Antibody characterization is important in research and biopharmaceutical development. All therapeutic proteins produced from current technologies have heterogeneities in the final product because various modifications occur during different stages of production. Due to these heterogeneities, thorough characterization is necessary for the reproducible and safe production of therapeutic proteins.
At Creative Biolabs, antibodies can be completely characterized using a variety of techniques.
Monoclonal antibodies and antibody drug conjugates (ADCs) are one of the most active areas of biologic drug therapies. Thanks to changes in the regulatory framework in the U.S., an increasing number of new monoclonal antibody (mAb) drug therapies are now entering commercial clinical trials. This trend is creating a strong demand for reliable, fast and efficient solutions for mAb characterization.
The basic mAb characterization workflows consist of the following types of analysis:
In addition, monoclonal and polyclonal antibody binding affinities can be characterized by Simple Western analysis. Relative specificity of the antibody to the target protein of interest can be monitored. Antibody clonal screening can be performed using nanoliter amounts of material.
Other optional Antibody Analysis Services:
Fig. 2 Identification of the core reactive epitope recognized by mAb-4F10 and homology analysis of the identified epitope in Glaesserella parasuis. (Qichao Chen, 2022)
Glaesserella parasuis, the pathogen of Glaser's disease, can invade piglets under stressful conditions, causing serious systemic infection and resulting in high mortality in weaned and nursed pigs. Cytolethal distending toxin (CDT) is a conservative virulence factor and is very important for the pathogenicity of G. parasuis. CdtB is the active subunit of CDT, which can lead to irreversible cell cycle arrest and apoptosis in eukaryotic cells. Here, the researchers screened monoclonal antibodies against CdtB. Among them, mAb 4F10 was found to specifically recognize all serotypes of G. parasuis without any reaction with other porcine bacterial pathogens. In addition, 4F10 can neutralize and inhibit the cytotoxicity of CdtB. Therefore, mAb 4F10 may be helpful for serological diagnosis, prevention, and control of the disease in the future.
One of the primary methods is ELISA (Enzyme-Linked Immunosorbent Assay), which is used to quantify the antigen-binding activity of an antibody and assess its concentration in various samples. Another important technique is Western Blotting, which helps in determining the molecular weight of an antigen and confirming the specificity of the antibody against its target protein. Further techniques include Flow Cytometry, which is employed to analyze the binding of antibodies to cells or receptors on the cell surface in a quantitative manner. This method is particularly useful for assessing the functionality of therapeutic antibodies in a cellular context. Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) are advanced technologies used to measure the binding kinetics and affinity of antibodies to their antigens, providing detailed insights into their interaction dynamics crucial for therapeutic development.
Antibody characterization is fundamental in the development of monoclonal antibodies (mAbs) for therapeutic purposes, ensuring that these biomolecules meet the stringent regulatory standards for safety and effectiveness. Characterization processes evaluate the binding specificity of monoclonal antibodies to ensure that they interact exclusively with their intended target, minimizing off-target effects and potential toxicity. This specificity is crucial for therapies aimed at targeting diseased cells without affecting healthy tissues. Another critical aspect is the assessment of the affinity and avidity of the antibodies, which influences their therapeutic dosage and administration frequency. High-affinity antibodies are often more effective at lower doses, reducing the risk of side effects and improving patient compliance. Additionally, antibody characterization involves evaluating the stability and aggregation tendency of mAbs, as these factors significantly affect their shelf life, efficacy, and safety. Techniques such as Differential Scanning Calorimetry (DSC) and Size-Exclusion Chromatography (SEC) are utilized to assess these properties.
Glycosylation analysis is a critical aspect of antibody characterization, especially for therapeutic antibodies, because the glycosylation patterns significantly influence the efficacy, stability, and immunogenicity of the antibody. Glycosylation, the attachment of sugar molecules to the antibody, can affect the antibody's mechanism of action, including its ability to engage with immune receptors and to elicit an immune response.
Techniques commonly used for glycosylation analysis include Mass Spectrometry (MS) and Capillary Electrophoresis (CE). Mass Spectrometry provides detailed information on the molecular weights and structures of glycan moieties attached to antibodies, offering insights into the heterogeneity and distribution of glycans. Capillary Electrophoresis is utilized to separate glycan species based on their charge and size, allowing for a comprehensive profiling of the glycosylation patterns.
The characterization of Antibody-Drug Conjugates (ADCs) is more complex than that of traditional monoclonal antibodies due to the addition of cytotoxic drugs to the antibody structure. ADC characterization not only involves the evaluation of the antibody's affinity and specificity, but also the stability of the linker and the drug-to-antibody ratio (DAR), which can significantly impact the therapeutic's efficacy and safety.
Techniques such as Hydrophobic Interaction Chromatography (HIC) and Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) are essential for analyzing the DAR and assessing the homogeneity of ADC preparations. HIC allows for the separation of ADC molecules based on differences in hydrophobicity which can indicate variations in drug loading, while RP-HPLC provides high-resolution profiles of ADCs, identifying species with different numbers of drug molecules attached.
Additionally, the stability of the linker between the drug and the antibody is crucial, as premature release of the drug can lead to toxicity and reduced efficacy. Stability tests, including thermal stability analysis and in vitro release studies, are conducted to ensure that the ADC will behave predictably in physiological conditions.
Functional activity assessment is pivotal in antibody characterization, especially for therapeutic antibodies, where it is essential to confirm that the antibody not only binds to its target but also performs its intended biological function. Functional assays are designed based on the mechanism of action of the antibody. For instance, if an antibody is designed to neutralize a virus, a neutralization assay would be conducted to test its ability to prevent the virus from infecting cells. Similarly, if an antibody is expected to induce cell death via apoptosis, cell-based assays can be employed to measure its cytotoxic effects.
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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.
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