Insulin Structure and Stability Assessed by Intrinsic Fluorescence and Simultaneous UV-vis Absorbance Spectroscopy Coupled with Chemometric Analysis

Abstract

Stability and aggregation of insulin is studied using simultaneous fluorescence excitation emission matrices (EEMs) and UV-vis absorbance spectroscopy. Insulin is a protein-hormone, produced by the pancreas and is necessary for basic metabolic processes. The different types of insulin therapeutics, used to treat approximately 1.25 million people in the US with Type 1 Diabetes, generally fall into two categories: short-acting and long-acting insulin. The difference between short-acting and long-acting insulin is one residue in the protein sequence. This residue change, 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. Changes in protein stability and structure, such as those important to the pharmacokinetics of insulin, are often 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. A novel instrument will measure both techniques simultaneously. Fluorescence EEMs are used in various fields to track changes in complex mixtures including water quality and food science, but the use of EEMs in biotherapeutics is relatively new and still being explored. In this study, the stability and structural differences of two commercially available insulin formulations are shown using fluorescence EEMs and simultaneous UV-visible absorbance spectroscopy. Using a novel instrument, absorbance spectra are simultaneously collected with each EEM to correct for non-linearities in fluorescence intensity, specifically resulting from inner-filter effects at high absorbance solutions. Component analysis from these automatically corrected fluorescence EEMs will be presented for tyrosine and phenylalanine residues between the two types of insulin, varying in pH and structure.