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

Nature Biomedical Engineering volume 4, pages 1044–1052 (2020)Cite this article

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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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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.

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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.

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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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Author notes

  1. These authors contributed equally: Jaeyeon An, Sungwan Kim.

Authors and Affiliations

  1. Center for Self-Assembly and Complexity (CSC), Institute for Basic Science (IBS), Pohang, Republic of Korea

    Jaeyeon An, Sungwan Kim, Annadka Shrinidhi, Junghyun Kim, Gihyun Sung, Kyeng Min Park & Kimoon Kim

  2. Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea

    Jaeyeon An & Kimoon Kim

  3. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea

    Hasanul Banna & Kimoon Kim

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Contributions

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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Correspondence to Kyeng Min Park or Kimoon Kim.

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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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