Analysis of Disulfide Bridges by Peptide Mapping of Biosimilars (CAT#: STEM-B-0004-ZJF)

Peptide mapping is a method for the identification of proteins, especially those obtained by rDNA technology. It involves chemical or enzymatic processing of proteins to form peptide fragments, which are then isolated and identified in a repeatable manner. This is a powerful test that is able to identify changes in almost any single amino acid that are caused by events such as a misread of a complementary DNA (cDNA) sequence or a point mutation. Peptide mapping is a comparison procedure because the information obtained confirms the primary structure of the protein, enables detection of whether the structure has changed, and demonstrates the consistency and genetic stability of the process when compared to reference substances of similar treatment. Each protein has unique characteristics that must be fully understood so that scientific and analytical methods allow the development of validated peptide maps that provide sufficient specificity.

Proteins, Biopharmaceutics

1. Reduction and alkylation of the protein: 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.
2. Digestion of the protein: 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.
3. 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 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.

• Chromatographic apparatus
• Mass spectrometry apparatus
• Sample material