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          傷寒Vi多糖柱層析純化工藝的優化

          摘要

          目的:優化傷寒Vi 多糖柱層析純化工藝,以替代傳統苯酚抽提、乙醇分級沉淀法。方法:采用DEAE 離子交換層析柱,分別在緩沖液(20 mmol/L Tris-ClpH 值為6. 07. 0 8. 0 的條件下純化1 批傷寒Vi 多糖粗糖,并與傳統苯酚抽提、乙醇分級沉淀法進行比較。按《中國藥典》三部(2010 版)要求檢測純化樣品的蛋白質含量、核酸含量、O-乙酰基含量、分子大小及內毒素含量,并計算樣品的多糖回收率。采用優化的DEAE 柱層析純化工藝純化3 批傷寒Vi 多糖粗糖,驗證該工藝的穩定性。結果DEAE 離子交換柱緩沖液(20 mmol /L Tris-ClpH 值為7. 0 8. 0 時,可有效將傷寒Vi 多糖的雜質與純化產物分離,多糖回收率分別為36. 52% 38. 35%,明顯高于傳統工藝的多糖回收率(15. 52%),且純化產物的內毒素含量(10 EU /μg)明顯低于傳統工藝(200 EU/μg)。以優化工藝(pH 7. 0)純化3 批傷寒Vi 多糖粗糖獲得的6 批精糖的蛋白質含量、核酸含量、O-乙酰基含量、分子大小、內毒素含量及多糖回收率差異較小,標準偏差均小于5%。結論優化了傷寒Vi 多糖柱層析的純化工藝,該工藝穩定性良好,可替代傳統苯酚抽提、乙醇分級沉淀法,應用于傷寒Vi 多糖的純化。
           
