Process Characterization Of The Chinese Hamster Ovary Cells Culture Process

Monoclonal antibody drugs are macromolecular protein drugs with biological activity expressed by animal cells.With the development and development of monoclonal drugs in recent years,the research on antibody production process has been gradually deepened,and the requirements for batch differences in production process have become increasingly strict.Process characterization is an important part of Quality by design(QbD).The concept of QbD is mainly to determine the Critical quality attribute(CQA)through risk assessment,and determine the Critical process parameter(CPP)and Key process parameter(KPP)that affect CQA and Key process attribute(KPA)through process characterization research.Then,the scope of CPP/KPP was systematically studied,and finally the Design space of parameters and the control strategy of process were determined.Depending on the process development process,characterization of the production process is usually required prior to phase III clinical studies.The purpose of the process characterization study is to improve the process stability,reduce the differences between batches,reduce the deviation in production,reduce the risk of failure,and ultimately ensure the uniformity of product quality.In this paper,the object of study for China's ongoing clinical?monoclonal antibody cell culture technique of the project,the purpose is for the commercial production stage production process scope set to provide guidance,better stability of cell culture technology,less risk of failure process.The cell culture process was carried out in a 500L reactor.After the proteins obtained were successively purified and prepared,the products would be applied to the treatment of diseases in which the tumor still has progress after radiotherapy and chemotherapy,which has great practical application value.Therefore,the purpose of this study was to evaluate the effects of CPPs and KPPs on antibody CQAs and KPAs in upstream cell culture.Finally,the reduced model is established to provide the experimental basis for the subsequent process research.At the same time,the influence relation of each key process parameter to key process attribute is obtained.First of all,through the risk assessment method to confirm the key quality attributes for antibody polymer,the main composition in charge different plasmids,antibody fragments,antibody type of sugar,and then through the various process steps of a direct impact on the key quality attributes to evaluate process steps,finally confirmed the key technology of properties for the ultimate prize of living cells density>15×10~6 cells/ml,live cells at harvest rate>80%,and harvest time protein antibody concentration>2.0 g/L.According to these indicators,the key process steps from cell resuscitation to the end of production tank were identified,and the key process steps were finally identified as:50L seed amplification reactor and 500L production reactor.To analyze the parameters of the two process steps,determine the key process parameters of the key process steps,process parameters that need to study as follows:the production tank on the cultivation of the culture temperature on pH value,production can reduce the density of living cells,production on the pot on the cooling time,the production of cooling temperature,in the production of cans on the initial inoculation density,production value of dissolved oxygen on the cans,production cans on the cultivation of the time,these parameters for exact experimental study carefully cells;In addition,the parameters that need to be studied on the seed pot include:seed density for seed transplanting,seed density before seed pot amplification,seed age,and the density of living cells diluted by seed cells in the seed pot.In order to carry out experimental research on a small-scale reactor that can represent a 500L reactor,we first started the establishment of a scale down model on a 2L tank and a5L tank.We used three strategy to scale down the 500L bioreactor 1.Power input equal 2.The blade tip velocity of the stirring blade is scale down by the method of equal speed;3.General technology(platform technology)method scale down.First after get the result of the experiment through direct visual method to judge the validity of the scale down model,found that the first round of experiments in cell growth,cell metabolism and protein production can accord with 500 L data,but on the glucose concentration and 500 L reactor have bigger difference,through the analysis of the causes of filling material after medium flow and strategy is optimized,confirmed the experiment again.After adjusting the feeding strategy,the Scale down model was verified by direct visual comparison method and unilateral t-test method,and the equivalence of the reduced model of 2L and 5L was confirmed.After the equivalence verification of the Scale down models of 2L and 5L cans was passed,the key process parameters of the production cans were studied on 2L cans,and the key process parameters of 50L seed cans were studied on 5L cans.The results are as follows:Eventually found that within the scope of the representation,the condition of low pH(6.8)set point,after prolonged culture,cells rate decline intensified,led to Final Viability is beyond control limits.Therefore,shortening the culture time is beneficial to reduce the risk of over-limit of the central control index,but it may lose part of the antibody production.It was found that seed passage and amplification treatments were in the characterization range,and although they had potential effects on CQAs and KPAs on the production tank,there was no risk of overloading.Finally,through the summary and analysis of the research results,we get the relationship between the studied parameters and Critical quality attributes(CQAs)and key process attributes(KPPs),and the influence of process parameters on key quality attributes and process attributes.