Development and validation of a quantitative real-time PCR method for determination of residual host DNA in recombinant human C1 esterase inhibitor (rhC1INH).

A quantitative real-time polymerase chain reaction (qPCR)-based method was established and validated for the detection of residual host DNA in recombinant human C1 esterase inhibitor (rhC1INH), a biopharmaceutical produced in the mammary glands of transgenic rabbits. DNA was extracted from rhC1INH samples using a sodium iodide-based extraction kit, and a TaqMan qPCR assay targeting the rabbit-specific L1Oc gene was optimized to evaluate its specificity, linearity, limit of quantification (LOQ), accuracy, precision, and robustness. The method exhibited high species specificity, with no cross-reactivity to human or other common mammalian DNA. A broad dynamic range of 0.001-100 pg/μL was achieved, with the standard curve showing excellent linearity (R2 > 0.99) and amplification efficiency of 90-110%. The LOQ was determined to be 0.001 pg/μL, satisfying the sensitivity requirements for trace DNA detection. Spike-and-recovery assays verified favorable quantification accuracy, with recovery rates ranging from 71.83% to 97.26% at low, medium, and high concentration levels. Intra-assay and inter-assay precision were assessed, and the relative standard deviation (RSD) was no more than 15.42% at all tested concentrations, which confirmed good method repeatability. Method robustness was validated by investigating the influences of different reagent batches and lysis temperature variations, with all corresponding indicators exhibited RSD values no higher than 30%. This qPCR method features high specificity, superior sensitivity, and convenient operation, and can serve as a reliable technical means for the quality control of rhC1INH and other transgenic rabbit mammary gland-derived biopharmaceuticals.