Protein aggregation is a constant risk during the manufacture, storage, and administration of biopharma-ceuticals.1–6
Users of biopharmaceuticals are potentially vulnerable to the harmful effects of aggregation, such as reduced drug bioactivity and possible immunogenic responses to the resulting protein particles.4,7,8
Different molecular mechanisms can cause protein aggregation. One mechanism is protein denaturation with subsequent misfolding or aggregation, resulting in amyloid fibril formation.1,5,6
Although some proteins are more susceptible than others, it is believed that most proteins can form amyloid fibrils under appropriate denaturing conditions.9
Intrinsic fibrillation of endogenous proteins and peptides is a common theme for a variety of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s.10,11
The mechanism of protein fibrillation is rooted in partial protein or peptide denaturation or misfolding, leading to fibril nucleus formation.12
This nucleus catalyzes a rapid and irreversible cascade of amyloid fibril growth. The structural hallmark of all amyloid fibrils is a stacked parallel b-sheet structure.13–15
The fibril-forming nature of regular human insulin is well characterized, and it is often used as a model protein for studying intrinsic protein fibrillation.16–25
Formation of insulin fibrils within pharmaceutical preparations reduces the bioactivity of the drug and may be one of the causes of occlusion in continuous insulin infusion sets.26
Either of these situations can result in diabetic ketoacidosis, a life-threatening complication.27
The key to avoiding insulin intrinsic fibrillation is maintenance of the hexameric state prior to injection, because the monomer is more prone to hydrophobic surface-induced denaturation and intrinsic fibrillation.21
Commercial insulin preparations are formulated with excipients to promote this high-order conformation and help maintain stability. In the presence of zinc, regular human insulin exists as a hexamer that is composed of three dimers. The phenolic excipients that are added to insulin preparations also promote a particularly stable hexameric conformation known as R-hexamer.28–30
Despite the attention paid to fibrillation in regular human insulin preparations, there are little published data available about the intrinsic fibrillation potential of the newer fast-acting insulin analogs in common use today. These analogs have strategic amino acid substitutions in the B chain (listed in
) designed to limit self-association into hexamers.31–33
These substitutions result in greater monomeric nature, leading to faster dissociation, absorption, and biological action after injection.34–38
The resulting changes in self-association may also have implications for the intrinsic fibrillation of the various analogs.
Amino Acid Substitutions in Fast-Acting Insulin Analogsa
The common feature of lispro and aspart is substitution of the B28 proline, which is critical in the formation of the monomer–monomer b-sheet interfaces involved in insulin dimerization.37
These amino acid substitutions prevent dimerization, increasing the monomeric nature of the analogs. However, lispro and aspart do form zinc–insulin hexamers in the presence of the phenolic excipients present in commercial pharmaceutical formulations.29,31
Allosteric interactions of the analogs with these phenolic excipients lead to hexamers resembling the human insulin R-hexamer. After injection, diffusion of these phenolic excipients, zinc dissociation, and insulin dilution contribute to rapid hexamer dissociation into active and readily absorbed monomers.
The glulisine analog is incapable of forming zinc–insulin hexamers because of steric and electrostatic effects from the B3 lysine substitution.37
Also, the B29 glutamic acid substitution reduces but does not prevent dimerization. Glulisine contains an intramolecular salt bridge between the N terminus of the A chain and the B29 glutamic acid, increasing monomer stability. Polysorbate 20, found in glulisine preparations, also acts as a surfactant that further limits surface-induced monomer denaturation at the hydrophobic air–water interface.39
Insulin analog formulations currently in use have been designed and extensively tested to maintain stability in their original primary containers when stored and used appropriately under the manufactures’ recommended storage and shelf-life conditions. However, improper storage, handling, or interactions with medical devices introduce the potential for deleterious changes in both the insulin formulations and the insulin protein. This article evaluates several methods to assess the intrinsic fibrillation of fast-acting insulin analogs and subsequently compares their intrinsic fibrillation rates in the presence of heat and agitation and without the stabilizing excipients that are added to the respective commercial formulations. Intrinsic fibrillation of the analogs was demonstrated by the binding of the fibril-specific dye thioflavine T (ThT), increasing turbidity, and electron microscopy. Complete insulin precipitation was confirmed by ultraviolet (UV) absorbance quantification of soluble insulin and gravimetric quantification of insoluble insulin. Subsequent analysis using an automated 96-well plate format with higher sampling frequency was used to provide a more accurate quantification of intrinsic fibrillation kinetics. This kinetic analysis showed the same relative order of insulin analog intrinsic fibrillation rates, but all were more stable than regular human insulin.
This article represents an early investigation of methods useful for examining the aggregation potential of insulin analogs and an initial comparison of preliminary intrinsic fibrillation rates between the common analogs under conditions designed to exacerbate aggregation.