Analysis of monoclonal antibodies (mAb) by LC-MS/MS – comprehensive qualitative characterization of biological drugs
What are monoclonal antibodies and why is their analysis crucial?
Monoclonal antibodies (mAbs) are a modern class of biological drugs used in the treatment of cancer, autoimmune and infectious diseases. Thanks to their high specificity and selectivity of action, these antibodies enable targeted treatment with limited side effects. Because they are produced in living cells, the process of making them is complex and requires rigorous quality control. Even subtle changes in the structure of a protein can affect its therapeutic efficacy, stability and safety application. Therefore, The analysis of monoclonal antibodies is a key part of every phase of their development, production and evaluation, ensuring efficacy, safety and compliance with the requirements of regulatory agencies
Critical Quality Attributes (CQA) – the basis for mAb assessment
A key element of this process is the identification and monitoring of the Critical Quality Attributes (CQA). i.e. properties Physicochemical, biological and structural, which have a significant impact on the quality, safety and efficacy of a biological product. The CQA assessment is required by regulatory agenciessuch as EMA and FDA, in accordance with the guidelines ICH Q6B. Among the most important parameters of the CQA are:
- amino acid sequence (confirmation of primary structure),
- molecular weight,
- post-translational modifications (e.g., glycosylation, oxidation, deamidation and dozens of others),
- process contaminants (e.g., HCP – host cell proteins),
- structural stability.
Detailed analysis of these parameters by MAM (Multiple Attribution Monitoring) methods makes it possible to ensure compatibility with the reference drug, comparative evaluation of production batches (lot-to-lot) and optimization of the manufacturing process.
Our offer – characteristics of monoclonal antibodies by LC-MS/MS
Our laboratory performs comprehensive analyses of monoclonal antibodies (mAb) using LC-MS/MS mass spectrometry. We work on a high-resolution ZenoTOF 7600 instrument (SCIEX), using advanced fragmentation techniques: CID (Collision-Induced Dissociation) and EAD (Electron-Activated Dissociation). We analyze the data using dedicated software: Biologics Explorer, Sciex OS, Skyline, Spectronaut, PEAKS Studio.
The scope of our LC-MS/MS analyses:
1. Peptide mapping
- Amino acid sequence analysis and confirmation of full protein sequence.
- High sequence coverage due to proteases (e.g., trypsin).
2. Structural analysis – disulfide bond analysis
- Confirmation of the presence of the expected disulfide bonds.
- Identification of the localization of disulfide bonds, including unknown and undesirable sites of their formation in the protein structure, is possible by using the EAD fragmentation technique.
3. Characteristics of post-translational modifications (PTMs)
- Detection and localization of modifications such as glycosylation, phosphorylation, oxidation, deamidation, acetylation and many others.
- Determination of the percentage of PTMs.
4. N-glycans profile analysis
Glycosylation is one of the key modifications affecting the stability, structure and biological activity of monoclonal antibodies. Therefore, detailed analysis of the N-glycosylation profile is an important part of the qualitative characterization of mAb. Gallate analysis includes:
- Analysis of glycosylated and deglycosylated form and after reduction of disulfide bridges
- Localization of typical and atypical glycosylation sites through EAD (Electron-Activated Dissociation).
- Use in biosimilarity studies and at the product development stage
5. Site occupancy
- Assessing the occupancy of glycosylation sites, that is, the percentage of protein molecules in a sample that are glycosylated at a given site.
- This parameter has a significant impact on the structure, stability, function and immunogenicity of the protein.
6. Intact Protein Analysis – total protein mass analysis
- Precise determination of the mass of the entire mAb molecule.
- Verification of compliance with the theoretical mass.
7. The TiPOS (Technical Proof of Similarity) study
- Comparison of synthesized protein (from different LOTs) with reference protein.
- Support for comparative studies of biosimilar drugs.
Why cooperate with our laboratory?
- Modern apparatus and specialized software,
- Experienced team with years of experience in protein analysis,
- Ability to customize the scope of analysis.
Contact us
Contact us for a free quote and to learn how we can support your project. We offer highly specific and sensitive LC-MS/MS characterisation of monoclonal antibodies to ensure the safety, efficacy and regulatory compliance of your biological product.
