Multimeric fusion single-chain variable fragments as potential novel high-capacity ligands

Research output: Contribution to journalArticle

Abstract

In basic and applied biotechnology, design of affinity ligands has become essential for high-capacity applications such as affinity-based downstream processes for therapeutic molecules. Here, we established a proof-of-concept for the use of multimeric fusion single-chain variable fragment (scFvs) as high-capacity ligands in affinity adsorbents. Mono- and di/tri-scFvs separated by Pro-rich negatively charged linkers were designed, produced, and immobilized to 6% cross-linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL−1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high-capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications.

Details

Authors
  • Laila I. Sakhnini
  • Anja K. Pedersen
  • Maria B. Dainiak
  • Leif Bülow
Organisations
External organisations
  • Novo Nordisk A/S
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Organic Chemistry

Keywords

  • affinity chromatography, binding capacity, peptide linker, recombinant fusion protein, single-chain variable fragment
Original languageEnglish
Pages (from-to)507-514
Number of pages8
JournalFEBS Open Bio
Volume10
Issue number4
Early online date2020 Jan 17
Publication statusPublished - 2020 Apr
Publication categoryResearch
Peer-reviewedYes