Niu D.-Y.,Chengdu Hongkong Biotechnology Co. |
Lian W.,Chengdu Hongkong Biotechnology Co. |
He J.,Chengdu Hongkong Biotechnology Co. |
Wu Z.-G.,Chengdu Hongkong Biotechnology Co. |
Ke X.,Chengdu Hongkong Biotechnology Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To develop a specific fluorescent quantitative PCR (Q-PCR) method for determination of residual Chinese hamster ovary (CHO) cell DNA. Methods: Genomic DNA of CHO cells was extracted by using DNeasy Tissue Kit and prepared into standard DNA. The reaction system and condition for Q-PCR were determined, and a standard curve was plotted, based on which a Q-PCR method for residual CHO cell DNA was developed and verified for specificity, accuracy and precision. The residual CHO cell DNA contents in six batches (Lot No. 1003b02, 1008b05, 1012b09, 1104b04, 1108b13 and 1112b24 of bulks of Conbercept were determined by the developed method. Results: The Ct value of established standard curve showed good linear relationship to the concentration of template DNA, with a correlation coefficient of more than 0.99. No specific amplification curves for residual HUVEC and HEK293 cell DNA were found. All the mean recovery rates of standard DNA at high(105 pg/ml), moderate(103 pg/ml) and low(101 pg/ml) concentrations were within the acceptable range of Q-PCR (50%-200%), with inter-CV values of 9.3%, 21.8% and 26. 8% respectively. The mean recovery rate of standard DNA at a concentration of 10° pg/ml was 111.6%, with a CV value of 20. 4%. The residual CHO cell DNA content in bulk of Conbercept was far less than 100 pg/dose. Conclusion: A specific Q-PCR method for determination residual CHO cell DNA content was successfully developed, which showed high specificity, accuracy and precision, and might be used as a routine method for residual CHO cell DNA content. Source