Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids

Ryan Kilgore; Wenning Chu; Dipendra Bhandari; David Fischler; Ruben G Carbonell; Michael Crapanzano; Stefano Menegatti

doi:10.1016/j.chroma.2022.463701

Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids

J Chromatogr A. 2023 Jan 4:1687:463701. doi: 10.1016/j.chroma.2022.463701. Epub 2022 Dec 5.

Authors

Ryan Kilgore¹, Wenning Chu¹, Dipendra Bhandari², David Fischler², Ruben G Carbonell³, Michael Crapanzano², Stefano Menegatti⁴

Affiliations

¹ Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States.
² LigaTrap Technologies, 1791 Varsity Dr., Suite 150, Raleigh, NC 27606, United States.
³ Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695, United States.
⁴ Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States; LigaTrap Technologies, 1791 Varsity Dr., Suite 150, Raleigh, NC 27606, United States; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695, United States. Electronic address: smenega@ncsu.edu.

PMID: 36502645
DOI: 10.1016/j.chroma.2022.463701

Abstract

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the C_H1-3 and C_L chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Q_max ∼ 20 mg of Fab per mL of resin) and dissociation constant (K_D = 2.16·10^-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC_10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC_10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.

Keywords: Affinity chromatography; Cell culture harvests; Fragment antigen binding; Monoclonal antibodies; Peptide ligands.

MeSH terms

Animals
Antibodies, Monoclonal*
CHO Cells
Chromatography, Affinity
Cricetinae
Cricetulus
Humans
Ligands
Molecular Docking Simulation
Peptides* / chemistry
Protein Binding

Substances

Ligands
Peptides
Antibodies, Monoclonal