B-221 Quantitation of DNA in Antibody-based and RNA-based Drugs by Quantitative Polymerase Chain Reaction (qPCR)

Abstract

Abstract Background Current FDA regulation defined acceptance level of 10 ng per dose of residual DNA in drug products poses great challenges for accurate quantification of residual DNA, particularly for the products used in high doses or formulated in complex matrixes. The purpose of this study is to develop and qualify a qPCR method for the quantification of Chinese Hamster Ovary (CHO) DNA in antibody-based drugs and for quantitation of human DNA in RNA-based drugs. Methods An 8-point standard curve consisting of 0.005, 0.05, 0.5, 5, 50, 500, 1000 and 5000 pg CHO DNA and a 5-point standard curve consisting of 0.3, 3, 30, 300 and 3000 pg human DNA was generated by serial dilutions. The antibody sample (300 µg) was spiked with 2.5, 10, and 50 pg CHO DNA. The RNA sample (1.6 µg) was spiked with 0.3, 3 and 30 pg human DNA. The samples were analyzed by qPCR using a Real-Time PCR instrument. Commercial PCR test kits (TaqMan™ Probe and TaqMan™ Universal Master Mix), and sequence specific forward and reverse primers were used. Results For CHO DNA in antibody product, the linearity slope and R2 are −3.5 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 2.5 pg, 10 pg and 50 pg CHO DNA in 300 µg antibody, 150 pg CHO DNA in formulation buffer, and 150 pg CHO DNA in 1000 pg human DNA are 70%, 76%, 86%, 93% and 95%, respectively. The precision of the assay for 2.5 pg, 10 pg, and 50 pg of CHO DNA spiked in 300 µg antibody are 6%, 7% and 2%, respectively. The QL of the assay is 2.5 pg CHO DNA per 300 µg of antibody product.For human DNA in RNA product, the linearity slope and R2 are −3.3 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 0.3 pg, 3 pg, and 30 pg human DNA in 1.6 µg RNA, 3 pg human DNA in formulation buffer, 1.5 pg human DNA in 150 pg CHO DNA are 113%, 104%, 102%, 97% and 88%, respectively. The precision of the assay for 0.3 pg, 3 pg and 30 pg human DNA spiked in 1.6 µg RNA are 6%, 5% and 2%, respectively. The QL of the assay is 0.3 pg human DNA per 1.6 µg of RNA product. Conclusion Two qPCR methods were developed to quantify residual DNA in drug products. One method is specific to quantitation of low levels of CHO DNA in antibody product. Another method is specific to quantitation of low levels of human DNA in RNA product.