The ICH Q6B Guidelines requires disulfide bridge analysis in biopharmaceutical proteins and antibodies. This include both determination of the number and the position of disulfide bridges.
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. This means non-reduced protein with intact disulfide bridges.
- Determination of exact molecular weight of reduced protein by UV-LC ESI-MS. This means disulfide bridges converted to free cysteine thiol groups.
- Calculation of the number of both disulfide bridges and free thiol groups. We do this 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. We use 2-4 enzymes followed by peptide mapping.
- Digestion of reduced protein/antibody. Here we also use 2-4 enzymes, followed by peptide mapping.
We conduct the analysis at conditions to keep scrambling effects to a minimum.