          傷寒是由傷寒沙門桿菌引起的一種急性腸道傳染病,該病經腸道感染網狀內皮細胞系統、腸道淋巴系統和膽囊而致急性全身性感染。我國自1998 年洪澇災害后,傷寒發病率逐漸升高。據2008 年中國衛生部統計,傷寒或副傷寒的感染率為118/10 萬,病死率約為0. 04 / 10 萬,該病是中國發病率前十位的傳染病之一[1]。WHO 推薦使用傷寒Vi 多糖疫苗預防傷寒的流行2。研究表明,大規模的使用傷寒Vi 多糖疫苗,可明顯降低傷寒的流行,減輕社會的經濟負擔3-4
          傷寒Vi 多糖的有效抗原為細胞壁外的莢膜多糖。傳統純化工藝多采用苯酚抽提、乙醇分級沉淀等方法,該方法所使用的提取試劑對環境污染嚴重,工藝的繁瑣導致多糖回收率偏低。本實驗對純化條件溫和的DEAE 陰離子交換層析法的pH 值進行了分析比較,建立一種步驟簡單、純化效果好、環境污染小的傷寒Vi 多糖柱層析純化工藝,并對該方法的穩定性進行驗證,以替代傳統苯酚抽提、乙醇分級沉淀法,應用于傷寒Vi 多糖的純化。
          1 材料及方法
          1. 1 樣品4 批傷寒Vi 多糖粗糖(批號為STVi20111206STVi 20111207STVi 20120101 STVi20120102)由本公司發酵研究室提供。
          1. 2 主要試劑及儀器Tris-Cl 和氯化鈉購自國藥集團化學試劑有限公司;AKTA explorer 層析系統、DEAE 層析填料和XK26/20 層析柱均購自GE 公司。
          1. 3 DEAE柱層析純化采用AKTA exploere 層析系統,DEAE 填料柱體積為45 ml,用20 mmol/L Tris-ClpH 6. 07. 0 8. 0)緩沖液充分平衡DEAE柱。稱取傷寒Vi 多糖粗糖(STVi 20111206300 mg,按5 mg/ml 溶解于20 mmol/L Tris-ClpH 6. 07. 0 8. 0)緩沖液中,經0. 45 μm 濾膜過濾后,按每毫升填料5 ~ 10 mg 樣品上樣于預處理好的DEAE 層析柱,流速為3 ~ 6 ml/min,用緩沖液A20 mmol/L Tris-ClpH 6. 07. 0 8. 0)]淋洗4 ~5 倍柱體積,用緩沖液B20 mmol/L Tris-Cl +0. 5 mol/L NaClpH 6. 07. 0 8. 0)]進行10 ~15倍柱體積的連續梯度洗脫。純化過程分別用紫外206260280 nm 波長檢測多糖、核酸及蛋白含量。收集穿透峰,除菌過濾后用注射用水透析48 h,置真空干燥,用于后續檢測。
          1. 4 傳統工藝純化參考《中國藥典》三部(2010 版)5傷寒Vi 多糖疫苗)的方法純化傷寒Vi 多糖粗糖(STVi20111206)。
          1. 5 純化樣品的檢測取適量純化產物,用注射用水溶解,依據《中國藥典》三部(2010 版)5對純化產物的蛋白質含量(應小于10 mg/g)、核酸含量(應小于20 mg/g)、O-乙酰基含量(應不低于2. 0 mmol/g)、分子大小(多糖分子的KD 值在0. 25 以前的洗脫液多糖回收率應在50%以上)及內毒素含量進行檢測,并進行覽鑒別試驗(Vi 多糖與傷寒Vi 血清產生明顯沉淀線)。按下式計算多糖回收率。
          多糖回收率(%= 精糖的收量(g/粗糖的投量(g)× 100%
          1. 6 DEAE柱層析穩定性的檢測采用優化的DEAE柱層析工藝純化3 批傷寒Vi 多糖粗糖(STVi 2011-1207STVi 20120101 STVi 20120102),并對純化產物進行相關項目的檢測,計算標準差(SD)和變異系數(CV),驗證該工藝的穩定性。
          2 結果
          2. 1 DEAE 柱層析純化檢測結果可見,上樣緩沖液為pH 6. 0 20 mmol/L Tris-Cl 時,目的產物流穿,但核酸和雜蛋白未結合在層析柱上,隨穿透峰一同穿出;上樣緩沖液為pH 7. 0 20 mmol/LTris-Cl 時,目的產物流穿,核酸和雜蛋白結合于層析柱上,并隨緩沖液B 梯度的增加逐漸被洗脫下來;上樣緩沖液為pH 8. 0 20 mmol/L Tris-Cl 時,純化效果與pH 7. 0條件下相似,見圖1
          2. 2 純化樣品的鑒定檢測結果表明,DEAE 柱層析純化緩沖液pH 值為6. 0 時無法有效去除傷寒Vi多糖中的雜蛋白及核酸,其純化產物的蛋白質含量是pH 7. 0 pH 8. 0 條件下的17. 86 15. 37 倍,核酸含量是pH 7. 0 pH 8. 0 條件下的5. 25 6. 06倍,內毒素含量遠低于傳統工藝,僅為傳統工藝的5%。見表1。傳統工藝純化多糖回收率為15%,而DEAE 柱層析工藝(pH 7. 0 pH 8. 0)純化多糖回收率分別為36. 52%和38. 35%,見表2。由于傷寒Vi 多糖中含O-乙酰基,在堿性條件下O-乙酰基易脫落,因此,本實驗選擇pH 7. 0 為層析純化的最佳條件。
          2. 3 DEAE 柱層析的穩定性檢測結果表明,以優化工藝純化3 批傷寒Vi 多糖粗糖獲得的6 批精糖的蛋白質含量、核酸含量、O-乙酰基含量、分子大小、內毒素含量及多糖回收率差異較小,標準偏差均小于5%。表明該方法具有良好的穩定性。見表3 和表4
           
