Purification Of Hamster Kidney Cell Rabies Vaccine By Gel And Ion Exchange Chromatography

The standard of human rabies vaccine protein contenthas been raised in Chinese Pharmacopoeia2010edition. It requirespurified total protein content and bovine serum protein residuescontent no higher than80μg/agent and50ng/agent.Before addingstabilizer into vaccine, the residual host cell protein of endproducts require no more than24μg/agent.Improve the separationand purification effect has always been one of the problems to beovercome for bio-pharmaceutical companies. We have learnt from thelong-term exploration experiments of application in chromatographysystem that: Gel Chromatography column (Sepharose4Fast Flow,4FF)provides good separation ability for the host protein and bovineserum protein with mild operating conditions. However, due to thehigh impurity content of samples, it is easy to block liquid inletduring actual production applications, and would prolongseparation time and influence separation ability. Ion ExchangeChromatography (Core700)which contains all the ability of highsensitivity, small size, easy to clean, heavy workload, and shortseparation time, widely used in the field of Bio-medicine and otherfields. This paper chooses a chapter of fumble experiment, combinethe Gel Filtration and Ion Exchange Chromatography with threedifferent sequences: Core700-Core700-4FF, Core700-4FF-4FF andCore700-4FF-Core700, to separate and purify experimental samples.The experiment was divided into two parts: the first part, selectthree batches of experimental samples, then purify and isolate eachbatch of samples according to the three above-mentioned combinationmethods. Depending on the content of total protein, host protein,bovine serum protein, and antigen, select the combination methodwhich achieve Chinese Pharmacopoeia standard. The experimental1shows when used in combination method Core700-4FF-4FF, separation effect is relatively higher than other methods. The second part:based on results of experiment1,use other three batches ofexperimental samples to do validation test according to thecombination method described above, test the content of finalpurified solution’s total protein, host protein, bovine serumprotein and antigen. This two-part experiment shows undercombination method Core700-4FF-4FF use, the detection result ofpurified samples meets the requirements of Chinese Pharmacopoeia2010edition. It can be considered as a reference of establishmentof purification process, and may also be used as a reference ofoperation and experiment for relevant technical personnel.Application research in vaccine purification with chromatographysystem has been a subject of concern, all of this factors influencevaccine purification effect which include sample volume, loadingrate, sample concentration, and the starting and ending points ofcollection.The joint use of Gel Filtration Chromatography and IonExchange Chromatography provide a better way to play thecharacteristics of two chromatography column, and optimizepurification effect of the vaccine. According to differentconditions, bio-pharmaceutical companies can use Core700and4FFin a reasonable and flexible way to ensure vaccine quality, improvethe efficiency of purification and increase economic efficiency forenterprises.