Analytical Chemistry, Regeneron Pharmaceuticals Inc.
Analytical Chemistry, Regeneron Pharmaceuticals Inc.;
J Vis Exp. 2024 Jan 19(203). doi: 10.3791/65544.
Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS). This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.
宿主细胞蛋白（HCPs）是杂质，即使在少量存在的情况下，也可能对治疗性蛋白产生不利影响。为了评估与药物产品相关的潜在风险，已经开发出了一些方法来鉴定低丰度的 HCPs。开发一种敏感的 HCP 检测方法的关键方法是在分析之前，利用液相色谱-质谱联用（LC-MS）同时富集 HCP 并去除单克隆抗体（mAbs）。本方案提供了使用市售的蛋白质组学富集珠富集宿主细胞蛋白的详细说明。这些珠子包含具有不同蛋白质特异性亲和力的六肽配体的多样化文库。该方案还包括有限的消化和随后使用纳升 LC-MS/MS 进行肽检测。通过采用这些技术，可以将低丰度的 HCP 富集超过 7000 倍，检测限低至 0.002ppm。值得注意的是，该方案使用 NIST mAb 以高置信度检测到 850 种 HCP。此外，它设计为用户友好型，并包括视频演示，以协助其实施。通过遵循这些步骤，研究人员可以有效地富集和检测 HCPs，提高药物产品风险评估的灵敏度和准确性。
