Disulfide bridge analysis

Determination of disulfide bridges in drug substance protein

Want to know number and position of disulfide bridges in your drug substance protein?

We offer an accurate determination of the number and position of protein disulfide bridges in your drug substance by disulfide mapping. Disulfide mapping consist of two parts as explained below.

The ICH Q6B Guidelines (Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) requires determination of number and position of protein disulfide bridges.

Our disulfide bridge Service includes:

Part 1: Determination of number of protein disulfide bridges and free sulfhydryl groups

  • Exact molecular weight of the native protein is  determined by UV-LC ESI-MS, ie. non-reduced protein with intact disulfide bridges.
  • Exact molecular weight of reduced protein determined by UV-LC ESI-MS, ie disufide bridges converted to free cysteine thiol groups.
  • The number of disulfide bridges and free thiol groups is calculated from the Mw shift between the native and the reduced protein.

Part 2: Position of protein disulfide bridges determined by peptide mapping of non-reduced and reduced protein

  • Non-reduced protein is digested with 2 enzymes and subjected to peptide mapping by nano LC-MS/MS.
  • Reduced protein is digested with 2 enzymes and subjected to peptide mapping by nano LC-MS/MS.

Send us a request to discuss your project.

Protein analysis by Alphalyse

Disulfide bridge mapping of drug substance

  • Number of disulfide bridges by intact mass before and after reduction
  • Position of disulfide bridges by peptide mapping
  • Comparison with Drug Substance sequence
  • Customized analysis based on specific protein property


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