Abstract B129: Novel miniprep system for rapid purification of recombinant proteins and antibodies on high capacity membranes

Recombinant protein production and purification is critical in a wide range of research settings including academic research institutions, biopharmaceutical organizations, and enzyme and agricultural industries. Protein fusion tags are widely used, in order to improve yields and enable purification and characterization of protein structure and function. Currently the most popular tag used for purification of proteins is the polyhistidine tag, which incorporates 6-10 histidine residues at either the N or C terminal of proteins. The polyhistidine tag binds specifically to immobilized metal ions such as Co2+, Ni2+, Cu2+ or Zn2+ allowing selective retention of the protein of interest to the matrix while removing unbound materials. The purified protein is then eluted under reduced pH or using imidazole-containing buffers. Typical purification methods using immobilized metal affinity chromatography (IMAC) columns require several hours to complete due to long column equilibration/binding times and slow diffusion of large macromolecules through the resin bed. Longer times in turn increase the possibility of protein degradation due to proteases, or loss of activity due to unfolding or denaturation. Membrane-based metal affinity systems have rapid flow induced mass transport, and short residence times; however, they have been plagued with low capacity due to small internal surface areas. Here, we describe a novel, nylon membrane based IMAC system which has protein binding capacity comparable to or better than resins at 75 mg or more per cm3 of membrane, due to chemically-enhanced surface area of pores. However, unlike traditional resin based systems, the entire purification process from loading the lysate to pure protein can be completed at room temperature in less than 5 minutes. We have assembled these membranes into spin columns and filtration devices such as 96 well plates, and demonstrate that the spin columns can purify his-tagged proteins produced in both bacterial and mammalian cells. These membranes function perfectly in the presence of additives such as glycerol and EDTA, reducing agents such as DTT, BME and TCEP, and under denaturing conditions (urea and guanidinium hydrochloride). Furthermore, we have now extended the high capacity membrane technology to immobilize proteins A and G, which allow extremely fast purification of antibodies from various matrices based on the affinity of these proteins for the Fc region of antibodies. The antibody purification can be accomplished in less than 10 minutes from start to finish, and with capacities up to 50 mg/mL. These novel membrane-based spinnable affinity columns will be useful for purifying a variety of recombinant proteins and antibodies in academic and industrial settings. Citation Format: Sayantan Mitra, Mike Vierra, Boris Levitan, Gia Jokadze, Andrew Farmer. Novel miniprep system for rapid purification of recombinant proteins and antibodies on high capacity membranes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B129.