当前位置：
X-MOL 学术
›
Anal. Chem.
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Raman Spectroscopy to Monitor Post-Translational Modifications and Degradation in Monoclonal Antibody Therapeutics.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-07-02 , DOI: 10.1021/acs.analchem.0c00627 Bethan S McAvan 1 , Leo A Bowsher 2 , Thomas Powell 2 , John F O'Hara 2 , Mariangela Spitali 2 , Royston Goodacre 3 , Andrew J Doig 4
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-07-02 , DOI: 10.1021/acs.analchem.0c00627 Bethan S McAvan 1 , Leo A Bowsher 2 , Thomas Powell 2 , John F O'Hara 2 , Mariangela Spitali 2 , Royston Goodacre 3 , Andrew J Doig 4
Affiliation
|
Monoclonal antibodies (mAbs) represent a rapidly expanding market for biotherapeutics. Structural changes in the mAb can lead to unwanted immunogenicity, reduced efficacy, and loss of material during production. The pharmaceutical sector requires new protein characterization tools that are fast, applicable in situ and to the manufacturing process. Raman has been highlighted as a technique to suit this application as it is information-rich, minimally invasive, insensitive to water background and requires little to no sample preparation. This study investigates the applicability of Raman to detect Post-Translational Modifications (PTMs) and degradation seen in mAbs. IgG4 molecules have been incubated under a range of conditions known to result in degradation of the therapeutic including varied pH, temperature, agitation, photo, and chemical stresses. Aggregation was measured using size-exclusion chromatography, and PTM levels were calculated using peptide mapping. By combining principal component analysis (PCA) with Raman spectroscopy and circular dichroism (CD) spectroscopy structural analysis we were able to separate proteins based on PTMs and degradation. Furthermore, by identifying key bands that lead to the PCA separation we could correlate spectral peaks to specific PTMs. In particular, we have identified a peak which exhibits a shift in samples with higher levels of Trp oxidation. Through separation of IgG4 aggregates, by size, we have shown a linear correlation between peak wavenumbers of specific functional groups and the amount of aggregate present. We therefore demonstrate the capability for Raman spectroscopy to be used as an analytical tool to measure degradation and PTMs in-line with therapeutic production.
中文翻译：
拉曼光谱监测单克隆抗体治疗中的翻译后修饰和降解。
单克隆抗体（mAbs）代表了生物治疗技术的迅速扩展的市场。mAb的结构变化可能会导致不良的免疫原性，降低的功效以及生产过程中材料的损失。制药行业需要快速，可就地适用于生产过程的新型蛋白质表征工具。拉曼（Raman）已被强调为一种适合该应用的技术，因为它具有丰富的信息，微创，对水背景不敏感并且几乎不需要样品制备。这项研究调查了拉曼在检测单克隆抗体中发现的翻译后修饰（PTM）和降解的适用性。IgG4分子已在一系列已知会导致治疗剂降解的条件下进行温育，包括改变的pH，温度，搅动，光和化学应力。使用尺寸排阻色谱法测量聚集，并使用肽图计算PTM水平。通过将主成分分析（PCA）与拉曼光谱和圆二色性（CD）光谱相结合，我们能够基于PTM和降解分离蛋白质。此外，通过识别导致PCA分离的关键带，我们可以将光谱峰与特定的PTM相关联。特别是，我们确定了一个峰，该峰在具有较高Trp氧化水平的样品中表现出偏移。通过按大小分离IgG4聚集体，我们显示了特定官能团的峰波数与存在的聚集体数量之间存在线性关系。
更新日期：2020-08-04
中文翻译：
拉曼光谱监测单克隆抗体治疗中的翻译后修饰和降解。
单克隆抗体（mAbs）代表了生物治疗技术的迅速扩展的市场。mAb的结构变化可能会导致不良的免疫原性，降低的功效以及生产过程中材料的损失。制药行业需要快速，可就地适用于生产过程的新型蛋白质表征工具。拉曼（Raman）已被强调为一种适合该应用的技术，因为它具有丰富的信息，微创，对水背景不敏感并且几乎不需要样品制备。这项研究调查了拉曼在检测单克隆抗体中发现的翻译后修饰（PTM）和降解的适用性。IgG4分子已在一系列已知会导致治疗剂降解的条件下进行温育，包括改变的pH，温度，搅动，光和化学应力。使用尺寸排阻色谱法测量聚集，并使用肽图计算PTM水平。通过将主成分分析（PCA）与拉曼光谱和圆二色性（CD）光谱相结合，我们能够基于PTM和降解分离蛋白质。此外，通过识别导致PCA分离的关键带，我们可以将光谱峰与特定的PTM相关联。特别是，我们确定了一个峰，该峰在具有较高Trp氧化水平的样品中表现出偏移。通过按大小分离IgG4聚集体，我们显示了特定官能团的峰波数与存在的聚集体数量之间存在线性关系。
京公网安备 11010802027423号