Kumar Amit, Baycin-Hizal Deniz, Wolozny Daniel, Pedersen Lasse Ebdrup, Lewis Nathan E, Heffner Kelley, Chaerkady Raghothama, Cole Robert N, Shiloach Joseph, Zhang Hui, Bowen Michael A, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.
Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases , National Institute of Health, Building 14A, Bethesda, Maryland 20892, United States.
J Proteome Res. 2015 Nov 6;14(11):4687-703. doi: 10.1021/acs.jproteome.5b00588. Epub 2015 Oct 13.
Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.
中国仓鼠卵巢（CHO）细胞是生产包括单克隆抗体在内的多种复杂生物治疗药物的首选宿主细胞系。我们对贴壁CHO细胞的废培养基进行了蛋白质组学和生物信息学分析。收集CHO-K1培养物的上清液，进行溶液内消化，然后进行液相色谱/液相色谱-串联质谱分析，从而鉴定出3281种不同的宿主细胞蛋白（HCP）。为了对它们进行功能分类，我们将多种生物信息学工具应用于我们研究中鉴定出的蛋白质，包括信号肽预测软件（SignalP）、亚细胞定位预测软件（TargetP）、分泌蛋白预测软件（SecretomeP）、跨膜结构域预测软件（TMHMM）、蛋白质亚细胞定位工具（WoLF PSORT）和跨膜区及信号肽预测软件（Phobius）。该分析提供了有关信号肽、跨膜结构域和细胞定位的信息，并表明分泌蛋白和细胞内蛋白都是上清液的组成部分。鉴定出的蛋白质定位于分泌途径，包括在细胞生长、增殖和折叠中起作用的蛋白质，以及参与蛋白质降解和清除的蛋白质。在合并预测为分泌型或具有信号肽的蛋白质后，我们鉴定出1015种蛋白质，我们将其称为CHO上清液蛋白质组（CHO-SO）或超级蛋白质组。作为这项工作的一部分，我们创建了一个名为GO-CHO的基于网络的公共工具，用于对CHO-SO中发现的蛋白质进行功能分类，并识别富集的分子功能、生物学过程和细胞成分。我们还使用了一种工具来评估高丰度HCP的免疫原性潜力。富集的功能包括催化活性和细胞骨架的结构成分。发现各种与运输相关的生物学过程，如囊泡介导的运输，高度富集。细胞外空间和囊泡外泌体相关蛋白被发现是最富集的细胞成分。超级蛋白质组还包含从经典和非经典分泌途径分泌的蛋白质。我们研究中描述的工作和数据库将使CHO研究群体能够快速识别其培养物中的高丰度HCP，从而有助于评估生物药物生产中使用的工艺和纯化方法。
