Get access

Design, synthesis, and assessment of a de novo affinity adsorbent for the purification of recombinant human erythropoietin

Authors

  • Graziella El Khoury,

    Corresponding author
    1. Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom
    • Correspondence to: G. El Khoury

    Search for more papers by this author
  • Yian Wang,

    1. Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom
    Search for more papers by this author
  • Di Wang,

    1. Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom
    Search for more papers by this author
  • Shaleem I. Jacob,

    1. Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom
    Search for more papers by this author
  • Christopher R. Lowe

    1. Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom
    Search for more papers by this author

ABSTRACT

This work describes the assessment of a de novo synthetic affinity ligand for recombinant human erythropoietin (rHuEPO), based on the multicomponent Ugi reaction. Four Ugi ligands were designed based on the X-ray crystallographic structure of the complex between human erythropoietin and site 1 of its cell-surface receptor (EPObp)2; screening of the ligands with pure rHuEPO samples identified a lead ligand (A9C10I8) immobilized on aldehyde-functionalized agarose beads, which was able to bind and elute erythropoietin, as determined by SDS–PAGE and Western blot analyses. Furthermore, small-scale affinity chromatography performed on the immobilized adsorbent showed its ability to isolate rHuEPO from a spiked mammalian cell supernatant with a purity of ∼80%, as estimated with gel densitometry. This approach could lead to the development of a cost-effective downstream process for rHuEPO, as an alternative to the current multi-step purification protocols. Biotechnol. Bioeng. 2013;110: 3063–3069. © 2013 Wiley Periodicals, Inc.

Ancillary