Development and verification of a quantitative real-time PCR method for detecting residual exogenous DNA in recombinant human hepatocyte growth factor naked plasmid injection
10.13200/j.cnki.cjb.004646
- VernacularTitle:重组人肝细胞生长因子裸质粒注射液外源性DNA残留量实时荧光定量PCR检测方法的建立及验证
- Author:
Bo ZHANG
1
Author Information
1. Beijing Northland Biotechnology Co., Ltd., Beijing 100085, China
- Publication Type:Journal Article
- Keywords:
Hepatocyte growth factor(HGF);
Recombinant naked plasmid;
Residual amount of exogenous DNA;
Quantitative real-time PCR(qPCR);
Quality control
- From:
Chinese Journal of Biologicals
2026;39(02):195-200
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a specific quantitative real-time PCR(qPCR) method for the detection of residual E.coli host DNA in recombinant human hepatocyte growth factor(rhHGF) naked plasmid injection products, and perform verification and preliminary application to provide a reliable basis for the quality control of the drug, and at the same time provide reference for the safety evaluation of similar gene therapy products.Methods The 16 SrRNA gene of E.coli was selected as the target sequence, and specific primers and probes were designed for qPCR to measure residual DNA. The specificity, linearity and range, lower limit of quantification(LLOQ), accuracy, repeatability and intermediate precision of the established method were verified. Additionally, exogenous DNA residues in three batches of rhHGF naked plasmid bulk solutions of injections were tested.Results The method demonstrated excellent linearity within the DNA concentration range of 0. 01-100 pg/??L, R~2= 0. 999, with an LLOQ of 0. 01 pg/??L. No specific amplification was observed for DNA of CHO cells. The recovery rates for high, medium, and low concentration spiked samples were 104. 0%, 105. 1%, and 100. 0%,respectively. The RSDs of repeatability and intermediate precision verification were both less than 30%. The residual exogenous DNA in all three batches of bulk solutions was below 2 ??g/mg plasmids.Conclusion The developed qPCR method exhibits high sensitivity, strong specificity, and reliable accuracy and intermediate precision, which is suitable for detecting residual exogenous DNA in rhHGF naked plasmid injections and other E.coli-expressed biopharmaceutical products.
