Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

Abstract

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products

“…Higher-resolution analytical methods are needed to ensure structural and compositional similarities between protein therapeutics containing the same drug substance. In addition to protein higher-order structure probed by NMR, circular dichroism (CD) or other approaches (2), sub-visible protein aggregation presents an immunogenicity risk to drug safety (3–5). For most solution systems, particle size measurements can be inferred from the measured diffusion coefficient of a molecule and its proportionality to hydrodynamic radius.…”

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli

“…The best known impurities of insulin and insulin analogs are desamido A21 and desamido B3 derivatives ( 6 ). Other related proteins of insulins identified during drug production or storage are: des-Thr-insulin which is an undesirable side-product, di-Arg B31-B32 -insulin, and Arg-insulin, which are intermediates during insulin production, N αB1 carbamoyl insulin, N αA1 carbamoyl insulin ( 7 – 9 ) acetylated lispro insulin ( 10 ), desPhe B1 -N-oxalyl-Val B2 insulin ( 11 ), desPhe B1 -, desPhe B1 -N-formyl-Val B2 - and pyroGlu B4 insulin ( 12 ), covalent insulin dimers ( 13 ), insulin fragments ( 14 ), misincorporated A9 (Ser → Asn) human insulin ( 14 ) and insulin with amino acid residues oxidized to 3,4-dihydroxyphenylalanine (DOPA) and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH) ( 15 ). Such impurities present in a protein substance and dosage forms cannot exceed acceptable amounts in order to meet the Pharmacopoeia requirements ( 16 ).…”

“…Multiple chromatographic separations and pooling of desired fractions are applied in order to collect a sufficient amount of material for further identification and characterization ( 11 ). This is often preceded by keeping a material under stress conditions in order to increase the content of impurities of interest ( 13 ).…”

Phylogenetic, sequence and structural analysis of Insulin superfamily proteins reveals an indelible link between evolution and structure-function relationship

“…Although, in the last few decades many structure, evolution and functional studies (12)(13)(14)(15) have been performed, to our knowledge, there is no study that involves sequence, structure, function and phylogenetic perspective encompassing all the four major families of insulin superfamily. With the surge of sequence information in the post genomics era and recently available structures of several of these ISP members (16)(17)(18)(19)(20) in few cases their complexes with receptors, we were curious to know, whether any novel insights could be derived from the cumulative analysis of these evolutionarily related but functionally diverse molecules having similar structural architecture.…”