Antibody Glycoengineering
CD BioGlyco can customize one-stop services for glycoengineered antibodies according to customers' R&D or cGMP production needs. We have confidence to be your essential research assistant in the field of glycobiology.
Glycoengineering in Antibodies
Monoclonal antibodies (mAbs) are currently the largest and fastest-growing category of biopharmaceuticals. Since 1984, more than 60 mAbs and fusion molecules has been approvedfor use in more than 30 cancers, autoimmune and cardiovascular diseases. It is estimated that by 2024, the global market value of mAbs will reach 138.6 billion dollars.
The clinical efficacy and safety of mAbs are affected by their glycosylation structure and composition. Glycosyl distribution is usually heterogeneous, depends largely on the manufacturing process, and is therefore susceptible to changes in cell culture conditions. Therefore, it is considered a key quality attribute of mAbs. Great efforts have been made in mammalian and non-mammalian cells to control the glycosylation of mAbs. However, it is still a challenge to obtain a specific glycoform that is completely homogeneous.
The latest innovations in chemoenzymatic glycoengineering technology will allow the production of mAbs with well-defined and uniform Fc glycoforms, thereby giving them the desired biological properties. This method has significant advantages, such as enhanced Fc effector function, improved safety, higher batch-to-batch consistency, reduced immunogenicity, and can be expressed economically. Overall, producing mAbs with the innovative glycoengineering technology will bring tangible benefits to patients and manufacturers.
Fig.1 Glycoengineering of therapeutic antibodies. (Li, et al., 2021)
Key Technologies
Mammalian host systems are the preferred expression platform because they can maximize the post-translational processing and functional activity of proteins.
- CD BioGlyco provides the most prominent host cell lines for commercially available products: Chinese Hamster Ovary (CHO) or Mouse Myeloma (NS0, SP2/0) cell lines, and other cell lines. We have carried out glycoengineering of different cell lines, and strictly controlled and optimized environmental factors and cell culture conditions.
Chemoenzymatic glycoengineering refers to the in vitro remodeling of glycans using enzymes such as endoglycosidase and sugar synthase.
- The solution we provide usually includes three steps: Deglycosylation of IgG by endo β-N-acetylglucosaminidase (ENGase, such as Endo S), while retaining the innermost GlcNAc at N297; the oxazoline derivatives with customized N-linked glycan structure as sugar donors are prepared by chemical methods; transglycosylation of the glycan oxazoline donor to the innermost GlcNAc acceptor.
Antibody Glycoengineering: Precision, Potency, and Performance
At CD BioGlyco, our expertise in antibody glycoengineering is built upon a foundation of cutting-edge technologies designed for precision and control. We leverage a suite of advanced methodologies to manipulate antibody glycosylation, ensuring optimal performance for diverse applications. Our services include:
- Project Design
After gaining a deep understanding of your project's unique needs, target antibody characteristics, and desired functional outcomes, our experts will work with you to develop the optimal glycoengineering strategy, taking into account factors such as antibody class, target activity, and desired glycoforms.
- Antibody Expression and Purification
Select the appropriate cell line to express your antibody of interest at high yield and quality. Purify using chromatography to ensure purity and integrity.
- Glycoengineering Modification
This is the core of our service, where precise glycoengineering modifications are performed. Depending on the agreed-upon strategy, this may involve:
Our antibody remodeling service enables precise modification of antibody glycans, such as G0 (lacking a terminal galactose), G1 (one terminal galactose), G2 (two terminal galactoses), and G2S2 (two galactoses and two sialic acids), to balance various effector functions and improve pharmacokinetic properties.
- Digestion of IgG Below the Hinge Region: Generates F(ab')_2 fragments by cleaving IgG antibodies below the hinge region, preserving the antigen-binding capabilities while removing the Fc region.
- Digestion of IgG in the Hinge Region: Offer digestion within the hinge region to produce Fab fragments, useful for studies requiring only the antigen-binding domains without the Fc portion.
- Digestion of Human IgG1 Above the Hinge Region: specific digestion of human IgG1 above the hinge region, yielding Fc and Fab fragments for detailed structural and functional analysis.
- Digestion of Flexible Linkers: provide targeted digestion of flexible linkers within engineered antibodies or fusion proteins, allowing for the separation of domains or the release of conjugated molecules.
Antibody Deglycosylation Service
We facilitate the complete or partial removal of glycans for studies on glycan-independent antibody functions or for specific conjugation strategies.
- Hydrolysis of All Fc-Glycans: This service involves the complete enzymatic removal of all N-linked glycans from the Fc region of antibodies, enabling the study of antibody functions devoid of glycan influence.
- Hydrolysis of Complex Type N-Glycans: We offer selective hydrolysis of complex type N-glycans, providing a controlled method to simplify glycan profiles for analytical purposes or to prepare antibodies for specific modifications.
Site-Specific Antibody Conjugation Service
Our site-specific antibody conjugation service enables the precise attachment of various payloads, such as Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, biotin, deferoxamine (DFO), and azide, to antibodies, yielding highly homogeneous and effective antibody-drug conjugates.
- Comprehensive Analysis
We utilize advanced analytical techniques such as Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography–tandem MS (LC-MS/MS), and hydrophilic interaction liquid chromatography (HILIC)-HPLC to meticulously characterize the glycan profiles of antibodies, confirm the success of the engineering process, and quantify the desired glycoforms.
