Insulin related compounds and identification

Area of Science:

• Biochemistry
• Protein Chemistry
• Recombinant Protein Production

Background:

• Insulin-related compounds (IRCs) are impurities that can arise during the recombinant production of human insulin.
• Understanding the origin and identity of these IRCs is crucial for ensuring the purity and safety of therapeutic insulin.

Purpose of the Study:

• To identify and characterize insulin-related compounds (IRCs) formed during human insulin expression and purification using recombinant Escherichia coli.
• To investigate the origins of these IRCs in different E. coli strains (B5K and H27R).
• To present methods for inhibiting the formation of IRCs.

Main Methods:

• High-Performance Liquid Chromatography (HPLC) was primarily used to identify and quantify IRCs.
• Analysis involved characterizing IRCs formed in E. coli B5K and E. coli H27R strains.
• Methods for inhibiting IRC formation were investigated and presented.

Main Results:

• Common IRCs (desamido and desthreonine human insulin) were found in both strains.
• E. coli B5K produced additional IRCs including Arg(A(0))-insulin, prepeptide-insulin, and Glu(A(22))-insulin, with amounts ranging from 0.1-0.3%.
• E. coli H27R produced human insulin fragment (B1-B21) (10-15%), A9(Ser→Asn) mutation insulin (0.2%), and Arg(B(31))-insulin (0.2%).
• Origins identified include incomplete enzyme reactions, mistranslation (amber mutation), and refolding processes involving β-mercaptoethanol.

Conclusions:

• Specific IRCs are generated in distinct E. coli strains due to variations in proinsulin fusion peptides and expression strategies.
• Incomplete enzymatic processing and translational errors are significant contributors to IRC formation.
• Effective strategies for inhibiting IRC formation involve optimizing enzyme concentrations, reaction rates, codon usage, and employing specific chemical treatments.