PRV CHO_gD悬浮细胞流加培养的研究OA

This study aims to lay the foundation for the production of a pseudorabies virus(PRV)vaccine by exploring bioreactor parameters for CHO_gD cells stably expressing PRV.CHO_gD cells were passaged using 125 mL shake flasks,seed cells were cultured in a 10 L bioreactor,and Fed-batch expression of CHO_gD cells was performed in a 50 L single-use bioreactor bag.Protein expression levels were then detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and high performance liquid chromatography(HPLC).The results showed that in the LP 50 bioreactor,the initial culture volume for CHO_gD cells was 30 L,the starting cell density was(1.0±0.1)×10 ⁶ cells/mL,the temperature was 37℃(from the start to day 4)and 32℃(from day 5 to day 17),the stirring speed was 65 r/min(from the start to day 10),the pH was 6.85～7.15,and the dissolved oxygen was 40％.Aeration:compressed air at an initial rate of 0.01 L/min,adjusted according to pH changes;O₂:0～3 L/min,linked to dissolved oxygen control;CO₂:0～2 L/min,linked to pH control.Feeding:4％CD Feed008.2 and 0.4％CD Feed009 were added on days 3,5,7,9,and 11.Glucose supplementation:when the glucose level fell below 3.0 g/L,it was raised to 6.0 g/L.SDS-PAGE analysis of three cell batches showed that protein expression peaked on day 15,reaching 3 815.60,3 739.80,and 3 811.20 mg/L,respectively.HPLC analysis showed that for batches 20240316 and 20240512,protein expression also peaked on day 15(3 610.65 and 3 683.5 mg/L),while for batch 20240224 it peaked on day 17(3 660.00 mg/L).This study indicates that antigen can be harvested from CHO_gD cells on day 15 of culture.