Studying the structural and biological function of glycans is essential to understand the mechanism of various biological activities. CD BioGlyco has developed a series of outstanding Enzymatic Cleavage methods by integrating various advanced technologies to provide Glycan Release services.

Overview of N-Glycan

N-Glycosylation is a common post-translational modification that affects glycoprotein structure and function. N-Glycans are attached to the nitrogen (N) atom in the asparagine (Asn) side chain of the sequon. Glycomic analysis of these intact N-glycans has the potential to lead to therapeutic targets and potentially to treat many genetic defects and their diseases. For example, comparing the glycan profiles of patients and healthy individuals to indicate disease and possible treatment pathways. Studying N-glycans relies on their intact release from glycoproteins, followed by mass spectrometry (MS) techniques analysis.

Fig.1 Workflow for the analysis of N-glycans in glycoproteins. (Regan, et al., 2019)

There are 2 common methods to release N-glycans: chemical release and enzymatic release. Peptide: N-glycosidase F (PNGase F) is the most widely used method for N-glycan release, which releases intact N-glycans to keep the peptide backbone intact. Endoglycosidase H (Endo H) only releases high-mannose N-glycans or hybrid N-glycans. Glycopeptidase A (PNGase A) cleaves all high-mannose, hybrid, and short-complex N-glycans, but fails to release glycans containing sialic acid residues. Hydrazinolysis and β-elimination are the two main chemical release techniques. Both methods are used to release intact glycans from the peptide backbone, but they result in the degradation of the peptide backbone. Therefore, enzymatic methods have become the method of choice for N-glycan release.

The potential applications of N-linked glycosylation in the pharmaceutical field are becoming increasingly apparent. Many therapeutic antibodies on the market are N-linked glycoproteins. In addition, researchers have genetically engineered therapeutic glycoproteins with human-like N-linked glycans. Therefore, the development of efficient glycan release services is necessary.

Key Technologies

CD BioGlyco employs a suite of highly specific glycosidases as core technologies for the enzymatic release of N-glycans. The choice of enzyme is critical and depends on the type of N-glycan, the source material, and the specific analytical goals. Our primary enzymes include:

• PNGase F: Cleave all types of Asp-linked glycans (high-mannose, hybrid, and complex) from glycoproteins
• Endo H: Cleave the chitobiose core of the glycan, leaving one GlcNAc residue still attached to the Asp on the protein.
• Endoglycosidases F1, F2, F3 (Endo F1, Endo F2, and Endo F3): A family of enzymes with distinct specificities.

Enzymatic Release N-Glycan at CD BioGlyco

N-Glycans are essential for physiological and pathological processes. A reliable method must be identified to better separate N-glycans from proteins before analysis. Conventional methods include enzymatic release methods and Chemical Release methods. At CD BioGlyco, we have developed an advanced glycan release platform to provide clients with efficient and accurate N-glycan release services on antibodies, fusion proteins, and other N-glycosylated proteins via PNGase F. We successfully hydrolyze the amide bond between the Asn and N-glycans (including high-mannose, complex, and hybrid N-glycans) to release intact glycans. In addition, we provide clients with fast and sensitive N-glycan quantification and characterization services.

Fig.2 The process of enzymatic release of N-glycan. (CD BioGlyco)

We also use Endo H and Endo F1, Endo F2, and Endo F3 to release N-glycans. Endo H specifically cleaves high mannose and some mixed N-glycans between two N-acetylglucosamine (GlcNAc) residues in the core region. It is particularly useful for distinguishing different N-glycan classes and studying glycoprotein processing in the endoplasmic reticulum. Endo F1, Endo F2, and Endo F3 have different specificities for different types of N-glycans. We understand that every research project has its own unique needs, so our services are highly customizable and provide comprehensive solutions covering a variety of biological sources. Our goal is to provide you with high-quality released N-glycans that are ideal for detailed structural characterization, quantitative analysis, and functional studies on a variety of analytical platforms.

Workflow

Publication Data

Applications

• Biopharmaceutical development: Essential for characterizing therapeutic glycoproteins (e.g., monoclonal antibodies, fusion proteins, vaccines) to ensure product consistency, efficacy, and safety.
• Disease pathogenesis: Enables detailed studies of altered glycosylation patterns in disease states, providing insights into disease mechanisms and potential therapeutic targets.
• Fundamental glycoscience research: Supports basic research aimed at elucidating glycan structures, biosynthesis pathways, and their functions in cellular processes, development, and host-pathogen interactions.
• Food science and nutrition: Used in the analysis of glycoproteins in food products for quality assessment, allergenicity studies, and understanding nutrient bioavailability.

Advantages

• Exceptional specificity and efficiency: Our protocols utilize highly purified glycosidases, ensuring the precise cleavage of N-glycans from their protein backbone without non-specific degradation. This enzymatic precision guarantees the integrity of the released glycans, which is crucial for accurate downstream analysis.
• Maximized glycan recovery: Compared to harsher chemical methods, enzymatic release is gentle, significantly reducing the risk of glycan degradation, desialylation, or other structural modifications. This gentle approach maximizes the recovery of intact, native N-glycans, providing a more complete and accurate representation of the glycome.
• High reproducibility and consistency: With many years of experience, CD BioGlyco has developed and optimized robust, standardized protocols for N-glycan release. Our stringent quality control measures at every step ensure high reproducibility across batches and consistent results.

Frequently Asked Questions

The development of the enzymatic cleavage of N-glycan is a complex process. CD BioGlyco has advanced technology platforms and professional research teams in the field of enzymatic release. Our fast and effective strategy will greatly accelerate the project progress of our clients. Please feel free to contact us for more detailed information.

Associated Services

To help you achieve your complete glycomics research goals, CD BioGlyco offers a suite of complementary services that seamlessly integrate with our enzymatic release of N-glycan service. These offerings ensure a comprehensive solution from sample preparation to in-depth data analysis.