The Development Of MDCK Cell-based Serum-free And High Cell Density Process For Influenza Virus Production

With the continuous emergence and prevalence of viral infectious diseases in the globe,the control and prevention of the rapid disease spread relies on the availability of effective and safe viral vaccines.In recent years,the safety,economy and timeliness of animal cell culture technology for viral vaccine production have become increasingly obvious.For influenza vaccines,it is even expected to supplement the traditional chicken embryo platform and boost the industry.Compared with serum-containing adherent cell culture,serum-free suspension culture has more advantages for its feasibility,economy and process stability.However,the virus productivity using suspension cells in high-cell-density serum-free culture still lacks competitivity compared with the chicken embryo platform.Additionally,the immunogenicity of influenza vaccines produced by animal cell culture is poorly understood.Therefore,to understand the effects of operating conditions and cultivation parameters on cell growth,virus production and virus related quality attributes in high cell density cultivation is particularly important for the development of a high-yield and high-quality process for influenza vaccine production using suspension cells.In this thesis,firstly,the effects of media,virus adaptation,key infection parameters in shake flasks and key operating parameters in bioreactors on the MDCK suspension cell growth and influenza virus propagation were investigated.Based on the process evaluation in bioreactors,an efficient dilution culture process for influenza vaccine production using MDCK suspension cells in serum-free medium was developed.Next,perfusion culture was attempted to be applied in the influenza vaccine production.Using MDCK suspension cells,a semi-perfusion model on the scale of shake flasks was developed.Furthermore,ATF perfusion culture in bioreactors were performed for the optimization of cell growth and virus production to overcome the "cell density effect" and develop a highly efficient high cell density process for influenza virus production.Finally,the process performance of dilution culture and perfusion culture as well as the quality attributes of influenza virus produced by two processes were compared,in which the glycosylation patterns of influenza virus protein were particularly analyzed to give a deeper understanding for the future large-scale manufacturing of influenza vaccines using MDCK suspension cells in high cell density cultures.Firstly,serum-free medium named Xeno-CDM1 was screened out in shake flasks to support high cell growth of MDCK suspension cells.Serial virus adaptation can significantly accelerate virus infection and replication process.On this basis,the operating conditions for cell growth and virus propagation were studied and optimized:MOI was 0.001,the trypsin concentration was 20-30 U/mL and the cell density was adjusted to 6.0 × 106 cells/mL by 1:1 or 1:2 medium dilution at time of infection.Under these optimized conditions,the HA titer over 3.60 log10(HAU/100 μL)and the cell specific virus yield over 12000 virions/cell were reached in shake flasks.The parameter optimization in bioreactors showed that the agitation and pH had an impact on cell growth and virus production and the stirring speed of 80 rpm and pH of 7.20 in the virus production phase were applied.Thus,a MDCK suspension cell-based process for influenza vaccine production was successfully developed in bioreactors with a cell growth to 1.0 ×107 cells/mL,the HA titer over 3.60 log10(HAU/100 μL)and the cell specific virus yield over 10000 virions/cell.After the downstream purification,both the removal of host cell DNA and proteins as well as the virus recovery met the standard.In addition,the optimal harvest point of 21-24 hpi was confirmed and it can be used as a key process attribute for future manufacturing.As cell concentration and cell-specific virus yield are two key factors for efficient influenza virus production with suspension cells,in this chapter perfusion culture was introduced.Firstly,a semi-perfusion model of MDCK cells was established in shake flasks,and it was found that MDCK cell growth exceeding 4 ×107 cells/mL and HA titer over 4.20 log10(HAU/100 μL)were obtained at a cell-specific perfusion rate of 60 pL/cell/d.In addition,MOI and trypsin concentration affected influenza virus propagation in high cell density cultivation and MOI of 0.001 and trypsin concentration of 20 U/mL were optimal,respectively.Next,for the "cell density effect" founded in the continuous ATF perfusion culture in bioreactors,further investigation revealed that the pH control and temperature reduction in the virus production phase had a significant impact on virus yield.In addition,the temperature reduction allowed more influenza virus to get into the permeate through the ATF membrane.To improve the perfusion process economy,proper perfusion rate control is crucial.Firstly,a decrease in the cell specific perfusion rate to 40 pL/cell/d contributed to higher virus yield and less medium consumption.Secondly,a capacitance probe was introduced to the perfusion culture for the online monitoring of perfusion rate.The results showed a linear correlation between the permittivity of capacitance probe and viable cell concentration,so it can be used for online monitoring of viable cell concentration in the growth phase of perfusion culture to achieve automated perfusion rate control.With all these improvements,a cost-effective and automated perfusion culture process for influenza vaccine production was developed,with the MDCK cell concentration over 4×107 cells/mL,HA titer of 4.37 log10(HAU/100 μL)and cell specific virus yield over 11000 virions/cell,which overcame the cell density effect.In last chapter,process performance of MDCK cell-based perfusion process and dilution process for influenza virus production was compared and the quality attributes of virus antigen HA protein derived from these two processes were analyzed.For the process performance,the results showed that with the same cell-specific virus yield,the virus titer was 5-8 times higher in the optimized MDCK cell-based perfusion process due to the increase in cell concentration than the dilution process.Despite of a slightly lower volumetric virus productivity,the space time yield of the MDCK cell-based perfusion process is way higher than that of the dilution process,making the perfusion process more suitable for an influenza pandemic outbreak.In addition,various virus strains tested in this chapter showed same performances in the improvement of virus yield in perfusion process,indicating that the established MDCK cell-based perfusion process can be applied for other types or subtypes of influenza viruses.Then,the study of influenza virus HA proteins derived from two processes suggested that the HA nucleic acid sequences derived from MDCK cells were consistent with the original virus strain derived from chicken embryos.Based on this,glycosylation modifications and heterogeneity were found on all the 8 potential N-glycosylation sites of HA protein,on which the glycoforms were mainly complex.Besides,process conditions had little effect on the glycosylation of HA protein.Last,the computer simulation showed that glycosylation in or near the antigenic sites may affect the virus antigenicity,but not the binding capacity of the receptor binding site of the HA protein to the sialic acid.The influence of glycosylation pattern on innate immunity and acquired immunity was further discussed.It is believed that the glycosylation of influenza HA protein has a potential impact on the immunogenicity of vaccines,which needs to be focused in future influenza vaccine development.In conclusion,in this thesis,an economical and efficient dilution process and a perfusion process for influenza virus production using MDCK suspension cells in high cell density culture were successfully developed,which gave a technical support for its large-scale manufacturing.In addition,studies on the quality attributes including glycosylation of influenza virus proteins produced by animal cell culture provided a scientific basis for our further understanding of the immunogenicity of influenza vaccines.