Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid.

Autor: Prabhala SV; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, United States., Marshall B; Eli Lilly and Company, Indianapolis, IN 46285, United States., Galiardi J; Pfizer Inc., Chesterfield, MO 63017, United States., Fan Y; Johnson & Johnson, 4560 Jinke Road, Shanghai 201210, China., Creamer E; Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, United States., Wood DW; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, United States; Protein Capture Science LLC, Columbus, OH 43212, United States. Electronic address: wood.750@osu.edu.
Jazyk: angličtina
Zdroj: Journal of chromatography. A [J Chromatogr A] 2024 Nov 08; Vol. 1736, pp. 465430. Date of Electronic Publication: 2024 Oct 09.
DOI: 10.1016/j.chroma.2024.465430
Abstrakt: Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTag™ affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTag™ for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99 %) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTag™ as a universal capture platform for diverse classes of recombinant therapeutic proteins.
Competing Interests: Declaration of competing interest DWW has recently founded a start-up company, Protein Capture Science, to commercialize intein-based purification resins, including the iCapTag™ system.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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