首页> 外文会议>Society of Photo-Optical Instrumentation Engineers;Conference on Biomedical Vibrational Spectroscopy: Advances in Research and Industry >Development of an insulin activity test based on cell culture monitoring by microdialysis and infrared spectrometry
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Development of an insulin activity test based on cell culture monitoring by microdialysis and infrared spectrometry

机译:开发基于微透析和红外光谱的细胞培养监测的胰岛素活性测试

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Dependent on various factors such as pH, temperature and shear forces, therapeutic insulins undergo a continuousprocess of chemical degradation during manufacturing and storage until administered by patients. Consequently, changesin secondary up to quaternary structures of the protein appear, with the consequence of a decrease in biological activitydue to partial misfolding of the monomers and finally their aggregation to fibrils. Infrared spectrometry has been appliedfor quantifying chemical degradation processes of therapeutic insulins, based on changes in secondary structure. For thedetermination of insulin potency, the glucose metabolism rate of cells from the human monocytic cell line MONOMAC-6 has been monitored under standardized conditions, providing a measure of biological insulin activity. For cellculture monitoring with a focus on substrate and metabolite concentrations, microdialysis has been used in combinationwith infrared spectrometry of the continuously sampled dialysates with duration up to 48 h. The dialysate spectra wereanalyzed by a classical least-squares (CLS) method with appropriate reference spectra, including the determination ofmicrodialysis recovery rates as obtained from perfusate losses of mannitol, which had been used as internal standard. Byanalysing the time dependent glucose utilization, the potency of tested insulins can be assessed without patient clampexperiments or animal testing.
机译:根据各种因素(例如pH,温度和剪切力),治疗性胰岛素会连续 在制造和存储过程中直至患者使用之前化学降解的过程。因此,变化 在蛋白质的二级至四级结构中出现,导致生物活性降低 由于单体的部分错误折叠,最后由于它们的聚集形成了原纤维。红外光谱已应用 用于根据二级结构的变化来量化治疗性胰岛素的化学降解过程。为了 人单核细胞系MONOMAC-细胞的胰岛素效力,细胞葡萄糖代谢率的测定 已在标准化条件下对6进行了监测,从而提供了生物胰岛素活性的量度。对于细胞 着重于底物和代谢物浓度的培养监测,微透析已结合使用 用红外光谱法对连续采样的透析液进行分析,持续时间长达48小时。透析液光谱为 采用经典最小二乘法(CLS)进行分析,并采用适当的参考光谱进行分析,包括确定 由灌洗液中甘露醇损失而获得的微透析回收率(已用作内标)。经过 分析随时间变化的葡萄糖利用情况,无需患者钳制即可评估测试胰岛素的效力 实验或动物测试。

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