Establishment And Validation Of Host Cell Residual DNA Detection Method In Therapeutic Antibody

The prevalence of antibody therapeutics in the drug market place has increased dramatically in recent years.The antibody therapeutics were produced from mammalian cell.The residual host cell DNA can not be removed completely during purification,and exist in antibody therapeutics.The potential risks associated with the presence of residual host cell DNA in antibody products are infectivity,oncogenicity,immunogenicity,and mutagenesis.To ensure that sufficient DNA removal is achieved during purification,it is essential to have an accurate and sensitive assay for host cell DNA.At present,some Q-PCR methods for measuring the residual host cell DNA have the disadvantages of low sensitivity,high cost,and inability to recognize false negatives.In the study,we successfully developed and qualified a highly accurate,specific,sensitive,low cost and efficient method for the quantitation of residual host cell DNA.In the study,CHO cells genomic DNA was extracted and prepared into the standard DNA of residual determination;high copy number repetitive sequences of CHO cell genome were used as targets,Taqman probe and primers were designed,the DNA dilution Buffer was optimized;the Q-PCR reaction system and conditions were determined,the stand curve was plotted;The method of determination of host cell residual DNA in antibody therapeutics was established,and validated and applied for the samples.The main contents and results are as follows:The high copy number repetitive Alu-equivalent sequences Clone 250 of CHO cell genome were used as targets,the Q-PCR method of host cell residual DNA determination in antibody therapeutics was established.To improve the sensitivity of the method,the CHO cells DNA standard dilution Buffer was optimized through the use of Carrier RNA（t RNA）.To monitor the false-negative amplification and PCR inhibiting effect in the Q-PCR reaction system,the internal control system was established,The method was comprehensively validated.No specific amplification curves for residual yeast,E.coli and human cell DNA were found.The relative standard deviations ofrepeatability were less than 30%.The recovery rates of standard DNA were 70%¹30 %,and within the acceptable range of Q-PCR（50% 200%）.The minimum detection limit of residual CHO cell DNA was 0.1 fg/μL,while the linear range was1×10^-43 × 10² pg/μL,with a correlation coefficient of more than 0.99,the amplification efficiency approached 100%.The method meet requirements of the determination of CHO cells residual DNA in antibody therapeutics.The determination of residual DNA in CHO cells was performed on samples of different purification steps of antibodies,which confirmed that different purification steps in the production process had a good removal effect on the host cell residual DNA.Among them,the removal effect of anion exchange chromatography was the most obvious.The purification process development optimization and process validation provided strong support.Conclusion:This study established and validated a method for the detection of DNA residues in CHO host cells in therapeutic antibody drugs,Compared to DNA hybridization and Fluorescence method of determination of host cell residual DNA in Chinese Pharmacopoeia（2015 edition）,the Q-PCR Methods showed good species specificity,high sensitivity,repeatability,accuracy and high throughput Compared with the Q-PCR method of host cell residual DNA assay reported in the literature,this method has higher sensitivity and low-cost,and can effectively eliminate the occurrence of false negative and amplification inhibition.The method has been applied to the IND Filing of antibody therapeutics and can accurately determine the residual DNA of CHO cells in antibodies to ensure the safety and quality control of antibody drugs.