Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.
Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.
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DOI：
10.1002/biot.201500550
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发表时间：
2016-08
影响因子：
4.7
通讯作者：
Smales, C. Mark
中科院分区：
文献类型：
--
作者：
Chiverton, Lesley M.;Evans, Caroline;Pandhal, Jagroop;Landels, Andrew R.;Rees, Byron J.;Levison, Peter R.;Wright, Phillip C.;Smales, C. Mark
很少有研究定义CHO宿主细胞蛋白（HCP）及其在整个下游纯化工艺中的通量。在此，我们应用定量iTRAQ蛋白质组学，在由蛋白A、阳离子和阴离子交换工艺组成的标准下游纯化程序中跟踪产生抗体（mAb）的CHO‐S细胞系的HCP谱。我们使用6个样品iTRAQ实验分析三个样品的技术重复，即培养物收获物（HCCF）、蛋白A流通液和蛋白A纯化液，并使用8个样品格式分析四种样品类型的技术重复; HCCF与蛋白A纯化液以及随后的阳离子和阴离子交换纯化进行比较。在6份样品iTRAQ实验中，8781个光谱与来自819种蛋白质（包括mAb链）的肽确信匹配。在6和8个样品实验中，鉴定了936种蛋白质。在8个样品的比较中，4187个光谱与来自219个蛋白质的肽确信地匹配。然后，我们使用iTRAQ数据来估计纯化步骤中单个蛋白质的相对变化。这些数据为基于关键HCP知识的工艺开发应用iTRAQ提供了基础。
There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO‐S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.
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