Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Purification of protein therapeutics via high-affinity supramolecular host–guest interactions


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: CB[7]-based affinity chromatography for purification of therapeutic proteins.
Fig. 2: Purification of AdA–Herceptin by CB[7] beads affinity purification.
Fig. 3: Comparison of monoclonal antibody recovery between CB[7] beads and protein A beads.
Fig. 4: Regeneration of CB[7] beads for affinity purification of Herceptin.
Fig. 5: Scalability and sterilizability of CB[7] beads for monoclonal antibody affinity purification.
Fig. 6: Purification of a non-monoclonal antibody target protein using CB[7] beads and regeneration of CB[7] beads for non-monoclonal antibody affinity purification.

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.


  1. 1.

    Strohl, W. R. & Knight, D. M. Discovery and development of biopharmaceuticals: current issues. Curr. Opin. Biotechnol. 20, 668–672 (2009).

    CAS  Google Scholar 

  2. 2.

    Carter, P. J. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp. Cell Res. 317, 1261–1269 (2011).

    CAS  Google Scholar 

  3. 3.

    Frokjaer, S. & Otzen, D. E. Protein drug stability: a formulation challenge. Nat. Rev. Drug Discov. 4, 298–306 (2005).

    CAS  Google Scholar 

  4. 4.

    Roger, S. D. & Goldsmith, D. Biosimilars: it’s not as simple as cost alone. J. Clin. Pharm. Ther. 33, 459–464 (2008).

    CAS  Google Scholar 

  5. 5.

    Jameel, F. & Hershenson, S. (eds). Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals (John Wiley & Sons, 2010).

  6. 6.

    Bhambure, R., Kumar, K. & Rathore, A. S. High-throughput process development for biopharmaceutical drug substances. Trends Biotechnol. 29, 127–135 (2011).

    CAS  Google Scholar 

  7. 7.

    Camejo, R. R., McGrath, C. & Herings, R. A dynamic perspective on pharmaceutical competition, drug development and cost effectiveness. Health Policy 100, 18–24 (2011).

    Google Scholar 

  8. 8.

    DiMasi, J. A. & Grabowski, H. G. The cost of biopharmaceutical R&D: is biotech different? Manage. Decis. Econ. 28, 469–479 (2007).

    Google Scholar 

  9. 9.

    Ho, R. J. Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs (John Wiley & Sons, 2013).

  10. 10.

    Hanke, A. T. & Ottens, M. Purifying biopharmaceuticals: knowledge-based chromatographic process development. Trends Biotechnol. 32, 210–220 (2014).

    CAS  Google Scholar 

  11. 11.

    Sekhon, B. S. Biopharmaceuticals: an overview. Thai J. Pharm. Sci. 34, 1–19 (2010).

    CAS  Google Scholar 

  12. 12.

    Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    CAS  Google Scholar 

  13. 13.

    Huse, K., Böhme, H.-J. & Scholz, G. H. Purification of antibodies by affinity chromatography. J. Biochem. Biophys. Methods 51, 217–231 (2002).

    CAS  Google Scholar 

  14. 14.

    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).

    CAS  Google Scholar 

  15. 15.

    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).

    CAS  Google Scholar 

  16. 16.

    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).

    CAS  Google Scholar 

  17. 17.

    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).

    CAS  Google Scholar 

  18. 18.

    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).

    CAS  Google Scholar 

  19. 19.

    DePalma, A. Affinity labels for protein purification. Genet. Eng. Biotechnol. N. 35, 24–26 (2015).

    Google Scholar 

  20. 20.

    Nfor, B. K. et al. Design strategies for integrated protein purification processes: challenges, progress and outlook. J. Chem. Technol. Biotechnol. 83, 124–132 (2008).

    CAS  Google Scholar 

  21. 21.

    Sofer, G. K. & Hagel, L. Handbook of Process Chromatography: A Guide to Optimization, Scale Up, and Validation Vol. 1 (Academic Press, 1997).

