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.Graphical abstract
Highlights
For the chemical analysis of commercial biopharmaceuticals for therapeutic usage and their bioactivity assessment, appropriate analytical methods are required, when concerned with proteins as the active ingredients and their excipients
Formulated products include a collection of protein- or peptide-based therapeutics, which involve a wide range of products such as vaccines, blood components, e.g., interferons or growth factors, and recombinant therapeutic proteins
In contrast to prevalent transmission spectroscopy, samples can directly be analyzed without any preparation
Summary
For the chemical analysis of commercial biopharmaceuticals for therapeutic usage and their bioactivity assessment, appropriate analytical methods are required, when concerned with proteins as the active ingredients and their excipients. Formulated products include a collection of protein- or peptide-based therapeutics, which involve a wide range of products such as vaccines, blood components, e.g., interferons or growth factors, and recombinant therapeutic proteins. Therapeutic proteins can have enzymatic or regulatory and pharmacological activities. Human insulins and their analogs, which are representative members of the category of life-. There are different challenges for the quantification and determination of the molecular stability in pharmaceuticals during production or later when obtained from pharmacies— especially after long-term storage under different environmental conditions that may be limiting their lifecycle These adverse storage conditions can be associated with breaking the storage cold chain or during transport, encountered with shaking and elevated temperatures [1]. First deteriorating effects can be protein misfolding with drastic changes in the protein
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