Insulin Structure and Stability Assessment

Multivariate analysis of different insulin proteins

Score plots of component EEMs from PARAFAC multivariate analysis for human insulin, insulin glargine, and insulin aspart at 4 mg/mL concentration of protein at different pH (4.5, 7.4, 8.5) and temperature. Each group of 5 points represents the data measured at temperatures 5, 20, 25, 30, 37 deg C in that order at each pH and insulin type.

Stability and aggregation of insulin are studied using simultaneous fluorescence excitation emission matrices (EEMs) and UV-Vis absorbance spectroscopy [1]. Insulin is a protein-hormone, produced by the pancreas and is necessary for basic metabolic processes. The different types of commercial insulin therapeutics generally fall into two categories: short-acting and long-acting insulin. The difference between some short-acting and long-acting insulin is, in some cases, only one to three residues in the protein sequence. This difference in sequence, along with controlled pH of storage and delivery, is used to either trigger or prevent the formation of insulin dimers and hexamers in the blood stream. The formation of these aggregates lets the body absorb insulin more slowly and the absence of aggregates makes it absorb more quickly. [2] Changes in protein stability and structure, such as those important to the pharmacokinetics of insulin, can potentially be measured using fluorescence emission spectra, UV-Vis absorbance spectra or sometimes both, using intrinsically fluorescent amino acids. Furthermore, UV-Vis spectrophotometers and fluorometers are typically separate instruments. Here we present a new and fast method for simultaneously generating the individual excitation and emission spectra for all fluorescent sample components providing information needed to correct the fluorescence spectra for the sample concentrationdependent inner filter effects (IFE), as already described [3].

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