Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells

Prabhu, Anuja; Shanmugam, Dhanasekaran; Gadgil, Mugdha

doi:10.1016/j.ymben.2022.09.003

Cited by 6 publications

(5 citation statements)

References 49 publications

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“…Therefore, engineering the amount of expressed galactosylated glycans on the mAb provides the possibility to actively modulate their therapeutic effects in patients and to ensure consistent and controllable product quality. Most approaches have focused mainly on overexpression or silencing of key genes in relevant metabolic pathways or variation of feeding‐strategies and media supplementation (Amann et al, 2018; Bydlinski et al, 2018; Chung, Wang, Yang, Ponce, et al, 2017; Gramer et al, 2011; Prabhu et al, 2018, 2022; Zhang et al, 2021). However, current genetic engineering approaches based on knock‐out and overexpression are often unable to fine‐tune the phenotype of glycosylation.…”

Section: Discussionmentioning

confidence: 99%

“…However, current genetic engineering approaches based on knock‐out and overexpression are often unable to fine‐tune the phenotype of glycosylation. Recent studies are therefore pursuing new approaches that allow tailored and tune‐able glycosylation (Klingler et al, 2023; Prabhu et al, 2022). A previous screening study identified miRNAs that modulate whole glycosylation pathways and thereby allowing expression of customized glycosylation patterns in CHO mAb production cells (Klingler et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

“…However, this hypothesis would need to be investigated by further studies, for example at the metabolite level, to investigate potential changes in the intracellular concentration of intermediates and key substrates such as UDP‐galactose or galactose. Nonetheless, engineering the synthesis pathways of the precursor nucleotide sugar is also a promising approach to regulate galactose levels on the glycan (Prabhu et al, 2022). In contrast to that, miRNAs increasing galactosylation displayed an upregulation of B4GALT1 which fits to the observed phenotypic effect of increased galactosylation.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, lower levels of terminally galactosylated glycoforms may lead to enhanced lectin‐mediated complement activation as well as increased antiviral activity (Ackerman et al, 2013; Malhotra et al, 1995) (Figure 1a). In addition to approaches that utilized optimized feeding strategies current cell line engineering approaches to modulate galactosylation are mainly focused on the overexpression or knock‐out of genes in relevant pathways (Amann et al, 2018; Bydlinski et al, 2018; Chung, Wang, Yang, Yin, et al, 2017; Gramer et al, 2011; Prabhu et al, 2022; Sha & Yoon, 2019). However, the use of small‐interfering RNAs (siRNAs) or microRNAs (miRNAs) as a tool to reduce the activity of relevant genes of the galactosylation pathway in CHO cells has not yet been explored and may represent a novel instrument for a more gradual modulation of glycan galactosylation that could be relevant, for example, for the production of biosimilars and new “bio‐better” therapeutic proteins.…”

Section: Introductionmentioning

confidence: 99%

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Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation

Klingler,

Schlossbauer,

Naumann

et al. 2023

Biotech & Bioengineering

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N‐linked glycosylation is one of the most important post‐translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytolysis (CDC) activation, serum half‐life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu‐miR‐452‐5p, mmu‐miR‐193b‐3p) or decreased (mmu‐miR‐7646‐5p, mmu‐miR‐7243‐3p, mmu‐miR‐1668, mmu‐let‐7c‐1‐3p, mmu‐miR‐7665‐3p, mmu‐miR‐6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess‐related parameters. Most important, stable plasmid‐based overexpression of these miRNAs represents a versatile tool for engineering N‐linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation

Klingler,

Schlossbauer,

Naumann

et al. 2023

Biotech & Bioengineering

View full text Add to dashboard Cite

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“…Galactosylation and fucosylation levels can be simultaneously and independently controlled by simply altering the availability of extracellular galactose and/or fucose. This modified CHO host platform can facilitate the quick synthesis of differently glycoengineered proteins for the assessment of biological activity in addition to attaining the manufacture of specific glycoforms (Prabhu et al, 2022).…”

Section: Glycoengineeringmentioning

confidence: 99%

CRISPR‐interceded CHO cell line development approaches

Amiri

Adibzadeh

Ghanbari

et al. 2023

Biotech & Bioengineering

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For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high‐quality biologics with human‐like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long‐term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR‐Cas systems, the use of CRISPR‐Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast‐track product development process with consistent, improved product yield and quality.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

Harvey

2024

Mass Spectrometry Reviews

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The use of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates is a well‐established technique and this review is the 12th update of the original article published in 1999 and brings coverage of the literature to the end of 2022. As with previous review, this review also includes a few papers that describe methods appropriate to analysis by MALDI, such as sample preparation, even though the ionization method is not MALDI. The review follows the same format as previous reviews. It is divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of computer software for structural identification. (2) Applications to various structural types such as oligo‐ and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other general areas such as medicine, industrial processes, natural products and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. MALDI is still an ideal technique for carbohydrate analysis, particularly in its ability to produce single ions from each analyte and advancements in the technique and range of applications show little sign of diminishing.

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Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells

Cited by 6 publications

References 49 publications

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation

CRISPR‐interceded CHO cell line development approaches

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

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