摘要
目的验证重组蛋白制品中大肠埃希菌宿主蛋白(host cell protein,HCP)残留检测试剂盒的适用性。方法对淄博云桥生物技术有限公司制备的大肠埃希菌HCP的ELISA检测试剂盒进行特异性、灵敏度、精密性、准确性验证,同时进行基质干扰试验和稀释回收试验。采用该试剂盒及一款市售进口试剂盒分别对本公司制备的3批重组碱性成纤维细胞生长因子(recombinant basic fibroblast growth factor,rbFGF)制品原液及纯化工艺中间样品的HCP残留量进行检测。结果试剂盒可特异性检测大肠埃希菌HCP残留量;线性范围为3.33~810 ng/ml;检测限和定量限均为3.33 ng/ml;试验内变异系数(CV)和试验间CV均<16%;低、中、高3个浓度样品的回收率分别为113.1%、113.7%和97.5%;原液样品基质对测定基本无影响,原液基质中DTT含量<5 mmol/L对测定结果的影响在可接受范围内;原液样品在测定HCP含量时存在干扰,稀释3倍时,干扰最小;该试剂盒与另一款市售试剂盒检测3批rbFGF制品原液及纯化工艺中间样品的HCP残留量基本一致。结论该试剂盒具有良好的特异性、灵敏度、准确性、精密度和线性,可用于本公司重组蛋白制品中大肠埃希菌HCP残留的检测。
Objective To verify the applicability of detection kit for residual E. coli host cell protein（HCP）in recombinant protein products. Methods The ELISA kit for E. coli HCP（Array Bridge Inc） was verified for specificity,sensitivity,precision and accuracy,and subjected to matrix interference test and sample dilution and recovery test. The residual E.coli HCPs in three batches of bulk and intermediates in purification process of recombinant basic fibroblast growth factor（rbFGF） manufactured by Zhuhai ESSEX Bio-Pharmaceutical Company Limited were determined by the kit,using an imported kit as control. Results The residual E. coli HCP content was determined by the kit specifically. The linear range of the kit was 3. 33 ~ 810 ng/ml,while both the detection and quantitative limits were 3. 33 ng/ml. Both the CVs in intra-and inter-assays were less than 16%,while the recovery rates of samples at low,moderate and high concentrations were 113. 1%,113. 7% and 97. 5% respectively. The matrix of bulk showed no effect on the determination of HCPs,while the effect of matrix containing not more than 5 mmol/L DTT was within the acceptable range. However,the bulk samples showed interference to the determination of HCP content,while those diluted by 3 folds showed little interference. The determination results of three batches of bulks and intermediates of rbFGF by the kit were basically consistent with that by the imported kit. Conclusion The kit showed high specificity,sensitivity,accuracy and precision,which might be used for the determination of residual E. coli HCP in recombinant protein products.
作者
邓春平
杨波
庄兰芳
李梦瑶
郑赞顺
曲伟
DENG Chun-ping YANG Bo ZHUANG Lan-fang LI Meng-yao ZHENG Zan-shun QU Wei(Zhuhai ESSEX Bio-Pharmaceutical Company Limited, Zhuhai 519085, Guangdong Province, China)
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第1期49-52,58,共5页
Chinese Journal of Biologicals
基金
珠海市战略性新兴产业重大项目(2012D0201990037)
