Can disulfide bridges influence the stability of my protein?
The activity of my biopharmaceutical protein is low, and I suspect that it is related to decreased protein stability. Could problems with the disulfide bridges be the cause?
Since disulfide bonds provide stability, problems with the protein’s stability and activity often correlate with an abnormal disulfide bridge pattern. For many proteins and peptides, disulfide bridges are even essential for optimal biologic function [1, 2].
Disulfide bonds are single covalent bonds that form by reaction between the sulfhydryl (-SH) groups of two cysteine residues. Disulfide bonds are crucial for the protein’s tertiary and quaternary structures [1, 2].
Many biopharmaceutical proteins are cross-linked by disulfide bonds, which increase the durability and protect from proteolytic degradation. Therefore, the disulfide bridge pattern might influence the efficacy and integrity of the final drug product [3, 4].
Optimize and document biologics process development
With disulfide bridges being so important for the final drug stability, mapping analyses can aid and optimize developmental process steps. If you can get an early insight into the integrity of a biotherapeutic you may also identify potential sources of error early on. You should also be aware that disulfide bonds can contribute to aggregation and must be monitored for patient safety [3-7].
If you are involved in biologics manufacturing it is important to document manufacturing consistency, demonstrate comparability and ensure patient safety. If you document a consistent disulfide-bonding pattern of your biologic, you are more likely to meet regulatory guidelines and meet your project timeline [5-7].
FDA and EMA guidelines: Mapping of disulfide bridges is critical
In the biopharmaceutical production, elucidating the cystine positioning is necessary to prevent disulfide scrambling and incorrect folding. This is also seen in guidelines from FDA and EMA, in which disulfide bond mapping is seen as critical. If you expect any cysteine residues, you must thus determine the number and positions of any free sulfhydryl groups and/or disulfide bridges [5-7].
European Medicines Agency (EMA):
“Sulfhydryl group(s) and disulfide bridges. If cysteine residues are expected, the number and positions of any free sulfhydryl groups and/or disulfide bridges should be determined, to the extent possible. Peptide mapping (under reducing and non-reducing conditions), mass spectrometry, or other appropriate techniques may be useful for this evaluation” 
Methods suited for analysis of activity and stability of biopharmaceutical proteins typically include identification of the number and/or position of disulfide bridges present . However, such methods should also be possible to detect mismatched or scrambled disulfide bridge formation.
Find out more about analysis of disulfide bridges at
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 Sevier et al: “Formation and transfer of disulphide bonds in living cells“, Nature Reviews Molecular Cell Biology, 2002
 Thornton, J.M.: “Disulphide bridges in globular proteins“, Journal of Molecular Biology, 1981
 Dobson, C.M.: “Protein folding and misfolding“, Nature, 2003
 Kosuri et al: “Protein Folding Drives Disulfide Formation“, Cell, 2012
 U.S. Department of Health and Human Services – Food and Drug Administration: “Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products“, 1999
 European Medicines Agency: “NOTE FOR GUIDANCE ON SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS“, 1999
 U.S. Department of Health and Human Services – Food and Drug Administration: “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product“, 2015