QPCR法与DNA探针杂交法检测重组人生长激素原液中残余DNA的比较
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摘要
目的对重组人生长激素(recombinant human growth hormone,rhGH)原液中残余DNA检测的实时荧光定量PCR(以下简称QPCR)法和DNA探针杂交法进行对比。方法提取3批rhGH原液中残余DNA,经SYBR GREENⅠ荧光染料法对残余DNA进行QPCR检测,对建立的方法进行准确度、精密度及引物特异性验证,并与《中国药典》三部(2015版)中规定的DNA探针杂交法检测的残余DNA含量进行比较。结果 QPCR法对3批rhGH原液残余DNA含量均可定量,灵敏度可达7. 5 fg。标准品DNA浓度在7. 5 fg/μL～750 pg/μL范围内线性关系良好,R2为0. 998,加标回收率为78. 40%～106. 45%。两种方法检测的3批rhGH原液残余DNA含量均小于《中国药典》三部(2015版)中规定的10 ng/剂量的要求。结论 QPCR法高效快捷,通过DNA与染料结合可实时监控PCR过程中的每一步反应,减小污染,对样品中残余DNA可进行准确定量,比DNA探针杂交法更适用于样品中残余DNA含量的检测。
Objective To compare the real-time fluorescent quantitative PCR(QPCR) and DNA hybridization in determination of residual DNA in bulk of recombinant human growth hormone(rhGH). Methods The residual DNAs in three batches of bulk of rhGH were extracted and determined by QPCR using SYBR GREEN I fluorescent dye. The developed method was verified for accuracy,precision and primer specificity,by which the determined residual DNA content was compared with that by DNA probe hybridization method in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results QPCR could be used to quantify the residual DNA in bulk of rh GH,of which the sensitivity was7. 5 fg. The linear range of standard DNA concentration was 7. 5 fg/μL ～ 750 pg/μL,with a R2 value of 0. 998,while the spike recovery was 78. 40% ～ 106. 45%. The residual DNA contents in three batches of bulk of rhGH determined by the two methods were less than 10 ng/dose specified in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion QPCR method is highly effective and rapid,of which each step may be performed under real-time monitoring by DNA in combination with dye so as to reduce pollution and accurately quantify the residual DNA in samples. Therefore,QPCR is more suitable for the determination of residual DNA in samples than digoxin hybridization.
Objective To compare the real-time fluorescent quantitative PCR(QPCR) and DNA hybridization in determination of residual DNA in bulk of recombinant human growth hormone(rhGH). Methods The residual DNAs in three batches of bulk of rhGH were extracted and determined by QPCR using SYBR GREEN I fluorescent dye. The developed method was verified for accuracy,precision and primer specificity,by which the determined residual DNA content was compared with that by DNA probe hybridization method in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results QPCR could be used to quantify the residual DNA in bulk of rh GH,of which the sensitivity was7. 5 fg. The linear range of standard DNA concentration was 7. 5 fg/μL ～ 750 pg/μL,with a R2 value of 0. 998,while the spike recovery was 78. 40% ～ 106. 45%. The residual DNA contents in three batches of bulk of rhGH determined by the two methods were less than 10 ng/dose specified in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion QPCR method is highly effective and rapid,of which each step may be performed under real-time monitoring by DNA in combination with dye so as to reduce pollution and accurately quantify the residual DNA in samples. Therefore,QPCR is more suitable for the determination of residual DNA in samples than digoxin hybridization.
引文
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[10] WHO Expert Committee on biological standardization. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks[S]. Geneva:World Health Organization,2010.
[11] SU Z,ZHOU C D,HUANG Z S,et al. Determination of residual host cell DNA in recombinant human interferonα2b substances by quantitative PCR[J]. Chin J Biochem Pharm,2016,36(4):193-195.(in Chinese)苏喆,周朝东,黄哲甦,等.荧光定量PCR法测定重组人干扰素α2b原液中宿主DNA的残留[J].中国生化药物杂志,2016,36(4):193-195.
