Digestion of Human IgG1 Above the Hinge Region
CD BioGlyco has been working on the Glycoengineering for many years. We have developed systematic solutions to provide customers with the digestion of human IgG1 above the hinge region services.
Although the importance of antibodies in therapy is well-known. However, when only part of the function of the antibody is required, such as blocking the biological activity of the molecule, only the part of the antibody can be used, and this part is derived from the smaller protein of the antibody called the antibody fragment. Among them, antigen-binding fragments (Fabs) have high-affinity binding properties. For therapeutic and basic research, mass production of Fab fragments with retained binding activity is critical.
While it is possible to engineer Fabs using bacterial expression systems, expression of Fabs containing essential disulfide bonds necessary for their activity and stability is challenging using an Escherichia coli (E. coli) system. Limitations in yield, structural, and functional are often encountered when producing Fab fragments in E. coli. In general, Fabs used in research (e.g., antibody mimetics, antibody-drug conjugates, bispecific antibodies) are typically obtained by enzymatic digestion of monoclonal antibodies using immobilized papain. Despite obtaining pure Fab, the use of immobilized papain to digest IgG has limitations such as long digestion times (often more than 8 hours), high cost, and limited scalability.
Fig.1 Representation of the structure of the IgG motif and related fragments. (Collins & Khalili, 2022)
We use a highly specific cysteine protease IgdE. Unlike non-specific proteases that cleave at multiple sites and generate a mixture of fragments, IgdE targets a single, highly specific site within the human IgG1 heavy chain. This cleavage occurs precisely above the hinge region, a flexible area connecting the Fab and Fc domains. The result is the generation of two intact, homogeneous Fab fragments and a single, homogeneous Fc fragment.
Proteolytic cleavage with papain is the most common method for producing Fab monovalent binders. However, this papain-mediated digestion often results in under- or over-digestion. And the desired Fab needs to be optimized (e.g., chromatographically purified).
The protease provided by CD BioGlyco is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification. The protease digests human IgG1 at one specific site above the hinge with no need for reducing conditions or co-factors, generating intact Fab and Fc fragments.
Our experts will discuss your unique project needs and key antibody information via email, phone, or meeting to understand your specific needs. These include the source and properties of the IgG1, desired antibody purity, and intended downstream applications. We use this information to select and optimize IgdE protease parameters to ensure high-yield and highly specific cleavage.
Upon receipt of the IgG1 sample, we perform an initial check to confirm its integrity. Protein concentration is measured using UV-Vis spectrophotometry, and any pre-existing aggregation or degradation is assessed using size exclusion chromatography (SEC).
We meticulously optimize key reaction parameters, including enzyme-substrate ratio, incubation time, and temperature, to ensure that IgdE protease cleaves the IgG1 protein only at the specific site above the hinge region.
After digestion, the Fab and Fc fragment mixture undergoes our advanced purification process, typically involving affinity or ion exchange chromatography. This step ensures that the final fragment is free of contaminants that could interfere with the results.
The purified Fab and Fc fragments undergo a final quality assessment, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the expected molecular weight and purity, and mass spectrometry (MS) to verify the accurate mass.
Journal: Analytical Chemistry
IF: 7.4
Published: 2023
Results: This study compares two hinge-cleaving proteases, IgdE and BdpK, for liquid chromatography-MS (LC-MS)-based IgG1 clonal profiling. Experiments showed highly similar qualitative and quantitative clonal repertoires post-digestion, validated by top-down electron transfer dissociation proteomics. BdpK detected more clones (~700 vs. ~350 with IgdE) and higher total ion current, primarily capturing low-abundant clones. Neither protease introduced detectable biases, but BdpK is preferred for its shorter digestion time, equal specificity, and efficiency in IgG1 clonal profiling and potential Fab de novo sequencing.
Fig.2 Experimental design for the comparison of the performance of BdpK and IgdE for LC-MS-based IgG1 Fab clonal profiling. (van Rijswijck, et al., 2023)
At CD BioGlyco, backed by our team's extensive expertise and a commitment to quality, we provide a reliable solution that saves you time and delivers reproducible results. Please feel free to contact us to learn more about our IgdE digestion service or to discuss your specific project needs.
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