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

Abstract 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, lead...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Krištof Bozovičar, Barbara Jenko Bizjan, Anže Meden, Jernej Kovač, Tomaž Bratkovič
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/034316d8ed774a0db32666981738785c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:034316d8ed774a0db32666981738785c
record_format	dspace
spelling	oai:doaj.org-article:034316d8ed774a0db32666981738785c2021-12-02T17:50:57ZFocused peptide library screening as a route to a superior affinity ligand for antibody purification10.1038/s41598-021-91208-02045-2322https://doaj.org/article/034316d8ed774a0db32666981738785c2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91208-0https://doaj.org/toc/2045-2322Abstract 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.Krištof BozovičarBarbara Jenko BizjanAnže MedenJernej KovačTomaž BratkovičNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Krištof Bozovičar Barbara Jenko Bizjan Anže Meden Jernej Kovač Tomaž Bratkovič Focused peptide library screening as a route to a superior affinity ligand for antibody purification
description	Abstract 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.
format	article
author	Krištof Bozovičar Barbara Jenko Bizjan Anže Meden Jernej Kovač Tomaž Bratkovič
author_facet	Krištof Bozovičar Barbara Jenko Bizjan Anže Meden Jernej Kovač Tomaž Bratkovič
author_sort	Krištof Bozovičar
title	Focused peptide library screening as a route to a superior affinity ligand for antibody purification
title_short	Focused peptide library screening as a route to a superior affinity ligand for antibody purification
title_full	Focused peptide library screening as a route to a superior affinity ligand for antibody purification
title_fullStr	Focused peptide library screening as a route to a superior affinity ligand for antibody purification
title_full_unstemmed	Focused peptide library screening as a route to a superior affinity ligand for antibody purification
title_sort	focused peptide library screening as a route to a superior affinity ligand for antibody purification
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/034316d8ed774a0db32666981738785c
work_keys_str_mv	AT kristofbozovicar focusedpeptidelibraryscreeningasaroutetoasuperioraffinityligandforantibodypurification AT barbarajenkobizjan focusedpeptidelibraryscreeningasaroutetoasuperioraffinityligandforantibodypurification AT anzemeden focusedpeptidelibraryscreeningasaroutetoasuperioraffinityligandforantibodypurification AT jernejkovac focusedpeptidelibraryscreeningasaroutetoasuperioraffinityligandforantibodypurification AT tomazbratkovic focusedpeptidelibraryscreeningasaroutetoasuperioraffinityligandforantibodypurification
_version_	1718379268320264192

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

Ejemplares similares