Challenges of Disulfide Bridge Analysis Case Study – Analysis of Products Containing Free Thiols

Some biopharmaceutical products have a mixture of disulfide bridges and free thiols within their sequence. An example of a molecule showing both bridging and free thiol is human Interferon-beta. The primary amino acid sequence of this glycoprotein is shown in Figure 1 below. The free sulfhydryl and disulfide bridging structure expected for the correctly bridged from are highlighted.

As mentioned above, care must be taken when handling such products since free thiol components can exist as the reactive S^- form at neutral and basic pH. This can induce scrambling of the bridges present in the same or other molecules by "attacking" disulfide bridges and thus shifting the disulfide bridge/free sulfhydryl structure. For example, the free thiol at Cys 17 in Interferon-beta at basic pH could "attack" the disulfide bridge between Cys residues 31 and 141 the result of which would be the two mis-matched structures shown in Figure 2 below. For more heavily bridged proteins this can lead to a cascade effect through the disulfide bridges.

Peptide mapping analysis following tryptic digestion would distinguish these forms but the very act of digesting with Trypsin at around pH8.4 could well induce disulfide bridge/free thiol scrambling. It is therefore important to prevent this scrambling if products with free thiols are to be handled at basic pH. Alkylating the free thiol prior to digestion at basic pH will lock the structure in place by preventing formation of the reactive S^- moiety for example, thus stabilizing the bridge pattern.

For new and innovative products, every attempt must be made to define the absolute structure of the disulfide bridge pattern and this often means that additional protein chemical techniques such as the sequential removal of amino acids from the N-terminus of a disulfide bridged peptide complex or further digestion of purified disulfide bridged peptide complexes could be required.

During biosimilar testing, disulfide bridge structural assessment is helped by the fact that the requirement is to show comparability with the Reference Material, rather than provide absolute assignment of the full disulfide bridge structure. In practice, this means that a comparative overview of the disulfide bridge structure (without the necessity of absolute definition of each disulfide bridge) coupled with the provision of orthogonal data can often be sufficient to provide evidence of comparability. These orthogonal assessments are performed to answer the following questions:

1. Are the secondary and tertiary structures of the Biosimilar and Reference Innovator Materials as determined by Circular Dichroism and NMR (1D and 2D) comparable?
2. Do the Biosimilar and Reference Innovator Materials behave similarly in terms of chromatographic and electrophoretic separation?
3. Is the functionality comparable across Biosimilar and Reference Innovator Materials?

The rationale here is that if the disulfide bridge structure of any given biopharmaceutical contains mismatched disulfide bridges, then the surface charge, surface hydrophobicity, tertiary structure and biological activity would all be impacted.

Therefore, a Denosumab comparability study could be performed by showing that similar levels of the 3 disulfide bridged peptides are present across multiple batches of Biosimilar and Reference Material along with confirming the absence of other disulfide bridged peptides and perhaps the supportive, orthogonal data mentioned above.

For a novel product, the disulfide bridged peptides would need to be collected and further characterized in an attempt to define the disulfide bridge structure to the extent possible. Characterization of the disulfide bridge structure is likely to include further sub-digestion of the collected disulfide bridged peptides, MS^e and MS/MS characterization and potential sequential removal of amino acids from the N-terminus to pull apart the disulfide bridge structure residue to residue.