Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography

IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology and Bioengineering Pub Date : 2024-09-13 DOI:10.1002/bit.28840
Roxana Disela, Tim Neijenhuis, Olivier Le Bussy, Geoffroy Geldhof, Marieke Klijn, Martin Pabst, Marcel Ottens
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Abstract

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry‐based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross‐validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.
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制备色谱过程中大肠杆菌宿主细胞蛋白质组保留的实验表征与预测
纯化重组生产的生物制药需要去除宿主细胞物质,如宿主细胞蛋白(HCP)。对于常见表达宿主大肠杆菌(E. coli)的裂解物,可以鉴定出超过 1500 种独特的蛋白质。目前,人们对制备色谱等纯化操作中单个 HCP 的行为了解有限。因此,我们旨在阐明大肠杆菌菌株 BLR(DE3) 中的单个 HCP 在层析过程中的洗脱行为。了解这种复杂的混合物并掌握每种 HCP 的色谱行为有助于提高合理设计纯化工艺的能力。具体来说,我们使用离子交换色谱(IEX)和疏水相互作用色谱进行了线性梯度实验,并结合基于质谱的蛋白质组学来绘制单个 HCP 的保留图谱。我们结合文献中有关蛋白质位置、功能和相互作用的知识,确定了洗脱行为的趋势。此外,我们还训练了将蛋白质三维结构与 IEX 期间的洗脱行为相关联的定量结构-性质关系模型。对于完整的数据集,构建了一个交叉验证 R2 为 0.55 的模型,如果只考虑单体蛋白质,R2 可提高到 0.70。最终,这项研究为加深对工艺的理解迈出了重要一步。
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来源期刊
Biotechnology and Bioengineering
Biotechnology and Bioengineering 工程技术-生物工程与应用微生物
CiteScore
7.90
自引率
5.30%
发文量
280
审稿时长
2.1 months
期刊介绍: Biotechnology & Bioengineering publishes Perspectives, Articles, Reviews, Mini-Reviews, and Communications to the Editor that embrace all aspects of biotechnology. These include: -Enzyme systems and their applications, including enzyme reactors, purification, and applied aspects of protein engineering -Animal-cell biotechnology, including media development -Applied aspects of cellular physiology, metabolism, and energetics -Biocatalysis and applied enzymology, including enzyme reactors, protein engineering, and nanobiotechnology -Biothermodynamics -Biofuels, including biomass and renewable resource engineering -Biomaterials, including delivery systems and materials for tissue engineering -Bioprocess engineering, including kinetics and modeling of biological systems, transport phenomena in bioreactors, bioreactor design, monitoring, and control -Biosensors and instrumentation -Computational and systems biology, including bioinformatics and genomic/proteomic studies -Environmental biotechnology, including biofilms, algal systems, and bioremediation -Metabolic and cellular engineering -Plant-cell biotechnology -Spectroscopic and other analytical techniques for biotechnological applications -Synthetic biology -Tissue engineering, stem-cell bioengineering, regenerative medicine, gene therapy and delivery systems The editors will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science. Submission of papers dealing with routine aspects of bioprocessing, description of established equipment, and routine applications of established methodologies (e.g., control strategies, modeling, experimental methods) is discouraged. Theoretical papers will be judged based on the novelty of the approach and their potential impact, or on their novel capability to predict and elucidate experimental observations.
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