Study on the Process Development of Anti-CD52 Monoclonal Antibody Prodution in CHO Cell Perfusion Culture

Author:Zheng Zuo

Supervisor:qi nian min

Database:Doctor

Degree Year:2018

Download:46

Pages:159

Size:5830K

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Antibody drugs,due to their strong specificity,high sensitivity and moderated side effects,gradually become mainstream drugs in the field of bio-medicine and are widely used in tumors,autoimmunity,immune rejection and other diseases.Large-scale mammalian cell culture technology has become the key technology of multinational pharmaceutical companies to produce monoclonal antibodies and has become a bottleneck restricting Chinese biopharmaceutical companies to catch up with the big companies.Perfusion culture is one of the most important means to break through the bottleneck and realize a robustness process of high cell density,high production and high quality of the product.In this study,we investigated the perfusion culture process of rCHO cells producing anti-CD52 monoclonal antibody,firstly carried out the preliminary small-scale process development,then comparatively studied the process of two phase pH perfusion culture in bioreactor,with quality attributes and biological potency analysis,and lastly studied the robustness of process within and between three consecutive batches of pilot plant scale perfusion culture.Objective:To establish high cell density,long culture time,high productivity,high quality and high biological potency CHO cell perfusion culture process of producing anti-CD52antibody.Methods:1.Conduct the batch culture research on rCHO cells to understand the characteristics of cell growth,metabolism and expression.2.Conduct the simulated perfusion culture of rCHO cells to explore the parameter’s impact on the cell growth,metabolism and production,which include initial glucose concentration in the medium,perfusion rate,temperature shift time,temperature of shift,determining the preliminary process parameters of perfusion culture.3.Conduct the studied of effect of pH on rCHO cells in bioreactor batch culture and innovatively comparative study the effect of two-stage pH and constant pH perfusion cultivation on cell growth,metabolism,production,quality attributes and biological potency of the product.4.Conduct the three consecutive batches in pilot scale perfusion culture of rCHO cells with optimum process parameters study,and the robustness between batch and batch,the consistency of critical quality attributes and biological potency of anti-CD52 antibody within batch were investigated.Results and discussion:1.Firstly,this study has fully understood the basic characteristics of the growth,metabolism and production of the cell by the batch culture of rCHO cells production anti-CD52 antibody.2.Secondly,this study shows that initial glucose concentration in the medium,perfusion rate,temperature shift time and temperature of shift have significant effect on the cell growth,metabolism and production during perfusion culture,and determine the optimum parameters:the initial glucose concentration is 10 g/L,the perfusion rate is 1.00VVD,temperature shift time is when the viable cell density is 25.0×10~6 cells/mL,temperature of shift is 34.0℃.3.The results of batch culture under different pH conditions showed that the optimal growth pH and the optimal expression pH were 7.15 and 6.85 respectively.In perfusion culture,the cell growth,metabolic,production,quality and biological potency of the product,and robustness of process under two stages pH(7.15-6.85)perfusion culture significantly better than constant pH(7.15)perfusion culture group.4.The rCHO cell growth and production were comparable between three consecutive perfusion culture batches,and the critical quality attributes and biological potency of the product in different periods within batches and between batches showed no difference.Conclusion:In this study,we developed the anti-CD52 antibodies CHO cells perfusion culture process,established the robustness,high cell density,long culture time,high productivity,high quality and high biological potency pilot production process.