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Rapid antibody glycoengineering in Chinese hamster ovary cells.

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Title: Rapid antibody glycoengineering in Chinese hamster ovary cells.
Authors: Marbiah, M
Kotidis, P
Donini, R
Gómez, IA
Jimenez Del Val, I
Haslam, SM
Polizzi, KM
Kontoravdi, C
Item Type: Journal Article
Abstract: Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.
Issue Date: 2-Jun-2022
Date of Acceptance: 1-Jun-2022
URI: http://hdl.handle.net/10044/1/97896
DOI: 10.3791/63872
ISSN: 1940-087X
Publisher: MyJove Corporation
Start Page: 1
End Page: 19
Journal / Book Title: Journal of Visualized Experiments
Volume: 184
Issue: 184
Copyright Statement: © 2022 JoVE Creative Commons Attribution 3.0 License
Keywords: 0601 Biochemistry and Cell Biology
1701 Psychology
1702 Cognitive Sciences
Publication Status: Published
Conference Place: United States
Online Publication Date: 2022-06-02
Appears in Collections:Chemical Engineering
Faculty of Natural Sciences
Faculty of Engineering

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