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.
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.
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.
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.
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).
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.
Please contact our scientists if you would like to discuss appropriate characterization strategies for your PEGylated biopharmaceutical.
In summary, disulfide bridge analysis is a complex undertaking where many factors should be considered, including:
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.