Monoclonal Antibody Characterization Service

Monoclonal antibody characterization is the comprehensive identification of the structure, function, and quality of monoclonal antibodies to ensure their accuracy, consistency, and stability. Monoclonal antibodies are a class of antibodies produced by a single B-cell clone, characterized by high specificity and affinity, allowing them to precisely recognize and bind to specific antigen epitopes. Monoclonal antibodies (mAbs) are Y-shaped three-dimensional structures typically composed of two heavy chains and two light chains. The Fv region, located within the variable regions of the heavy and light chains, is responsible for specific antigen recognition. Within the Fv region, the complementarity-determining regions (CDRs) serve as the core antigen-binding sites, consisting of three highly variable short peptide segments that determine the antibody's specificity and affinity. The Fab region of the antibody is primarily responsible for antigen binding, Monoclonal antibody characterization is is essential since the Fc region mediates effector functions such as immune system activation. mAbs often exhibit heterogeneity, such as C-terminal lysine processing, glycosylation, oxidation, and deamidation, which can impact their function and stability.

 



Figure 1. The Structure of Monoclonal Antibody

 

Monoclonal antibody characterization is crucial because its quality directly affects the efficacy and safety of the product. Monoclonal antibody characterization ensures the correct structure and proper folding of higher-order structures, confirming that the antibody functions as intended and preventing adverse effects caused by incorrect modifications or impurities. Monoclonal antibody characterization addresses critical issues such as purity and impurity analysis, verification of antigen-binding properties, and evaluation of effector functions. Additionally, it detects variations in the production process, supporting process optimization and quality control. The results of monoclonal antibody characterization provide critical data for antibody drug development, manufacturing, and regulatory submission, ensuring product consistency, safety, and efficacy.

 

Below are the content of monoclonal antibody characterization:

1. Structural and Physiochemical Characterization

Structural and physicochemical characterization according to the ICH Q6B guidelines for biopharmaceutical products should include amino acid sequence analysis, N-terminal and C-terminal sequencing, peptide mapping, identification of free sulfhydryl groups and disulfide bonds, carbohydrate content, glycosylation, glycan structure, and post-translational modifications such as oxidation and deamidation. mAbs typically have an N-glycosylation site on each heavy chain located in the Fc region, and the presence or absence of these sites must be verified in mAbs. Secondary, tertiary, and quaternary structures are collectively referred to as higher-order structures (HOS), which determine the 3-D shape and proper folding of biopharmaceuticals. Incorrect 3-D conformation can affect protein function and may lead to inhibition of antigen binding, exposure of immunogenic epitopes, and protein aggregation. The structure and physicochemical properties analysis in monoclonal antibody characterization involves various analytical techniques and instruments.

 



Fojtík, L. et al. Anal Chem. 2024. 

Figure 2. Quantification of the Modification for Aromatic Residues of Trastuzumab

 

2. Immunological Properties Characterization

It is essential to analyze the binding assay between the antibody and antigen as well as the identification of CDRs. Additionally, epitopes and molecules bearing their associated epitopes are identified through immunohistochemical methods. The measurement of affinity, avidity, and immunoreactivity is carried out using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Both ELISA and SPR are considered complementary techniques, providing consistent results in the moloclonal antibody characterization. These techniques can also be used to study mAb-antigen complexes and provide affinity values in the form of equilibrium dissociation constants. The biological activity and pharmacokinetics of mAbs inherently depend on their binding to the target antigen, and controlling this binding is crucial for mAb characterization. SPR technology has extensive applications in achieving monoclonal antibody characterization, as it can measure receptor binding, antigen binding, and the active concentration required for binding. SPR also aids in determining epitope specificity, which is a critical component of monoclonal antibody characterization.

 



Petrotchenko, EV. et al. J Proteome Res. 2023.

Figure 3. Determination of Monoclonal Antibodies Epitope through Integrated Structural Proteomics Methods

 

3. Biological Activities Characterization

Monoclonal antibody characterization involves the analysis of its biological activity through appropriate in vitro assays (enzymatic/radioisotope/fluorescence methods) and in vivo experiments. Detailed analyses are also conducted on antibody-dependent cellular cytotoxicity (ADCC), cytotoxicity (apoptosis), complement binding and activation, as well as neonatal Fc receptor binding activity, to determine the mechanism of action and significance of the product's effector functions. The mechanisms underlying the biological activity of mAbs include immune-mediated cytotoxicity, direct cytotoxicity, immune system modulation, and vascular system disruption. Due to various advancements in mAb development, they are now used for the treatment of a wide range of cancers and autoimmune diseases, with the added benefit of reducing the side effects commonly observed in traditional therapeutic strategies. However, extensive research is still needed to identify new targets for mAbs and improve their therapeutic potential. mAbs are a part of multidisciplinary treatment approaches for cancer patients and have been shown to significantly improve the quality of life for those undergoing cancer treatment.

