Quantitation of Residual Host Cell DNA in Protamine Sulfate Drug Product by qPCR Assay

Publication date: Available online 31 July 2018Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Cynthia Sommers, Barry Rosenzweig, Lida Oum, Karol Thompson, David A. KeireAbstractProtamine sulfate (PS) is an FDA approved drug used to reverse heparin-induced anticoagulation in patients. Protamine sulfate is a mixture of primarily four ~4 kDa arginine-rich cationic polypeptide chains derived from chum (Oncorhynchus keta) salmon sperm. Because the presence of residual host cell salmon DNA (resDNA) in PS drug product can pose safety concerns, processing steps during PS manufacturing are designed to target the reduction of these impurities. However, given protamine’s positively charged structure, isolating and measuring negatively charged residual DNA is challenging. Here, the development of a sensitive detection method using real-time quantitative polymerase chain reaction (qPCR) assay for a multicopy gene (5S ribosomal DNA) using custom-designed primers and TaqMan probes is described. The PS qPCR standard curve was accurate over a linear range of 0.0025 to 156.25 pg/μL using protease-digested research grade salmon sperm DNA (neat) as the reference standard. DNA present in PS drug products was extracted using an optimized two-hour procedure achieving ~85% recovery values from 1 to 125 pg reference DNA spiked into PS (1 mg) samples. The procedure lower limit of quantitation (LLOQ) of 5 pg of DNA per mg of PS or 250&thi...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research

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