title: 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.
authors.affiliation: Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK.|Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kinna
authors.affiliation: Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK.
authors.forename: Berend
authors.initials: B
authors.lastname: Tolner
authors.affiliation: Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK.
authors.forename: Enrique Miranda
authors.initials: EM
authors.lastname: Rota
authors.affiliation: Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Nigel
authors.initials: N
authors.lastname: Titchener-Hooker
authors.affiliation: Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Nesbeth
authors.affiliation: Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK. k.chester@ucl.ac.uk.
authors.forename: Kerry
authors.initials: K
authors.lastname: Chester
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000302:isolation & purification / Q000378:metabolism; D002470:Cell Survival; D002846:Chromatography, Affinity / Q000379:methods*; D003412:Cricetulus; D008863:Microspheres; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism
chemical_list: D002272:Carcinoembryonic Antigen; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride
keywords: Chinese hamster ovary cells; IMAC; downstream processing; integrated processing; radial flow chromatography; recombinant protein