Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Biotechnol Bioeng. 2013 Jan;110(1):240-51. doi: 10.1002/bit.24607. Epub 2012 Aug 6.
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.
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物。尽管需要去除和监测 HCP，但在下游加工 (DSP) 过程中，这些 HCP 的性质和命运仍未得到很好的描述。我们应用蛋白质组学方法研究了单克隆抗体产生细胞系上清液中 HCP 在早期 DSP 期间的动力学和命运，包括离心、深度过滤和蛋白 A 捕获层析。所选的主要澄清技术显示会影响进入后续下游步骤的 HCP 谱。MabSelect 蛋白 A 层析去除了大部分污染蛋白，但使用 2D-PAGE，我们不仅可以观察到洗脱液中的抗体物种（重链和轻链），还可以观察到污染物 HCP。这些数据表明，二次澄清的选择会影响蛋白 A 层析后的 HCP 谱，因为当比较深度过滤器时，特定 HCP 的存在和丰度会出现差异。从无细胞系培养上清液的蛋白 A 洗脱部分中鉴定出几种细胞内定位的 HCP，包括伴侣蛋白 Bip/GRP78、热休克蛋白和酶烯醇酶。我们证明，早期 DSP 步骤的选择会影响最终的 HCP 谱，并且 2D-PAGE 可用于监测和鉴定蛋白 A 层析后的 HCP。这种方法可用于筛选细胞系或宿主，以选择 HCP 谱减少的细胞系或宿主，或鉴定难以去除的 HCP，以便应用细胞工程方法减少或消除这些 HCP。
