Focused peptide library screening as a route to a superior affinity ligand for antibody purification

Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to li...

Full description

Saved in:
Bibliographic Details
Published in:Scientific reports 2021-06, Vol.11 (1), p.11650-11650, Article 11650
Main Authors: Bozovičar, Krištof, Jenko Bizjan, Barbara, Meden, Anže, Kovač, Jernej, Bratkovič, Tomaž
Format: Article
Language:English
Subjects:
631/45
631/57
631/61
631/92
Affinity
Affinity chromatography
Antibodies
Denaturation
Developmental stages
Directed evolution
Humanities and Social Sciences
Immunoglobulin G
Leaching
Libraries
Ligands
multidisciplinary
Next-generation sequencing
Peptides
Phages
Protein A
Science
Science (multidisciplinary)
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-021-91208-0