Research Of Microcarrier Pretreatment And The Optimization Of Aluminum Hydroxide Adjuvant Processing In The Vaccine Production

As new arriving vaccines in recent years,Sabin inactivated poliomyelitis vaccine and enterovirus 71 inactivated vaccines are the powerful weapons to eliminate poliomyelitis and preventative children with hand-foot-and-mouth disease caused by EV71 globally.The both are live vaccines.In order to raise vaccine's immunogenicity,kinds of adjuvants are used to augment the immune effect.In one hand,recently,with the microcarrier culture technique popularization,cell yield can be increased effectively and the antigens are increased.In the other hand,as the traditional immune adjuvant,Aluminum hydroxide can augment the immune effect,and decrease the using of antigens.These two materials,solved the low productivity and high price of the inactivated vaccines,provided the possibility of low-price and abundant vaccines are supplied to the developing countries,even the whole world.This article researches how the microcarrier pretreatment in production of sIPV affect the sterile temperature and Vero cell attachment rate.At the same time,the article optimizes the preparing conditions of aluminum hydroxide adjuvant in production of EV71.All these researches with a view to guide to preparing and producing microcarrier and aluminum hydroxide,make the two materials used better into production of Sabin inactivated poliomyelitis vaccine and enterovirus 71 inactivated vaccines and be laying foundations for increasing vaccines productivity and decreasing production cost.I:How the microcarrier pretreatment in production of sIPV affect the sterile temperature and Vero cell attachment rate.At present,commercial microcarriers are stored and soled as dry powder.The powder is pretreatedbefore cell culture,which is hydrated,imbibed and washed with PBS(no Ca2+,Mg2+),then steam sterilized.Butduring the progress,microcarrier could be affected and then influence the adherence rate of Vero cell.In thisstudy,the Cytodexl was pretreated by PBS(no Ca2+,Mg2+)at three concentration:1:20(50g·L-1),1:40(25 g·L-1),1:60(16.7g·L-1),each concentration with three different loading as 720,2400 and 6000mL,andthen steam sterilized at 121?,to observe the effect of sterilization temperature caused by different concentrationand loading.After the sterilization,the samples were observed under microscope,the microcarrier samples wereused for the 7L bioreactor culture of Vero cells.After 12h,the adherence rate of each group was recorded.The results showed that the heating rate was affected by the concentration and loading amount when the carrier was pretreated.When the concentration was less than 50g·L-1,the sterilization was less than 212min above 121?,the Cytodexl's physical characteristics and Vero cell adhesion rate were not affected(P>0.05).During the cultureof Vero cells,in addition to the adherent rate,whether the sterile control could meet the requirement under thedifferent sterilization temperatures caused by different pretreatment conditions is more important.It is provided inour study that.?:How to optimize the preparing conditions of aluminum hydroxide adjuvant in production of EV71.Prepare aluminum hydroxide adjuvant with ammonia water method.The items are for optimizing including the way of dropping ammonia-water and dialysis method.The aluminum hydroxide adjuvant products of 3 batches before optimization and 3 batches after optimization were compared by size testing,sedimentation analysis,ammonium ion testing,aluminum content testing and aluminum adsorption effect testing.Results:The average size of aluminum hydroxide adjuvant after optimization was markedly smaller than before(P<0.05),as well as the variation coefficient was smaller than before.For the 3 batches after optimization,sedimentation rate was 0ml,and the ammonium ion testing was 100%qualified.Aluminum and aluminum hydroxide contents were evidently more than before(P<0.05)increased by 24.7%and 26.1%respectively.For all of the semi-manufactures in the 6 batches,the antigen percentage in supernatant was 1.25%.There was no difference of adsorption effect to enterovirus 71 antigen among the 6 batches of aluminum hydroxide adjuvant(P<0.05).The improvement of the technique might increase the quality and safety of aluminum hydroxide adjuvant product,decrease batches variation,and increase the production efficiency.And the effectiveness of aluminum hydroxide adjuvant would not be affected.The safe and effective self-made aluminum hydroxide is significative for being not dependent on imported ones.So is for breaking the price-fixing by import manufacturer.It creates a chance to decrease the vaccine price.