The 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. In 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. These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.

Malaria is the mosquito-borne lethal infectious disease.The pfs25 protein was proved to be the antigen for transmission-blocking vaccine.The aim of our study is to construct eukaryotic expressive vector for expression of Plasmodium falciparum transmission-blocking vaccine antigen pfs25 in CHO cell.In this study,the novel vector,in which the expression cassette was integrated with weaken SV40 promoter and dihydrofolate reductase(dhfr),was constructed and pfs25 gene was cloned into the new vector.The novel vector was designed and accomplished based on the pBudCE4.1 vector and named as pCMV-WD.It included the artificially synthetica weaken SV40 promoter,dhfr expression cassette and newly designed multiclone sites(MCS).The vector was applied especially on the CHO cell.The pfs25 gene was inserted into the pCMV-WD vector and recombinant plasmid pfs25/pCMV-WD was constructed,and then transformed into the host CHO-DHFR-cell by electroporation.Positive CHO cells expressed pfs25 gene were obtained by selection of deleting hypoxomthine,thymioline and adding zeocin antibiotics in medium.The recombinant pfs25 protein was identified by ELISA and Western Blot.The positive reaction was found in the Western Blot assay.Seven positive CHO cell lines expressed pfs25 gene were obtained,and the 0.1 mg/mL pfs25 was detected in the culture medium of 5A4 CHO cell line.The pfs25 gene was expressed successfully in the CHO cell by the eukaryotic expressive vector construction.