The activity of my biopharmaceutical protein is low, and I suspect that it is related to decreased protein stability. What could be the cause?
Since disulfide bonds provide stability, problems with the protein’s stability and activity often correlate with an abnormal disulfide bridge pattern. For many proteins and peptides, disulfide bridges are even essential for optimal biologic function.
Disulfide bonds are single covalent bonds that form by reaction between the sulfhydryl (-SH) groups of two cysteine residues. Disulfide bonds are crucial for the protein’s tertiary and quaternary structures.
Many biopharmaceutical proteins are cross-linked by disulfide bridges, which increase the durability and protect from proteolytic degradation. Therefore, the disulfide bridge pattern might influence the efficacy and integrity of the final drug product.
Optimize and document biologics process development
With disulfide bridges being so important for the final drug stability, mapping analyses can aid and optimize developmental process steps. If you can get an early insight into the integrity of a biotherapeutic you may also identify potential sources of error early on. You should also be aware that disulfide bonds can contribute to aggregation and must be monitored for patient safety.
If you are involved in biologics manufacturing it is important to document manufacturing consistency, demonstrate comparability and ensure patient safety. If you document a consistent disulfide-bonding pattern of your biologic, you are more likely to meet regulatory guidelines and meet your project timeline.
FDA guidelines: Disulfide bridge mapping is critical
In the biopharmaceutical production, elucidating the cystine positioning is necessary to prevent disulfide scrambling and incorrect folding. This is also seen in recent biosimilars guidelines from FDA and EMA, in which disulfide bond mapping is seen as critical. If you expect any cysteine residues, you must thus determine the number and positions of any free sulfhydryl groups and/or disulfide bridges.
Methods suited for analysis of activity and stability of biopharmaceutical proteins typically include identification of the number and/or position of disulfide bridges present. However, such methods should also be possible to detect mismatched or scrambled disulfide bridge formation.