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Biotechnol Bioeng. 2016 Jan;113(1):130-40. doi: 10.1002/bit.25705. Epub 2015 Sep 3.

IMAC capture of recombinant protein from unclarified mammalian cell feed streams.

Author information

1
Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK.
2
Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
3
Department of Oncology, University College London, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK. k.chester@ucl.ac.uk.

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.

KEYWORDS:

Chinese hamster ovary cells; IMAC; downstream processing; integrated processing; radial flow chromatography; recombinant protein

PMID:
26174988
PMCID:
PMC4737217
DOI:
10.1002/bit.25705
[Indexed for MEDLINE]
Free PMC Article

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