定量PCR法和DNA杂交法检测聚乙二醇修饰尿酸酶原液中DNA残留量的比较

Comparison of Quantitative PCR and DNA Hybridization for Detection of Residual DNA in PEGylated Uricase Stock Solution

关键词:  DNA残留量  定量PCR法  探针杂交法  PEG修饰尿酸酶

Key words:  residual DNA  quantitative PCR  DNA hybridization  PEGylated uricase

摘要:

目的 建立快速准确的聚乙二醇修饰重组尿酸酶(PEGylated uricase，PEG-UHC)原液DNA残留量测定法，为其他多位点PEG修饰蛋白药物的DNA残留量检测提供参考。方法 参照中国药典2020年版三部“外源性DNA残留量检测法”第一法DNA探针杂交法和第三法定量PCR法，检测PEG-UHC原液DNA残留量；对定量PCR法进行检测限度、线性范围、准确度和精密度等方法学验证；进行3批次PEG-UHC原液DNA残留量测定。结果 探针杂交法DNA残留量测定线性范围为1×10^-4~0.1 ng·μL^-1，3批次PEG-UHC原液未见明显显色，DNA残留量均低于每剂10 ng。PCR法检测限度为1×10^-5 ng·μL^-1，DNA浓度在1×10^-4~100 ng·μL^-1内线性关系良好(R²=0.999)。不同浓度的标准DNA在PEG-UHC原液中的回收率范围为92.57%~114.17%，表明PEG偶联物对定量PCR影响较小。3批次PEG-UHC原液中DNA残留量分别为每剂0.143，0.187，0.154 ng，符合国家药品监督管理局限量要求。结论 DNA探针杂交法为传统定性检测，耗时较长，准确度较低。定量PCR法操作简便快速、灵敏度高，且可定量分析，适于PEG-UHC等多位点PEG修饰蛋白药物DNA残留量测定。

Abstract:

OBJECTIVE To develop fast and accurate method for determining the residual DNA in PEGylated uricase (PEG-UHC) stock solution, and provide reference values for residual DNA quantification in other multiple-sites PEGylated protein drugs. METHODS The Quantitative PCR and DNA hybridization methods were used to determine the residual DNA based on "Exogenous residual DNA testing methods" in Chinese Pharmacopoeia 2020 edition Vol III. The detection limit, linearity, range, accuracy and precision of the quantitative PCR method were further evaluated. The residual DNA in three batches of PEG-UHC stock solution were determined by the above two methods. RESULTS The concentration ranges of DNA hybridization assay were 1×10^-4-0.1 ng·μL^-1, and there were no coloration upon the addition of PEG-UHC stock solution, indicating the quantities of residual DNA were below 10 ng per does in the three batches. The developed quantitative PCR had high detection limit up to 1×10^-5 ng·μL^-1 of DNA with good linearity(R²=0.999) in the concentration range of 1×10^-4-100 ng·μL^-1. The recoveries by adding different concentrations of standard DNA to PEG-UHC solution were 92.57%-114.17%, indicating no interference with PEG-UHC. The residual DNA of three batches of PEG-UHC stock solution was 0.143, 0.187, 0.154 ng per dose, respectively. All the contents met the National Medical Products Adiministration requirements for residual DNA. CONCLUSION The DNA hybridization assay is traditional qualitative methods, suffering from drawbacks of long time-consuming and low accuracy. The quantitative PCR assay has advantages of simple operation, high sensitivity, and can be used for quantitative analysis of residual DNA in multiple-sites PEGylated protein drugs.