The ICH Q6B Guidelines requires a disulfide bridge analysis for biopharmaceutical proteins and antibodies. This include both determination of the number and the position of disulfide bridges.
Our disulfide bridge analysis includes:
Part 1: Determination of number of disulfide bridges and free sulfhydryl groups
- First we determine the exact molecular weight (MW) of the native protein by UV-LC ESI-MS. This means non-reduced protein with intact disulfide bridges.
- We also determine the exact molecular weight of reduced protein by UV-LC ESI-MS. This means bridges converted to free cysteine thiol groups.
- Then we calculate 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
- Then follows a digestion of non-reduced protein/antibody. We use 2-4 enzymes followed by peptide mapping.
- We also digest the reduced protein/antibody with 2-4 enzymes. Finally, we perform peptide mapping, and present all the results in a detailed report.
We conduct the analysis at conditions that keep scrambling effects to a minimum.
Do you want to combine this analysis with other characterization analyses? Then check out our pages on Molecular weight determination • De-novo sequencing of mAb • Peptide mapping • Glycosylation analysis.