What are Disulfide Bridges?

Disulfide bridges are sometimes called disulfide bonds or S-S bonds. They are covalent links between the Sulphur atoms of two cysteine amino acids and their formation stabilizes the tertiary and higher order structure of proteins.

What is the function of disulfide bridges?

Different biologics have different numbers and formations of disulfide bridges that should be fully assessed during protein characterization studies (providing that the expected primary amino acid sequence of the product contains the amino acid cysteine). This is also a requirement of the regulatory document ICH Topic Q6B:

If, based on the gene sequence for the desired product, 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.

Disulfide Bridge Analysis: Methodology

BioPharmaSpec scientists are skilled in the analysis and interpretation of free sulfhydryl groups and disulfide bridges. Peptide mapping post-digestion with a specific protease, followed by analysis using on-line LC/ES-MS and/or LC/ES-MS/MS prior to and following reduction, provides the data necessary for a full assessment of disulfide bridges and free thiols.

During this experiment, a proteolytic agent (enzyme, chemical digest or combination of the two) is designed to digest between the Cysteine residues in the primary amino acid sequence. The resulting peptide mixture, potentially containing disulfide bridged peptides and free thiol containing peptides, is analyzed by mass spectrometry prior to and following reduction.

This method, used widely today for analysis of disulfide bridges, was discovered and reported by BioPharmaSpec CSO Prof. Howard R.Morris in 1985 in this paper.

BioPharmaSpec also provides the Ellman’s test which derivatizes sulfhydryls using (5,5′-dithio-bis-[2-nitrobenzoic acid]). The resulting product is analyzed by UV spectrophotometry at 412nm to determine the levels of free thiols. The Ellman’s test is an important part of any full Cysteine containing product assessment.

Challenges in Disulfide Bridge Analysis

There are many examples of biopharmaceuticals where it is difficult or indeed impossible to cleave between the predicted Cysteine/Cystine residues. In these cases, BioPharmaSpec scientists may purify larger disulfide bridged species, followed by sub-digestion and re-analysis. Another solution is to use manual Edman degradation to sequentially remove amino acids for further mass spectrometric analyses.


Linaclotide is only 14 amino acids long but contains three disulfide bridges when it is correctly produced.

Linaclotide has a very tightly bound structure, due to the high number of disulfide bonds relative to the size of the molecule. This creates challenges for structural characterization because the structure must be fragmented in order to allow mass spectrometric analysis. BioPharmaSpec has developed robust methods for elucidation of the disulfide structure in Linaclotide samples. Please contact us to learn more.

Disulfide Bridge Scrambling in Insulin

Incorrect crosslinking, known as disulfide bridge scrambling, will result in misfolded protein which can be inactive, immunogenic or susceptible to aggregation.

Peptide mapping can be used to assess samples for scrambled bridges. This is a key technique performed during the characterization of Insulin, which is known to have impurities related to S-S scrambling.

Insulin is digested under non-reducing conditions and analysed by LC/ES-MS using a Q-TOF mass spectrometer. Bridging patterns can be assessed in MS mode and signals verified by fragment ions generated by MSe

An assessment of disulfide bridge mis-matching or scrambling is particularly important for products manufactured using E. coli cell systems where the disulfide bridging has to be created post translationally using a chemical process, rather than happening co-translationally (as is the case in mammalian cells).

With all Cysteine containing biopharmaceuticals, disulfide bridge analysis should also be used to assess for other Cysteine containing peptide forms such as mismatched disulfide bridged peptides, trisulfide bridges and thioether linkages. The regulatory guidelines define product containing these forms as product-related impurities that must be identified.

Regulators pay particular attention to the consistency of disulfide bridge patterns (as well as Post Translational Modifications (PTMs) and oligosaccharide profiles) to ascertain whether you have control of your manufacturing process and can produce consistent batches of product.

Disuflide Bridges Form Higher Order Structure

S-S bridging is the first step in forming the correct 3-dimensional shape, or higher order structure, of the biopharmaceutical. Any mismatching of disulfide bridging would lead to a portion of the biopharmaceutical having the wrong 3-dimensional shape and possibly being less active or immunogenic.

Full assessments of secondary and tertiary structure include protein characterization techniques such as Circular Dichroism (CD), Fourier Transform-Infra Red (FT-IR), Intrinsic and Extrinsic Fluorescence and 1D and 2D NMR. Correlating data from these characterization methods with disulfide bridge data will provide an overall assessment of the Higher Order Structure of your biopharmaceutical.