Workflow
Case Study
Background
The carbohydrate structures attached to the conserved N-glycosylation site (Asn297) of therapeutic mAbs are critical modulators of effector functions, such as ADCC and serum half-life. Natural antibody production in mammalian cell lines typically yields a highly heterogeneous mixture of glycoforms, often dominated by core-fucosylated structures that significantly weaken binding affinity to FcγRIIIa receptors, thereby limiting therapeutic potency. Achieving custom, homogeneous glycoform profiles (such as pure A2G2 structures) with near-complete core-defucosylation is a major hurdle in biopharmaceutical engineering, requiring highly efficient, scalable, and trace-less enzymatic remodeling platforms.
Our Solution
CD BioGlyco deployed an advanced, multi-stage chemoenzymatic remodeling and endoglycosidase-catalyzed ligation platform to achieve high-fidelity glycan trimming, core-fucose removal, and homogeneous glycan elongation on target IgG antibodies.
- Precision Glycan Trimming & Core Defucosylation: The heterogeneous native N-glycans of the original IgG antibody were trimmed down to the innermost GlcNAc core utilizing immobilized Endo-S2 endoglycosidase. To drastically enhance ADCC potential, a specialized fucosidase (LpAlFc) was introduced to the IgG-GlcNAc intermediate pool under optimized kinetic conditions, selectively cleaving the rigid α-1,6-linked core fucose residues.
Fig.3 IgG-GlcNAc preparation. (CD BioGlyco)
- In-Process Quality Control (QC) Monitoring: To rigorously monitor reaction progress without introducing analytical ambiguity, intermediate fractions were digested with IdeS protease to isolate specific Fc fragments, followed by high-resolution LC-MS to quantify exact defucosylation rates.
- Homogeneous Glycan Elongation via Oxazoline Ligation: Following affinity purification using a Protein G column, the core-defucosylated IgG-GlcNAc was subjected to synthetic remodeling. Utilizing a highly engineered endoglycosidase mutant, Endo S2 (D184M) defined A2G2 N-glycan-oxazoline donors were sequentially ligated onto the GlcNAc acceptor site in a strictly controlled micro-reaction matrix.
Fig.4 IgG-A2G2 preparation. (CD BioGlyco)
Results
The integrated glycoengineering workflow demonstrated exceptional reaction efficiency and structural control, delivering a premium-grade, uniformly remodeled antibody candidate:
- Exceptional Defucosylation Yield: Integrated LC-MS analysis of the IdeS-cleaved Fc fragments confirmed that the specialized LpAlFc processing achieved a remarkable 92.42% core-fucose removal rate, transforming the heterogeneous antibody pool into a highly potent, low-fucose therapeutic candidate.
Fig.5 LC-MS spectrum of IgG-GlcNAc. (CD BioGlyco)
- High-Efficiency Transglycosylation: Real-time MS monitoring of the glycosynthase-mediated remodeling verified that the multi-stage addition of the A2G2 oxazoline donor achieved a seamless 92.20% sugar conversion rate, yielding a highly homogeneous antibody population bearing precisely tailored, symmetrical biantennary complex N-glycans.
Fig.6 LC-MS spectrum of IgG-A2G2. (CD BioGlyco)
Publication Data
Journal: Antibodies
IF: 2.7
Published: 2020
Results: This study combined Fc protein-engineering and Fc glyco-engineering (afucosylation) to enhance CD19 antibodies' complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The researchers generated four versions of a CD19 antibody based on tafasitamab's V-regions: a native IgG1, an Fc protein-engineered version with the EFTAE modification, and afucosylated (Fc glyco-engineered) versions of both to promote ADCC. The double-engineered antibody showed stronger C1q binding (boosting CDC) and higher affinity to FcγRIIIA (enhancing ADCC) than native or single-engineered versions, demonstrating improved dual effector functions for better cancer immunotherapy efficacy.
Fig.2 Generation of Fc engineered CD19 antibodies. (Roßkopf, et al., 2020)
Applications
- Autoimmune diseases: Engineering antibodies with modified glycan profiles (e.g., increased sialylation) to reduce inflammation and modulate immune responses in conditions like rheumatoid arthritis and lupus.
- Pharmacokinetic optimization: Modifying glycan structures to extend antibody half-life in circulation, reducing dosing frequency, and improving compliance.
- Biosimilar development: Characterizing and matching glycan profiles of innovator biologics to ensure comparability and efficacy of biosimilar products.
Advantages
- Our streamlined workflow and extensive experience allow for rapid project execution, from initial consultation to final product delivery. This efficiency helps accelerate your drug development timelines.
- We provide robust and comprehensive glycan analysis using state-of-the-art MS and chromatographic techniques. This detailed characterization ensures the quality, purity, and precise glycoform composition of every antibody.
- Recognizing that each project is unique, we offer highly flexible and customized glycoengineering strategies tailored to your specific antibody and goals.
Frequently Asked Questions
CD BioGlyco is your trusted partner in advancing biopharmaceutical innovation through precision antibody glycoengineering. From precise glycan remodeling to site-specific conjugation, our comprehensive services are designed to meet the most demanding research and development challenges. Please feel free to contact us; our team of experienced specialists is eager to collaborate with you and provide tailored solutions.
Associated Services
References
- Li, S.; et al. Glycoengineering of therapeutic antibodies with small molecule inhibitors. Antibodies. 2021, 10(4): 44. (Open Access)
- Roßkopf, S.; et al. Enhancing CDC and ADCC of CD19 antibodies by combining Fc protein-engineering with Fc glyco-engineering. Antibodies. 2020, 9(4): 63. (Open Access)