[12] WANG L,LI M,GUO W,et al. Determination of residual host cell DNA in monoclonal antibody products by magnetic bead based extraction combined with quantitative PCR[J].Chin J Biologicals,2014,27(4):555-558.(in Chinese)王兰,李萌,郭玮,等.磁珠分离结合定量PCR法检测单克隆抗体产品中CHO细胞DNA残留量[J].中国生物制品学杂志,2014,27(4):555-558.
[13] WANG L,GAO K,BI H,et al. Comparison of fliuorescence and DNA hybridization methods for the determination of residual DNA in recombinant products[J]. Chin J Pharm Anal,2009,29(7):1063-1066.(in Chinese)王兰,高凯,毕华,等.荧光法和DNA杂交法检测重组技术产品中残余DNA的比较[J].药物分析杂志,2009,29(7):1063-1066.
[14] WHO. WHO Study Group on cell substrates for production of biologicals[R]. WHO meeting report,Geneva,2007-06-11
[2] CHEN B,ZHU W. Advances on human growth hormone[J]. J Biol,2004,21(1):9-11.(in Chinese)陈蓓,朱威.人生长激素研究进展[J].生物学杂志,2004,21(1):9-11.
[3] LIU J H,LIU Y L,LIU C,et al. Separation,purification and structural confirmation of recombinant human growth hormone[J].Chin J Biologicals,2015,28(11):1153-1156.(in Chinese)刘景会,刘玉林,刘晨,等.重组人生长激素的分离纯化及其结构确证[J].中国生物制品学杂志,2015,28(11):1153-1156.
[4] ISASSKSON O G P,LINDAHL A,NILSSON A,et al. Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth[J]. Endocr Rev,1987,8(4):426-438.
[5]朱兰香.儿童生长激素缺乏症临床综合分析[J].医学论坛杂志,2008,29(19):46-47.
[6] PARYAN M,KHODAYAR M,KIA V,et al. Development of an in-house TaqMan real-time PCR-based method to detect residual host cell DNA in HBV vaccine[J]. Appl Biochem Biotechnol,2016,179(3):375-382.
[7] WANG L,WANG J Z. Issue on quality control of residual DNA in biological products[J]. Chin J N Drugs,2011,20(8):678-683.(in Chinese)王兰,王军志.关于生物制品残余DNA质量控制问题[J].中国新药杂志,2011,20(8):678-683.
[8] DOEHMER J. Residual cellular DNA as a potential transforming factor[J]. Dev Biol Stand,1987,68(1):33-41.
[9] Chinese Pharmacopoeia Commission. Pharmacopoeia of Peoples Republic of China(VolumeⅡ)[S]. Beijing:Chinese Med Sci Press,2015:788.(in Chinese)国家药典委员会.中华人民共和国药典(二部)[S].北京:中国医药科技出版社,2015:788.
[10] WHO Expert Committee on biological standardization. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks[S]. Geneva:World Health Organization,2010.
[11] SU Z,ZHOU C D,HUANG Z S,et al. Determination of residual host cell DNA in recombinant human interferonα2b substances by quantitative PCR[J]. Chin J Biochem Pharm,2016,36(4):193-195.(in Chinese)苏喆,周朝东,黄哲甦,等.荧光定量PCR法测定重组人干扰素α2b原液中宿主DNA的残留[J].中国生化药物杂志,2016,36(4):193-195.
[12] WANG L,LI M,GUO W,et al. Determination of residual host cell DNA in monoclonal antibody products by magnetic bead based extraction combined with quantitative PCR[J].Chin J Biologicals,2014,27(4):555-558.(in Chinese)王兰,李萌,郭玮,等.磁珠分离结合定量PCR法检测单克隆抗体产品中CHO细胞DNA残留量[J].中国生物制品学杂志,2014,27(4):555-558.
[13] WANG L,GAO K,BI H,et al. Comparison of fliuorescence and DNA hybridization methods for the determination of residual DNA in recombinant products[J]. Chin J Pharm Anal,2009,29(7):1063-1066.(in Chinese)王兰,高凯,毕华,等.荧光法和DNA杂交法检测重组技术产品中残余DNA的比较[J].药物分析杂志,2009,29(7):1063-1066.
[14] WHO. WHO Study Group on cell substrates for production of biologicals[R]. WHO meeting report,Geneva,2007-06-11
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