Abstract
BackgroundThe presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving.ResultsIn this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3–105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065–0.452% and 0.471–1.312%, respectively.ConclusionsThese results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.
Highlights
The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk
Biological products such as recombinant protein, antibody and vaccine are all expressed from the hosts of bacterial, yeast, animal cells, and continuous cell lines in the process of production, such as recombinant hepatitis B vaccine(CHO cell), Vero cell rabies vaccine, monoclonal antibody and some recombinant therapeutic proteins [1,2,3,4,5]
The objective of this paper is to develop a method for detecting the residual Chinese hamster ovary cells (CHO) cell DNA based on TaqMan Real-Time PCR
Summary
The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving Biological products such as recombinant protein, antibody and vaccine are all expressed from the hosts of bacterial, yeast, animal cells, and continuous cell lines in the process of production, such as recombinant hepatitis B vaccine(CHO cell), Vero cell rabies vaccine, monoclonal antibody and some recombinant therapeutic proteins [1,2,3,4,5]. Three methods(DNA hybridization assay, Threshold® assay, and quantitative PCR)have been recommended by regulatory agencies for residual host cell DNA quantitation [14, 15] Among these methods, qPCR is considered to be the most practical for residual DNA quantitation due to their sensitivity, accuracy, precision, and timesaving features. There are some methods that can directly detect residual DNA by no-extraction, these methods may result in great differences due to its applicability for different proteins [17, 18]
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