A combination of multiple LC-MS approaches for the comprehensive characterization of recombinant herpes zoster
Abstract:
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.
