化学
表征(材料科学)
工作流程
大小排阻色谱法
生物过程
纳米技术
多角度光散射
色谱法
生物系统
光散射
计算机科学
化学工程
散射
数据库
材料科学
生物化学
物理
光学
工程类
生物
酶
作者
Rodell C. Barrientos,Andrew N. Singh,Ophelia Ukaegbu,Mohamed Hemida,Heather Wang,Imad A. Haidar Ahmad,Hang Hu,Zachary D. Dunn,Emmanuel Appiah‐Amponsah,Erik L. Regalado
标识
DOI:10.1021/acs.analchem.3c05969
摘要
The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.
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