Methods comparison of two-dimensional gel electrophoresis for host cell protein characterization

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Abigail King, Yiwei Zhao, Alexandru Lazar, Margeaux Capron, Niranjan Thiruvur, Xinrong Liu
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Abstract

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non-traditional method of 2D-DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP-based applications.

Abstract Image

用于鉴定宿主细胞蛋白质的二维凝胶电泳方法比较。
二维电泳(2DE)是一种基于尺寸和电荷的凝胶蛋白质分离方法,常用于生物技术和制药公司药物开发过程中宿主细胞蛋白质(HCPs)的表征。HCPs 是在生物药品生产过程中由宿主细胞产生的异源蛋白质混合物。我们比较了不同的凝胶电泳方法,包括传统的二维 SDS-PAGE 银染色法、SYPRO Ruby 荧光染料染色法以及二维差分凝胶电泳法(2D-DIGE),以确定它们在表征 HCPs 方面的相对能力。在传统的二维凝胶中,无论是否存在产品蛋白,SYPRO Ruby 的灵敏度都高于银染色法。银染色法在对酸性蛋白质染色时明显优于碱性蛋白质,而 SYPRO Ruby 在对不同等电点的蛋白质成像时更为一致。非传统的 2D-DIGE 方法在比较具有相似蛋白质特征的样本时具有高分辨率和可重复性,但由于其动态范围较窄,在成像 HCP 斑点时受到限制。总之,2DE 是一种分离和表征 HCP 的强大工具,可通过为每种特定应用选择最佳染色剂或方法进行优化。在可能的情况下,结合使用两种或两种以上不同的 2DE 染色方法,可提供最全面的覆盖范围,以支持 HCP 等复杂混合物的表征。不过,在只能使用一种染色方法的情况下,对于大多数基于 HCP 的应用,SYPRO Ruby 被证明是更可靠、更灵敏、更易用的传统染色方法。
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来源期刊
Biotechnology Progress
Biotechnology Progress 工程技术-生物工程与应用微生物
CiteScore
6.50
自引率
3.40%
发文量
83
审稿时长
4 months
期刊介绍: Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries. Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.
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