Cell Glycoengineering by Manipulating Heterogeneity

Services Published Data

Glycan Heterogeneity in Glycoproteins

Heterogeneity stems from the various and incomplete enzymatic modifications during N-glycan biosynthesis and processing. As a result, both native and recombinant glycoproteins produced in various cell lines, especially mammalian cells, yield mixtures with varying proportions of different glycoforms. Macro-heterogeneity in recombinant glycoproteins arises due to variations in glycosylation site occupancy, for example, glycosylation sites might be skipped, or additional sites might be occupied. Micro-heterogeneity means the existence of distinct glycans at the same glycosylation site, allowing individual sites to possess glycans with considerable structural diversity.

Fig.1 Glycan heterogeneity. Fig.1 Glycan macro- and micro-heterogeneity.^{1, 3}

Cell Glycoengineering by Manipulating Heterogeneity at Creative Biolabs

By leveraging cutting-edge technologies and an expert technical team, Creative Biolabs has proposed various genetic strategies to manipulate heterogeneity in various cell lines and provides high-quality Glycoengineering Services for our clients worldwide. Our methods for reducing N-glycan heterogeneity and producing uniform glycan forms mainly involve multiple genetic strategies, such as Overexpression, and Knockin/Knockout, to amplify or diminish the availability of precursors crucial for the N-glycosylation processes.

Manipulating macro-heterogeneity

We use different strategies to overcome differences in N-glycosylation site occupancy, such as overexpression of LmSTT3 in Pichia pastoris, and mutation within the N-glycosylation consensus sequence from Asn-X-Ser to Asn-X-Thr, which has demonstrated improved efficiency of glycosylation in various eukaryotic systems.

Manipulating micro-heterogeneity

We counteract undesired glycans and simplify the complexity of glycosylation through genetic editing of glycosyltransferases and glycoside hydrolases.

For example, combined knockout of GnT genes, mgat4A/4B/5, enable the production of rhEPO featuring homogenous bi-antennary N-glycans from CHO cells. Introduction of ST6Gal-I into CHO ST3Gal4/6 knockout cells can produce N-glycans characterized by exclusive α-2,6 sialylation.

Applications of Cell Glycoengineering for Minimizing Glycan Heterogeneity

The presence of heterogeneity in the N-glycan structures of therapeutic proteins introduces difficulties in protein purification, product consistency, and batch-to-batch reproducibility. Furthermore, excessive heterogeneity can pose a safety risk due to the potential for non-human glycans to trigger undesirable immune responses. Manipulating heterogeneity to achieve uniform glycan structures is crucial for maintaining the potency and safety of glycoprotein biologics, as well as regulating their pharmacokinetic properties and improving therapeutic efficacy.

Features of Our Services

Optional genetic approaches for manipulating heterogeneity
Precise technology for genetic editing
Highly efficient and stable methods for knockout/knockin
Customized strategies for different cell line systems
Professional team experienced in cell line glycoengineering

Published data

Carbohydrates, especially glycans, play multiple key roles in organisms. They are not only an important source of energy but also provide structural support by participating in the formation of cell walls. They also have a vital effect on the cell recognition process and the immune process. In this study, the authors used CRISPR/Cas9 gene knockout technology to gene edit multiple glycosylation pathways involved in glycan chain extension or synthesis in HEK293F cells. They characterized the changes in glycans after gene editing through cell binding experiments. The results of the study showed that this precise gene editing technology ensured stability and accuracy during the cell glycoengineering process while achieving stable production of extracellular vesicles.

Fig.2 Cell-based glycoengineering strategy.^{2, 3}

Creative Biolabs offers a range of genetic approaches to controlling the heterogeneity in glycosylation and directing the biosynthetic process toward desired glycan forms. Our scientists have amassed a wealth of expertise in cell line glycoengineering. Please feel free to contact us for more details.

References

Čaval, Tomislav, Albert JR Heck, and Karli R. Reiding. "Meta-heterogeneity: evaluating and describing the diversity in glycosylation between sites on the same glycoprotein." Molecular & Cellular Proteomics 20 (2021).
Tian, Weihua, et al. "Cell-based glycoengineering of extracellular vesicles through precise genome editing." New Biotechnology 83 (2024): 101-109.
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