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

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

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.

• Jezik: Angleški jezik
• Ključne besede: biochemistry, biophysics, biotechnology, chemical biology
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: FFA - Fakulteta za farmacijo
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2021
• Št. strani: 12 str.
• Številčenje: Vol. 11, art. 11650
• PID: 20.500.12556/RUL-145024
• UDK: 543.544+577.27
• ISSN pri članku: 2045-2322
• DOI: 10.1038/s41598-021-91208-0
• COBISS.SI-ID: 65576707
• Datum objave v RUL: 30.03.2023

Gradivo je del revije

• Naslov: Scientific reports
• Skrajšan naslov: Sci. rep.
• Založnik: Nature Publishing Group
• ISSN: 2045-2322
• COBISS.SI-ID: 18727432

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: peptidne knjižnice, ELISA, imunoglobulini, kromatografija, analizna kemija

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P4-0127
• Naslov: Farmacevtska biotehnologija: znanost za zdravje

• Financer: Drugi - Drug financer ali več financerjev
• Program financ.: University of Ljubljana Innovation Fund
• Številka projekta: 820-1/2020-33