Digestion of IgG in the Hinge Region
CD BioGlyco is a world-leading independent biotechnology company, focusing on high-quality innovative Antibody Digestion Services. Our advanced technology platforms and high-quality services will fully meet your needs for the digestion of IgG in the hinge region. We have the confidence to be your essential research assistant in the field of antibody glycoengineering.
IgG monoclonal antibodies require the synergy of the antigen binding and Fc (effector) domains for their efficient function in the removal of pathological cells. The hinge regions linking these domains exhibit binding effects to Fc receptors on immune effector cells, resulting in pathological cell lysis. In recent years, proteolytic enzymes with no negative impact on human physiology have been successfully used to generate IgG fragments for reagent and therapeutic use. It was subsequently noted that proteases also cleaved human IgG specifically in the hinge region.
Pepsin-mediated digestion allows different sites in the hinge to split IgG, resulting in producing a fragment that contains two binding sites F(ab')2. This fragment is cleaved into two identical univalent fragments that are almost identical with Fab. FC fragments generated by pepsin digestion are usually unable to recover.
CD BioGlyco leverages state-of-the-art enzymatic and chemical digestion technologies to achieve highly specific and reproducible cleavage of IgG. Our primary method utilizes a recombinant cysteine protease immunoglobulin G-degrading enzyme from Streptococcus pyogenes (IdeS). This enzyme exhibits exceptional specificity, cleaving IgG antibodies with high efficiency and precision below the hinge region, yielding intact Fc and F(ab')2 fragments.
CD BioGlyco provides a wide range of IgG digestion services to universities, research institutes, and companies worldwide to meet diverse R&D needs. Our services specialize in IgG antibodies derived from multiple species, including mouse, rat, goat, sheep, and rabbit, as well as human IgG. This flexibility ensures that our targeted approaches are suitable for a wide range of studies, such as intact quality analysis of antibody fragments, comparative studies of biosimilars, and detailed characterization of antibody-drug conjugates (ADCs). We specialize in selecting the appropriate protease for optimal cleavage based on your specific IgG type and desired cleavage site. Our services include:
Upon receipt of your antibody, we determine its purity and concentration using methods such as spectrophotometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to ensure optimal digestion efficiency and subsequent analytical success.
Enzymatic digestion using the protease IdeS under optimized conditions (enzyme-substrate ratio, temperature, pH, and incubation time) ensures specific cleavage within the hinge region, generating the desired fragments (e.g., Fc and F(ab')2), while maximizing yield, specificity, and batch-to-batch consistency.
After digestion, the resulting fragments are purified using advanced chromatographic techniques such as size exclusion chromatography (SEC) or ion exchange chromatography (IEX) to remove enzymes and any residual uncleaved IgG, yielding highly pure Fc and F(ab')2 fragments for subsequent high-resolution analysis.
Purified fragments undergo intact mass analysis by mass spectrometry (MS) to confirm fragment identity and homogeneity and detect any post-translational modifications. We also use advanced chromatographic methods to further assess fragment purity and structural integrity.
We provide a comprehensive report that includes raw data, detailed methodology, and clear, expert interpretation of the results. Our expert team is ready to discuss your findings, provide valuable insights, and help you leverage the data to advance your project goals.
Journal: Molecular & cellular proteomics
IF: 5.5
Published: 2015
Results: This study identifies partial O-glycosylation in the hinge region of human IgG3 for the first time. IgG3 was analyzed using advanced MS (nanoLC-ESI-IT-MS/MS, CE-MS/MS) and recombinant mutants. Researchers found O-glycans (non-, mono-, disialylated core 1-type) attach specifically to threonine residues in the hinge's triple repeats. Serum-derived IgG3 showed ~10% glycosylation site occupancy, while monoclonal versions had ~13%, with little individual variation in O-glycan structures, unlike the variable N-glycosylation in the Fc region. Glycosylation is randomly distributed but not clustered, possibly protecting the hinge from proteases. Pooled plasma IgG3 had altered glycosylation due to purification, and IgG4 only showed hinge O-glycosylation in Fc constructs, not intact antibodies, linking glycosylation to hinge accessibility.
Fig.1 A schematic overview of IgG3, which consists of two heavy chains and two light chains. (Plomp, et al., 2015)
CD BioGlyco has developed an advanced Glycoengineering Platform for research in glycoscience. Our professional research teams provide customers with high-quality services for the digestion of IgG in the hinge region according to your needs. If you are interested in our services, please contact us for more details without any hesitation.
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