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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2019
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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. |
format | Online Article Text |
id | pubmed-6816383 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
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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