Quantification of Residual Host Cell DNA in Protamine Sulfate Drug Product Using Droplet Digital PCR

摘要

Protamine Sulfate (ProS) is the only FDA approved anticoagulation reversal agent for unfractionated heparin. ProS is biologically derived from chum salmon sperm and therefore may be contaminated with residual salmon sperm DNA. Because residual host cell DNA (resDNA) may pose a safety concern to patients, the amount should be closely monitored during manufacturing and strictly regulated in final drug products. The regulatorily defined acceptance level (10 ng per dose) of resDNA in drug products poses great challenges for accurate quantification of resDNA, particularly for the products used in high doses or formulated in complex matrixes. resDNA quantification in the presence of the cationic arginine-rich ProS structure can be challenging due to the polyanionic resDNA binding to ProS which may render isolation and analysis of resDNA problematic. In this work, a droplet digital polymerase chain reaction (ddPCR) method was adapted to quantify resDNA in ProS drug product.The ddPCR reaction was tolerant to matrix interferences thus eliminating the need for DNA extraction required with traditional PCR assay. In addition, the method provided absolute DNA quantification without the need for calibration curves. The developed ddPCR assay had a lower limit of quantitation (LLOQ) of 1.67 pg of DNA per mg of ProS (1.67 ppb) or 83 pg per 50 mg dose of ProS, a wide dynamic range spanning three orders of magnitude and an average % recovery of 82.5% with inter-day coefficient of variations (CV) < 20% across the entire concentration range. Overall, the ddPCR analytical procedure replaced the need for comprehensive DNA extraction with a simple in-plate protease digestion of ProS and was 120-fold more sensitive than the acceptable amount of resDNA. Five lots of ProS drug products were analyzed using this method and none tested positive for resDNA contamination above the method's LLOQ. The method may also be promising for quantifying resDNA in other challenging protein drug products which have a propensity to bind DNA or have other matrix-related complexities.