Development And Optimization Of CHO Cell Serum-free Culture Process Based On The Concept Of Quality By Design

Antibody drugs are widely used in in the clinical treatment of malignant tumors and autoimmune diseases due to their advantages of high specificity and high safety,and are the most popular field of drug development in the world.The production of antibody drugs primarily involves three main production units:cell culture,purification,and formulation.These procedures are complicated and influenced by many factors.Quality by Design(Quality by Design,QbD)is a quality design concept recommended by the U.S.Food and Drug Administration(FDA),which aims to design the production process for products with predetermined quality based on science and quality risk management,and consistently produce final products with expected quality.However,the application of QbD concept in antibody drug development is still in its infancy,and most of the cases are used in cell culture process development.Due to the various components of serum-free medium and the complicated development process,QbD is rarely used in the development of serum-free medium.It is a tremendous problem and challenge to make the statistical analysis,optimization and control of complex and diverse medium components based on the QbD concept.Therefore,this paper attempts to apply the QbD concept to establish an algorithm model that can analyze and predict the key components of the culture medium,and apply it to the development of serum-free culture medium oriented by the critical quality attributes of antibodies.We establish the method for the design and optimization of serum-free culture process of CHO cell based on the quality attributes of antibodies through combination of the algorithm model,statistical tools and risk classification.Firstly,the serum-free medium that affects the critical process attributes and critical quality attributes of the humanized antibody ACTE802 were investigated.33 groups of serum-free media with different formulations were designed by Simplex Lattice,and fed-batch culture was carried out in cell culture shaking tubes.The culture results showed that the highest peak viable cell density was 10.4×106 cells/ml,the highest integral of viable cell concentration to time(IVCC)was 62.37×109 cells day/L,and the highest titer was 3.98 g/L.The results of antibody quality test showed that the main peak content of charge variants was 57.24%-73.52%,the content of acidic charge variants was 21.96%-27.64%,the content of basic charge variants was 1.79%-16.46%,the proportion of low molecular weight fragments was 2.50%-6.28%,the glycoforms of GOF range from 74.4%to 92%,and the glycoforms of Man5 range from 0.1%to 1.0%.Based on these results,the locally weighted regression algorithm(LOESS algorithm)was used to statistically analyze the importance of medium components,and 16 key components were predicted to affect antibody titer and critical quality attributes.Furthermore,the results of deterministic screening design(DSD)confirmed that the total of 13 key components have the significant impact on antibody titer,charge variants,low molecular weight fragments,and glycosylation modifications.The accuracy of LOESS prediction over 80%.Then,the gaussian mixture model(GMM)combined with Gradient Boosting Decision Tree(GBDT)(GMM-GBDT)was used to establish the prediction model of antibody titer and critical quality attributes.On the one hand,3 new formulations were generated by the Uncertainty Estimates method.,and we compared these 3 new formulations on 2L bioreactor.The results showed that the optimal formulation was BMpro2.The antibody titer reached 4.16g/L using BMpro2,which was 21%higher than that of the control group.The acidic charge variants content was reduced from 27.29%to 18.09%,the proportion of low molecular weight fragment decreased from 8.41%to 3.31%,and the other quality attributes met the preset standards.On the other hand,combined with the Local Interpretable Model-Agnostic Explanations(LIME algorithm)to investigate the impact of the fluctuation of the effective concentration of key medium components on the critical process attributes and critical quality attributes.No significant fluctuation was founded that when the concentration of these key components fluctuates within±5%,the antibody titer and critical quality attributes are both within±10%of the test results of the control group.This result has also been verified by a single factor experiment.Obviously,the algorithm model based on QbD could analyze and predict the effect of key components of the medium on the critical quality attributes of antibodies.The serum-free medium development using the algorithm model has excellent process performance and robustness.It could reduce the failure risk of key process attributes and critical quality attributes when the medium components fluctuate in the design space.Based on the serum-free culture medium developed above,we observed that inoculation density,culture temperature,pH,feeding strategy,and culture period had high risk to the process performance of fed-batch culture process through fishbone diagram and causality matrix analysis.Therefore,these parameters were defined as potential critical process parameters.The results of partial factorial design experiments further confirmed that culture temperature,inoculation density and feeding strategy had significant effects on process attributes and critical quality attributes(P<0.05),and they were identified as the critical process parameters for the development,optimization and operation of ACTE802 cell culture process.Therefore,the design space of the inoculation density,culture temperature and feeding strategy was explored by response surface center compound experimental design,and we determined the design space of cultivation temperature,inoculation density and feeding strategy were 36.5-37.5?,1.0-1.8×106 cells/ml and 26%-34%,respectively.Furthermore,the total of 15 batches of culture experiments were completed on a 2 L bioreactor scale.The antibody titer reached 4.10±0.44 g/L,the content of acidic charge variants was 16.15±1.39%,the content of basic charge variants was 7.63±2.75%,the aggregates ratio was 1.14%±0.28%,the low molecular weight fragment ratio was 3.36±1.02%,the glycoform of GOF range was 77.00±5.64%,the glycoform of Man5 range was 0.70±0.19%.All these results were within the design range of the critical quality attributes of the product,and it verifying the robustness of the process.Finally,the serum-free medium and cell culture process determined above were scaled up to the pilot scale of 200 L bioreactor,and the process was scaled up using the principle of equal power(P/V equal)amplification.6 batches of 200 L bioreactor culture experiments were successfully completed,the average titer of 200 L bioreactor was 4.04 g/L,the average aggregates ratio was 1.12%,the average low molecular weight fragment ratio was 4.15%,the content of average acidic charge variants was 16.37%,the content of basic charge variants was 5.73%,the average glycoform of GOF was 80.96%,and the average glycoform of Man5 was 0.82%.All these results were all within the Mean±1.5SD range of the corresponding data in the 2 L bioreactor.Partial least squares regression(PLS)model and principal component analysis(PCA)model were established based on the process data,process attributes and critical quality attributes of 15 batches of 2 L bioreactor to evaluate the process performance of 6 batches of 200 L bioreactor.The results of PLS analysis showed that the parameter trajectories of 6 batches of 200 L pilot scale cell culture process were all within the control limit of 2 L culture process parameter trajectory(Average±3SD),and the results of PCA analysis showed that the batch points of the 200 L bioreactor were all distributed within the 95%confidence interval of the data of the 2 L bioreactor.These results shows that the process performance and product quality of the 200 L bioreactor were consistent with that of the 2 L bioreactor.The process has good robustness and scalability,and can satisfy the pre-designed key process attributes and critical quality attribute requirements.In this study,we applied the concept of QbD to the process development of antibody drugs creatively.For cell culture production unit,a variety of statistical methods can be used to characterize the relationship between medium composition,culture process parameters and critical process attributes/critical quality attributes.This process developed based on QbD concept could establish the process from shaking tube to pilot scale cell culture rapidly,and reduce the risk of cell culture production unit failure.This study provides a theoretical basis for the industrial production of innovative ACTE802 antibody.At present,this project has successfully entered phase II clinical trials.In addition,it also provides important guidance for the development and establishment of other antibody drug cell culture processes.