Study Recombinant Human Erythropoietin(CHO-rhEPO)Cell Mass Amplification

The research on amplification conditions of recombinant human erythropoietin (rhEPO) cell and biological reactor has been studied in this project. In order to implement large-scale amplification of Chinese hamster ovary cells in the packed bed bioreactor and the ball-to-ball method (porous), which is used for expressing EPO, a series of research on the purity, activity, stability and preservation of serum-free supernatant in the expression prodcut has been done.The large-scale amplification of Chinese hamster ovary cells has been carried out in two ways, which are the packed bed bioreactor and the ball-to-ball method (porous). Through the comparion of medium preparation, seed production, culture process control and product inspection, the packed bed bioreactor has been confirmed as the best way for the large-scale amplification of Chinese hamster ovary cells because it has more superiorities than the packed bed bioreactor, such as less workload, less contamination chance during seed culture and so on.The general process:The stoste first through the Perfusate, reach the blue gel chromatography, and then obtain concentrated product after ion exchange I chromatography, and then C4reversed phase chromatography, this has been further optimized, the obtained product by ion exchange chromatography and II molecular sieve chromatography, finally to achieve the increase of the objective product purity requirements. There are several parameters to determine whether the product is pure, such as endotoxin and exogenous DNA content, CHO protein and so on. The purified EPO original purity, activity, separation and recovery rate of product quality index,has reached the European Pharmacopoeia requirements (EP7.0). Our study shows that it is possible to use bioreactors for recombinant human erythropoietin production of mass culture.The sample from Purification method Ⅱ has been diluted untile the EPO content is20μg/ml, using the buffer which is composed of sodium citrate, citric acid and sodium chloride with a pH value of7.0. Then0.25%(w/v) Human serum albumin (HSA) should been added. After being filtrated with0.22μm filter membrane, sterilization examination and activity test have been carried out. According to analytical data, we can use the buffer mentioned above to adjust the solution to2000μg/ml of activity unit and make this as the final product. After adding the stabilizer compatible with the human tissue, optimal preservation condition has been studied. Research found that in the condition of 2-8℃, one year is stable (activity, physical properties and pH values have not significantly changed); in the condition of ambient temperature, the rentation period is at most4weeks; and the product should avoid high temperature and not be frozen. These information is valuable for the effective conservation after large-scale cultivation of EPO and also for the more extensive clinical application.