Screening And Optimization Of Serum-free Medium For CPV ID3 Monoclonal Antibody Cell Line

The Canine Parvovirus Disease is one of the most serious infectious diseases endangering the dog industry in China.Injection of Canine Parvovirus Monoclonal Antibody(CPV McAb)is one of the most effective treatments for the Canine Parvovirus Disease.There are many disadvantages in culturing cells with serum-containing medium to produce monoclonal antibodies.However,the culturing process of cell suspension culture in serum-free medium is easy to scale up and has high stability.In order to achieve the safe and efficient production of CPV McAb from cultured cells using serum-free medium,this study used an adherent cultured CPV ID3 cell line that stably expresses CPV McAb to conduct suspension acclimation and serum-free medium selection optimization tests.So as to provide basic data support for the efficient industrial production of CPV McAb.First,using commercial serum-free medium to acclimate the CPV ID3 cells cultured in serum-containing medium into suspension cells that can be cultured in serum-free medium by continuous subculture method.The main bank of serum-free suspension cells of CPV ID3 cell line was established.The CPV ID3 cell lines were cultured by batch culture and constant rate flow plus glutamine culture respectively.By comparing the growth datas in the culture process,the constant rate flow plus glutamine culture method was determined as the culture method in the serum-free medium screening optimization experiment.On this basis,the Simplex Centroid Design method and the Central Composite Design method in the Design Expert software were used to carry out the serum-free medium screening optimization test.And using the best medium-based formula SBMY medium to continuously subculture cells and comparing with other commercial media to verify the optimal medium.The SBMY serum-free medium was verified to maintain stable and continuous passage of the CPV ID3 cell line,with a maximum viable cell density of 8.87×10~6cells/m L.For the purpose of increasing the antibody titer,SBMY medium is used as the basic medium to carry out the screening test of the feed medium and its feeding strategy.After comparison,the best feed medium combination and the corresponding best feeding strategy were selected.From the second day after inoculation,the Max FA accounting for 1.50%of the initial culture volume,the Max Fb accounting for 0.15%of the initial culture volume,the wheat hydrolysate accounting for 1.00%of the initial culture volume,and the 200 m M glutamine accounting for 1.25%of the initial culture volume were added daily.Finally,the serum-free medium combination and its feeding strategy were verified and screened by the shake flask and 3 L bioreactor.The antibody titer obtained in the reactor system and the shake flask system can reach 1:3840.And it exceeded the highest antibody titer of 1:640 obtained by perfusion culture with the original serum-containing medium.In summary,the screening and optimization of a serum-free medium combination suitable for the CPV ID3cell line to efficiently produce CPV McAb.