Antibody Digestion Service
CD BioGlyco's advanced Glycoengineering Platform closely follows the development trend in science and technology. We aim to provide customers with unparalleled support services for antibody digestion.
Antibodies are powerful tools for protein and molecular detection and purification. While intact antibodies (usually IgG or IgM) are suitable for most immunoassay applications, the performance of certain steps is enhanced by the use of antibody fragments such as Fab and F(ab')2.
The discovery that the digestion of antibodies with certain proteolytic enzymes yields predictable fragments prompted earlier studies of antibody structures. For example, papain cleavage of the heavy chain CH2 domain above the disulfide bond linking the heavy chain produces three distinct fragments. These resulting fragments consist of the constant and variable regions of the heavy chains (VH and CH1) and light chains (VL and CL) linked by disulfide bonds. Each Fab binds one antigen. The remaining fragment, consisting of CH2 and CH3 heavy chain units, is easily crystallized and is called Fc (crystalline fraction). Subsequent studies showed that the Fc portion of antibodies was responsible for binding to receptors on immunocompetent cells. The IgG heavy chain is cleaved using pepsin at the site below the disulfide bond connecting the two heavy chains. The resulting fragment consists of two Fabs linked together by heavy chain disulfide bonds. The large molecular weight fragment is called F(ab')2. Each F(ab')2 can bind two antigens.
In fact, achieving antibody fragmentation is challenging and requires optimization of enzyme-mediated protein digestion. In addition, a sufficient amount of antibodies needs to be prepared to achieve the desired efficiency. Therefore, fragmentation is only performed when there is a specific need and sufficient antibody is available.
Fig.1 Structure of a typical full-size IgG antibody molecule showing its various domains. (Rodrigo, et al., 2015)
Our core expertise lies in utilizing recombinant enzymes and engineered proteases that cleave at specific, well-defined sites within the antibody structure. This enables us to consistently generate high-purity Fab, F(ab')2, and Fc fragments without affecting the integrity of the glycan structures or the antigen-binding sites.
CD BioGlyco has developed a range of enzymes and related kits for antibody digestion. Our services include antibody dialysis, digestion with enzymes, and purification of proteolytic fragments. Digested antibodies generate homogeneous subunits that facilitate the development and analysis of biopharmaceuticals. In addition, we offer custom antibody fragmentation antibody development services.
Our digestion services of IgG below the hinge region focus on the Digestion of Mouse IgG2a and IgG3 below the Hinge Region. We have optimized protocols specifically for the unique structural characteristics of mouse IgG isotypes. Our methods ensure efficient and precise cleavage of mouse IgG2a and IgG3 antibodies, yielding high-quality fragments for mouse-specific research.
At CD BioGlyco, our digestion services for IgG below the hinge region are as follows:
We understand your specific research goals, antibody characteristics, and desired fragments. We then design a customized enzymatic digestion strategy, selecting the optimal enzyme, buffer, and reaction conditions to ensure precise cleavage.
Using our proprietary protocol, we perform controlled enzymatic digestion of your antibody, carefully monitoring the reaction progress to achieve the desired level of cleavage, minimize off-target proteolysis, and maximize the yield of the desired fragment.
Advanced chromatographic techniques such as affinity and size exclusion chromatography are used to separate Fab, F(ab')2, and Fc fragments from digestion enzymes and residual uncleaved antibody.
Comprehensive characterization using techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) verifies fragment identity, purity, and integrity.
A detailed report is provided, including detailed experimental procedures, quality control data, and analytical results.
Journal: Antibodies
IF: 2.7
Published: 2015
Results: This article explores antibody fragments and their purification via Protein L affinity chromatography. Antibody fragments, including Fabs, scFvs, and dAbs, offer unique biopharmaceutical benefits but lack the Fc region, rendering Protein A chromatography ineffective. Protein L, derived from Peptostreptococcus magnus, binds to kappa light chain variable regions without disrupting antigen binding, making it ideal for purifying many such fragments. Compared to peptide tags (e.g., 6His), Protein L avoids altering fragments or requiring tag cleavage, with robust performance. The study concludes that Protein L has the potential to become a key unit operation in antibody fragment processing, akin to Protein A in monoclonal antibody purification, with further improvements via recombinant technology anticipated.
Fig.2 IgG and various fragments thereof together with detail of the antigen binding Fab region. (Rodrigo, et al., 2015)
CD BioGlyco aspires to become the trusted solution provider of your first choice. Our team of scientists has many years of experience in Antibody Glycoengineering. We provide customers with strong support for antibody digestion. For further details, please don't hesitate to contact us.
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