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

Bozovičar, Krištof (author), Jenko Bizjan, Barbara (author), Meden, Anže (author), Kovač, Jernej (author), Bratkovič, Tomaž (author)

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.

• Language: English
• Keywords: biochemistry, biophysics, biotechnology, chemical biology
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2021
• Number of pages: 12 str.
• Numbering: Vol. 11, art. 11650
• PID: 20.500.12556/RUL-145024
• UDC: 543.544+577.27
• ISSN on article: 2045-2322
• DOI: 10.1038/s41598-021-91208-0
• COBISS.SI-ID: 65576707
• Publication date in RUL: 30.03.2023

Record is a part of a journal

• Title: Scientific reports
• Shortened title: Sci. rep.
• Publisher: Nature Publishing Group
• ISSN: 2045-2322
• COBISS.SI-ID: 18727432

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: peptidne knjižnice, ELISA, imunoglobulini, kromatografija, analizna kemija

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0127
• Name: Farmacevtska biotehnologija: znanost za zdravje

• Funder: Other - Other funder or multiple funders
• Funding programme: University of Ljubljana Innovation Fund
• Project number: 820-1/2020-33