Antibody Glycosylation

Service Description

Antibody glycosylation is an important biotechnology that is widely used to study the structure-function relationship of antibodies. As a leading biotechnology company, we provide high-quality antibody glycosylation analysis services to improve the accuracy and validity of your research. Through in-depth analysis of antibody glycosylation, you can monitor the functional changes of antibodies, optimize treatment effects, and explore the potential of antibodies in disease treatment, providing better solutions for biopharmaceutical development.

What is antibody glycosylation?

Antibody glycosylation is the process of transferring carbohydrates to antibodies in response to glycosyltransferases and involves a variety of glycoforms, including O-glycosylation and N-glycosylation.

Glycosylation can stabilize the CH2 domain of the antibody, preventing its thermal instability from increasing and affecting the conformation and stability of the antibody. The level of glycosylation affects the ability of the antibody to bind to FcγR (Fc receptor), which in turn affects ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) effects. Certain glycoforms, such as sialylation, can affect the immunogenicity of antibodies, and high mannose forms may lead to increased immunogenicity.

Cell lines, media, culture processes, and media components can all affect the level of glycosylation. The selection of host cells (e.g., CHO cells, NS0 cells) has an important impact on glycosylation modifications. Structural characterization of glycosylation often requires a combination of bioactivity and physicochemical analysis methods, such as bioactivity assays and mass spectrometry.

In summary, antibody glycosylation is a post-translational modification that has an important impact on antibody function, involves a variety of glycoforms, and is affected by a variety of factors such as cell expression systems and production processes.

Antibody Glycosylation Site

Antibody glycosylation sites can be divided into two main forms: N-glycosylation and O-glycosylation:

N-glycosylation Site: N-glycosylation occurs at the CH2 asparagine (Asn) residue of the Fc fragment crystallizable of the antibody. Common N-glycosylation site sequences include asn-x-ser/thr (x is any amino acid that is non-proline), where x can be serine (Ser) or threonine (Thr). N-glycosylation is the main type of antibody glycosylation modification, which has an important impact on the biological activity and stability of antibodies.

O-glycosylation Site: O-glycosylation occurs on serine (Ser) or threonine (Thr) residues. O-glycosylation is not as common as N-glycosylation, but has some effect on antibody performance.

These glycosylation sites are important in the quality control and research of antibody drugs, as they can affect properties such as drug stability, biological activity, and immune response. These glycosylation sites can be confirmed and analyzed by mass spectrometry, liquid chromatography, and other techniques.

Applications of Antibody Glycosylation

Antibody glycosylation has a wide range of applications in the biomedical field, mainly including the following aspects:

Disease Diagnostics & Biomarker Development: Glycosylation patterns can reveal biomarkers of disease. For example, in many types of cancer, the glycoprotein glycan chain structure on the surface of tumor cells is altered, and these alterations can serve as biomarkers of the disease. For example, CA125 is a glycoprotein marker in ovarian cancer, and its glycosylation status is closely related to disease stage and prognosis.

Disease Treatment and Drug Development: Glycosylation affects protein function and cell-to-cell interactions, making glycosylation pathways and associated enzymes potential drug targets. The glycosylation pattern of antibody drugs has an important impact on their therapeutic efficacy. By modulating the glycosylation status of an antibody, its efficacy can be enhanced or side effects reduced. For example, removing fucosylation from an antibody can enhance its ability to bind to the FcγRIIIa receptor, thereby increasing ADCC (antibody-dependent cell-mediated cytotoxicity) activity.

Research on the Pathogenesis and Progression of Diseases: Glycosylation modifications may affect cell growth, migration, and immune escape mechanisms, which are particularly important for the aggressiveness and metastasis of cancer. By studying the mechanisms of glycosylation modifications, it is possible to better understand the progression of the disease and potential therapeutic strategies.

Glycosylation Analysis Methods: Glycosylation analysis is key to ensuring the quality of antibody drugs. At different stages of product development, researchers use a variety of analytical methods to characterize the structure of sugars, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and others. These methods not only determine the substance of the finished drug product and the real-time quality attributes of the drug product, but also enable quality identification in the early stages of monoclonal antibody development.

In summary, glycosylation plays a key role in the development, production, and clinical application of antibody drugs, involving many aspects such as diagnosis, treatment, and mechanism research of diseases.

Fig.1 Application of anti-glycan monoclonal antibody. (Gillmann, Kara M., et al., 2023)

Our Services

Comprehensive Glycosylation Profiling: Detailed glycan structure analysis using state-of-the-art MS and HPLC, ensuring accurate characterization of N-glycans and O-glycans in your antibodies.

Glycoengineering for Enhanced Therapeutics: Tailored glycoengineering services to optimize glycosylation patterns, enhancing therapeutic activity such as ADCC and CDC.

Process Monitoring and Glycosylation Control: Real-time glycosylation monitoring during antibody production, ensuring batch consistency and quality for clinical-grade antibodies.

Functional Impact Studies: In-depth analysis of how glycosylation modifications impact antibody stability, pharmacokinetics, and immunogenicity using in vitro and in vivo assays.

Consultation and Strategy Development: Expert consulting on glycosylation strategies for your therapeutic antibody pipeline, ensuring compliance with regulatory guidelines and optimizing therapeutic performance.

Our Competitive Advantages

Advanced Analytical Technologies: We employ the latest mass spectrometry and chromatography techniques for high-resolution glycan profiling, ensuring precise and reproducible results.

Glycoengineering Expertise: Our experienced team has deep expertise in glycoengineering, offering cutting-edge solutions to enhance antibody efficacy and safety profiles.

