Abstract

Research Article

Efficient Sequential Chromatographic Purification of a Recombinant Nanobody-Fc Fusion Designed for Treatment of Severe Fever with Thrombocytopenia Syndrome

Simona Serban*, Long Liu, Yan Liu, Xiaoju Lei, Cheng Zhang, Yanjun Li, Xiaokang Kou and Alessandra Basso

Published: 29 January, 2025 | Volume 9 - Issue 1 | Pages: 001-006

Severe fever with thrombocytopenia syndrome (SFTS) is caused by a virus that induces acute infections. Despite its expansion beyond China, where it first appeared in 2009, no specific drug exists to treat the disease. The discovery that antibodies targeting the SFTS virus surface glycoprotein (Glycoprotein N, GN) significantly enhance patient survival has driven the development of antibodies, particularly nanobodies. Nanobodies targeting the GN protein are a promising therapeutic approach. This paper presents a systematic study of the purification process for a recombinant nanobody-Fc fusion designed to treat the SFTS virus HB29. The study evaluated a sequential purification approach using affinity (AFF), ion exchange (IEC), and hydrophobic interaction chromatography (HIC) techniques to gradually remove impurities. The results demonstrate that this approach achieves an overall yield of more than 50% and a total purity of 95%. Efficient nanobody purification methods, as outlined here, can pave the way for novel treatments to manage this disease.

Read Full Article HTML DOI: 10.29328/journal.aac.1001053 Cite this Article Read Full Article PDF

Keywords:

Nanobody-Fc fusion; Chromatography; SFTS; Affinity chromatography; Protein A

References

  1. Casel MA, Park SJ, Choi YK. Severe fever with thrombocytopenia syndrome virus: emerging novel phlebovirus and their control strategy. Exp Mol Med. 2021;53:713–722. Available from: https://doi.org/10.1038/s12276-021-00610-1
  2. Ren X, Sun J, Kuang W, Yu F, Wang B, Wang Y, et al. A broadly protective antibody targeting glycoprotein Gn inhibits severe fever with thrombocytopenia syndrome virus infection. Nat Commun. 2024;15:7009–7023. Available from: https://www.nature.com/articles/s41467-024-51108-z
  3. Kim KH, Kim J, Ko M, Chun JY, Kim H, Kim S, et al. An anti-Gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus. PLOS Pathog. 2019;21:1–21. Available from: https://doi.org/10.1371/journal.ppat.1007375
  4. Xilin W, Zhiwei W, Yanlei L, Yi P. A SFTSV detection kit. CN 110684102A. 2019.
  5. Liu M, Li L, Jin D, Liu Y. Nanobody—A versatile tool for cancer diagnosis and therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2021;13:e1697. Available from: https://doi.org/10.1002/wnan.1697
  6. Salvador JP, Vilaplana L, Marco MP. Nanobody: outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem. 2019;411:1703–1713. Available from: https://doi.org/10.1007/s00216-019-01633-4
  7. Bannas P, Hambach J, Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol. 2017;8:1603. Available from: https://doi.org/10.3389/fimmu.2017.01603
  8. Jovčevska I, Muyldermans S. The Therapeutic Potential of Nanobodies. BioDrugs. 2020;34:11–26. Available from: https://doi.org/10.1007/s40259-019-00392-z
  9. Jin B, Odongo S, Radwanska M, Magez S. NANOBODIES: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci. 2023;24:5994. Available from: https://doi.org/10.3390/ijms24065994
  10. Stevens TA, Tomaleri GP, Hazu M, Wei S, Nguyen VN, et al. A nanobody-based strategy for rapid and scalable purification of human protein complexes. Nat Protoc. 2024;19:127–158. Available from: https://doi.org/10.1038/s41596-023-00904-w
  11. Haddad M, Soukkarieh C, Khalaf HE, Abbady AQ. Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant Nanobodies. Open Life Sci. 2016;11:1–9. Available from: https://doi.org/10.1515/biol-2016-0001
  12. Liu Q, Lu Y, Cai C, Huang Y, Zhou L, Guan Y, et al. A broad neutralizing nanobody against SARS-CoV-2 engineered from an approved drug. Cell Death Dis. 2024;15:458. Available from: https://www.nature.com/articles/s41419-024-06802-7
  13. Ji M, Hu J, Zhang D, Huang B, Xu S, Jiang N, et al. Inhibition of SFTSV replication in humanized mice by a subcutaneously administered anti-PD1 nanobody. EMBO Mol Med. 2024;16:575–595. Available from: https://doi.org/10.1038/s44321-024-00026-0
  14. Basso A, Serban S, Gu TN, Liu L, Li YJ. Innovation in mAb purification using affinity chromatography resins based on proprietary rProtein A. TWENTYFOURSEVENBIOPHARMA. 2024;1:66–68. Available from: https://247biopharma.com/article/innovation-in-mab-purification-using-affinity-chromatography-resins-based-on-proprietary-rprotein-a/
  15. Yanjun L, Gang L, Tongnian G, Jiantao Z, Long L, Xiaoju L, et al. Polypeptide, fusion type polymer protein and application thereof. CN115850408A. 2022.
  16. Chen T, Guo G, Tan G, Wang Y, Li Y. Antibody aggregate removal using a mixed-mode chromatography resin. Methods Mol Biol. 2021;2178:345–354. Available from: https://doi.org/10.1007/978-1-0716-0775-6_23
  17. Sakata M, Yamaguchi Y. Affinity chromatography removes endotoxins. BioPharm Int. 2005;18. Available from: https://www.biopharminternational.com/view/affinity-chormatography-removes-endotoxins
  18. Li Y. Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins. Protein Expr Purif. 2017;134:96–103. Available from: https://doi.org/10.1016/j.pep.2017.04.006
  19. Tang S, Tao J, Li Y. Challenges and solutions for the downstream purification of therapeutic proteins. Antibody Ther. 2024;7:1–12. Available from: https://doi.org/10.1093/abt/tbad028

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