           
          3 討論
          通常在離子交換層析中,pH、離子強度等條件是純化效果的關鍵因素,本實驗采用DEAE 柱層析純化多糖是使純化產物經層析柱后流穿,雜質結合于層析柱上,因此,離子強度對該純化方法無重要影響。本實驗重點是對層析柱的pH 值進行了優化。
          實驗結果表明,在緩沖液(20 mmol/L Tris-ClpH 6. 0 條件下未能將目的產物與雜質分離,在pH 7. 08. 0 條件下多糖流穿,大部分核酸及蛋白等雜質結合于層析柱上,并隨緩沖液B 梯度的增加而被逐步洗脫下來,由于傷寒Vi 多糖中含O-乙酰基,在堿性條件下O-乙酰基易脫落,因此,選擇pH 7. 0 作為傷寒Vi 多糖的最佳柱層析純化條件。
          為避免疫苗生產過程中苯酚等有毒化學試劑及高濃度乙醇、甲醇等易燃易爆的有機溶劑對疫苗質量的影響6-7,采用現代純化技術對傳統的生產工藝進行改進,減少或棄用有毒化學試劑,以提高疫苗產品質量。Pato 8探討了流腦C 群莢膜多糖的柱層析純化工藝,國內也報道層析法對流腦A 群莢膜多糖去雜蛋白的有效性9
          傷寒Vi 多糖疫苗的有效抗原部分為莢膜多糖,通常提取莢膜多糖的方法是以Gotschlich 法為基礎10,傳統傷寒Vi 多糖的提取工藝多采用苯酚抽提、乙醇分級沉淀等方法5-6,11,但苯酚為有毒化學試劑,為該疫苗的使用帶來一定的安全隱患,因此,本實驗通過采用DEAE 離子交換層析,確定了緩沖液20 mmol/LTris-Cl pH 7. 0 DEAE 層析柱工藝的最佳條件,中性pH 值確保了多糖結構的穩定性,提高了純化產物的質量指標,且明顯降低了純化產物的內毒素含量。
          本實驗采用優化的DEAE 柱層析工藝對3 批粗糖進行了純化,實驗結果顯示,所得到的6 批精糖質量均符合規定,蛋白質含量、核酸含量、O-乙酰基含量、分子大小、內毒素含量及多糖回收率差異較小,標準偏差均小于5%,表明該工藝具有良好的穩定性,易于工藝放大,提高了疫苗的安全性及產品的質量標準,可替代傳統苯酚抽提、乙醇分級沉淀法,應用于傷寒Vi 多糖的純化。
           
           
          參考文獻
          [1] 王江,劉慶云,謝崢,等. 傷寒疫苗的研究進展[J]. 微生物學免疫學進展,2010,38(2):70-74.
          [2] WHO. Typhoid vaccines:WHO position paper[J]. Wkly Epidemiol Rec,2008,83(6):49-59.
          [3] Khan MI,Ochiai RL,Clemens JD. Population impact of Vi capsular polysaccharide vaccine[J]. Expert Rev Vaccines,2010,9(5):485-496.
          [4] Schwartz E. A cluster-randomized effectiveness trial of Vi typhoid vaccine in India[J]. N Engl J Med,2009,361(22):2191-2192.
          [5] 國家藥典委員會. 中華人民共和國藥典(三部)[S]. 北京:中國醫藥科技出版社,2010:38-40
          [6] Kothari S,Kothari N,Lee E,et al. Development of an efficient and scalable method for processing and purification of Vicapsular polysaccharide[J]. Procedia Vaccinol,2010,2(1):78-81.
          [7] Hamidi A,Beurret MF. Process for producing a capsular polysaccharide for use in conjugate vaccines[P]:USA,US Patent Application 20070065460,2007-03-22.
          [8] Pato TP,Barbosa Ade P,Da Silva Junior JG. Purification of capsular polysaccharide from Neisseria meningitidis serogroup Cby liquid chromatography [J]. J Chromatogr B Analyt Technol Biomed Life Sci,2006,832(2):262-267.
          [9] 解紅艷,王濟源,劉靜,等. 層析法去除A 群流腦多糖疫苗粗糖中雜蛋白[J]. 中國生物工程雜志,2010,30(8):88-92.
          [10] Gotschlich EC,Liu TY,Artenstein MS. Human immunity to the meningococcus III. Preparation and immunochemical properties of the group A,group B,and group C meningococcal polysaccharides[J]. J Exp Med,1969,129(6):1349-1365.
          [11] 朱為,榮家康,江元翔,等. A 群和C 群腦膜炎球菌莢膜多糖提制工藝研究[J]. 藥物生物技術,2011,18(1):56-60.
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