Disulfide Bridge Analysis

Creative Biolabs has established a platform to analyze the disulfide bridge of therapeutic drug candidates, strictly following the ICH Topic Q6b guideline. When cysteine residues are expected for the desired protein based on the gene sequence, the number and positions of any free sulfhydryl groups and/or disulfide bridges should be confirmed. The stability of proteins is crucially enhanced by naturally occurring disulfide cross-links. A disulfide bond can increase as much as 5-6 kcal/mol to the stability of the folded protein at optimal temperature.

Disulfide bridge analysis

Disulfide bonds are formed in proteins when they become mature in the cell. The maturation process occurs in the endoplasmic reticulum, Golgi complexes and mitochondrial inter-membrane space in eukaryotic cells, and also in the periplamsic space in bacteria. Disulfide bonds are formed by the assistance of a class of enzymes, known as oxidoreductases. Disulfide bonds stabilize the protein’s 3D structure and are critical for their biological function. In biopharmaceutical industry, disulfide bond match and mismatch are highly concerned and need to be confirmed. Some disulfide bonds contribute a functional role and have been classified as catalytic or allosteric bonds. The catalytic bonds locate in the active sites of oxidoreductases, which mediate thiol/disulfide exchange in other proteins. The allosteric bonds control the function of the protein in that they reside by mediating a change when they are reduced or oxidized. The redox state of the allosteric disulfides is controlled by the catalytic disulfides.

Monoclonal antibodies (mAbs) are one of the fastest growing families of pharmaceutical products. They contribute greatly in the treatment of various conditions, e.g. cancer, infectious diseases, allergies, inflammation and auto-immune diseases. Since mAbs can demonstrate significant heterogeneity, extensive analytical characterization is requested to achieve approval for a new mAb as a therapeutic product. Creative Biolabs provide a series analysis of sulfhydryl groups and disulfide bonds by Matrix-assisted laser (LC/ESI-MS, Q-TOF). The basic strategy for analysis includes the identification of disulfide-linked peptides in digests of proteins and characterization of their half-cystine peptide constituents. All the proteins are characterized in house in 2-3 weeks.

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