Systematic stability testing of insulins as representative biopharmaceuticals using ATR FTIR-spectroscopy with focus on quality assurance

Abstract

. Significance: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity. Aim: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation. Approach: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis. Results: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity. Conclusions: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.

Chemical Bonding of Biomolecules to the Surface of Nano-Hydroxyapatite to Enhance Its Bioactivity

“…The absorption band at 1740 cm −1 in the FT-IR spectrum (Figure 18c) is attributed to the carbonyl groups of PLGA. In addition, the absorption bands at 1650 cm −1 and 1550 cm −1 (Figure 18b) were due to the presence of amide I and amide-II groups, which were formed on the bonding of insulin to nHA-S [94]. The FT-IR spectrum of the PLGA/nHA-I scaffold (Figure 18d) was also having all characteristic peaks of nHA, PLGA, and insulin.…”

Engineering Flexible Metal‐Phenolic Networks with Guest Responsiveness via Intermolecular Interactions

“…The presence of Fe−O stretching vibrations at 625 cm −1 and the two characteristic peaks at amide I and amide II indicated the incorporation of INS within the capsules (Figure 3g). [19] The fluorescein isothiocyanate‐labeled insulin (FINS) enabled the visualization of the capsules (Figure 3h), and energy‐dispersive X‐ray (EDX) mapping results revealed the presence of elements C, N, O, S, and Fe (Figure 3i), confirming the formation of FINS@MPN capsules.…”

Subcutaneous amyloidoma models for screening potential anti-fibrillating agents in vivo

“…Diabetic patients under insulin therapy often develop insulin-derived amyloidoma at the site of repeated injections ( D’Souza et al., 2014 ; Mayhew et al., 2017 ; Nagase et al., 2009 ; Nilsson, 2016 ) because commercially available injectable insulin formulations (especially fast-acting analogs) with phenolic preservatives are susceptible to heat-induced fibrillation ( Ahmad et al., 2003 ; Carter and Heinemann, 2018 ; Delbeck and Heise, 2021 ; Woods et al., 2012 ). This iatrogenic amyloidosis with associated subcutaneous insulin resistance ( Nagase et al., 2014 ; Nakamura et al., 2019 ) fuelled biomedical research towards developing anti-amyloidogenic insulin formulations.…”