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.

中文翻译：

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使用 ATR FTIR 光谱对作为代表性生物药物的胰岛素进行系统稳定性测试，重点是质量保证

意义：生物活性蛋白代表了用于治疗应用的生物制药产品中最重要的成分类别。它们的生产通常是通过基因工程微生物通过生物技术实现的。对于作为救生药物代表的胰岛素的质量保证，需要能够对小瓶或批次中的总蛋白质进行定量的分析方法。化学和物理因素，例如不稳定的温度或储存时的剪切速率暴露，会导致胰岛素的错误折叠、成核和随后的原纤维形成。这些过程与生物活性的降低并行的假设是有效的。目的：红外 (IR) 光谱已成功用于蛋白质错误折叠和原纤维形成的二级结构分析。方法：使用胰岛素干膜傅里叶变换红外光谱结合衰减全反射 (ATR) 技术和随后的数据分析（如带移测定、光谱带解卷积、和主成分分析。结果：对模型胰岛素样品进行了胰岛素光谱的系统研究，这些样品可作为原始制剂或通过超滤纯化的激素获得。胰岛素样本分别在不同温度下储存，即分别在 0°C、20°C 和 37°C 下最多可保存三个月。每周 ATR 测量允许监测激素二级结构变化，这被认为与胰岛素生物活性呈负相关。结论：