The Optimization Of Process Control Strategy For Hybridoma Large-scale Culture And Lyophilization Strategy For Antibody Preparation

Aim:On-line analysis and control are very important for the optimization and achievement of high cell concentration and product yield in animal cell culture.Among various parameters,Oxygen uptake rate(OUR) is a sensitive indicator of cell growth and metabolism during large-scale culture in bioreactors.Through real time measurement of OUR,the metabolic state of cell can be shown in time and from this analysis the culture strategy can be regulated correctly. Furthermore, the analysis of OUR can provide some useful information to guide the optimization of culture technology. Relative studies have shown that OUR had become a necessary part for analyzing culture process with the further study of large-scale culture.Under some conditions, a linear relationship between OUR and density of live cells at least in exponential growth period will be displayed. And the amount of ATP produced can be estimated based on the amount of lactate produced.So the physiological state of cells can be estimated by measuring ATP and an optimization for on-line monitoring strategy of cultivation can be carried out through ATP balancing, too.For this reasons,one aim of my work is to establish and optimize the feed-back control strategy based on OUR and applythe optimized strategy to batch or perfusion culture of hybridoma HAb18 for higher efficiency of HAb18 IgG production.From another aspect, the importance of correct disposal for products should be emphasized when the purpose is to get target products(antibody, cytokine,enzyme,etc.) which exist in suspension and are damaged easily by surrounding factors.Denaturation can be found in antibody molecule under the effects of water,heat,oxygen and other chemical factors.Therefor, a correct method for the conservation of purified antibody is very important. Among various conserviation methods,lyophilization can keep the physical and chemical characters and biological activity of products during the procedure.For this reason,another aim of my work is to optimize the lyophilization strategy for purified antibody to prolong the term of validity,i.e. improve the culture efficiency by decreasing the lost of products. Methods:1. Study on the relationship between OUR and hybridoma growth and metabolism in fed-batch culture.(1) A hybridoma cell line (HAb18) was cultured in batch model in 5L Biostat B bioreactor.The DO level was set to 30% and 70% of air saturation respectively to study the effect of DO on cell growth.(2)Two bioreactors were set to the same set point:stirrer speed at 60 rpm,temperature at 37℃,surface aeration rate was 1.5L/min and pH at 7.0-7.2.Under thus conditions,the volumetric oxygen transfer coefficient KLa was determined from the change of DO(60%-30%),which recorded in a 5min interval.(3)The hybridoma HAblS was cultured in batch model in 5L Biostat B bioreactor. The relationship between OUR and hybridoma growth andmetabolism was studied.2. Study on the relationship between OUR and hybridoma growth andmetabolism in perfusion culture.The hybridoma HAb18 was cultured in perfusion model in 5L CelliGen Plus bioreactor, work volume 3.5L.The production of antibody was studied in a glucose-indicated perfusion model. The perfusion rate was adjusted to keep the gluose concentration at 1.5g/L and the range was 0~5L/d. 3. Study on feed-back control model based on OUR in perfusion culture. (1)The hybridoma HAb18 was cultured in 75mL flasks and divided into several groups with different lactate or ammonia concentrations.The density of cell in all of the flasks were determined every day to study the effects of byproducts on cell growth..(2)The hybridoma HAb18 was cultured in perfusion model in 5L CelliGen Plus bioreactor.The nutrients fed strategy based on OUR were studied.During the culture,OUR was determined on-line and 0.5L fresh medium was fed at the moment that A OUR=0.(3)The amino acid fed strategy based on OUR was studied in the 5L CelliGen Plus bioreactor.The perfusion rate was maintained at 1.0 volume/day from 4th day,and an multiple amin...