Different glycan residues have different effects on antibody effector function. CD BioGlyco provides clients with efficient Antibody Remodeling services through chemoenzymatic glycoengineering methods.

Structure of the IgG-Fc Glycans

The Crystallizable fragment (Fc) region of immunoglobulin G (IgG) has glycans at asparagine (Asn) 297, which exerts antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of IgG influence. IgG-Fc glycans typically consist of a core complex biantennary heptose nucleus. Highly heterogeneous glycans can be obtained by adding fucose, galactose, and sialic acid residues to the biantennary heptose core. IgG-Fc glycans are divided into G0, G1, and G2 according to the number of galactose residues in the outer arms of the glycan. Each class of glycans is in turn divided into four subclasses based on the presence or absence of core fucose and bisected N-acetylglucosamine (GlcNAc).

Influence of the Fc Glycan on Antibody Effector Functions

Each component of IgG-Fc glycans affects the effector function of the antibody differently. The high mannose content varies from 5-9 in the Fc glycans linked to the core GlcNAc. It is found that high mannose-type Fc glycans have a positive effect on ADCC activity, but negatively affected the CDC activity of IgG molecules. Fucose residues are added to the GlcNAc residues in the core of IgG-Fc glycans, reducing antibody interaction with Fc receptor IIIa (FcRIIIa) and ADCC activity. Human serum contains approximately 10% bisecting GlcNAc residues. The addition of bisected GlcNAc residues has been reported to enhance the binding affinity of IgG to FcγRIIIa, resulting in a 10~30-fold higher ADCC activity. Human endogenous IgG-Fc glycans have 0, 1, or 2 terminal galactose moieties, respectively. Terminal galactose residue content has been shown to have a significant effect on CDC of IgG, but does not appear to have any effect on ADCC. Sialic acid is usually linked to the terminal galactose of human serum IgG via α-2,3 or α-2,6 linkages. Fc sialylation increases the anti-inflammatory response to intravenous Ig (IVIG).

Fig.1 Schematic representation of IgG glycosylation. (Sjögren, et al., 2020)

Remodeling Antibodies with G2 Glycoforms

Different glycan residues have different effects on antibody effector function. To improve the safety and efficacy of therapeutic antibodies, chemoenzymatic glycoengineering is regarded as one of the most promising approaches to synthesizing homogeneous glycoforms of a given glycoprotein. CD BioGlyco integrates various advanced technologies and has developed efficient Antibody Glycoengineering strategies. We provide clients with homogeneous Fc G2 glycoforms of human IgG (including human IgG1, IgG2, and IgG4) through chemoenzymatic glycoengineering methods. Our workflow is as follows:

• Antibody Preparation and Pre-Analytical Assessment

Upon receipt of the provided antibody material, initial quality control checks are performed to identify any aggregates, fragments, or co-purifying impurities that may interfere with downstream enzymatic reactions. Antibody concentration is accurately determined using UV-visible spectrophotometry (A280).

• Fc Glycan Trimming

The antibody is incubated with a highly specific IgG-cleaving endoglycosidase (e.g., Endo S or Endo S2) to cleave the glycosidic bond at Asn297 in the Fc region, located between the two innermost GlcNAc residues. Small samples are analyzed by mass spectrometry (MS) at various time points to confirm complete deglycosylation and monitor reaction progress. The deglycosylated antibody is then purified by affinity chromatography, size exclusion chromatography (SEC), or other purification methods to ensure purity.

• G2 Glycan Ligation

The purified deglycosylated antibody (containing a core GlcNAc) is conjugated to a preassembled activated oxazoline form of the G2 glycan. Intact MS technology is used to monitor the conversion of GlcNAc antibodies to the G2 glycosylation form in real time or intermittently. Common purification techniques include SEC, anion exchange chromatography (AEX), or hydrophobic interaction chromatography (HIC), which separate different glycan forms based on subtle differences in charge or hydrophobicity.

Workflow

Publication Data

Applications

• This service provides researchers with homogeneous G2-glycosylated human IgG. This is crucial for deeply understanding how specific glycan structures affect antibody function, eliminating research hurdles caused by glycan heterogeneity in traditional production.
• By precisely altering antibody glycan structures, this service helps researchers systematically optimize the safety and efficacy of therapeutic antibodies.
• They enable researchers to precisely engineer conjugation sites on the antibody Fc region, leading to uniform drug-to-antibody ratios (DAR) and protecting antigen-binding and Fc effector functions.

Advantages

• We employ highly optimized and efficient antibody glycoengineering strategies that streamline the complex process of modifying Fc glycans.
• Our service utilizes a rapid chemoenzymatic glycoengineering method, significantly reducing the time required to generate antibodies with specific glycoforms.
• We possess the capability to produce human IgG1, IgG2, and IgG4 subclasses with highly homologous glycoforms. This versatility ensures that researchers achieve precise glycan modifications across different IgG subclasses.

Frequently Asked Questions

CD BioGlyco has been recognized by scientists from several countries in the glycoengineering field. We are willing to share our knowledge and experience in remodeling antibodies with G2 glycoforms. If you are interested in remodeling antibody services with G2 glycoforms, please contact us for more details without any hesitation.

Associated Services

To further support your antibody development and glycoengineering needs, CD BioGlyco offers a range of complementary services and specialized options related to our G2 glycan remodeling expertise.