Waters Corporation, Milford, MA, USA.
MAbs. 2012 Jan-Feb;4(1):24-44. doi: 10.4161/mabs.4.1.18748.
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 (MSE methodology). The MSE 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.
提供了用于鉴定和定量生物治疗蛋白中宿主细胞蛋白 (HCP) 的分析方法，其浓度跨度超过 5 个数量级。HCP 分析方法包括两种类型：使用全面的在线二维液相色谱与高分辨率质谱联用 (2D-LC/MS) 进行 HCP 鉴定，然后通过液相色谱、多重反应监测 (LC-MRM) 进行高通量 HCP 定量。前者被描述为“发现”测定法，后者被描述为“监测”测定法。纯化的生物治疗蛋白（例如单克隆抗体）在用还原和烷基化试剂处理后用胰蛋白酶消化，并且在用高 pH（pH 10）进行的一维分步梯度反相 (RP) 色谱中对消化产物进行分级，然后在二维中在低 pH（pH 2.5）进行高分辨率分离。当肽从二维洗脱时，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（MSE 方法）。使用经过验证的无标签定量技术（“Hi3”方法），使用 MSE 数据来鉴定和定量混合物中的蛋白质。还挖掘了相同的数据集，以随后在三重四极杆质谱仪上以高通量方式（20 分钟 LC-MRM 分析）监测选定 HCP 的浓度。该分析方法应用于六种 PTG1 样品中低丰度 HCP 的鉴定和定量，PTG1 是一种重组嵌合抗磷酸酪氨酸单克隆抗体 (mAb)。从 PTG1 样品中共鉴定出 33 种 HCP，其中选择 21 种 HCP 同工型用于 MRM 监测。在样品中掺入同位素标记肽后，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 Hi3 肽的 TOF 定量进行比较，并且 TOF 和 MRM 数据集相关性良好。结果表明，这些测定法提供了详细的有价值的信息，有助于了解纯化方案对生物制药样品中 HCP 杂质的性质和浓度的相对贡献，并且这些测定法可用作生物制药行业中 HCP 分析的通用方法。
