Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry
Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry
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DOI：
10.4161/mabs.4.1.18748
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发表时间：
2012-01-01
期刊：
影响因子：
5.3
通讯作者：
Chen, Weibin
中科院分区：
文献类型：
--
作者：
Doneanu, Catalin E.;Xenopoulos, Alex;Chen, Weibin
本发明提供了用于鉴定和定量浓度超过5个数量级的生物素蛋白中的宿主细胞蛋白（HCP）的测定法。HCP测定包括两种类型：使用综合在线二维液相色谱结合高分辨率质谱（2D-LC/MS）进行HCP鉴定，然后通过液相色谱多反应监测（LC-MRM）进行高通量HCP定量。前者被描述为“发现”测定，后者被描述为“监测”测定。纯化的双歧杆菌蛋白（例如，单克隆抗体）在还原和烷基化后用胰蛋白酶消化，并且在高pH（pH 10）下使用反相（RP）色谱法通过在第一维上的阶梯梯度分级分离，随后在第二维上在低pH（pH 2.5）下进行高分辨率分离。当肽从第二维洗脱时，使用四极杆飞行时间质谱仪通过在低能量和高能量之间交替碰撞池能量来同时检测肽及其片段（MS方法）。MS数据用于使用经证实的无标记定量技术（“Hi 3”方法）鉴定和定量混合物中的蛋白质。挖掘相同的数据集，随后开发目标肽和转换，用于以高通量方式在三重四极杆质谱仪上监测选定HCP的浓度（20 min LC-MRM分析）。该分析方法用于鉴定和定量PTG 1（一种重组嵌合抗磷酸酪氨酸单克隆抗体（mAb））6份样品中的低丰度HCP。从PTG 1样品中共鉴别出33种HCP，其中选择21种HCP亚型进行MRM监测。在样品中加标同位素标记的肽后，在两个不同的LC-MRM平台上对三种选定HCP进行绝对定量。最后，将MRM定量结果与基于H13肽的基于TOF的定量进行比较，并且TOF和MRM数据集相当好地相关。结果表明，该测定提供了详细的有价值的信息，以了解纯化方案的性质和浓度的HCP杂质在生物制药样品中的相对贡献，和该测定可用作HCP分析在生物制药行业的通用方法。
Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MS' methodology). The MS' data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.