In vitro glycoengineering (IVGE) technology specifically and effectively changes the type of glycosylation of biopharmaceuticals, thus affecting their biological activity. CD BioGlyco has developed an advanced Glycoengineering platform to provide clients with IVGE services to solve their problems in the development of recombinant protein drugs and monoclonal antibodies.

Glycosylation of Proteins

Glycosylation is a common post-translational modification that occurs on the side chains of many amino acid residues of proteins in many different ways. Depending on the linkage established with the protein, protein glycosylation is divided into two general types: 1) Attached of N-glycans to the nitrogen atom of the asparagine (Asn) side chain; 2) Attached of O-glycans to the oxygen atom of the serine (Ser) or threonine (Thr) side chain. Glycans bound to glycoproteins typically consist of as many as a dozen monosaccharides. These glycans interact with amino acid residues, which in turn affect protein folding efficiency and conformational stability.

Fig.1 The two most common types of protein glycosylation. (Ma, et al., 2020)

Studies have found that glycosylation affects the pharmacokinetics, pharmacodynamics, therapeutic activity, and biomanufacturing of protein therapeutics. The absorption, distribution, metabolism, and excretion of protein therapeutics are closely related to the glycosylation process. In terms of pharmacodynamics and biological activity, it has been shown that the activity of protein drugs such as recombinant human hyaluronidase depends on glycosylation. The production of protein drugs relies on glycocompatible production systems, such as the Chinese hamster ovary cell (CHO) expression system.

Glycoengineering

Glycoengineering is a method to improve protein properties by changing protein glycosylation. In the early days, scientists added or removed target sugars to proteins through in vivo genetic techniques and in vitro enzymatic methods. With the development of recent years, glycoengineering technology has not been limited to improving the properties of proteins through naturally occurring glycans and glycosidic linkages. Researchers have also applied unnatural glycans and site-selective conjugation chemistry to glycoengineering to improve the therapeutic effects of drugs.

Fig.2 Glycoengineering strategies for recombinant glycoprotein therapeutics. (Narimatsu, et al., 2021)

IVGE: Redefining Glycosylation Control

Glycoengineering has attracted more and more scientists' attention as a method to improve the efficacy of protein drugs. At CD BioGlyco, we have established an advanced IVGE platform to provide our clients with protein glycoengineering and antibody glycoengineering services. We specifically and efficiently change the type of glycosylation through IVGE technology, thereby altering the biological activity of the drug. Our protein glycoengineering strategy is mainly by changing the number and location of glycosylation sites or changing the glycan structure of individual glycosylation sites. We also provide clients with high-quality antibody remodeling, digestion, deglycosylation, and site-specific conjugation services.

• Preliminary Glycoprotein Analysis

Comprehensive glycan profiling of the client's purified glycoproteins using techniques such as mass spectrometry (MS) and liquid chromatography (LC) is performed to determine the initial glycoform profile.

• Strategic Glycan Remodeling

Based on the characterization data and the client's project goals, we will develop a specific glycosylation remodeling plan. This plan outlines the precise enzymatic reactions required to achieve the desired glycosyl structure. This may involve using glycosidases to remove existing heterogeneous glycans or using a combination of enzymes to construct new, well-defined glycosyl structures.

• In Vitro Enzymatic Reactions

Purified glycoproteins are incubated with the selected enzyme and activated glycosubstrate under optimized reaction conditions (such as temperature, pH, and cofactors). Our robust and scalable processes ensure high reaction efficiency and minimal side reactions, resulting in rapid generation of the target glycoforms.

• Purification and Separation

Following the enzymatic reaction, the modified glycoprotein is purified to remove residual enzyme, unreacted substrate, and any byproducts. We utilize state-of-the-art purification techniques, including affinity and ion exchange chromatography, to ensure the final product is highly pure and free of contaminants.

• Quality Control and Validation

A range of analytical methods, including high-resolution MS and functional analysis, is used to confirm the integrity of the protein backbone and the homogeneity of the glycan structure.

Workflow

Publication Data

Applications

• Biopharmaceutical development: IVGE is essential for developing next-generation therapeutic proteins, vaccines, and antibodies with optimized safety, stability, and efficacy profiles.
• Biosimilar development: Our services help in creating biosimilars with glycan profiles that closely match or even surpass those of the reference product, ensuring comparability and functional equivalence.
• Glycobiology research: IVGE allows researchers to generate well-defined glycoprotein standards for studying the biological roles of specific glycans, including their impact on protein folding, receptor binding, and cellular signaling.

Advantages

• Our enzymatic approach enables the creation of a single, defined glycoform, eliminating the batch-to-batch variability and heterogeneity inherent in cell-based systems.
• The enzymatic reactions are robust and easily scalable, making them suitable for projects ranging from early-stage research to large-scale biopharmaceutical manufacturing.
• We enable the rational design of glycans to improve key attributes such as serum half-life, solubility, and receptor binding affinity.

Frequently Asked Questions

CD BioGlyco is a leading service provider specializing in glycoengineering services. We have established a professional IVGE platform. If you are interested in our IVGE services, please contact us for more details without any hesitation.

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