| Production of the valuable protein using the mammal celllarge-scale culture technology is now one of bio-engineeringdomain important topics. The mammal cell large-scale culturetechnology only then starts in the 60's to appear, but hasobtained the development immediately which progresses byleaps and bounds. So far, oneself has the commercializationproduction product which the nearly hundred examplessucceeds, but these products mostly are the immune bodiesclass, mainly is by the hybrid lump cell aerosol culture system.Regarding Attaching Dependent Cell System, Also some manyliving thing aspect and the project technology aspect questionis restricting its development. Because the present high valueprotein product mainly is the ADCS system produces, thereforediscusses the new culture method, creates the appropriategrowth and the working condition, the research cell pathways,reduces has the inhibitory action to the cell metabolismby-product production, enhances the goal protein the content,lengthens the culture the time, enhances the productionefficiency to appear especially importantly.The culture of animal cell in vitro has to proteindecorating with the product butcher exocrine function. But, theanimal cell multiplies the time long, the pathways complex, tothe nutrition request high, to the external environment liketemperature, PH, dissolves the oxygen, the osmotic pressure,the shearing force sensitivity strongly, the cell condition is easyto change, the product density low status question, increasedthe animal cell culture difficulty greatly. So far has developedthe many kinds of biological reactor, and widely adopted onenew culture method -to pour into the culture method,Enhances the cell density by the time, then enhancementproduction efficiency.The massive research indicated that, pours culture is onefeasible culture method. This method causes the culturemedium to pour into continuously, has provided the fullnutrition ingredient for the cell, and may carry off themetabolite, maintains a stable cell environment. At the sametime, the cell retains in the reactor system, may achieve thevery high cell density. Speaking of ADCS, so long as well theelective culture system, the solution or avoids the cell stoppingup the question, successfully carries on the cell high densitylarge-scale culture is completely feasible.The 30L bioreactor may use in half class adding a raid ofculture and harvesting the cell nutrient fluid continuously andsupplements the new nutrient fluid, trades the fluid speed to bepossible expresses according to the cell number and the cellularform and HBsAg adjusts. In slightly tries in the craftfoundation, we select the continuous culture method, the celldisposable meet the type and quantity to join for 5×105 themicrocarrier, receive trade the fluid speed in 15 days are 11L/d,after 15 days changes 22L/d, and changes to 33L/ d while theHBsAg titer expression is 1: 256 , and to D.O.Culturecondition and so on PH has done the research to the HBsAgsecretion influence.In CHO-C28 cell culture, supplies glucose quantity veryimportant, both must meet the cell growth need, and mustcontrol its quantity, by guards against the lactic acid and so onthe excessive production, is disadvantageous to the cell growth.When agitation speed is 55rpm, 60rpm or 70rpm, althoughthe shearing force do not tremendous influence to the cellattaching, but the D.O. value is very difficult to maintain;When agitation speed is 90rpm, the D.O. value maintenance isgood, but the cell falls off too many;Only then when 75-80rpm,The D.O. value maintenance is good, the cell is affectedslightly the shearing force, the cell falls off few, thereforecultures, the later period rotational speed should control in75-80rpm.The experimental result indicated that, the continuousculture 60 days, the CHO cell growth pattern is good, assumesthe clew shape, the rate of cell attaching on the 30th day is 70%,falls off few. On the 18th day the cell density reaches themaximum value, is 7.0×106cell/ml, later equally will maintainat 5.0×106cell/ml, the RPHA titer may reach 1: 512, theaverage in 1: 128 ~ 1: 256 levels, the HBsAg average dailyoutput is 30mg/d.This craft has replaced in the original craft extensionbottle by the biological reactor and the micro carrier, expressesHBsAg with the cytogenetics convention method after thehepatitis B gene transformation the CHO-C28 cell line and themicro carrier continuous cultivation the 60d CHO-C28 cell linecarries on the genetics examination, finally indicated, the twochromosome is 20, had not discovered the chromosomemultiple the change, showed hands over the associationglucosan take DEAE as structure Cytodex, the surface replacestransfers the bottle to culture the CHO cell is successful.The application of biology reactor duplicates ten, eachtime the cycle is about 60 days, the RPHA titer of HBsAg mayreach about 1: 512 levels, was indicating pours culture waycontinuously, the speed of pour flows need adjust, D.O. valueand PH value of the CHO-C28 cell to the 30L biology reactor isfeasible, preliminary establishment major and medium scalebiology reactor production hepatitis B gene vaccine new craft.
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