Mass spectrometry is one of the most important techniques for antibody drug analysis. It is divided based on its applications in analyzing primary structures and advanced structures.
The significant improvement in the performance of the mass spectrometer is mainly based on the development of two ionization technologies: one is the medium-assisted laser desorption/ionization technology, and the other is the electrospray ionization technology. The emergence of these two ionization technologies enables mass spectrometry, which can only detect small molecules, to detect biological macromolecules. In the past, mass spectrometry technology was mainly used for the characterization of primary structures and sequences, but it is now increasingly used for the analysis of advanced structures.
Mass spectrometry can be used for primary structure analysis of antibody drugs
1. Precise molecular weight determination of intact antibody drugs
The molecular weight of an antibody drug can be detected directly by high-resolution MALDI-TOF or ESI-MS. Through the detection of molecular weight after desugaring, preliminary qualitative analysis of antibody drugs can be performed, and it can be used as an analytical method for routine drug release. The analysis of antibody drugs before deglycation can obtain the information of glycosylation type and the distribution of glycosylation level of antibody drugs, which is of great significance for quickly understanding the relationship between production process and drug quality.
2. Drug-antibody coupling ratio (DAR)
For lysine-linked antibody-conjugated drugs, a C4 chromatographic column and coupled mass spectrometry were used to separate the deglycosylated samples. The number of couplings is judged according to the increase in the mass of the coupled drug molecules of different numbers. When using mass spectrometry, not only the exact drug-antibody coupling ratio can be given, but the distribution of linking different small molecule drugs and the distribution of the empty link heads of the byproducts of the reaction process can also be given.
The mass map of peptide fragments obtained after protein is digested by specific enzymes and protease hydrolyzes. Since different antibody drugs have different amino acid sequences, the peptide fragments produced after the protein is hydrolyzed by enzymes are also different, and the mass number of the peptide mixture has unique characteristics. LC-ESI-MS can be used to identify the primary mass number of peptide fragments, and LC-ESI-MS/MS can further confirm each peptide fragment to improve the mapping accuracy.
4. Post-translational modification analysis
Post-translational modification (PTM) of proteins is very important for the biological functions of antibody drugs. Common PTMs include phosphorylation, deamidation, methionine oxidation, glycosylation modification, N-terminal pyroglutamic acid cyclization, and C-terminal lysine excision. The mass spectrometer can detect the molecular weight deviation of proteins and peptide fragments, which can identify the types of protein PTMs in a high-sensitivity, high-throughput, and high-precision manner.
5. N-terminal amino acid sequence detection
Conventional N-terminal amino acid detection can be done by the Edman degradation method, but antibody drugs sometimes exhibit N-terminal cyclization. In this case, the Edman degradation method needs to deblock the antibody first. However, the N-terminal amino acid sequence can be directly measured by mass spectrometry, and the relative proportion of N-terminal cyclization can be detected at the same time.
Mass spectrometry can be used for advanced structural analysis of antibody drugs
1. Hydrogen/deuterium exchange mass spectrometry (HDX-MS)
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can be used to analyze protein conformation, solution kinetics, and epitope mapping. Among the high-order structure and dynamic structure technology, HDX-MS has proven to be suitable for the conformational analysis of monoclonal antibodies and monoclonal antibody-antigen complexes.
2. Ion mobility mass spectrometry (IM-MS)
Ion mobility is a method of selective separation based on the charge and shape of the protein. It can distinguish between proteins and peptides of the same molecular weight, and can be used to detect simple and high-level protein structures.
3. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS)
FTICR-MS has high resolution and is currently recognized as a powerful tool for proteomics research, especially for the identification of complete protein, up/down-regulation, and post-translational modification (PTM).
