Challenges of Disulfide Bridge Analysis Case Study – IgG2 Monoclonal Antibodies

The expected disulfide bridge patterns of the various isoforms of the IgG2 product Denosumab mean that multiple bridged peptides may be produced. Figure 1 below shows part of the hinge region, which is packed with Cysteine residues, and the three different bridged isoforms naturally occurring in the antibody.

a)

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b)

c)

d)

Defining the disulfide bridge structure of the hinge region is particularly challenging, with the expected tryptic peptides being:

a)

b)

c)

This complexity means that a strategy built around recovering and detecting a wide range of disulfide bridged peptides, in terms of molecular weight and hydrophobicity, is essential to provide a full picture of the disulfide bridge pattern of an IgG2 product. This also highlights the fact that certain Cysteine residues in some samples may form multiple bridges, all of which are correct and part of the natural product.