Insulin related compounds and identification

Insulin-related compounds (IRCs), which originate during the expression and purification of human insulin using recombinant Escherichia coli, were purified and identified. We investigated the identity of IRCs and their origin. We also presented methods for inhibiting IRC formation. The strains used in this report were E. coli B5K and E. coli H27R. E. coli B5K had a 6-amino acid-fused peptide at the N-terminus of proinsulin, and E. coli H27R had a 28-amino acid-fused peptide at the N-terminus of proinsulin. We investigated the identity of IRCs and their origin by mainly using High Performance Liquid Chromatography (HPLC). The well-known IRCs, desamido human insulin and desthreonine human insulin, formed in both strains. In addition to these two IRCs, the B5K strain produced three different IRCs, ArgA(0)-insulin (IRC 1), prepeptide-insulin (IRC 2), and GluA(22)-insulin (IRC 3). The amounts of IRC 1, IRC 2, IRC 3 were approximately 0.1–0.3% after final purification step. Among these IRCs, ArgA(0)-insulin, prepeptide-insulin, and desthreonine insulin originated from incomplete enzyme reaction. GluA(22)-insulin was formed when we used a double stop codon during the expression of preproinsulin; that is, it was formed by the misreading of the first stop codon through the amber mutation. The major IRCs of the H27R strain were human insulin fragment (B1–B21) (IRC 4), and A9(Ser → Asn) amino acid single mutation human insulin (IRC 5), ArgB(31)-insulin (IRC 6). Human insulin fragment (B1–B21) was formed by β-mercaptoethanol, which was added during refolding. It formed when the disulfide bonds between A-chain and B-chain of human insulin were cut by β-mercaptoethanol, followed by cleavage of the B-chain by trypsin and carboxypeptidase B. A9(Ser → Asn) amino acid single mutation human insulin originated from the mistranslation of A9 serine, such that asparagine was translated instead of serine. ArgB(31)-insulin originated from incomplete enzyme reaction. The amount of IRC 4 was 10–15% after enzyme reaction. The amounts of IRC 5, IRC 6 were around 0.2% after final purification step. We present methods for inhibiting the formation of IRCs by controlling the amount of enzyme, controlling the rate of enzyme reaction, using a single stop codon, using hydrogen peroxide (H2O2) to inhibit β-mercaptoethanol, and modifying the A9 codon.

► We report six human insulin-related compounds from two different expression strains. ► Insulin-related compounds were purified and identified using analytical methods. ► An analytical HPLC method was introduced to detect desthreonine human insulin, which is a major impurity during human insulin production. ► The formation mechanisms of insulin-related compounds were studied based on the identities of the related compounds. ► Taken together, our results present several insulin-related compounds and effective analytical methods for detecting them.