototype with 1 L chamber volume. Using 100 g of carrier, a purification performance of approx. 12 g His-GFP per hour could be achieved with an eluate purity of 96% and a yield of 93% for the whole process. We demonstrate how varying processing parameters enhances the final results and provide evidence of the potential of HGMS to become a real alternative to conventional downstream processes. These pilot scale experiments show that the combination of high performance nanocarriers and optimized separator design offers an attractive system for technical implementation. Almost no nanoparticle loss took place during the experiments. The demonstration of liter scale processing with nanoparticles is important because, due to their higher surface-to-volume ratio in comparison to microparticles, it is now possible to achieve higher capacities. Furthermore, the applied nanoparticles can be seen as low-cost, very stable carriers compared to common polymer microparticles.