Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

Abstract Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processi...

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Autores principales:	Ngan T. B. Nguyen, Jianer Lin, Shi Jie Tay, Mariati, Jessna Yeo, Terry Nguyen-Khuong, Yuansheng Yang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d6016d0862bc4687baedcd7b078f5685
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spelling	oai:doaj.org-article:d6016d0862bc4687baedcd7b078f56852021-12-02T16:07:04ZMultiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans10.1038/s41598-021-92320-x2045-2322https://doaj.org/article/d6016d0862bc4687baedcd7b078f56852021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92320-xhttps://doaj.org/toc/2045-2322Abstract Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.Ngan T. B. NguyenJianer LinShi Jie TayMariatiJessna YeoTerry Nguyen-KhuongYuansheng YangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ngan T. B. Nguyen Jianer Lin Shi Jie Tay Mariati Jessna Yeo Terry Nguyen-Khuong Yuansheng Yang Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
description	Abstract Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.
format	article
author	Ngan T. B. Nguyen Jianer Lin Shi Jie Tay Mariati Jessna Yeo Terry Nguyen-Khuong Yuansheng Yang
author_facet	Ngan T. B. Nguyen Jianer Lin Shi Jie Tay Mariati Jessna Yeo Terry Nguyen-Khuong Yuansheng Yang
author_sort	Ngan T. B. Nguyen
title	Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
title_short	Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
title_full	Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
title_fullStr	Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
title_full_unstemmed	Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans
title_sort	multiplexed engineering glycosyltransferase genes in cho cells via targeted integration for producing antibodies with diverse complex-type n-glycans
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d6016d0862bc4687baedcd7b078f5685
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Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

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