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Abstract
PurposeIdentification of human insulin analogs’ impurity with a mass shift +14 Da in comparison to a parent protein.MethodsThe protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing.ResultsThe misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs.ConclusionsAlthough there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.
Highlights
Human insulin is a protein hormone produced in pancreatic beta cells
These proteins are biosynthesized in E. coli or yeast as a precursor polypeptide which is cleaved by trypsin and carboxypeptidase B (CPB) to achieve a two-chain protein [3]
Recombinant human insulin and its analogs are produced at IBA by the precursor method using E. coli expression system by the similar procedure, as described in a literature [22]
Summary
Human insulin is a protein hormone produced in pancreatic beta cells. In type 1 and sometimes in type 2 diabetes, insulin must be administered exogenously. Contemporary preparations are based on recombinant human insulin or its analogs produced by rDNA technology [1,2]. These proteins are biosynthesized in E. coli or yeast as a precursor polypeptide which is cleaved by trypsin and carboxypeptidase B (CPB) to achieve a two-chain protein [3]. Productrelated impurities result from post-translational modifications and degradation events induced by the manufacturing process conditions. Properties of these modified entities are different from those of the desired protein and may affect safety and efficacy of a drug product [5]. Other related proteins of insulins identified during drug production or storage are: des-Thr-insulin which is an undesirable side-product, di-ArgB31-B32-insulin, and Arginsulin, which are intermediates during insulin production, N αB1 carbamoyl insulin, N αA1 carbamoyl insulin [7,8,9] acetylated lispro insulin [10], desPheB1-N-oxalyl-ValB2 insulin [11], desPheB1-, desPheB1-N-formyl-ValB2- and pyroGluB4 insulin [12], covalent insulin dimers [13], insulin fragments [14], misincorporated A9 (Ser → Asn) human insulin [14] and insulin with amino acid residues oxidized to 3,4dihydroxyphenylalanine (DOPA) and 2-amino-3-(3,4dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH) [15]
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