Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery
Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery
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DOI：
10.3390/ijms20071729
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发表时间：
2019-04-08
影响因子：
5.6
通讯作者：
Menegatti, Stefano
中科院分区：
文献类型：
--
作者：
Lavoie, R. Ashton;di Fazio, Alice;Menegatti, Stefano
生物制造中质量设计（QbD）概念的日益整合要求详细和定量地了解杂质的概况及其对产品安全性和有效性的影响。特别有价值的是测定由用于生产的重组细胞分泌的宿主细胞蛋白（HCP）以及目的产物的残留水平。尽管HCP通常被称为单一杂质，但其包含具有不同丰度、大小、功能和组成的多种物质。由于这些杂质的细胞系间、产品间和批次间差异，其清除是一个复杂的问题。通过基于蛋白质组学的方法对HCP监测的改进已经导致鉴定出一部分“有问题”HCP，这些HCP在产品捕获和产品精制步骤中特别难以去除，并且即使在痕量浓度下也会损害产品稳定性和安全性。本文描述了能够捕获广谱中国仓鼠卵巢（CHO）HCP的合成肽配体与允许高级混合模式结合的肽物质组合的开发。筛选固相肽文库，以鉴定和表征捕获CHO HCP同时显示与人IgG最小结合的肽，此处用作模型产品。四聚体和六聚体配体的特点是多极或疏水/正氨基酸组合物被认为是最有效的。四聚多极配体表现出最高的靶向结合率（HCP清除率与IgG损失的比率），是工业上用于IgG抛光的商业混合模式和阴离子交换树脂的两倍以上。与IgG相比，所有测试的肽树脂均显示优先结合HCP，表明其在流通模式或弱分配模式色谱中的潜在用途。
The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of "problematic" HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.