Highly Efficient Production of an Influenza H9N2 Vaccine Using MDCK Suspension Cells

Author: Yixiao Wu, Hanjing Jia, Hanzhang Lai, Xuping Liu*, Wen-Song Tan.
Bioresources and Bioprocessing, 2020, 7: 63

Abstract
The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

利用MDCK懸浮細胞高效生產H9N2流感疫苗
H9N2亞型禽流感疫苗的使用是控制禽流感病毒在禽類間傳播的有效途徑，而細胞培養生產平台能夠克服傳統雞蛋平台的局限性並對其替代，從而實現疫苗生產工藝的升級。無血清懸浮細胞培養技術的發展，特別是高產懸浮細胞株的開發和培養策略的優化，能夠有助於提高病毒的產量。本研究將一株貼壁型MDCK細胞系進行了無血清懸浮馴化，批培養條件下最高細胞密度可達12×106 cells/mL。隨後對H9N2流感病毒在MDCK懸浮細胞系中的擴增情況進行了評估，並從MOI和感染時的細胞密度等方面對感染條件進行了優化。此外，為感染期提高病毒滴度，本研究考察了不同的補料策略，併發現採用1:2培養基稀釋策略時能獲得最大的HA滴度13 log2 (HAU/50μL)。進一步對比經操作條件優化的生物反應器和搖瓶培養體系，MDCK細胞的性能和病毒產量相當，但在反應器中單位細胞的病毒產量更高，達到13000 virions/cell以上。最後，生物反應器中收穫並經純化後的H9N2病毒，也即MDCK細胞生產的疫苗能夠在雞體內誘導高滴度的中和抗體。綜上，這些結果顯示了MDCK細胞高效生產平台在禽流感疫苗生產應用中的廣闊前景。