Intrinsic fibrillation of fast-acting insulin analogs

Abstract

Background: Aggregation of insulin into insoluble fibrils (fibrillation) may lead to complications for diabetes patients such as reduced insulin potency, occlusion of insulin delivery devices, or potentially increased immunological potential. Even after extensive investigation of fibril formation in regular human insulin, there are little published data about the intrinsic fibrillation of fast-acting analogs. This article investigates and compares the intrinsic fibrillation of three fast-acting insulin analogs--lispro, aspart, and glulisine--as a function of their primary protein structure and exclusive of the stabilizing excipients that are added to their respective commercial formulations.

Methods: The insulin analogs underwent a buffer exchange into phosphate-buffered saline to remove formulation excipients and then were heated and agitated to characterize intrinsic fibrillation potentials devoid of excipient stabilizing effects. Different analytical methods were used to determine the amount of intrinsic fibrillation for the analogs. After initial lag times, intrinsic fibrillation was detected by an amyloid-specific stain. Precipitation of insulin was confirmed by ultraviolet analysis of soluble insulin and gravimetric measurement of insoluble insulin. Electron microscopy showed dense fibrous material, with individual fibrils that are shorter than typical insulin fibrils. Higher resolution kinetic analyses were carried out in 96-well plates to provide more accurate measures of lag times and fibril growth rates.

Results: All three analogs exhibited longer lag times and slower intrinsic fibrillation rates than human insulin, with glulisine and lispro rates slower than aspart. This is the first study comparing the intrinsic fibrillation of fast-acting insulin analogs without the stabilizing excipients found in their commercial formulations.

Conclusions: Data show different intrinsic fibrillation potentials based on primary molecular structures when the formulation excipients that are critical for stability are absent. Understanding intrinsic fibrillation potential is critical for evaluating insulin analog stability and device compatibility.

Figure 1

Near-UV CD of fast-acting insulin analogs (3.5 mg/ml) (A) in marketed formulations and (B) after transfer to PBS using gel filtration columns. Spectra were corrected for buffer excipients alone and are the average of three scans. Standard deviations are not shown for clarity but were ≤ 1.0 mdeg for each point in (A) and ≤ 0.2 mdeg for each point in (B).

Figure 2

Intrinsic fibrillation of fast-acting insulin analogs in PBS monitored by ThT fluorescence with constant shaking at 37 or 45 °C: (A) lispro, (B) glulisine, and (C) aspart.

Figure 3

Intrinsic fibrillation of fast-acting insulin analogs in PBS at 37 °C, measured by ThT fluorescence, UV absorbance of soluble protein, gravimetric determination of insoluble insulin mass, and turbidity at 600 nm for (A) insulin lispro, (B) insulin glulisine, and (C) insulin aspart. A single representative replicate from n = 3 is shown for each analog. OD, optical density.

Figure 4

Transmission electron microscopy images of fast-acting insulin fibrils formed at 45 °C after agitation of insulin solutions in PBS for 5 days: (A, D) aspart, (B, E) glulisine, and (C, F) lispro. Magnification is 140,000–190,000x for (A–C) (100 nm ruler) and 7200x for (D–F) (2 mm ruler).

Figure 5

Intrinsic fibrillation kinetics of recombinant human insulin and three fast-acting insulin analogs monitored by turbidity (optical density at 600 nm). Plots are the averages of four or five replicates per analog. OD, optical density.

Figure 6

Intrinsic fibrillation lag times and fibril growth rates for recombinant human insulin and insulin analogs. Results are averages of four or five replicates per insulin type. Error bars are ± 1 standard deviation for both lag time (x error) and growth rate (y error).