Abstract
Efficient purification is crucial to providing large quantities of recombinant therapeutic proteins, such as monoclonal antibodies and cytokines. However, affinity techniques for manufacturing protein therapeutics that use biomolecule-conjugated agarose beads that harness specific biomolecular interactions suffer from issues related to protein denaturation, contamination and the need to maintain biomolecule-specific conditions for efficient protein capture. Here, we report a versatile and scalable method for the purification of recombinant protein therapeutics. The method exploits the high-affinity and controllable host–guest interactions between cucurbit[7]uril (CB[7]) and selected guests such as adamantylammonium. We show that the Herceptin (the brand name of trastuzumab, a monoclonal antibody drug used to treat breast cancer) and the much smaller cytokine interferon α-2a can be purified by site-specifically tagging them with adamantylammonium using the enzyme sortase A, followed by high-affinity binding with CB[7]-conjugated agarose beads and the recovery of the protein using a guest with a stronger affinity for CB[7]. The thermal and chemical stability of CB[7] beads and their scalability, recyclability and low cost may also make them advantageous for the manufacturing of biosimilars.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets are too numerous to be readily shared publicly but are available for research purposes from the corresponding author on reasonable request.
References
Strohl, W. R. & Knight, D. M. Discovery and development of biopharmaceuticals: current issues. Curr. Opin. Biotechnol. 20, 668–672 (2009).
Carter, P. J. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp. Cell Res. 317, 1261–1269 (2011).
Frokjaer, S. & Otzen, D. E. Protein drug stability: a formulation challenge. Nat. Rev. Drug Discov. 4, 298–306 (2005).
Roger, S. D. & Goldsmith, D. Biosimilars: it’s not as simple as cost alone. J. Clin. Pharm. Ther. 33, 459–464 (2008).
Jameel, F. & Hershenson, S. (eds). Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals (John Wiley & Sons, 2010).
Bhambure, R., Kumar, K. & Rathore, A. S. High-throughput process development for biopharmaceutical drug substances. Trends Biotechnol. 29, 127–135 (2011).
Camejo, R. R., McGrath, C. & Herings, R. A dynamic perspective on pharmaceutical competition, drug development and cost effectiveness. Health Policy 100, 18–24 (2011).
DiMasi, J. A. & Grabowski, H. G. The cost of biopharmaceutical R&D: is biotech different? Manage. Decis. Econ. 28, 469–479 (2007).
Ho, R. J. Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs (John Wiley & Sons, 2013).
Hanke, A. T. & Ottens, M. Purifying biopharmaceuticals: knowledge-based chromatographic process development. Trends Biotechnol. 32, 210–220 (2014).
Sekhon, B. S. Biopharmaceuticals: an overview. Thai J. Pharm. Sci. 34, 1–19 (2010).
Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).
Huse, K., Böhme, H.-J. & Scholz, G. H. Purification of antibodies by affinity chromatography. J. Biochem. Biophys. Methods 51, 217–231 (2002).
Svensson, H. G., Hoogenboom, H. R. & Sjöbring, U. Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties. Eur. J. Biochem. 258, 890–896 (1998).
Elgundi, Z., Reslan, M., Cruz, E., Sifniotis, V. & Kayser, V. The state-of-play and future of antibody therapeutics. Adv. Drug Deliv. Rev. 122, 2–19 (2017).
Ey, P., Prowse, S. J. & Jenkin, C. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A–sepharose. Immunochemistry 15, 429–436 (1978).
Duhamel, R. C., Schur, P. H., Brendel, K. & Meezan, E. pH gradient elution of human IgG1, IgG2 and IgG4 from protein A–sepharose. J. Immunol. Methods 31, 211–217 (1979).
Bloom, J. W., Wong, M. F. & Mitra, G. Detection and reduction of protein-A contamination in immobilized protein-A purified monoclonal-antibody preparations. J. Immunol. Methods 117, 83–89 (1989).
DePalma, A. Affinity labels for protein purification. Genet. Eng. Biotechnol. N. 35, 24–26 (2015).
Nfor, B. K. et al. Design strategies for integrated protein purification processes: challenges, progress and outlook. J. Chem. Technol. Biotechnol. 83, 124–132 (2008).
Sofer, G. K. & Hagel, L. Handbook of Process Chromatography: A Guide to Optimization, Scale Up, and Validation Vol. 1 (Academic Press, 1997).
Welte, K. et al. Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc. Natl Acad. Sci. USA 82, 1526–1530 (1985).
Block, H. et al. Immobilized-metal affinity chromatography (IMAC): a review. Methods Enzymol. 463, 439–473 (2009).
Bornhorst, J. A. & Falke, J. J. Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326, 245–254 (2000).
Müller, K. M., Arndt, K. M., Bauer, K. & Plückthun, A. Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins—evaluation of two anti-His-tag monoclonal antibodies. Anal. Biochem. 259, 54–61 (1998).
Khan, F., He, M. & Taussig, M. J. Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on Ni- nitrilotriacetic acid surfaces. Anal. Chem. 78, 3072–3079 (2006).
Kim, K., Murray, J., Selvapalam, N., Ko, Y. H. & Hwang, I. Cucurbiturils (World Scientific, 2018).
Barrow, S. J., Kasera, S., Rowland, M. J., del Barrio, J. & Scherman, O. A. Cucurbituril-based molecular recognition. Chem. Rev. 115, 12320–12406 (2015).
Isaacs, L. Stimuli responsive systems constructed using cucurbit[n]uril-type molecular containers. Acc. Chem. Res. 47, 2052–2062 (2014).
