The ICH Q6B Guidelines requires disulfide bridge analysis with determination of number and the position of disulfide bridges in biopharmaceutical proteins and antibodies.
Part 1: Determination of number of disulfide bridges and free sulfhydryl groups
- Determination of exact molecular weight (MW) of the native protein by UV-LC ESI-MS, i.e. non-reduced protein with intact disulfide bridges
- Determination of exact molecular weight of reduced protein by UV-LC ESI-MS, i.e. disulfide bridges converted to free cysteine thiol groups
- Calculation of the number of both disulfide bridges and free thiol groups from the MW shift between the native and the reduced protein
Part 2: Position of disulfide bridges determined by peptide mapping of both non-reduced and reduced protein
- Digestion of non-reduced protein/antibody with 2-4 enzymes followed by peptide mapping
- Digestion of reduced protein/antibody with 2-4 enzymes followed by peptide mapping
Analysis is conducted at conditions to keep scrambling effects to a minimum.
Do you want to combine this analysis with other characterization analyses? For example: Molecular weight determination • De-novo sequencing of mAb. • Peptide mapping.