Challenges of Disulfide Bridge Analysis Case Study – Insulin and Analogs

Dealing with the Challenges of Disulfide Bridge Analysis in Biopharmaceuticals

If we consider the six Cysteines spread across the A- and B-chains of Insulin (and Insulin analogs), there is potential for three disulfide bridge structures (assuming that no free thiols are present and that the A- and B-chains are linked by a single disulfide bridge). On this basis, the potential disulfide bridge patterns are shown in Figure 1 below:

schematic outlining the principles of disulfide bridge analysis

possible disulfide bridge combinations for Insulin (and analogs)

Residues 6 and 7 on the B-chain are both Cysteine, meaning that it is not possible to proteolytically cleave between them. So how do you obtain data to satisfy the regulators that, for example, your biosimilar Insulin Glargine has the same disulfide bridge pattern as the innovator reference product?

The answer to defining the structure at a molecular level comes from the strategy outlined above in Figure 2: a digestion strategy needs to be employed which can release disulfide bridged peptides. In our experience with Insulin and analogs, non-specific proteases are best at accomplishing this. The peptides released can then be assessed using MS, MSe and/or MS/MS to provide data to confirm the disulfide bridge structure(s) present and also to provide comparative data with the reference. In essence, a robust digestion strategy accompanied by high end mass spectrometric analysis is able to define which of the disulfide bridge structures above are present and hopefully show that the expected disulfide bridge structure in Figure 2 is present.

correct and expected disulfide bridge structure for Insulin (and analogs)