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An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates

The XpressCF+(®) cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of...

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Autores principales: Hanson, Jeffrey, Groff, Dan, Carlos, Abi, Usman, Hans, Fong, Kevin, Yu, Abigail, Armstrong, Stephanie, Dwyer, Allison, Masikat, Mary Rose, Yuan, Dawei, Tran, Cuong, Heibeck, Tyler, Zawada, James, Chen, Rishard, Hallam, Trevor, Yin, Gang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10045668/
https://www.ncbi.nlm.nih.gov/pubmed/36978695
http://dx.doi.org/10.3390/bioengineering10030304
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author Hanson, Jeffrey
Groff, Dan
Carlos, Abi
Usman, Hans
Fong, Kevin
Yu, Abigail
Armstrong, Stephanie
Dwyer, Allison
Masikat, Mary Rose
Yuan, Dawei
Tran, Cuong
Heibeck, Tyler
Zawada, James
Chen, Rishard
Hallam, Trevor
Yin, Gang
author_facet Hanson, Jeffrey
Groff, Dan
Carlos, Abi
Usman, Hans
Fong, Kevin
Yu, Abigail
Armstrong, Stephanie
Dwyer, Allison
Masikat, Mary Rose
Yuan, Dawei
Tran, Cuong
Heibeck, Tyler
Zawada, James
Chen, Rishard
Hallam, Trevor
Yin, Gang
author_sort Hanson, Jeffrey
collection PubMed
description The XpressCF+(®) cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50–100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+(®) reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.
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spelling pubmed-100456682023-03-29 An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates Hanson, Jeffrey Groff, Dan Carlos, Abi Usman, Hans Fong, Kevin Yu, Abigail Armstrong, Stephanie Dwyer, Allison Masikat, Mary Rose Yuan, Dawei Tran, Cuong Heibeck, Tyler Zawada, James Chen, Rishard Hallam, Trevor Yin, Gang Bioengineering (Basel) Article The XpressCF+(®) cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50–100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+(®) reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms. MDPI 2023-02-28 /pmc/articles/PMC10045668/ /pubmed/36978695 http://dx.doi.org/10.3390/bioengineering10030304 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hanson, Jeffrey
Groff, Dan
Carlos, Abi
Usman, Hans
Fong, Kevin
Yu, Abigail
Armstrong, Stephanie
Dwyer, Allison
Masikat, Mary Rose
Yuan, Dawei
Tran, Cuong
Heibeck, Tyler
Zawada, James
Chen, Rishard
Hallam, Trevor
Yin, Gang
An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title_full An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title_fullStr An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title_full_unstemmed An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title_short An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates
title_sort integrated in vivo/in vitro protein production platform for site-specific antibody drug conjugates
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10045668/
https://www.ncbi.nlm.nih.gov/pubmed/36978695
http://dx.doi.org/10.3390/bioengineering10030304
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