Study Of Monoclonal Antibody Production In Chinese Hamster Ovary Cell Culture By Disposable Turbulent Bioreactor

In the past decade,the demand for certain types of biological drugs,such as monoclonal antibodies（mAbs）,has greatly increased worldwide.Monoclonal antibodies are used as drugs for the treatment of cancer and autoimmune diseases,and have demonstrated significant efficacy.Most therapeutic monoclonal antibodies are recombinant proteins expressed by mammalian cells and can produce post-translational modifications similar to human antibody proteins.Currently,the mainstream method for large-scale production of monoclonal antibodies is to use bioreactors to culture suspended CHO cells（Chinese hamster ovary cells）as hosts to express heterologous monoclonal antibodies.In this process,the bioreactor needs to accurately control the parameters such as temperature,dissolved oxygen,and PH,and mix the culture fluid to provide a uniform physical and chemical environment,nutrient concentration,and cell distribution.For bioreactors,mixing,oxygen supply,and carbon dioxide removal are issues that need to be addressed.At present,the most commonly used bioreactor for suspended CHO cell culture in the industry are stirred bioreactors and bubbling bioreactors.For the weaker CHO cells that lack cell wall protection,the shear stress of stirring and bubbling are the limitation of stirred stirred bioreactors and bubbling bioreactors.JYSS（Jinyi Shengshi）disposable turbulent bioreactor is based on disposable technology and has a simple design principle.It does not use interventional stirring devices and bubbling ventilation,avoiding the shear damage caused by stirring and bubbling to CHO cells,has great potential for CHO cell culture.In this thesis,we study the production of monoclonal antibodies by culturing CHO cells in JYSS disposable turbulent bioreactor,including the determination of reactor parameters,the study of cell growth and metabolism in the reactor,and product detection.Through the simulation of cell culture process testing,including mixing time,K_La,shear force,explore the speed range of JYSS disposable turbulent bioreactor to culture CHO cells to produce monoclonal antibodies.In terms of mixing time,the mixing time of JYSS-2L（working volume）above 50rpm is less than 20s,the mixing time of JYSS-50L above 40rpm is less than 40s,and the mixing time of JYSS-300L above 25rpm is less than90s.In terms of KLa,under 0.01V/V/M（gas volume/liquid volume/min）oxygen supply,K_La of JYSS-2L and JYSS-50L exceed 20h^-1 at 55rpm and 40rpm respectively,and K_La of JYSS-300L reaches 10-20h^-1 at a speed of 25rpm-30rpm.In terms of shear force,from 2L to 300L working volume,within the normal working speed range,the average shear rate of the fluid in the JYSS bioreactor is less than 20s^-1.Based on these test data,we judge that JYSS-2L can provide good mixing and oxygen transfer effects at a moderate speed range of 50rpm-70rpm.the monoclonal antibody production process（PH,temperature,dissolved oxygen,medium composition,culture time,etc.）developed and optimized in the 2L working volume Sartourius（Sart-2L）stirred bioreactor is transferred to JYSS-2L.At the optimal speed of 60 rpm obtained through optimization,under the same process conditions（PH,temperature,dissolved oxygen,medium composition,culture time,etc.）,JYSS-2L disposable bioreactors are used to culture CHO cells with a peak viable cell density achieving 13.9*10⁶cells/mL,the final target product monoclonal antibody output reaches 2416mg/L.The control,Sart-2L（Sartourius 2L stirred bioreactor）,reaches a peak viable cell density of 13.5*10⁶cells/mL,and the target product monoclonal antibody output of 2464mg/L.In terms of cell byproduct accumulation,the accumulation of carbon dioxide in JYSS-2L is significantly lower than that of Sart-2L,which is about 30%-50%lower.Lactate accumulation,and ammonia accumulation in two reactors show high consistency.In terms of product quality,polymer,charge variant,structural integrity and binding activity are mainly considered.The target product polymer content harvested in the JYSS-2L reactor is less than 1%,the main peak of the charge variant detection reaches about 70%,the complete structure content is 89.7%,and the relative binding activity is 142%.Except the relative binding activity is slightly higher than the Sart-2L（123%）,the several other types of quality of products in JYSS-2L is almost the same as control.According to the conversion of inner diameter and eccentricity（same rotational linear velocity）,combined with mixing time,Kla and fluid oscillation intensity in the reactor,the rotation speed range of JYSS-50L,JYSS-300L is locked Between 40-50rpm and25-30rpm respectively.After optimisation,for CHO cell culture,the optimal running speed of JYSS-50L and JYSS-300L is 40rpm and 25rpm respectively.Under thess parameter,CHO cell culture in JYSS-50L,the peak viable cell density is 13.0*10⁶cells/mL,and the product concentration is 2343mg/L.In terms of CHO cell culture in JYSS-300L,the peak density of viable cells is 9.7*10⁶cells/mL,which is lower than counterparts in JYSS-2L and JYSS-50L;the product concentration is 1958mg/L,which is lower than counterparts in JYSS-2L and JYSS-50L.However,in terms of cell viability,cell viability in JYSS-300L is superior to cell viability in JYSS-2L and JYSS-50L.Even at the end of the culture period,the cell viability in JYSS-300L remains 99%.In terms of product quality,the monoclonal antibody product harvested in JYSS-300L is superior to counterparts in JYSS-2L and JYSS-50L in structural integrity and binding activity.In general,the JYSS disposable turbulent bioreactor is suitable for culturing CHO cells to produce monoclonal antibodies.However,the phenomenon of extended mixing time,reduced oxygen transfer,decreased cell density and product concentration in large-scale applications requires further research.