9V型肺炎球菌多糖蛋白结合物原液中游离多糖含量检测方法的建立
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摘要
目的建立测定9V型肺炎球菌多糖蛋白结合物原液中游离多糖含量的检测方法。方法对9V型肺炎球菌多糖溶液进行不同浓度的脱氧胆酸钠(NaDOC)酸沉处理,选择仅沉淀结合物而不沉淀多糖的最佳处理条件,筛选出所能沉淀蛋白的最大浓度;对建立的方法进行准确度、精密度、专属性及线性验证。结果 9V型肺炎球菌多糖蛋白结合物原液的最佳NaDOC酸沉处理条件为2%NaDOC-0. 05 mol/L HCl;蛋白浓度低于200μg/m L时,NaDOC酸沉处理条件沉淀蛋白的效果最佳。准确度试验的回收率在90%～99%之间;实验内及实验间均具有良好的精密度(CV均小于10%);在10～100μg/mL范围内采用蒽酮硫酸法测定糖浓度的曲线,线性良好,r~2> 0. 998;NaDOC的终浓度在0. 25%以下,对蒽酮硫酸法检测无干扰。结论 NaDOC酸沉淀法能很好地分离结合物原液中的结合物与游离多糖,具有良好的重复性和准确性,可用于测定9V型肺炎球菌多糖蛋白结合物原液中的游离多糖含量。
Objective To develop a method for determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate. Methods Type 9 V pneumococcal polysaccharide solution was treated with sodium deoxycholate and hydrochloric acid,at various concentrations. The condition for treatment was optimized,under which the conjugate was completely precipitated while the polysaccharide was not precipitated. On the basis of this,the maximum protein concentration for precipitation was screened. The developed method was verified for accuracy,precision,specificity and linearity. Results The conjugate and protein in type 9 V pneumococcal polysaccharide conjugate was completely precipitated with 2% sodium deoxycholate-0. 05 mol/L bulk of hydrochloric acid at a protein concentration of less than 200 μg/m L. The recovery rate of polysaccharide in accuracy test was 90% ～ 99%. The developed method showed high precision in both intra-and inter-assays,with CVs of less than 10%. The curve for determination of polysaccharide concentration by anthrone-sulfuric method showed good linearity within a concentration range of 10 ～100 μg/mL,with an r~2 value of more than 0. 998. The sodium deoxycholate at a final concentration of less than 0. 25%showed no interference to anthrone-sulfuric acid method. Conclusion The conjugate and free polysaccharide in bulk of type 9 V pneumococcal polysaccharide conjugate was separated effectively by precipitation with sodium deoxycholate and hydrochloric acid. The developed method showed high reproducibility and accuracy,which might be used for quantitative determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate.
Objective To develop a method for determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate. Methods Type 9 V pneumococcal polysaccharide solution was treated with sodium deoxycholate and hydrochloric acid,at various concentrations. The condition for treatment was optimized,under which the conjugate was completely precipitated while the polysaccharide was not precipitated. On the basis of this,the maximum protein concentration for precipitation was screened. The developed method was verified for accuracy,precision,specificity and linearity. Results The conjugate and protein in type 9 V pneumococcal polysaccharide conjugate was completely precipitated with 2% sodium deoxycholate-0. 05 mol/L bulk of hydrochloric acid at a protein concentration of less than 200 μg/m L. The recovery rate of polysaccharide in accuracy test was 90% ～ 99%. The developed method showed high precision in both intra-and inter-assays,with CVs of less than 10%. The curve for determination of polysaccharide concentration by anthrone-sulfuric method showed good linearity within a concentration range of 10 ～100 μg/mL,with an r~2 value of more than 0. 998. The sodium deoxycholate at a final concentration of less than 0. 25%showed no interference to anthrone-sulfuric acid method. Conclusion The conjugate and free polysaccharide in bulk of type 9 V pneumococcal polysaccharide conjugate was separated effectively by precipitation with sodium deoxycholate and hydrochloric acid. The developed method showed high reproducibility and accuracy,which might be used for quantitative determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate.
引文
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[12] YI S S,DENG H Q,GAO Y J,et al. Method development for determination of free polysaccharide content in Streptococcus pneumonia type 19A polysaccharide conjugate[J]. Int J Biologicals,2017,40(4):157-160.(in Chinese)伊姗姗,邓海清,高宇皎,等. 19A型肺炎链球菌多糖结合物中游离多糖含量测定方法的建立[J].国际生物制品学杂志,2017,40(4):157-160.
