The global population faces persistent threats from influenza viruses, with vaccination remaining the most cost-effective preventive measure against influenza. Mammalian cell-based influenza vaccine production has garnered significant attention due to its enhanced safety profile, process controllability, and ability to circumvent adaptive mutations commonly associated with traditional egg-based vaccines, and the particular promise of suspension cell-based production systems for their convenience, economic viability, and scalability potential. Despite years of development and an increasing number of approved animal substrate-based vaccines, numerous challenges persist, especially the incomplete understanding of influenza virus amplification features in the producing cell lines. In previous research, we developed a high-titer suspension MDCK cell-based influenza virus production process and established a high-generation MDCK cell line (MDCK-HG). This study demonstrated enhanced productive capacity of MDCK-HG cells across diverse operational conditions. The maximum hemagglutination titer achieved 15.02 Log