Antibodies are molecules made up of 4 polypeptide chains: two heavy (H - high-chain) linked to each other by a specific type of chemical bond - a disulfide bridge, and two light (L - light-chain) linked by an identical bond to the heavy chains. Monoclonal antibodies (mABs) are used in various forms - as free molecules, in the form of a drug-antibody combination, and in the form of an immunoconjugate, a drug-antibody-biopolymer combination.
Their main use is in highly specific targeted therapy for cancer, cardiovascular, autoimmune and nervous system diseases. They are also used in the treatment of osteoporosis, Alzheimer's disease or transplantation. The range of applications of monoclonal antibodies is systematically expanded. The introduction of a new antibody to the market requires complex product characterization, which involves numerous proteomic analyses, which are performed by LC-MS/MS techniques, among others.
The characterization of monoclonal antibodies involves many aspects, including, but not limited to, a suite of analyses related to confirmation of the structure, chain sequence and degree of modification of the antibody. Post-translational modifications (PTMs) are chemical changes that occur after the translation process. These changes generally result in an increase in the immunogenicity of the drug. For this reason, it is essential to monitor modifications as critical quality attributes (CQAs) during both the production and storage of the drug. To ensure monitoring of CQA points, the primary structure of the protein is characterized based on peptide mapping (peptide mapping) analysis. The protein under study is subjected to proteolytic digestion using enzymes such as trypsin, chymotrypsin, Lys-C, Glu-C. As a result of various proteolytic enzymes, a wide range of peptides is obtained. Ultimately, coverage of the entire sequence of the protein under study is obtained.
Bioanalytic: Characteristics of monoclonal antibodies.At LabExperts, proteomic analyses are performed on a ZenoTOF 7600 high-resolution LC-MS/MS system (SCIEX). For MS/MS analyses, we use Collision induced dissociation (CID) and Electron-Activated Dissociation (EAD) fragmentation. This provides us with data for unambiguous identification of protein-building amino acids. We process the results using Sciex OS, Skyline and Spectronaut software.
We perform:
- Whole protein analyses - Intact - to determine the exact mass and confirm the theoretical mass;
- N-glycosylation profile determination (based on MS spectrum reconstruction):
- Glycosylated form;
- Deglycosylated;
- Glycosylated with reduced disulfide bridges;
- Deglycosylated with reduced disulfide bridges.
- Peptide maps:
- Protein digestion with various proteases so that sequence coverage is close to 100%
- Analysis of PTMs:
- Location of modifications
- [%] of modifications compared to the unmodified sequence
- TiPOS testing - (technical proof of similarity) - technical confirmation of the similarity of the protein synthesized by the customer (from several LOTs) with the target protein (provided as a template).
In order to maintain the highest possible analytical standards, we perform monoclonal antibody analyses under the regime of an internally developed and implemented laboratory quality system based on GLP principles.
See also our other proteomics services: Protein identification, Protein quantitative analysis
Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies.
Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies.