[13] MIAO X,YU X B,FAN F F,et al. Development and verification of a method determination of free polysaccharide content in Haemophilus influenzae type a conjugate[J]. Chin J Biologicals,2016,29(6):623-627,631.(in Chinese)苗鑫,于旭博,范锋锋,等. a型流感嗜血杆菌结合物游离多糖含量测定方法的建立及验证[J].中国生物制品学杂志,2016,29(6):623-627,631.
[14] LIU A P,REN Z Y,LIU Q,et al. Development of a determination method for free polysaccharide content in serotype 6B pneumococcal polysaccharide conjugate[J]. Chin J New Drugs,2017,26(24):2937-2941.(in Chinese)刘爱萍,任珍芸,刘倩,等. 6B型肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖测定方法的建立[J].中国新药杂志,2017,26(24):2937-2941.
[2]张惟杰.复合多糖生化研究技术[M].上海:上海科学技术出版社,1987:6-7.
[3] Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Repubic of China(VolⅣ)[S]. Beijing:China Med Sci Press,2015:96-97.(in Chinese)国家药典委员会.中华人民共和国药典(四部)[M].北京:中国医药科技出版社,2015:96-97.
[4] Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Repubic of China(VolⅢ)[S]. Beijing:China Med Sci Press,2015:153-155.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:153-155.
[5] Centers for Disease Control and Prevention(CDC). Progress in introduction of pneumococcal conjugate vaccine-worldwide,2000-2008[J]. MMWR Morb Mortal Wkly Rep,2008,57(42):1148-1151.
[6] WEIL OLIVIER C. Ten years of experience with the pneumococcal conjugate 7-valent vaccine in children[J]. Med Mal Infect,2013,43(8):309-321.
[7] VAN DER LINDEN M,FALKENHORST G,PERNICIARO S,et al. Effectiveness of pneumococcal conjugate vaccines(PCV7and PCV13)against invasive pneumococcal disease among children under two years of age in Germany[J]. PLoS One,2016,11(8):e0161257. doi:10.1371/journal.pone.0161257.
[8] SHEN J,LIANG F,FANG M L,et al. Establishment of a sodium deoxycholate precipitation method for determination of the free polysaccharide in Haemophilus influenza type b conjugate vaccine[J]. Int J Biologicals,2014,37(5):223-226.(in Chinese)沈坚,梁芳,方曼莉,等.测定b型流感嗜血杆菌结合疫苗游离多糖的脱氧胆酸钠沉淀法的建立[J].国际生物制品学杂志,2014,37(5):223-226.
[9] GUO Y Y,ANDERSON R,MCLVER J,et al. A simple and rapid method for measuring unconjugated capsular polysaccharide(PRP)of Haemophilus influenza type b in PRP-tetanus toxoid conjugate vaccine[J]. Biologicals,1998,26(1):33-38.
[10] LEI Q P,SHANNON A G,HELLER R K,et al. Quantification of free polysaccharide in meningococcal polysaccharidediphtheria toxoid conjugate vaccines[J]. Dev Biol(Basel),2000,103:259-264.
[11] WANG J,YU X B,LIU F L,et al. Development and validation of a determination method for free polysaccharide content in bulk of group A meningococcal polysaccharide conjugate[J].Chin J Biologicals,2014,27(10):1295-1298,1303.(in Chinese)王晶,于旭博,刘方蕾,等. A群脑膜炎球菌多糖结合物原液中游离糖含量测定方法的建立及其验证[J].中国生物制品学杂志,2014,27(10):1295-1298,1303.
[12] YI S S,DENG H Q,GAO Y J,et al. Method development for determination of free polysaccharide content in Streptococcus pneumonia type 19A polysaccharide conjugate[J]. Int J Biologicals,2017,40(4):157-160.(in Chinese)伊姗姗,邓海清,高宇皎,等. 19A型肺炎链球菌多糖结合物中游离多糖含量测定方法的建立[J].国际生物制品学杂志,2017,40(4):157-160.
[13] MIAO X,YU X B,FAN F F,et al. Development and verification of a method determination of free polysaccharide content in Haemophilus influenzae type a conjugate[J]. Chin J Biologicals,2016,29(6):623-627,631.(in Chinese)苗鑫,于旭博,范锋锋,等. a型流感嗜血杆菌结合物游离多糖含量测定方法的建立及验证[J].中国生物制品学杂志,2016,29(6):623-627,631.
[14] LIU A P,REN Z Y,LIU Q,et al. Development of a determination method for free polysaccharide content in serotype 6B pneumococcal polysaccharide conjugate[J]. Chin J New Drugs,2017,26(24):2937-2941.(in Chinese)刘爱萍,任珍芸,刘倩,等. 6B型肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖测定方法的建立[J].中国新药杂志,2017,26(24):2937-2941.