FT-IR versus EC-QCL spectroscopy for biopharmaceutical quality assessment with focus on insulin—total protein assay and secondary structure analysis using attenuated total reflection

Abstract

For the quality control of biopharmaceutical products, which contain proteins as the most important active ingredients, shelf life may be limited due to inappropriate storage conditions or mechanical stress. For insulins as representatives of life-saving pharmaceuticals, analytical methods are needed, which are providing additional information than obtained by assays for total protein quantification. Despite sophisticated formulations, the chemical stability may be challenged by temperatures deviating from recommended conditions or shear rate exposure under storage, leading to misfolding, nucleation, and subsequent fibril formation, accompanied by a decrease in bioactivity. A reliable method for insulin quantification and determination of secondary structure changes has been developed by attenuated total reflection (ATR) Fourier-transform infrared spectroscopy of insulin formulations by a silver halide fiber-coupled diamond probe with subsequent dry-film preparation. A special emphasis has been placed on the protein amide I band evaluation, for which spectral band analysis provides unique information on secondary structure fractions for intact and misfolded insulins. Quantitative measurements are possible down to concentrations of less than 0.5 mg/ml, whereas the dry-film preparation delivers high signal-to-noise ratios due to the prior water evaporation, thus allowing a reliable determination of secondary structure information.

Metasurface-enhanced infrared spectroscopy in multiwell format for real-time assaying of live cells

“…Traditionally limited to Fourier transform infrared (FTIR) spectroscopy but recently also expanded to include quantum cascade laser (QCL)-based wavelength-scanning spectroscopy, IR spectroscopy has seen expanding areas of applications in biology and life sciences. 1,2 These include, to name just a few, the analysis of protein samples for research or quality control, [3][4][5] the analysis of biofluid samples such as blood serum and plasma for disease screening, [6][7][8] the spectro-chemical imaging of histology samples as "digital staining", [9][10][11][12] and the spectroscopy and spectro-chemical imaging of fixed cells, live cells, or cell lysates to study cell biology. [13][14][15][16][17] IR spectroscopy-based monitoring of live cells in real time is particularly appealing because it enables quantitative measurements of dynamic cellular changes, such as growth, metabolism, differentiation, and cell-drug interaction, without missing any key biological events.…”

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

“…The ATR IR-spectroscopic method presented here offers a multi-purpose approach, combining the protein’s secondary structure analysis with concentration quantification and identification of insulin subclasses, respectively. 19 Combined with the aforementioned in vitro cell test, which offers insight into the biopotency of selected samples, this method could replace the current section for insulin quality control in international Pharmacopoeias. In comparison to quality assurance by HPLC methods, ATR IR-spectroscopy of protein-enriched insulin dry-films can detect early stages of protein misfolding, secured by reliable validation protocols based on multivariate data analysis, as described here.…”

“…4(b) , other excipient compounds such as phenol or m-cresol are found at low concentrations not contributing significantly to the dry-film spectrum; the same applies to glycerol within the protein amide I and II band interval, as demonstrated earlier by us. 19 However, still some traces of water are left over and visible from the deformation vibration band at .…”

Inspecting Insulin Products Using Water Proton NMR. I. Noninvasive vs Invasive Inspection

“…Specifically, for insulin pens, ifwNMR detects abnormal R 2 ( 1 H 2 O) value of a pen, based on the R 2 ( 1 H 2 O) value alone, one cannot specify whether the aberration is caused by insulin dose deviation, insulin aggregation, insulin misfolding, or insulin chemical degradation. These specific scenarios need to be clarified by invasive analytical techniques; the compendial HPLC method or biopotency method for insulin dose; 33,34 size exclusion chromatography, dynamic light scattering and microflow imaging for insulin aggregates, 19,35 small-angle X-ray scattering, X-ray absorption and transmission electron microscopy for insulin fibrillation, 36 Raman and FT-IR spectroscopy for insulin misfolding and total quantification, 37,38 and LC-MS for insulin chemical degradation. 39 From the quality control standpoint, noninvasive analytical techniques, like wNMR, are best implemented at the drug product (DP) level while invasive analyses are best implemented at the drug substance (DS) level.…”

“…The data show that even the simple procedure of sample transfer can be detected by w NMR, presumably because w NMR is sensitive to O 2 , which is paramagnetic. Techniques that are insensitive to O 2 , such as FT-IR that is equally efficient for liquid and dry-film preparations, 38,40 might not have detected that the insulin pen solution has experienced being transferred out of and then back into the pen. This sensitivity to O 2 is useful in detecting drug product tampering.…”