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Engineering protein glycosylation in CHO cells to be highly similar to murine host cells
Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9978007/ https://www.ncbi.nlm.nih.gov/pubmed/36873370 http://dx.doi.org/10.3389/fbioe.2023.1113994 |
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author | Gupta, Shivani Shah, Bhavana Fung, Coral Shek Chan, Pik Kay Wakefield, Devin L. Kuhns, Scott Goudar, Chetan T. Piret, James M. |
author_facet | Gupta, Shivani Shah, Bhavana Fung, Coral Shek Chan, Pik Kay Wakefield, Devin L. Kuhns, Scott Goudar, Chetan T. Piret, James M. |
author_sort | Gupta, Shivani |
collection | PubMed |
description | Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development. |
format | Online Article Text |
id | pubmed-9978007 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-99780072023-03-03 Engineering protein glycosylation in CHO cells to be highly similar to murine host cells Gupta, Shivani Shah, Bhavana Fung, Coral Shek Chan, Pik Kay Wakefield, Devin L. Kuhns, Scott Goudar, Chetan T. Piret, James M. Front Bioeng Biotechnol Bioengineering and Biotechnology Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development. Frontiers Media S.A. 2023-02-16 /pmc/articles/PMC9978007/ /pubmed/36873370 http://dx.doi.org/10.3389/fbioe.2023.1113994 Text en Copyright © 2023 Gupta, Shah, Fung, Chan, Wakefield, Kuhns, Goudar and Piret. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Gupta, Shivani Shah, Bhavana Fung, Coral Shek Chan, Pik Kay Wakefield, Devin L. Kuhns, Scott Goudar, Chetan T. Piret, James M. Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title | Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title_full | Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title_fullStr | Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title_full_unstemmed | Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title_short | Engineering protein glycosylation in CHO cells to be highly similar to murine host cells |
title_sort | engineering protein glycosylation in cho cells to be highly similar to murine host cells |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9978007/ https://www.ncbi.nlm.nih.gov/pubmed/36873370 http://dx.doi.org/10.3389/fbioe.2023.1113994 |
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