Covalent structures biopharmaceutical

Many polypeptides undergo covalent modification after (or sometimes during) their ribosomal assembly. The most commonly observed such PTMs are listed in Table 2.7. Such modifications generally influence either the biological activity or the structural stability of the polypeptide. The majority of therapeutic proteins bear some form of PTM. Although glycosylation represents the most common such modification, additional PTMs important in a biopharmaceutical context include carboxylation, hydroxylation, sulfation and amidation these PTMs are now considered further. 

Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics 2004, 57, 19-34. 

Once a peptide map is characterized using online MS, the chromatographic profile alone can serve as a routine analytical tool to monitor the protein s primary structure and covalent modifications, and is often used for batch release or stability testing of biopharmaceuticals. However, whenever in-depth characterization of a protein is needed, such as that required for comparability studies or reference material characterization, the peptide map should be coupled with MS to ensure a thorough examination of all peptides in the map. 

The sulfhydryl group of the Cys residue can form a covalent disulfide bond with another sulfhydryl. The other sulfhydryl may be from another Cys residue in the same protein, or from a small molecule such as glutathione or free cysteine (glutathionylation or cysteinylation). The formation of disulfide bonds in specific configurations is often important to the structure, function, or stability of the biopharmaceutical protein. Therefore, the confirmation of the disulfide connectivity is necessary. The analysis of disulfide bond structure is discussed in more detail in Section II.A.2. 

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