 



Dobson, CL. et al. Sci Rep. 2016.

Figure 4. Analysis of Biophysical and PK properties of MEDI-578 and MEDI1912 (Monoclonal Antibody)

 

4. Purity and Contaminants Characterization

Monoclonal antibodies (mAbs) typically exhibit various sources of heterogeneity, such as C-terminal lysine processing, oxidation, deamidation, fragmentation, isomerization, disulfide bond mismatches, glycosylation, and N-linked oligosaccharides. These heterogeneities result in a complex purity/impurity profile comprising multiple molecular entities or variants. Monoclonal antibody characterization comprehensively analyzes these heterogeneities by determining a range of physicochemical properties, including molecular weight or size, isomeric patterns, extinction coefficients, electrophoretic profiles (based on size and charge), chromatographic data (based on size, charge, hydrophobicity/hydrophilicity), and spectroscopic data (for structural analysis, including secondary and tertiary structures). Additionally, microbial species and endotoxins must be appropriately inhibited or completely removed, while monocyte activation tests and other analyses are introduced to eliminate pro-inflammatory contaminants, such as peptidoglycans.

 



 Leal-Lopes, C. et al. N Biotechnol. 2023.

Figure 5. Monoclonal Antibodies Purity and in vitro Functionality Analysis by SEC and SDS-PAGE

 

5. Quantification Characterization

Physicochemical and immunochemical assays have been used to determine the quantity of mAbs. The observed quantities are directly correlated with the results of biological assays. Quantitative analysis of mAbs using instrumental methods such as colorimetric assays, HPLC, or ion chromatography (IC) has been previously reported.

 



 Schäfer, A. et al. Anal Bioanal Chem. 2024. 

Figure 6. HPLC–MS/MS-based Quantification of Human Monoclonal Antibodies 

 

Monoclonal antibody characterization involves evaluating the interaction between the antibody and the purified antigen in customized binding assays to determine the immunological properties of the developed antibody. It also includes functional assessments through various in vitro cell-based assays, which elucidate cytotoxicity and effector functions. The quantity of the antibody, a critical parameter, is determined using calorimetric assays or chromatographic methods. Furthermore, characterization requires the identification and both quantitative and qualitative evaluation of process- and product-related impurities and contaminants. To meet regulatory guidelines, each new batch of mAbs must undergo identity, heterogeneity, impurity content, and activity testing before release.

 

Services at MtoZ Biolabs

MtoZ Biolabs offers professional monoclonal antibody characterization service using high-resolution mass spectrometry (LC-MS/MS) and advanced techniques to comprehensively characterize antibody amino acid sequences, post-translational modifications (such as glycosylation, oxidation, and deamidation), and higher-order structures (HOS). Our service includes purity analysis, heterogeneity evaluation, impurity detection, and antigen-binding validation, ensuring structural integrity and functional consistency of the antibodies. Our platform supports diverse analytical needs, providing efficient and accurate antibody identification solutions to advance antibody drug development and production quality control. If you are interested in our service, please contact us freely.

 

Service Advantages

1. High Sensitivity and High-Resolution  

MtoZ Biolabs leverages advanced LC-MS/MS technologies (such as Orbitrap and Q-TOF) to perform comprehensive and precise monoclonal antibody characterization, including intact protein analysis, peptide mapping, and detailed detection of post-translational modifications (e.g., glycosylation, oxidation, and deamidation). This service overcomes the challenges of detecting low-abundance antibodies in complex biological matrices, providing customers with highly sensitive and high-resolution results.

 

2. Comprehensive Characterization   

MtoZ Biolabs offers qualitative and quantitative analyses of monoclonal antibody heterogeneity, including C-terminal lysine processing, disulfide bond mismatches, glycosylation, and aggregate formation. This comprehensive heterogeneity characterization helps customers understand the impact of antibody variations on function and stability, aiding in quality control and regulatory compliance during antibody development.

 

3. Efficient Impurity Detection  

Through optimized LC-MS methods, MtoZ Biolabs accurately detects impurities from the production process, including host cell proteins, endotoxins, and antibody degradation products. This rigorous impurity assessment complies with ICH Q6B guidelines, helping customers ensure the safety, purity, and consistency of monoclonal antibody products, while providing reliable data for preclinical research and regulatory submissions.

 

Deliverables

1. Comprehensive Experimental Details

2. Materials, Instruments, and Methods

3. Relevant Liquid Chromatography and Mass Spectrometry Parameters

4. The Detailed Information of Monoclonal Antibody Characterization

5. Mass Spectrometry Image

6. Raw Data