A Droplet Digital PCR Method for CHO Host Residual DNA Quantification in Biologic Drugs

Abstract

The Chinese Hamster Ovary (CHO) cells are the preferred host for manufacturing therapeutic biomolecules in the biopharmaceutical industry. Host residual DNA (hrDNA) is an impurity and needs to be monitored in the purified drug to ensure purity and safety. Currently, real-time quantitative PCR (qPCR) based methods are widely employed for quantification of hrDNA, however, digital PCR technology promises higher assay sensitivity and precision. Here, we report a method where the protein drug is digested with a protease, the protease is denatured and the CHO primers and fluorescent-tagged probe from Bio-Rad Laboratories, Inc. and droplet digital PCR (ddPCR) mix are added to the reaction and nanoliter-sized droplets are generated. The droplets are then subjected to end-point PCR followed by analysis for fluorescence. Compared to qPCR, the ddPCR method shows increased sensitivity, with high precision and accuracy of determination. Additionally, the method eliminates DNA extraction step and the requirement of DNA standards in routine sample testing. The method was successfully applied to hrDNA quantification in several biologic drugs under development at Merck.

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.…”