Analytical Chemistry, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
Analytical Chemistry, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
J Pharm Biomed Anal. 2021 Jun 5;200:114069. doi: 10.1016/j.jpba.2021.114069. Epub 2021 Apr 20.
Host cell proteins (HCPs) are process-related impurities expressed by the host cells used for production of therapeutic proteins. Although an extensive purification process removes most of the HCPs, residual HCPs are commonly present in protein therapeutics. If not well-controlled, certain HCPs may be present in the product, impacting drug stability, and potentially affecting product safety leading to safety risks for patients. Therefore, as a critical quality attribute, the levels of HCPs must be closely monitored during drug development and determined in the final drug substance at release. Liquid chromatography-mass spectrometry (LC-MS) as an orthogonal approach to traditional ELISA for HCP analysis has shown tremendous value in drug process development and analytical characterization by providing additional critical information, such as protein identity and relative abundance of individual HCPs. To meet the challenges in HCP analysis during drug development, especially downstream process development, which entails fast turnaround time and robustness while identifying high level of HCPs and their clearance trend for further purification development, we have developed HCP-automated iterative MS (HCP-AIMS): it is a simple, automated and robust HCP analysis workflow with deep and unbiased identification and relative quantification capability. This HCP-AIMS approach only requires easy direct digestion of the samples without enrichment or pre- treatment. With the fully automated precursor ion exclusion in MS/MS mode, low abundance HCP peptides could be selected for MS/MS analysis in iterative replicates, and therefore, the identification of HCPs at low abundance can be achieved. Using an in-house mAb with various levels of spiked-in HCPs as well as the NIST mAb, we were able to achieve unbiased identification and quantitation of HCPs as low as 10 ppm level. Furthermore, robustness of the HCP-AIMS approach was also confirmed for the feasibility of large-scale and high-throughput analysis.
宿主细胞蛋白（HCP）是在生产治疗性蛋白时使用的宿主细胞表达的与生产过程相关的杂质。尽管经过广泛的纯化过程可以去除大部分 HCP，但蛋白质治疗药物中仍普遍存在残留的 HCP。如果控制不当，某些 HCP 可能会存在于产品中，影响药物稳定性，并可能对产品安全性产生影响，从而给患者带来安全风险。因此，作为关键质量属性，在药物开发过程中必须密切监测 HCP 水平，并在放行时确定最终药物物质中的 HCP 水平。液相色谱-质谱联用（LC-MS）作为传统 ELISA 分析 HCP 的正交方法，通过提供额外的关键信息，如蛋白质的身份和单个 HCP 的相对丰度，在药物工艺开发和分析特性方面显示出了巨大的价值。为了应对药物开发过程中 HCP 分析的挑战，特别是下游工艺开发的挑战，需要快速周转时间和稳健性，同时识别高水平的 HCP 及其清除趋势，以进一步进行纯化开发，我们开发了 HCP-自动化迭代 MS（HCP-AIMS）：它是一种简单、自动化和稳健的 HCP 分析工作流程，具有深度和无偏的鉴定和相对定量能力。这种 HCP-AIMS 方法只需要简单的直接消化样品，无需富集或预处理。通过在 MS/MS 模式下自动进行前体离子排除，可以选择低丰度的 HCP 肽进行迭代重复的 MS/MS 分析，从而可以实现低丰度 HCP 的鉴定。使用各种水平的掺入 HCP 的内部 mAb 以及 NIST mAb，我们能够实现 HCP 的无偏鉴定和定量，达到低至 10 ppm 水平。此外，还证实了 HCP-AIMS 方法的稳健性，可用于大规模和高通量分析。
