Proteomic analysis of host cell protein fouling during bioreactor harvesting

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Da Zhang, S. Ranil Wickramasinghe, Andrew L. Zydney, John P. Smelko, Abdullah Loman, April Wheeler, Xianghong Qian
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Abstract

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

生物反应器收获过程中宿主细胞蛋白质污垢的蛋白质组分析。
中国仓鼠卵巢(CHO)细胞是治疗性蛋白质生产中最常用的细胞系之一。生物反应器收获过程中的膜堵塞是治疗蛋白质下游纯化的主要限制因素。宿主细胞蛋白(HCP)是下游纯化过程中最具挑战性的杂质。本研究的重点是使用反向不对称商用膜 BioOptimal™ MF-SL 在 CHO 生物反应器收集过程中识别 HCP 污物。为了研究细胞澄清过程中的污物和污垢行为,首次开发了一种新型反冲洗工艺,可有效洗脱污膜过滤器中几乎所有的 HCP 和 DNA。利用二维凝胶电泳(2D SDS-PAGE)成功鉴定了生物反应器收获物和污损膜上主要 HCP 的等电点(pIs)和分子量(MWs)。此外,还利用基质辅助激光解吸电离质谱(MALDI-MS)鉴定出了 8 种 HCP。这些 HCP 大部分是酶或与外泌体相关联,这两种物质都能形成亚微米大小的颗粒,从而导致过滤器堵塞。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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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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