A Direct Droplet Digital PCR Method for <i>E. coli</i> Host Residual DNA Quantification

Abstract

Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.

A Novel Method for Removing Polyethyleneimine from Biopharmaceutical Samples: Improving Assay Sensitivity of Residual DNA Qpcr

“…was treated with a number of combinations including: sarkosyl (1% w/v) + NaOH (5 mM); SDS (1% w/v) + NaOH (5 mM); and sarkosyl (1% w/v) + NaOH (1 mM) + NaCl (150 mM) [11] following the experiment procedure outlined in Figure 1. In this set of experiments, droplet digital PCR (ddPCR) [13,14] was used without DNA extraction because ddPCR is faster and less labor-intensive than Wako DNA extraction combined with qPCR. Sample dilutions tested were 1:500 and 1:100.…”