Xue Andong, Kong Linlin, Li Jialin, Jiao Yuxin, Hu Zhishang, Fu Bin, Wang Guibin, Zhang Wanjun, Li Jianheng, Qin Weijie
Hebei University, Baoding, 071002, China.
State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 102206, China.
Anal Chim Acta. 2025 Jan 15;1335:343445. doi: 10.1016/j.aca.2024.343445. Epub 2024 Nov 21.
Host Cell Proteins (HCPs) are impurities expressed in host cells during the biopharmaceutical production process, whichmay compromise product quality and potentially leading to immunogenic reactions or other adverse effects. Mass spectrometry (MS)-based strategy is more and more considered as a promising method for HCPs analysis, since it is capable of simultaneously quantifying thousands of proteins in a single test. However, considering the large excess biopharmaceutical product protein present in the system and the extremely low abundance of HCPs, sensitive MS methods are urgently needed in HCPs analysis.
In this work, we introduced a novel approach that leveraged host cell lysate as a boosting channel to enhance the MS signal of the residue HCPs in biopharmaceutical products using isobaric TMT labeling, thereby elevating the low-abundant HCPs to detectable and quantifiable levels of current MS without using enrichment or depletion method to avoid disturbance of the original concentration of the HCPs. Our method surpassed previous benchmarks by identifying a significantly higher number (23844 unique peptides for 3475 proteins) compared to existing records (4541 unique peptides for 848 proteins) for HCPs analysis in RM8671 NIST monoclonal antibody (mAb), demonstrating unparalleled sensitivity and robustness. Furthermore, our workflow successfully identified 44 of 48 UPS1 proteins across a concentration range of 0.32-4.15 ppm in monoclonal antibodies (mAbs), proving its effectiveness for in-depth HCPs analysis in biopharmaceuticals.
Present even at sub-ppm levels, HCPs may compromise the stability and safety of product proteins and alter pharmacokinetics or neutralization of therapeutic effects. Our MS signal enhancing method presented an advancement in HCP analysis, combining improved sensitivity and increased scale of HCPs with a streamlined and robust workflow. This method allowed HCPs quantification at <1 ppm level without disturbance of the original HCPs concentration, which is still rare in the field.
宿主细胞蛋白（HCPs）是生物制药生产过程中宿主细胞表达的杂质，可能会影响产品质量，并有可能引发免疫反应或其他不良反应。基于质谱（MS）的策略越来越被认为是一种有前景的HCPs分析方法，因为它能够在一次检测中同时对数千种蛋白质进行定量。然而，考虑到系统中存在大量过量的生物制药产品蛋白以及HCPs的极低丰度，HCPs分析迫切需要灵敏的质谱方法。
在这项工作中，我们引入了一种新方法，利用宿主细胞裂解物作为增强通道，通过等压TMT标记增强生物制药产品中残留HCPs的质谱信号，从而在不使用富集或去除方法以避免干扰HCPs原始浓度的情况下，将低丰度HCPs提升至当前质谱可检测和可定量的水平。我们的方法在RM8671 NIST单克隆抗体（mAb）的HCPs分析中，与现有记录（848种蛋白质的4541个独特肽段）相比，鉴定出了显著更多的数量（3475种蛋白质的23844个独特肽段），超越了先前的基准，证明了无与伦比的灵敏度和稳健性。此外，我们的工作流程在单克隆抗体（mAbs）0.32 - 4.15 ppm的浓度范围内成功鉴定出了48种UPS1蛋白中的44种，证明了其在生物制药中深入分析HCPs的有效性。
即使在亚ppm水平存在，HCPs也可能会影响产品蛋白的稳定性和安全性，并改变药代动力学或治疗效果的中和作用。我们的质谱信号增强方法在HCP分析方面取得了进展，结合了提高的灵敏度、增加的HCPs检测规模以及简化且稳健的工作流程。该方法允许在不干扰原始HCPs浓度的情况下对<1 ppm水平的HCPs进行定量，这在该领域仍然很少见。