Assaf, K. I. & Nau, W. M. Cucurbiturils: from synthesis to high-affinity binding and catalysis. Chem. Soc. Rev. 44, 394–418 (2015).
Shetty, D., Khedkar, J. K., Park, K. M. & Kim, K. Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host–guest complexes and their applications. Chem. Soc. Rev. 44, 8747–8761 (2015).
Liu, S. et al. The cucurbit[n]uril family: prime components for self-sorting systems. J. Am. Chem. Soc. 127, 15959–15967 (2005).
Jeon, W. S. et al. Complexation of ferrocene derivatives by the cucurbit[7]uril host: a comparative study of the cucurbituril and cyclodextrin host families. J. Am. Chem. Soc. 127, 12984–12989 (2005).
Cao, L. et al. Cucurbit[7]uril-guest pair with an attomolar dissociation constant. Angew. Chem. Int. Ed. 53, 988–993 (2014).
Sigwalt, D. et al. Unraveling the structure–affinity relationship between cucurbit[n]urils (n= 7, 8) and cationic diamondoids. J. Am. Chem. Soc. 139, 3249–3258 (2017).
Li, M. et al. Autophagy caught in the act: a supramolecular FRET pair based on an ultrastable synthetic host–guest complex visualizes autophagosome-lysosome fusion. Angew. Chem. Int. Ed. 57, 2120–2125 (2018).
Kim, K. L. et al. Supramolecular latching system based on ultrastable synthetic binding pairs as versatile tools for protein imaging. Nat. Commun. 9, 1712 (2018).
Lee, D.-W. et al. Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host–guest binding pair. Nat. Chem. 3, 154–159 (2011).
Park, K. M., Murray, J. & Kim, K. Ultrastable artificial binding pairs as a supramolecular latching system: a next generation chemical tool for proteomics. Acc. Chem. Res. 50, 644–646 (2017).
Murray, J. et al. Enrichment of specifically labeled proteins by an immobilized host molecule. Angew. Chem. Int. Ed. 56, 2395–2398 (2017).
Li, M. et al. Bio-orthogonal supramolecular latching inside live animals and its application for in vivo cancer imaging. ACS Appl. Mater. Inter. 11, 43920–43927 (2019).
Ayhan, M. M. et al. Comprehensive synthesis of monohydroxy–cucurbit[n]urils (n = 5, 6, 7, 8): high purity and high conversions. J. Am. Chem. Soc. 137, 10238–10245 (2015).
Miskolczy, Z. & Biczók, L. Kinetics and thermodynamics of berberine inclusion in cucurbit[7]uril. J. Phys. Chem. B 118, 2499–2505 (2014).
Sung, G. et al. Supra-blot: an accurate and reliable assay for detecting target proteins with a synthetic host molecule–enzyme hybrid. Chem. Commun. 56, 1549–1552 (2020).
Ghosh, S. K. et al. Superacid-mediated functionalization of hydroxylated cucurbit[n]urils. J. Am. Chem. Soc. 141, 17503–17506 (2019).
Guimaraes, C. P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1787–1799 (2013).
Hou, Y. Q., Yuan, J. S., Zhou, Y., Yu, J. & Lu, H. A concise approach to site-specific topological protein-poly(amino acid) conjugates enabled by in situ-generated functionalities. J. Am. Chem. Soc. 138, 10995–11000 (2016).
Bonam, S. R., Partidos, C. D., Halmuthur, S. K. M. & Muller, S. An overview of novel adjuvants designed for improving vaccine efficacy. Trends Pharmacol. Sci. 38, 771–793 (2017).
Tundup, S., Srivastava, L., Nagy, T. & Harn, D. CD14 influences host immune responses and alternative activation of macrophages during Schistosoma mansoni infection. Infect. Immun. 82, 3240–3251 (2014).
da Silva, T. A. et al. CD14 is critical for TLR2-mediated M1 macrophage activation triggered by N-glycan recognition. Sci. Rep. 7, 7083 (2017).
Hawe, A., Kasper, J. C., Friess, W. & Jiskoot, W. Structural properties of monoclonal antibody aggregates induced by freeze–thawing and thermal stress. Eur. J. Pharm. Sci. 38, 79–87 (2009).
Carrington, J. C. & Dougherty, W. G. A viral cleavage site cassette—identification of amino-acid sequences required for tobacco etch virus polyprotein processing. Proc. Natl Acad. Sci. USA 85, 3391–3395 (1988).
Tisminetzky, G. S. & Baralle, F. E. Process for the production of alpha interferon of therapeutical degree. European Patent Application EP1310559A1 (2003).
Acknowledgements
This work was supported by the Institute for Basic Science (no. IBS-R007-D1). We thank Y. T. Chang and S. H. Ryu for support with THP-1 cells and commercial Herceptin, respectively.
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K.M.P. and K.K. conceived and supervised the project. J.A. and S.K. performed all the protein experiments. A.S. synthesized and analysed CB[7] beads. J.K. performed protein experiments, G.S. synthesized CB[7]–HRP. J.A. and H.B. synthesized adamantane derivatives. J.A., S.K., A.S., J.K., K.M.P. and K.K. wrote the manuscript.
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An, J., Kim, S., Shrinidhi, A. et al. Purification of protein therapeutics via high-affinity supramolecular host–guest interactions. Nat Biomed Eng 4, 1044–1052 (2020). https://doi.org/10.1038/s41551-020-0589-7
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DOI: https://doi.org/10.1038/s41551-020-0589-7