In-depth monitoring of host cell proteins in influenza vaccines throughout multi-step purification processes

Influenza viruses, characterized by antigenic drift and shift, can cause seasonal epidemics or pandemics. According to World Health Organization (WHO) data, approximately 650,000 global annual deaths are attributed to influenza-associated respiratory diseases, highlighting the urgent need for vaccine protection in high-risk populations. Residual host cell protein (HCP) may elicit allergic reactions or compromise antigen stability, with their permissible limits stringently regulated by international health authorities. This study employed liquid chromatography-tandem mass spectrometry (LC-MS) to systematically evaluate a downstream purification process involving ultrafiltration concentration coupled with Capto™ Core 700 multimodal chromatography and anion-exchange chromatography (AEC). Key results demonstrated that ultrafiltration concentration of the viral harvest significantly reduced HCP content, achieving a removal rate of 90.32%. Subsequent purification steps further eliminated HCP, with gel filtration chromatography and AEC providing removal rates of 92.47% and 61.52%, respectively. Concurrently, the principal antigen hemagglutinin (HA) increased from 2.21% to 8.61% in the viral bulk. HCP residuals were reduced to 535.67 ng per dose, while residual recombinant nuclease fell below the detection limit of 0.312 ng/mL. Proteomic profiling indicated substantial changes in dominant HCP species following ultrafiltration, with Annexin A2 (ANXA2), Actin cytoplasmic 2 (ACTG), and IF rod domain-containing protein predominating in the concentrate. The subsequent purification maintained consistent HCP profiles, primarily composed of ANXA2, ACTG, and Peroxiredoxin-1 (PRDX1). Overall, ultrafiltration critically reduces HCPs and process-related impurities, while multimodal chromatography effectively enriches key antigens (HA/NA). The established LC/MS platform might provide a robust analytical framework for influenza vaccine quality control and process optimization.