Abstract
ABSTRACTFusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc.
Highlights
Expression of recombinant proteins in yeasts, bacteria, and mammalian cells is routine practice at industrial scale
Having established that His-tagged proteins could be recovered from buffered media, we focused on recovery of the target protein after expression by Chinese Hamster Ovary (CHO)-carcinoembryonic antigen (CEA) cells
Once the optimum buffer composition had been chosen, we investigated the effect of media clarification on His-tagged CEA recovery in the radial flow column and compared this with recovery from a commercial axial flow column (HisTrap)
Summary
Expression of recombinant proteins in yeasts, bacteria, and mammalian cells is routine practice at industrial scale. In the past a substantial percentage of these costs derived from fermentation, whereas recently production costs have become more heavily weighted toward downstream processing (Gonzalez et al, 2003). This is largely due to improvements in specific productivity, better feeding strategies, and protein engineering; combining to produce cell densities in excess of 1 Â 107 cells/mL and yields of 8 g/L (Farid, 2007; Hacker et al, 2009). These advances have in turn created new challenges for downstream processing, leading to a focus on developing high capacity methods for the primary capture of protein from high cell density feeds
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