RÉSUMÉ
@#Objective To develop and verify a quantitative real-time PCR method for determination of the content of host cell DNA residues in severe acute respiratory symptom coronavirus 2(SARS-CoV-2) inactivated vaccine(Vero cells),in order to better control the safety of products.Methods DNA was extracted from inactivated SARS-CoV-2 vaccine(Vero cells) bulk by magnetic bead separation method,and the DNA residues of host cells were quantitatively analyzed by probetype PCR.The linear range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy of the developed method were verified,and the host cell DNA re sidues of 5 batches of inactivated SARS-CoV-2 vaccine(Vero cells)were determined by this method.Results DNA standard curve showed good linearity in the range of 300～0.003 pg/μL(each R~2> 0.99);Relative standard deviations(RSD) of repeatability and intermediate precision verification were less than 20%;The quantitative limit was 0.001 pg/μL;Sample dilution and purified liquid dilution had no interference to detection;The results of 60 min incubation at 53,55,57 ℃ and 56,60,64 min incubation at 55 ℃ showed no significant difference;The recoveries of accuracy verification were 79%～83%,RSD <5%.This method had good adaptability in detecting DNA residues in the bulk of inactivated SARS-CoV-2 vaccine(Vero cells).Conclusion The quantitative realtime PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) has been successfully developed,of which the linearity and range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy meet the acceptable standards,and are suitable for the detection and quality control of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells).
RÉSUMÉ
Objective To control residual DNA by optimizing methodology during the production of rabies vaccine using Vero cells as a vector .Methods The antigen recovery rate was assessed by linked immunosorbent assay-sandwich technique while the residual DNA was detected by DNA probe hybridization method .Antigen recovery and removal of DNA were the main indexes for evaluateing ultrafiltration , the vital part of rabies vaccine production .Three key factors in ultrafiltration were assessed: selection of membrane packages , ultrafiltration pressure and the concentration ratio .Then protamine was used to pretreat ultrafiltrates .Based on the two indicators mentioned above , the effect of protamine pretreat-ment on the ultrafiltrate was evaluated .Results and Conclusion The optimum condition of ultrafiltration was obtained on the basis of the general antigen recovery rate , DNA removal rate and actual production .The primary parameters of ultrafil-tration were as follows:7.5 ×105 ultrafiltration membrane packages, 20 times concentrated, 15 psi ultrafiltration pressure. After pretreatment with protamine , ultrafiltration has proved to be a molecular sieve in intercepting DNA ,while protamine can tangle the fragmented DNA and form a larger molecular segment , which is believed to be more conducive to ultrafiltra-tion interception .