  22. 22.

    Welte, K. et al. Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc. Natl Acad. Sci. USA 82, 1526–1530 (1985).

    CAS  Google Scholar 

  23. 23.

    Block, H. et al. Immobilized-metal affinity chromatography (IMAC): a review. Methods Enzymol. 463, 439–473 (2009).

    CAS  Google Scholar 

  24. 24.

    Bornhorst, J. A. & Falke, J. J. Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326, 245–254 (2000).

    CAS  Google Scholar 

  25. 25.

    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).

    Google Scholar 

  26. 26.

    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).

    CAS  Google Scholar 

  27. 27.

    Kim, K., Murray, J., Selvapalam, N., Ko, Y. H. & Hwang, I. Cucurbiturils (World Scientific, 2018).

  28. 28.

    Barrow, S. J., Kasera, S., Rowland, M. J., del Barrio, J. & Scherman, O. A. Cucurbituril-based molecular recognition. Chem. Rev. 115, 12320–12406 (2015).

    CAS  Google Scholar 

  29. 29.

    Isaacs, L. Stimuli responsive systems constructed using cucurbit[n]uril-type molecular containers. Acc. Chem. Res. 47, 2052–2062 (2014).

    CAS  Google Scholar 

  30. 30.

    Assaf, K. I. & Nau, W. M. Cucurbiturils: from synthesis to high-affinity binding and catalysis. Chem. Soc. Rev. 44, 394–418 (2015).

    CAS  Google Scholar 

  31. 31.

    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).

    CAS  Google Scholar 

  32. 32.

    Liu, S. et al. The cucurbit[n]uril family: prime components for self-sorting systems. J. Am. Chem. Soc. 127, 15959–15967 (2005).

    CAS  Google Scholar 

  33. 33.

    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).

    CAS  Google Scholar 

  34. 34.

    Cao, L. et al. Cucurbit[7]uril-guest pair with an attomolar dissociation constant. Angew. Chem. Int. Ed. 53, 988–993 (2014).

    CAS  Google Scholar 

  35. 35.

    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).

    CAS  Google Scholar 

  36. 36.

    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).

    CAS  Google Scholar 

  37. 37.

    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).

    Google Scholar 

  38. 38.

    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).

    CAS  Google Scholar 

  39. 39.

    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).

    CAS  Google Scholar 

  40. 40.

    Murray, J. et al. Enrichment of specifically labeled proteins by an immobilized host molecule. Angew. Chem. Int. Ed. 56, 2395–2398 (2017).

    CAS  Google Scholar 

  41. 41.

    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).

    CAS  Google Scholar 

  42. 42.

    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).

    CAS  Google Scholar 

  43. 43.

    Miskolczy, Z. & Biczók, L. Kinetics and thermodynamics of berberine inclusion in cucurbit[7]uril. J. Phys. Chem. B 118, 2499–2505 (2014).

    CAS  Google Scholar 

  44. 44.

    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).

    CAS  Google Scholar 

  45. 45.

    Ghosh, S. K. et al. Superacid-mediated functionalization of hydroxylated cucurbit[n]urils. J. Am. Chem. Soc. 141, 17503–17506 (2019).

    CAS  Google Scholar 

  46. 46.

    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).

    Google Scholar 

  47. 47.

    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).

    CAS  Google Scholar 

  48. 48.

    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).

    CAS  Google Scholar 

  49. 49.

    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).

    Google Scholar 

  50. 50.

    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).

    Google Scholar 

  51. 51.

    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).

    CAS  Google Scholar 

  52. 52.

    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).

    CAS  Google Scholar 

  53. 53.

    Tisminetzky, G. S. & Baralle, F. E. Process for the production of alpha interferon of therapeutical degree. European Patent Application EP1310559A1 (2003).

Download references


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.

Author information




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.

Corresponding authors

Correspondence to Kyeng Min Park or Kimoon Kim.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, figures and tables.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing