Abstract
Protamine sulfate (PS) is an approximately 4 kDa cationic polypeptide derived from chum salmon used to reverse heparin-induced anticoagulation in patients. 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. Suitable resDNA methods for PS require the generation of host DNA reference materials, efficient DNA extraction procedures and assay sensitivity and accuracy as high as possible. Here, optimization data are shown for the extraction of DNA present in PS drug products and for the generation of reference standard from protease-digested research grade chum salmon DNA. The lower limit of quantitation (LLOQ) for the reference standard determined from protease-digested DNA (0.0025–156.25 pg/μL) was 0.0025 pg/μL. The extraction procedure LLOQ, determined from DNA (0.01–1.25 pg/μL) spiked into PS samples, was 5 pg DNA per mg PS. The data supporting the LLOQs were evaluated using acceptance criteria of 70–130% recovery with % correlation coefficient (CV) ≤ 25% for DNA concentrations and curve metrics (slopes, R2 and y-intercepts) within 2SD of the mean. The data presented here complement a broader study (Sommers et al., 2018) [1] and are particularly useful for the development of resDNA methods for challenging drug products.
Highlights
Protamine sulfate (PS) is an approximately 4 kDa cationic polypeptide derived from chum salmon used to reverse heparin-induced anticoagulation in patients
Because the presence of residual host cell salmon DNA in PS drug product can pose safety concerns, processing steps during PS manufacturing are designed to target the reduction of these impurities
Drug Product Safety Residual host cell DNA Tables and Figures Real-time quantitative polymerase chain reaction assay for a multicopy gene (5S ribosomal DNA) using custom-designed primers and TaqMan probes Analyzed Saline, PS or TE buffer samples were spiked with protease-digested salmon DNA (0.01–1.25 pg/μL)
Summary
We tested the RecoverAlls kit, the PrepSEQs kit and Arg-C digestion methods and found the RecoverAlls kit most amenable for optimization given its ease, throughput and high DNA recoveries from saline (PS excipient) solutions [1]. The RecoverAlls kit was further optimized by evaluating 1) digestion times, 2) elution volumes with TE buffer, 3) RNase requirement and 4) column drying conditions. Salmon DNA (0.002–1.25 pg/μL) was spiked into TE buffer samples to evaluate the role of elution volume on % recovery. The DNA recovery curve, following two 100 μL elutions showed greater linearity (R2 1⁄4 0.999) and. Alignment with DNA concentrations assuming 100% recovery compared with the linearity (R2 1⁄4 0.971) and curve alignment following a single 100 μL elution (Appendix A)
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