测定重组肠道病毒71型疫苗(汉逊酵母)原液纯度的高效分子排阻色谱法的建立及方法的验证
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摘要
目的建立检测重组肠道病毒71型(enterovirus 71,EV71)(汉逊酵母)病毒样颗粒(virus-like particle,VLP)纯度的高效分子排阻色谱法(SEC-HPLC),并进行验证。方法建立SEC-HPLC法检测EV71 VLP纯度,并对该方法的专属性、精密度、检测限、定量限、耐用性及拖尾因子等指标进行验证。结果空白溶剂在设定的积分范围内无特异峰出现。重复性试验中原液参比品保留时间和峰面积相对标准偏差(RSD)分别为0. 14%和0. 21%,3批分析原液保留时间RSD均<0. 3%,峰面积RSD均<0. 8%;中间精密度试验原液参比品和3批分析原液保留时间RSD均<0. 2%;峰面积RSD均<2. 0%。检测限为样品浓度2. 5μg/mL,进样体积20μL;定量限为样品浓度10μg/mL,进样体积20μL。耐用性试验中原液参比品在不同浓度流动相中连续进样3次,保留时间和峰面积RSD均<2. 0%。原液参比品和3批分析原液目的峰的拖尾因子为1. 210±0. 01,RSD为0. 90%。结论建立的SEC-HPLC方法专属性、精密度、耐用性良好,适用于EV71(汉逊酵母)VLP纯度的检测。
Objective To develop and validate a size exclusion column-high performance liquid chromatography(SECHPLC)for determination of purity of virus-like particles(VLPs)of recombinant enterovirus type 71(EV71). Methods The purity of EV71 VLPs was determined by SEC-HPLC,and the method was validated for specificity,precision,detection limit,quantitative limit,durability and tailing factor. Results No specific peaks appeared within the range set points of blank solvent. The RSDs of retention time and peak area of bulk reference in reproducibility test were 0. 14% and 0. 21%respectively,while those of retention time of three batches of bulk were less than 0. 3%,and those of peak area were less than 0. 8%. However,the detection limits of sample concentration and volume were 2. 5 μg/mL and 20 μL,while the quantitative limits were 10 μg/mL and 20 μL,respectively. In durability test,the bulk reference was loaded to the mobile phase at different concentrations for 3 times,of which the RSDs of retention time and peak area were less than 2. 0%.The tailing factors of bulk reference and three batches of bulk were 1. 210 ± 0. 01,with a RSD of 0. 90%. Conclusion The developed SEC-HPLC method showed high specificity,precision and durability,which was suitable for determination of purity of recombinant EV71(Hansenula)VLPs.
Objective To develop and validate a size exclusion column-high performance liquid chromatography(SECHPLC)for determination of purity of virus-like particles(VLPs)of recombinant enterovirus type 71(EV71). Methods The purity of EV71 VLPs was determined by SEC-HPLC,and the method was validated for specificity,precision,detection limit,quantitative limit,durability and tailing factor. Results No specific peaks appeared within the range set points of blank solvent. The RSDs of retention time and peak area of bulk reference in reproducibility test were 0. 14% and 0. 21%respectively,while those of retention time of three batches of bulk were less than 0. 3%,and those of peak area were less than 0. 8%. However,the detection limits of sample concentration and volume were 2. 5 μg/mL and 20 μL,while the quantitative limits were 10 μg/mL and 20 μL,respectively. In durability test,the bulk reference was loaded to the mobile phase at different concentrations for 3 times,of which the RSDs of retention time and peak area were less than 2. 0%.The tailing factors of bulk reference and three batches of bulk were 1. 210 ± 0. 01,with a RSD of 0. 90%. Conclusion The developed SEC-HPLC method showed high specificity,precision and durability,which was suitable for determination of purity of recombinant EV71(Hansenula)VLPs.
引文
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[7]李国顺,郭林,方玉松,等.肠道病毒71型类病毒颗粒在汉逊酵母中的表达及其免疫原性[J].中国生物制品学杂志,2015,28(9):906-911.LI G S,GUO L,FANG Y S,et al. Expression of virus-like par-t icles of enterovirus 71 in Hansenula polymorpha[J]. Chin J Biologicals,2015,28(9):906-911.
[8] State Pharmacopoeia Committee. European pharmacopoeia 6. 0[S]. EDQM,2150-2153.
[9] EFFIO C L,OELMEIER S A,HUBBUCH J. High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography[J]. Vaccine,2016,34(10):1259-1267.
[10] YANG Y,LI H,LI Z,et al. Size-exclusion HPLC provides a simple,rapid,and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens[J].Vaccine,2015,33(9):1143-1150.
[2] ZHANG C,KU Z,LIU Q,et al. High-yield production of recombinant virus-like particles of enterovirus 71 in Pichia pastoris and their protective efficacy against oral viral challenge in mice[J]. Vaccine,2015,33(20):2335-2341.
[3] WANG J Z. The research,development and quality control of the biotechnological pharmacy[M]. Beijing:Science Press,2002:99.(in Chinese)王军志.生物技术药物研究开发和质量控制[M].北京:科学出版社,2002:99.
[4] RIBARSKA J,JOLEVSKA S,PANOVSKA A,et al. Studying the formation of aggregates in recombinant human granulocytecolony stimulating factor(rHuG-CSF),lenograstim,using sizeexclusion chromatography and SDS-PAGE[J]. Acta Pharm,2008,58(2):199-206.
[5] Chinese Pharmacopoeia Commission. Pharmacopoeia of People’s Repubic of China(VolⅣ)[S]. Beijing:China Med Sci Press,2015:59-61.(in Chinese)国家药典委员会.中华人民共和国药典(四部)[S].北京:中国医药科技出版社,2015:59-61.
[6] Chinese Pharmacopoeia Commission. Pharmacopoeia of People’s Repubic of China(VolⅣ)[S]. Beijing:China Med Sci Press,2015:374-377.(in Chinese)国家药典委员会.中华人民共和国药典(四部)[S].北京:中国医药科技出版社,2015:374-377.
[7]李国顺,郭林,方玉松,等.肠道病毒71型类病毒颗粒在汉逊酵母中的表达及其免疫原性[J].中国生物制品学杂志,2015,28(9):906-911.LI G S,GUO L,FANG Y S,et al. Expression of virus-like par-t icles of enterovirus 71 in Hansenula polymorpha[J]. Chin J Biologicals,2015,28(9):906-911.
[8] State Pharmacopoeia Committee. European pharmacopoeia 6. 0[S]. EDQM,2150-2153.
[9] EFFIO C L,OELMEIER S A,HUBBUCH J. High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography[J]. Vaccine,2016,34(10):1259-1267.
[10] YANG Y,LI H,LI Z,et al. Size-exclusion HPLC provides a simple,rapid,and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens[J].Vaccine,2015,33(9):1143-1150.