Swedish Orphan Biovitrum AB, Stockholm, Sweden.
Swedish Orphan Biovitrum AB, Stockholm, Sweden.
J Pharm Biomed Anal. 2020 Jun 5;185:113256. doi: 10.1016/j.jpba.2020.113256. Epub 2020 Mar 12.
Host cell proteins (HCPs) in biotherapeutics can be identified by the use of enzymatic digestion and LC-MS/MS analysis. However, the major challenge is that HCPs are often present at very low levels in relation to the protein drug (low ppm-levels). In this study, the ProteoMiner™ Enrichment Kit (Bio-Rad) was evaluated as a strategy to enable identification of HCPs by LC-MS/MS by enrichment of low-abundant HCPs and a simultaneous depletion of the high-abundant product protein. A recombinant protein produced in Chinese hamster ovary (CHO) cells was spiked with six standard proteins at varying concentrations (10-1000 ppm). The sample was split into two aliquots; one that was prepared with the ProteoMiner™ Enrichment Kit and one control, where the enrichment procedure was omitted. The ProteoMiner™ Enrichment Kit was combined with the ProteoMiner Sequential Elution Large-Capacity Kit (Bio-Rad), eluting the proteins into four fractions. The samples were then digested with trypsin and analyzed with LC-MS/MS. In addition, multiple reaction monitoring (MRM) was applied to obtain an estimate of the protein abundance of HCPs and spiked proteins. The results demonstrated that with the untargeted LC-MS/MS method, 30 HCPs and four of the six spiked standard proteins were identified in the four fractions. The spiked standard proteins were identified down to 30 ppm in the ProteoMiner treated samples, while no HCPs and only the most abundant standard protein (≈1000 ppm) were identified in the non-enriched control sample. MRM assays were developed for 14 out of the 30 identified HCPs. All targeted HCPs and five of the six standard proteins were detected in all fractions as well as in the control sample by MRM. There was an acceptable agreement between estimated concentrations of spiked standard proteins and expected values. An 80-700 fold enrichment of individual HCPs was observed in the fractions. In conclusion, the results clearly demonstrated that the ProteoMiner technology can be used for enriching HCPs in biotherapeutics, enabling their identification by LC-MS/MS.
宿主细胞蛋白 (HCP) 可通过酶解和 LC-MS/MS 分析来鉴定。然而，主要的挑战在于 HCP 相对于蛋白药物（低 ppm 级）的含量通常非常低。在本研究中，评估了 ProteoMiner™ 富集试剂盒（Bio-Rad）作为一种通过 LC-MS/MS 鉴定 HCP 的策略，该策略通过富集低丰度 HCP 并同时耗尽高丰度产物蛋白来实现。用六种标准蛋白（浓度 10-1000 ppm）对在中国仓鼠卵巢 (CHO) 细胞中生产的重组蛋白进行了加标。将样品分为两份；一份用 ProteoMiner™ 富集试剂盒处理，另一份为对照，其中省略了富集步骤。ProteoMiner™ 富集试剂盒与 ProteoMiner 顺序洗脱大容量试剂盒（Bio-Rad）联合使用，将蛋白质洗脱成四个馏分。然后用胰蛋白酶对样品进行消化，并进行 LC-MS/MS 分析。此外，还应用了多重反应监测 (MRM) 来估算 HCP 和加标蛋白的丰度。结果表明，在非靶向 LC-MS/MS 方法中，在四个馏分中鉴定出 30 种 HCP 和六种加标标准蛋白中的四种。在用 ProteoMiner 处理的样品中，六种加标标准蛋白中的四种可鉴定至 30 ppm，而在未经富集的对照样品中，未鉴定到 HCP，仅鉴定到最丰富的标准蛋白（≈1000 ppm）。针对 30 种鉴定出的 HCP 中的 14 种开发了 MRM 检测方法。通过 MRM，在所有馏分以及对照样品中均检测到所有靶向 HCP 和六种标准蛋白中的五种。加标标准蛋白的估计浓度与预期值之间具有可接受的一致性。在馏分中观察到单个 HCP 的富集倍数为 80-700 倍。总之，结果清楚地表明 ProteoMiner 技术可用于富集生物制药中的 HCP，从而通过 LC-MS/MS 进行鉴定。
