ABSTRACT
@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.
ABSTRACT
Objective To develop and verify a method for determination of residual host cell DNA in recombinant human interferon α2b substances, which is used for the quality control of the product.Methods The residual host cell DNA was extracted by wako DNA extractor kit and determined by SYBRGreen based q-PCR using standard DNA as control.The residual host cell DNA was analyzed according to the standard curve.The developed method was verified by primer specifity, results accuracy and precision and used for determination of 3 batches of interferon substances. Results The minimum quantitative limit of residual host cell DNA by the developed method was 12 fg/μL, while the linear range was 12 fg/μL-120 ng/μL, with a correlation coefficient (r) of 0.998.The designed primers were specific to the DNA templates.The recovery rates of spiked samples with different DNA quantity were between 50%-200%.The residual host cell DNA determined by this method were not more than the limit, which were complied with the requirements for residual host cell DNA in Chinese Pharmacopeia ( volume III,2010 edition and 2015 edition) .Conclusion The wako DNA extractor kit could successfully solved the technical difficulties of sample pretreatment during residual DNA assay.The q-PCR method was simple, rapid and accurate for quantitation of residual host cell DNA in interferon substances.