Customized Services Across Development Stages: Whether in the early R&D phase or large-scale production, we provide customized glycosylation services to meet the specific needs of your project.

Regulatory Compliance and Quality Assurance: Adherence to stringent regulatory standards (FDA, EMA) ensures that your antibody's glycosylation meets global quality and safety requirements.

Seamless Workflow and Efficiency: Our streamlined processes from sample preparation to data analysis allow us to deliver timely, cost-efficient results without compromising quality.

Client-Centered Approach: We prioritize close collaboration with our clients, offering transparent communication, personalized solutions, and expert guidance at every stage of the project.

Case Study

Case Study 1

In traditional ADCs (Antibody-Drug Conjugates), drug molecules are chemically and randomly attached to antibody surface residues (typically Lys or Cys), which may interfere with epitope binding and targeting, and lead to instability in overall product heterogeneity, colloidal status, and pharmacokinetics. To address this issue, the researchers developed a new method to specifically incorporate functionalized sialic acid into Fab glycans of mAbs using the previously unreported bacterial sialyltransferase AST-03. The specificity of the bacterial sialyltransferase AST-03 for Fab glycans was first demonstrated with cetuximab with naturally occurring Fab glycans. Subsequently, glyco-engineered cetuximab ADCs were demonstrated to have similar growth inhibition potency to cetuximab ADCs, in which the drug is conjugated to random Cys residues. In summary, the authors developed a novel method that can improve conjugate homogeneity and generate ADCs without compromising target specificity, while retaining the effector function of Fc glycans.

Fig.2 Overview of sialyltransferases conjugated with antibody glycans with different specificities. (Jaramillo, Maria L., et al., 2023)

Case Study 2

Changes in antibody glycosylation are strongly associated with multiple disease states, alloimmune, and autoimmune responses. However, existing methods often fail to provide high-resolution glycosylation information for specific antigens, resulting in a limited understanding of antibody glycosylation changes in disease. In response to this problem, professors David Falck and Manfred Wuhrer from Leiden University Medical Center in the Netherlands proposed the GlYcoLISA method, which is designed to achieve high efficiency and accuracy in glycosylation analysis of antibodies to specific antigens. Immunosorbent assay (ELISA) combined with liquid chromatography-mass spectrometry (LC-MS) in 96-well plates enables a comprehensive and detailed analysis of glycosylation of antibodies at high throughput. In the figure, the flow of GlYcoLISA first involves a classic 96-well plate ELISA for the purification of antibodies to specific antigens from clinical samples. The result of this step is a high-throughput affinity purification of the antibody by ELISA, which provides the basis for subsequent analysis. Next, LC-MS was applied for high-resolution analysis of IgG Fc N-glycosylation. The detailed steps of LC-MS are not directly shown in the figure, but the role of this key technique in glycosylation analysis is highlighted. The significance of GlYcoLISA is that it overcomes the limitations of traditional methods and makes glycosylation analysis of antibodies to specific antigens more feasible. By combining high-throughput sample processing in 96-well plates with high-resolution analysis by LC-MS, this method provides a comprehensive understanding of antibody glycosylation while maintaining efficiency. The application of automated data processing strategies further accelerates the process of obtaining and interpreting results. 

Fig.3 Schematic diagram of GlYcoLISA's workflow. (Falck, David, and Manfred Wuhrer., 2024)

FAQ

1. What does glycosylation do to an antibody?

Glycosylation modifies the antibody's structure, primarily affecting the Fc region. It can alter the antibody's interactions with immune receptors, enhancing its effector functions like antibody-dependent cellular cytotoxicity (ADCC) and complement activation, while also influencing stability and serum half-life.

2. What is the purpose of glycosylation?

Glycosylation improves protein properties such as stability, solubility, and immune system interactions. In antibodies, it enhances therapeutic efficacy by modulating receptor binding, prolonging circulation time, and regulating immune responses.

3. What is glycosylation in immunology?

In immunology, glycosylation refers to the addition of sugar molecules to proteins, including antibodies. It is critical for regulating immune responses, cellular recognition, and antigen presentation, and it helps fine-tune the immune system's ability to respond to pathogens.

4. What is the importance of IgG glycosylation?

IgG glycosylation is essential for its functional activity. The glycan structures in the Fc region affect interactions with Fc receptors and complement proteins, which regulate immune system activation, therapeutic potency, and anti-inflammatory properties.

5. How can glycosylation improve the therapeutic efficacy of antibodies?

Glycosylation can enhance the therapeutic efficacy of antibodies by optimizing their binding to immune cells, boosting ADCC, and prolonging serum half-life, leading to more effective disease targeting and improved clinical outcomes.

6. What methods are used to analyze antibody glycosylation?

Common methods include mass spectrometry (MS), liquid chromatography (LC), and capillary electrophoresis. These techniques allow precise analysis of glycan composition, glycosylation sites, and overall structural characterization of antibodies.

7. Can glycosylation patterns be engineered for specific antibody therapies?

Yes, glycoengineering can be used to modify glycosylation patterns to optimize antibody therapies. By tailoring the glycan structures, therapeutic antibodies can be designed to improve immune response, increase stability, and enhance specific interactions with immune cells.

8. How does glycosylation influence the safety and efficacy of monoclonal antibodies?

Glycosylation affects both the safety and efficacy of monoclonal antibodies by determining their immunogenicity, clearance rates, and effector functions. Proper glycan profiles ensure reduced adverse reactions and optimized therapeutic performance.