【正文】 78Table 11 Liquid Material Consumption Table 表格 12所用液體配料表3％DMEM culture media 3％DMEM培養(yǎng)液DMEM powder DMEM粉末Glycine甘氨酸LProline L脯氨酸Sodium bicarbonate powder 碳酸氫鈉粉末37gWater For Injection 注射用水至10LNew born bovine serum 新生牛血清3％Total 總計
10L4％DMEM culture media 4％DMEM培養(yǎng)液DMEM powder DMEM粉末Glycine甘氨酸LProline L脯氨酸Sodium bicarbonate powder 碳酸氫鈉粉末37gWater For Injection 注射用水至10
New born bovine serum 新生牛血清4％Total 總計10L5％DMEM culture media 5％DMEM培養(yǎng)液DMEM powder DMEM粉末Glycine甘氨酸LProline L脯氨酸Sodium bicarbonate powder 碳酸氫鈉粉末3
gWater For Injection 注射用水至10LNew born bovine serum 新生牛血清5％Total 總計10L10％DMEM culture media 10％DMEM培養(yǎng)液DMEM powder DMEM粉末Glycine甘氨酸LProline L脯氨酸Sodium bicarbonate powder 碳酸氫鈉粉末37
Water For Injection 注射用水至10LNew born bovine serum 新生牛血清10％Total 總計10LSolution A for ultraspeed centrifugation 超離所需液體ASodium Chloride 氯化鈉500gEdetate 乙二胺四乙酸3
2gWater For Injection 注射用水至8LTotal 總計8LSolution B for ultraspeed centrifugation 超離所需液體BSodium Chloride 氯化鈉500gEdetate 乙二胺四乙酸Potassium bromide 溴化鉀3500gWater For Injection 注射用水至8.
LTotal 總計Solution D for ultraspeed centrifugation 超離所需液體DPotassium bromide溴化鉀5000gWater For Injection 注射用水至8.
LTotal 總計 trypsin solution Sodium Chloride 氯化鈉85gTypsin蛋白胰酶25gWater For Injection 注射用水至10LTotal 總計10L75% ammonium sulfate solution 75%硫酸銨溶液Ammonium sulfate硫酸銨Water For Injection 注射用水至10LTotal 總計10LGlutamine 谷氨酰胺
Glucose 葡萄糖10gWater For Injection 注射用
至10LTotal 總計10LMTX solution MTX溶液Methotrexate甲氨蝶呤454mgSodium hydroxide氫氧化鈉20gWater For Injection 注射用水至10LTotal 總
10L8% sodium bicarbonate8%碳酸氫鈉Sodium bicarbonate碳酸氫鈉800gWater For Injection 注射用水至10LTotal 總計
0L20mmol/L phosphate buffer solution (pH )20mmol/L磷酸鹽緩沖液（）Disodium hydrogen phosphate磷酸氫二鈉17Sodium dihydrogen phosphate磷酸二氫鈉Water For Injection 注射用水至10LTotal 總計10L5% formaldehyde solution 5%甲醛溶液Formaldehyde 甲醛500mlWater For Injection 注射用水至10LTotal 總計10L1mol/L hydrochloric acid solution 1mol/L鹽酸溶液Hydrochloric acid 鹽酸1LWater For Injection 注射用水至10LTotal 總計1
L% sodium chloride solution %氯化鈉溶液Sodium chloride氯化鈉85gWater For Injection 注射用水至10LTotal 總計10LSodium hydroxide氫氧化鈉40gWater For Injection 注射用水至10LTotal 總計10L List of Production Plants生產(chǎn)車間一覽Table 13 List of Production Plants 表格 14生產(chǎn)車間一覽表Group 組別Operation Room Name操作間名稱No.編號Classification 潔凈級別Temperature and Humidity Requirements 溫濕度要求Cell 細胞Operation Room (5)1 操作間(5)116XBC03CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～65％Operation Room (5)2 操作間(5)216XBC04A/CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～65％Thermostatic room (2) 恒溫室(2)16XBC05CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度35～37℃，相對濕度45％～65％Production 生產(chǎn)Operation Room (1) 操作間(1)16SCA/C01A/CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～65％Operation Room (2) 操作間(2)16SCA/C02A/CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～65％Operation Room (3) 操作間(3)16SCA/C03A/CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～65％Thermostatic room (1)1 恒溫室(1)116SCC04CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度35～37℃，相對濕度45％～65％Thermostatic room (1)2 恒溫室(1)216SCC05CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度35～37℃，相對濕度45％～65％Purification 純化Rough extraction room粗提間16CHC01CTemperature: 18 to 26℃ and relative humidity 45% to 65% 溫度18～26℃，相對濕度45％～。For the consumption of aluminum chloride, it = aluminum hydroxide consumption 247。The contents of merthiolate and residual bovine serum protein conform to requirements in Volume III of CP 2010. 硫柳汞含量、牛血清白蛋白殘留量等符合《中華人民共和國藥典》2010版三部要求。 Sterilization of Purified Product 純化產(chǎn)物除菌 After acceptable results are obtained, HBsAg purified products from a same cell digestion batch are bined. And the bulk is obtained after UF, concentration and sterile filtration. 同一細胞消化批來源的HBsAg純化產(chǎn)物檢定合格后合并，經(jīng)超濾、濃縮、。1℃保溫72小時。 Formaldehyde treatment 甲醛處理Add 5% formaldehyde solution to the HBsAg purified product. The final concentration of formaldehyde is 200μg/ml. Maintain the temperature at 36℃177。經(jīng)除菌過濾后收集的液體即為純化產(chǎn)物。濃縮樣品進行Sepharose 4 FF凝膠過濾層析，上樣量為柱體積的4%～6%，流動相為pH ，洗脫流速為480ml/h，根據(jù)波長為280nm的紫外吸光度值收集HBsAg峰。兩次溴化鉀密度梯度離心收集HBsAg的方法相同。以每分鐘25000轉(zhuǎn)的轉(zhuǎn)速離心22小時，根據(jù)波長為280nm的紫外吸光度值收集HBsAg峰。u Potassium bromide density gradient centrifugation (twice) 溴化鉀密度梯度離心（兩次）HBsAg is separated with two potassium bromide density gradient centrifugations. The density ratios of the two density gradient centrifugation were both: 采用兩次溴化鉀密度梯度離心HBsAg，兩次離心時密度梯度液的比重均為：Solution A:A液:；Solution B: B液:；Solution C: C液:；Solution D: D液:。u Ammonium sulfate precipitation 硫酸銨沉淀After UF concentration, 312g of ammonium sulfate is added to each liter of the obtained culture media. It is then allowed to stand at room temperature for 4 to 5 days after stirring to allow HBsAg to precipitate. 超濾濃縮后的培養(yǎng)液每升加入312g硫酸銨，攪拌均勻后，室溫放置4～5天，使HBsAg沉淀。 Purification純化u Filtration過濾The harvested culture media go first through a filter and then a filter to remove any cell residues. ，過濾去除細胞碎片。1）次，每1～2批收獲液可合并純化。此后，每48小時更換一次維持液，并收獲培養(yǎng)液。 Harvesting收獲After passage to the No. 32 generation and the cells have grown into a pact single layer adhering to the wall (Day 5 to 7), discard the growth liquor, add the maintenance solution, discard the incubation liquor 48 hours later and add t