Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company , Indianapolis, Indiana 46285, United States.
Anal Chem. 2017 May 16;89(10):5436-5444. doi: 10.1021/acs.analchem.7b00304. Epub 2017 Apr 26.
Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm of HCP with molecular weight >60 kDa, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2D-UPLC/IM/MS method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.
残留宿主细胞蛋白 (HCP) 存在于重组 DNA 技术衍生的生物制药中，可能对患者构成潜在安全风险，或影响产品稳定性。因此，下游纯化工艺旨在证明能够有效去除这些杂质。ELISA 通常使用多克隆抗 HCP 抗体作为捕获、检测和定量试剂，用于在工艺开发过程中监测 HCP 去除情况，但该技术存在局限性。最近，LC-MS 用于残留 HCP 分析已成为支持纯化工艺开发的强大工具。然而，质谱需要克服巨大的动态范围，以检测生物制药样品中残留 HCP 的低 ppm 水平。我们描述了一种简单而强大的方法，通过结合使用胰蛋白酶消化的新型样品制备程序和 shotgun 蛋白质组学方法来表征单克隆抗体中的残留 HCP。与传统方法不同，该样品制备方法在保持抗体基本完整的同时消化 HCP。因此，MS 检测 HCP 的动态范围比传统的胰蛋白酶消化样品制备方法低 1 到 2 个数量级。HCP 加标实验表明，我们的方法可以检测到 0.5 ppm 分子量 >60 kDa 的 HCP，如 rPLBL2。将我们的方法应用于分析源自鼠细胞系表达系统的高纯度 NIST 单克隆抗体标准品 RM 8670，可检测到 60 种鼠 HCP，比之前使用 2D-UPLC/IM/MS 方法报道的多两倍。一种用于 70 次分析、450 天的对照单克隆抗体证明了我们的方法具有稳健性。
