IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Abstract

Fusion‐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.

Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

“…For a method/process to be of industrial relevance, the polishing of the target macromolecule being produced and purified is a crucial step. In this case, and considering the potential application of the biopharmaceutical in the ALL treatment, the elimination of the (His) 6 ‐tag from the biopharmaceutical can be achieved through the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the (His) 6 ‐tag is cleaved off by DPPI, which catalyses a stepwise excision of a wide range of dipeptides from the N‐terminus of a peptide chain .…”

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…Protein A-ligand-based affinity chromatography is the most robust, efficient, and prevalently used mAb capture technology, owing to its selectivity and high affinity to various human Fc, as well as its efficient dissociation of captured mAbs at a low pH for reuse. Fragment mAbs and recombinant formats based on Fabs and single chain variable fragments (scFv) are unable to take advantage of protein A affinity capture, and alternatives have been developed, such as capturing proteins G, M, and L, which bind at different mAb epitopes; ion exchange chromatography; and polyhistidine tagged capture through immobilized metal chromatography [100,101,102]. A further advantage of affinity chromatography in manufacture is the integration of a viral inactivation step by holding the low pH-eluted mAb product prior to further purification steps; however, this applies the mAb product to low pH at high concentrations, which can induce mild mAb instability, aggregation, and the formation of acidic variants [5,22,100,103,104].…”

Role of lipoprotein structure and dynamics in disease development: from atherosclerosis to Covid-19

“…The Histidine residues in the tag form coordinate bonds with the metal ions, allowing the protein to bind tightly to the column. After washing away unbound proteins and contaminants, the Histag-tagged protein can be eluted from the column by using a competitive agent such as imidazole that disrupts the histidine-metal ion interaction 137 .…”