The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process
The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process
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DOI：
10.1002/bit.24607
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发表时间：
2013-01-01
影响因子：
3.8
通讯作者：
Smales, C. Mark
中科院分区：
文献类型：
--
作者：
Hogwood, Catherine E. M.;Tait, Andrew S.;Smales, C. Mark
用于临床的重组蛋白产品如单克隆抗体（mAb）必须清除宿主细胞杂质如宿主细胞蛋白（HCP）、DNA/RNA和高分子量免疫原性聚集体。尽管需要去除和监测HCP，但在下游加工（DSP）期间这些HCP的性质和命运仍然很难表征。我们应用蛋白质组学方法研究了早期DSP期间mAb生产细胞系上清液中HCP的动态和命运，包括离心、深层过滤和蛋白A捕获色谱。所选择的初步澄清技术显示会影响进入后续下游步骤的HCP特征。MabSelect蛋白A色谱法去除了大部分污染蛋白，但是使用2D-PAGE，我们不仅可以观察到抗体中的抗体种类（重链和轻链），还可以观察到污染的HCP。这些数据表明，二次澄清的选择影响蛋白A色谱后的HCP谱，因为当比较深层过滤器时，特定HCP的存在和丰度均出现差异。许多位于细胞内的HCP被确定在蛋白A洗脱馏分从包括伴侣Bip/GRP 78，热休克蛋白，和酶烯醇化酶的pGEX细胞系培养上清液。我们证明，早期DSP步骤的选择会影响所得HCP图谱，并且2D-PAGE可用于蛋白A层析后HCP的监测和鉴定。这种方法可用于筛选细胞系或宿主，以选择具有降低的HCP谱的那些，或鉴定有问题且难以去除的HCP，使得细胞工程方法可应用于降低或消除此类HCP。Biotechnol. Bioeng. 2013; 110：240251。(C)2012 Wiley Periodicals，Inc.
Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240251. (C) 2012 Wiley Periodicals, Inc.