Peptide Mapping of Biosimilars
Peptide Mapping Analysis
- Reduction and alkylation of the protein of interest
- This step is used to reduce disulfide bridges and block the generated free thiol. Reduction of disulfide bridges opens the protein structure and allows proteolytic digestion to be more efficient. In the case of multi-chain proteins which have disulfide bridges linking the chains (e.g. monoclonal antibodies) this step will also break the bridges between the chains.
- Digestion of the protein using enzymes
- This step breaks the protein down into peptides. The enzymes used are chosen based on the theoretical sequence of the protein and a knowledge of how the enzymes will theoretically digest the protein. In this way, an informed choice of enzymes(s) can be made that should result in the best peptide mapping analysis.
- Analysis of the digested peptides using on-line Reverse Phase-High Performance Liquid Chromatography with Ultraviolet (UV) and Electrospray-Mass Spectrometric detection (LC/ES-MS).
- LC separates the peptides based on polarity (reverse phase-LC).
- Mass spectrometer generates mass information for each peptide as it elutes from the LC column. Fragment ion information is also generated, which allows sequence information from the peptides to be determined. This sequence information serves to confirm peptide identity and provides primary sequence information.
- The mass spectrometers at BioPharmaSpec are also capable of generating fragmentation information from the peptides as they enter the mass spectrometer source from the LC column.
- This is either selective (MS/MS) or non-selective (MSe), depending on the requirements of the study and can be considered as a form of tandem mass spectrometry.