Disulfide bonds are sometimes called S-S bonds or disulfide bridges. Disulfide bridges affect the bioactivity of biologics, like peptides, proteins, and antibodies.
Yaohai Bio-Pharma offers disulfide bonds analysis services mainly based on Liquid Chromatography-Mass Spectrometry (LC-MS).
We have been involved in the Protein Structural Characterization of various large molecules, including Recombinant Subunit Vaccines, Nanobodies/VHHs/ Single-domain Antibodies (sdAbs), Antibody Fragments, Hormones/Peptides, Cytokines, Growth Factors (GF), Enzymes, Collagens, etc.
Regulatory Requirements for Disulfide Bonds
There are different numbers and formations of disulfide bridges in different biologics, which should be fully assessed during protein characterization studies if the expected primary amino acid sequence of the product contains the amino acid cysteine. This is also required from the regulatory document ICH Topic Q6B.
Analytical Methods
| Analysis |
Methods |
| Disulfide bonds |
Liquid Chromatography-Mass Spectrometry (LC-MS) after digestion |
For the necessary data of full assessment of disulfide bridges and free thiols, scientists from Yaohai Bio-pharma conduct peptide mapping post-digestion with a specific protease. Thereafter analyse using on-line LC/ Electrospray Ionization Mass Spectrometry (ES-MS) and/or LC/ES-MS/MS prior to and following reduction.
Yaohai Bio-pharma can also conduct Ellman's test, which is an important part of any fully Cysteine-containing product assessment. Within Ellman's test, we derivatize sulfhydryls using (5,5’-dithio-bis- [2-nitrobenzoic acid]). To determine the levels of free thiols, the resulting product is analyzed by UV spectrophotometry at 412nm.
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