Terminal sialic acid residues affect antibody efficacy. CD BioGlyco has developed effective Antibody Remodeling strategies. We provide our clients with remodeling antibody services with homogeneous G2S2 glycoform.

Antibody Glycosylation

Antibodies are an important part of the human immune system and are involved in defending against foreign substances and pathogens. All human antibodies are glycoproteins with varying degrees of glycosylation, carrying at least one or more conserved N-glycans in the crystallizable fragment (Fc) domain. These glycans are often present as mixtures of heterogeneous glycoforms and have profound effects on the biological function and therapeutic efficacy of antibodies. The N-glycans of immunoglobulin G (IgG) contain a complex biantennary heptasaccharide core structure consisting of four N-acetylglucosamine (GlcNAc) and three mannoses, as well as other possible monosaccharides such as galactose, bisecting GlcNAc, fucose, and sialic acid.

Fig.1 Structure of IgG. (Boune, et al., 2020)

Effect of Terminal Sialylation on Antibody

Sialic acid is linked to the terminal galactose of human serum IgG via α-2,3 or α-2,6 bonds, accounting for about 11%-15% of IgG-Fc glycans. It is found that the sialic acid residues at the Fc terminus are highly dynamic and do not strongly interact with the protein. However, the anti-inflammatory properties of Fc-sialylated glycans have attracted more and more researchers' attention. Fc sialylated intravenous Ig (IVIG) is used not only to treat immunodeficiency diseases but also to treat autoimmune diseases such as idiopathic thrombocytopenic purpura. In addition, Fc sialylation affects the effector function of antibodies. Sialylated IgG has a reduced affinity for Fcγ receptor III (FcγRIII), resulting in reduced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Remodeling Antibody Service with G2S2 Glycoforms

Sialylated Fc not only contributes to the anti-inflammatory properties of the antibody but affects the effector function of the antibody. To better study sialylated IgG, it is necessary to modify the heteroglycan of a given glycoprotein by chemoenzymatic glycoengineering strategies. CD BioGlyco integrates multiple advanced technologies to develop efficient Antibody Glycoengineering strategies. We provide our clients with human IgG (including human IgG1, IgG2, and IgG4) of the homogeneous Fc G2S2 glycoform. Our workflow is as follows:

• Glycan Trimming and Preparation
  • Select an appropriate IgG-specific endoglycosidase, such as EndoS or EndoS2, to precisely cleave the Fc glycans, ensuring the efficient and complete removal of the sugar chains down to the innermost GlcNAc residue.
  • Optimize the reaction conditions for the endoglycosidase, including temperature, pH, enzyme-to-substrate ratio, and incubation time, to maximize trimming efficiency and minimize any potential off-target enzymatic activity.
  • Perform rigorous quality control on the deglycosylated IgG using analytical techniques like mass spectrometry (MS) to confirm complete glycan removal and verify the integrity of the protein backbone.
  • Isolate the core GlcNAc-modified IgG through chromatographic methods to remove the cleaved glycans, excess enzyme, and any reaction byproducts.
• G2S2 Glycosylation and Characterization
  • Synthesize high-purity G2S2 glycosyloxazoline, ensuring its structural integrity and reactivity for efficient enzymatic transfer.
  • Optimize the glycosynthase-catalyzed reaction conditions, including enzyme concentration, substrate molar ratios, reaction temperature, and buffer composition, to drive the efficient and specific transfer of the G2S2 glycosyloxazoline to the core GlcNAc.
  • Monitor the progress of the glycosylation reaction in real-time or at set intervals using analytical techniques like MS to ensure optimal conversion to the G2S2 glycosylated product.
  • Quench the glycosylation reaction at the optimal time to prevent over-glycosylation or unwanted side reactions, thereby preserving the homogeneity of the desired product.
  • Purify the humanized IgG with the homologous G2S2 glycosyl form using appropriate chromatographic techniques to separate it from unreacted starting materials, enzymes, and any minor byproducts.
  • Rigorously characterize the final G2S2-remodeled IgG using a suite of advanced analytical techniques, including intact MS, peptide mapping, and glycopeptide analysis, to confirm the precise site-specific incorporation of the G2S2 glycan and verify its overall structural integrity.

Workflow

Publication Data

Applications

• Our service enables the production of human IgG antibodies with precisely controlled levels of sialylation, specifically the G2S2 glycoform.
• By providing highly homogeneous sialylated IgG, our service facilitates detailed studies into how sialic acid residues influence key antibody effector functions.
• The remodeling process generates a defined glycan structure, providing a highly specific and uniform site for conjugation.
• By enabling the production of IgG with enhanced sialylation, researchers explore its potential to modulate immune responses and suppress inflammation.

Advantages

• Our service uniquely delivers human IgG antibodies bearing highly homogeneous G2S2 glycoforms, ensuring exceptional batch-to-batch consistency and purity.
• We utilize a cutting-edge chemoenzymatic approach for glycan remodeling, combining the precision of enzymatic reactions with the versatility of chemical modifications.
• We integrate the latest scientific advancements to offer custom solutions for complex glycan modifications, enabling the development of antibodies with optimized effector functions, improved pharmacokinetics, or enhanced targeting capabilities.

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