Based On The Industrial Production Of Therapeutic Monoclonal Antibody Preparation And Amplification Process Upstream Research

Objective: According to a series of laboratory studies, set the upstream preparationparameters of the therapeutic monoclonal antibody. Then set the process for commercialproduction by checking, correcting and supplementing laboratory data and large-scaleindustrial equipment capacity and accuracy which can be achieved,Methods: By screening different basal medium and feed medium, obtain theoptimized process in laboratory phase. In scale up phase, measure key parameters, such asbioreactor’s KLa, stirring paddle line speed and other parameters, based on the industrialequipment’s instruction and user manual, to establish the operating range. And then checkand correcting the process obtained in laboratoryResults:1. Using different basal media, same culture method, the viable medium#7is85E6/ml, and11#medium is only18E6/ml, the difference of cell density is about5times.Other medium, the viable cell density≤40E6cells/ml, there are four kinds;from40E6cells/ml to50E6cells/ml, there are five kinds; from50E6cells/ml to70E6cells/ml,there are seven kinds; from70E6cells/ml to85E6cells/ml, there are five kinds.2. In different basal medium, the titer of the protein of the differs.5#and16#medium, respectively,1.7g/L and1.6g/L. But8#medium protein only0.2g/L.#11and#9in the first12days as a result of living rate is reduced to60%or less, worthless totest the titer.3. Whether chemically defined peptide mixtures or traditional protein hydrolyzate,will not impact cell growth, and there is no significant difference to the control group.`4. Media1-cd4and media in the experimental group2-cd3has the highest titer.Furthermore, addition of a chemical composition defined in the experimental grouphydrolyzate, its titer in the different shake flask experiments of the same, more stable performance.5. fed-batch culture has a positive effect to express the target protein, add any kindof fed-batch culture in different combinations on the basis of media and hydrolyzate caneffectively increase the titer of protein. The media1-cd4-F1plus group is2.45g/L.6. within2L bioreactor impeller tip speed1.6m/s, cell viability has begun todecline, slightly lower impeller speed to1.4m/s, this time did not find the cell viabilitydecreased significantly.P7. Scale up the formula: Ne nd5V l32Voutput per unit volume consistentwith the principles of power amplification, consider cell viability, in the50L scale, paddlestirring speed should be controlled at≤194RPM, while200L scale, stirring speed shouldbe controlled at≤145RPM.8. In the bench scale2L bioreactors, stirring speed100rpm, temperature37℃,aeration rate of20ml/min, the reactor was added75%basal medium+25%feed medium,measured KLa=0.002s-1=7.2h-1.9. Test KLa values in2L bioreactor,50L bioreactor,200L bioreactor at a tip speedof0.3m/s,0.6m/s, at1.2m/s condition.10. Test the mixing time of concentrated solution of sodium chloride, to verify themixing time to meet the needs of the cell culture.11. Accroding to the KLa value, the air will meet the demand for oxygen in the cells,and the ventilation should be <0.1vvm to avoid bubbles.Conclusion: in monoclonal antibody production process, the change of productionscale, will lead to changes in protein production conditions, which will cause changes inprotein characteristics. The changes in protein characteristics will affect the quality ofmedicine. Considering the high cost of biological, it is impossible to develop process inindustrial-scale. Usually, the process is scaled up by amplifying the key parameters toprepare proteins has the same characteristics as in the pilot scale. Therefore, theeffectiveness, safety and quality controllability of the medicine can be guaranteed.