Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics

Kotidis, Pavlos; Marbiah, Masue; Donini, Roberto; Gómez, Itzcóatl A.; Val, Ioscani Jiménez del; Haslam, Stuart M.; Polizzi, Karen M.; Kontoravdi, Cleo

doi:10.1007/978-1-0716-1685-7_7

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“…The sense and antisense sequences for each construct are shown (respectively), and the GC content of each structure is displayed. Reprinted from Kotidis et al 52 .…”

Section: Dsirna Targetmentioning

confidence: 99%

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Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Marbiah

Kotidis

Donini

et al. 2022

JoVE

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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 laserinduced 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

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“…The sense and antisense sequences for each construct are shown (respectively), and the GC content of each structure is displayed. Reprinted from Kotidis et al 52 .…”

Section: Dsirna Targetmentioning

confidence: 99%

“…Biological triplicates performed for each experiment were mixed after harvesting to reduce the burden of downstream analysis. Reprinted from Kotidis et al 52 . Please click here to view a larger version of this figure.…”

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confidence: 99%