Dealing with the Challenges of Disulfide Bridge Analysis in Biopharmaceuticals

Following on from our January 2021 blog, "Dealing with the Challenges of N-Glycan Structures in Complex Glycoproteins" series, this article covers another challenge in biopharmaceutical characterization: disulfide bridge analysis. Any biopharmaceutical product containing the amino acid Cysteine requires an assessment of the free sulfhydryl and disulfide bridge pattern within the product.

Disulfide bridges are directly involved in ensuring that a protein attains its correct 3-dimensional shape, which is critical for the protein's functionality. An incorrect disulfide bridge pattern will render the incorrectly folded portion of a biopharmaceutical inactive, potentially immunogenic and susceptible to aggregation. Products containing mis-matched disulfide bridges are referred to as "product-related impurities" in the regulatory guidance.

  1. What type of organism is used in the manufacture of the product?
  2. How many Cysteine residues are present in the expected primary amino acid sequence?


Disulfide bridge analysis of biopharmaceuticals manufactured in E.coli or a prokaryotic cell type

It is important to understand that biopharmaceutical products manufactured in a prokaryotic cell line such as E. coli (e.g.  Ranibizumab, G-CSF and various insulins) may have been solubilized and refolded i.e. coaxed to form the correct disulfide bridge pattern in vitro. This is because proteins that are recombinantly manufactured in E. coli cells and end up within inclusion bodies contain mainly inactive, misfolded protein in their insoluble form. The inherent 3-dimensional shape of the protein promotes formation of the correct disulfide bonds and requires an oxidative environment and appropriate cellular machinery for this to occur (as found in the Endoplasmic Reticulum (ER) of mammalian cells). However, as with any in vitro process, the process itself can result in mis-folded or incorrect product. For this reason, the regulators place considerable scrutiny on analysis of the disulfide bridge pattern of products manufactured in this way.

Disulfide bridge analysis of biopharmaceuticals manufactured in yeast, insect or mammalian cells

In yeast, insect and mammalian cells, disulfide bridges are introduced into the product as part of the normal cellular processes e.g. in mammalian cells this occurs enzymatically and co-translationally in the ER. This means that the end product should theoretically have the "correct" disulfide bridge pattern. However, there are still some potential issues to consider.

  1. Products containing odd numbers of Cysteine residues may well have free thiols present in the final product (e.g. β-interferon). The free Cysteine residue can exist as a reactive S-, particularly at neutral and basic pH. This reactive site can catalyse disulfide bridge scrambling.
  2. Any post translational contact between the protein and a reductant can potentially break disulfide bridges and cause scrambling and/or formation of disulfide bridges with other Cysteine containing molecules and even Cysteine itself (which can be present in relatively high quantities in cell lysates and cell media).

For these reasons and as an assessment of product fidelity, the regulators require drug developers to assess (to the extent possible) the free sulfhydryl/disulfide bridge pattern of all biopharmaceuticals.

Analytical strategy for disulfide bridge analysis

The ideal strategy for disulfide bridge analysis involves proteolytic digestion between each of the Cysteine residues in the expected primary amino acid sequence with on line LC/ES-MS of the products prior to and following reduction (Figure 1).

schematic outlining the principles of disulfide bridge analysis

High energy MSe and/or MS/MS analysis of the non-reduced material can also be used to confirm assignments of disulfide bridged and free thiol containing peptides.

For proteins with relatively low numbers of Cysteine spread throughout the sequence, this strategy will provide all the necessary data. Additional challenges are presented when a larger number of Cysteine residues are packed into smaller proteins or regions of the protein backbone.

Case Study 1 - Insulin and Analogs

Case Study 2 - IgG2 Monoclonal Antibodies

Case Study 3 - Analysis of Products Containing
Free Thiols


In summary, disulfide bridge analysis is a complex undertaking where many factors should be considered, including:

  1. the origin of the material (i.e. the recombinant cell production),
  2. the potential presence of free thiols within the expected primary amino acid sequence and,
  3. the number and location of Cysteine residues within the expected primary amino acid sequence (i.e. is it possible to proteolytically cleave the protein between each of the Cysteine residues present).

This knowledge will allow development of a strategy to confirming the disulfide bridge pattern (with the assessment of any free sulfhydryls) to the extent required by the various regulators.