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Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for...

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Autores principales: Ollier, Romain, Wassmann, Paul, Monney, Thierry, Ries Fecourt, Christelle, Gn, Sunitha, C A, Vinu, Ayoub, Daniel, Stutz, Cian, Gudi, Girish S, Blein, Stanislas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816383/
https://www.ncbi.nlm.nih.gov/pubmed/31462177
http://dx.doi.org/10.1080/19420862.2019.1660564
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author Ollier, Romain
Wassmann, Paul
Monney, Thierry
Ries Fecourt, Christelle
Gn, Sunitha
C A, Vinu
Ayoub, Daniel
Stutz, Cian
Gudi, Girish S
Blein, Stanislas
author_facet Ollier, Romain
Wassmann, Paul
Monney, Thierry
Ries Fecourt, Christelle
Gn, Sunitha
C A, Vinu
Ayoub, Daniel
Stutz, Cian
Gudi, Girish S
Blein, Stanislas
author_sort Ollier, Romain
collection PubMed
description Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.
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spelling pubmed-68163832019-11-05 Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development Ollier, Romain Wassmann, Paul Monney, Thierry Ries Fecourt, Christelle Gn, Sunitha C A, Vinu Ayoub, Daniel Stutz, Cian Gudi, Girish S Blein, Stanislas MAbs Report Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented. Taylor & Francis 2019-09-12 /pmc/articles/PMC6816383/ /pubmed/31462177 http://dx.doi.org/10.1080/19420862.2019.1660564 Text en © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
spellingShingle Report
Ollier, Romain
Wassmann, Paul
Monney, Thierry
Ries Fecourt, Christelle
Gn, Sunitha
C A, Vinu
Ayoub, Daniel
Stutz, Cian
Gudi, Girish S
Blein, Stanislas
Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title_full Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title_fullStr Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title_full_unstemmed Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title_short Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development
title_sort single-step protein a and protein g avidity purification methods to support bispecific antibody discovery and development
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816383/
https://www.ncbi.nlm.nih.gov/pubmed/31462177
http://dx.doi.org/10.1080/19420862.2019.1660564
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