首页|生物制剂工艺特异性残留宿主蛋白ELISA检测方法的建立及验证

生物制剂工艺特异性残留宿主蛋白ELISA检测方法的建立及验证

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目的 建立基因重组生物制剂中工艺特异性大肠埃希菌残留蛋白双抗体夹心ELISA检测方法,并进行验证.方法 以大肠埃希菌表达胰高血糖素样肽(glucagon-like peptide,GLP)的生产与纯化工艺为特定工艺模型,采用同一工艺用于空载大肠埃希菌(不表达重组蛋白的正常大肠埃希菌)残留蛋白的截取.将获得的残留蛋白作为免疫原分别免疫1只雌性新西兰白兔和6只雌性昆明小鼠,以兔免疫血清纯化IgG抗体为包被抗体,鼠免疫血清为第二夹心抗体,抗鼠IgG-HRP为酶标二抗,建立工艺特异性大肠埃希菌残留蛋白双抗体夹心ELISA方法.对建立的方法进行特异性、准确性、精密性验证,并确定方法的检测限.同时采用建立的方法和市售大肠埃希菌宿主蛋白残留检测试剂盒对纯化的GLP制剂进行残留蛋白定量测定.结果 对已知浓度的工艺特异性残留蛋白进行系列梯度稀释后,建立的ELISA方法检测的灵敏度可达338 pg/mL;该方法检测CHO和酵母细胞裂解蛋白无交叉反应,检测低、中、高3个浓度样品的回收率均在80%~120%范围内,检测低、中、高浓度空载大肠埃希菌截留液标准品的试验内和试验间CV均<15%.针对GLP制剂中的残留蛋白,与建立的方法检出的GLP制剂中残留蛋白总量相比,约有62%的残留蛋白未被市售非工艺特异性ELISA试剂盒检出,这些残留蛋白应为本工艺特异的残留蛋白.结论 建立的双抗体夹心ELISA法检测工艺特异性大肠埃希菌残留蛋白灵敏度高,特异性强,准确性和精密性良好,可满足残留蛋白在生物制剂中低于0.01%~0.1%标准的检测要求.
Development and verification of an ELISA method for detection of process-specific residual host protein in biological preparation
Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein).One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen.Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and anti-mouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established.The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined.Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL.No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80%—120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than 15%.For the residual protein in GLP preparation,about 62%of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than 0.01%—0.1%in biological preparations.

Process specificityEscherichia coli(E.coli)Host cell protein(HCP)Enzyme-linked immunosorbent assay(ELISA)

田易晓、王新月、窦向雅、韩翠翠、高珂、邵东燕、段苏杨、白岳丘、赵雯、黄庆生

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西北工业大学生命学院空间生物科学与生物技术重点实验室,陕西西安 710072

工艺特异性 大肠埃希菌 宿主蛋白 酶联免疫吸附测定

国家重点研发计划陕西省重点研发计划

2018YFF010121042021ZDLSF01-06

2024

中国生物制品学杂志
中华预防医学会,长春生物制品研究所有限责任公司

中国生物制品学杂志

CSTPCD
影响因子:0.417
ISSN:1004-5503
年,卷(期):2024.37(2)
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