Long Zhen, Wei Chen, Zhu Xiang, Luo Xi, Li Xiangjun, Zhao Suli
ThermoFisher Scientific Corporation, Beijing 100080, China.
National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 102629, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Sep 1;1263:124712. doi: 10.1016/j.jchromb.2025.124712. Epub 2025 Jun 26.
Recombinant herpes zoster (HZ) vaccines were reported as the most effective strategy to reduce the burden of herpes zoster and related complications. To ensure the quality, safety, and efficacy of recombinant HZ vaccines, comprehensive characterization of its antigen, recombinant glycoprotein E (RgE), is critical. However, due to the extreme complexity and heterogeneity of RgE, a single analytical platform is insufficient for comprehensive characterization. This study developed an innovative LC-MS-based workflow that combines multiple-parallel-protease digestion, various separation techniques (nano-flow LC, high-flow LC, and native separation), and high-resolution mass spectrometry (HR-MS) with multiple ion activation methods to achieve a comprehensive analysis of RgE. As a result, 21 and 21 O-glycosylation and phosphorylation sites were identified by EThcD-induced LC-MS/MS. Two N-glycosylation sites (N207 and N406) were identified with various N-glycans. The most abundant N-glycan for N207 was A2S2F. The top five N-glycans detected for N406 were also identified. Additionally, five disulfide linkages were detected for RgE. They were Cys 195-Cys 205, Cys 401-Cys 411, Cys 177-Cys 189, Cys 365-Cys 374, and Cys 356-Cys 382. For host cell protein (HCP) analysis, a high-sensitivity nano LC-MS/MS approach was applied, identifying 50 host cell proteins (HCPs), including two high-risk proteins, clusterin and peroxiredoxin-1. Additionally, molecular weight measurements obtained under both native and denatured conditions agreed well with the amino acid sequence acquired by peptide mapping. Following the optimization of the LC-MS workflow using RgE, the method was applied to evaluate recombinant zoster vaccines produced through different manufacturing processes. The results demonstrated that this workflow is highly effective for characterizing new recombinant zoster vaccines and may also serve as a valuable tool for the development of other complex recombinant vaccines.
重组带状疱疹（HZ）疫苗被报道为减轻带状疱疹及其相关并发症负担的最有效策略。为确保重组HZ疫苗的质量、安全性和有效性，对其抗原重组糖蛋白E（RgE）进行全面表征至关重要。然而，由于RgE极其复杂且具有异质性，单一的分析平台不足以进行全面表征。本研究开发了一种基于液相色谱-质谱联用（LC-MS）的创新工作流程，该流程结合了多平行蛋白酶消化、多种分离技术（纳流LC、高流LC和天然分离）以及具有多种离子激活方法的高分辨率质谱（HR-MS），以实现对RgE的全面分析。结果，通过电子转移高能碰撞解离（EThcD）诱导的LC-MS/MS鉴定出21个O-糖基化位点和21个磷酸化位点。通过各种N-聚糖鉴定出两个N-糖基化位点（N207和N406）。N207最丰富的N-聚糖是A2S2F。还鉴定出了N406检测到的前五种N-聚糖。此外，检测到RgE的五个二硫键。它们分别是Cys 195-Cys 205、Cys 401-Cys 411、Cys 177-Cys 189、Cys 365-Cys 374和Cys 356-Cys 382。对于宿主细胞蛋白（HCP）分析，采用了高灵敏度的纳流LC-MS/MS方法，鉴定出50种宿主细胞蛋白（HCPs），包括两种高风险蛋白，簇集素和过氧化物酶1。此外，在天然和变性条件下获得的分子量测量结果与通过肽图谱获得的氨基酸序列吻合良好。在使用RgE对LC-MS工作流程进行优化后，该方法被应用于评估通过不同生产工艺生产的重组带状疱疹疫苗。结果表明，该工作流程对于表征新型重组带状疱疹疫苗非常有效，也可能成为开发其他复杂重组疫苗的有价值工具。
Zotero 插件