A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual DNA qPCR
Open Access
- 31 May 2020
- journal article
- research article
- Published by Informa UK Limited in BioTechniques
- Vol. 68 (6), 353-358
- https://doi.org/10.2144/btn-2020-0011
Abstract
Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e.g., 1:10,000) can overcome the interference of PEI, but at the cost of DNA assay sensitivity. Diluting samples poses a significant risk to the assay sensitivity needed to satisfy regulatory requirements on the quantitation of residual genomic DNA present per dose (i.e., 10 ng/dose). Removing PEI while retaining DNA, by the use of sodium dodecyl sulfate, heparin and/or sarkosyl can overcome the interference of PEI and allow a more accurate quantitation of residual DNA. METHOD SUMMARY A method to remove PEI from samples by the addition of heparin and sarkosyl or sodium dodecyl sulfate and NaOH is described. Different samples were spiked with DNA and the recovery quantitated by Wako/Kingfisher DNA extraction and qPCR or droplet digital PCR, as outlined in Figure 1.Keywords
This publication has 13 references indexed in Scilit:
- Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical ProductsBiological Procedures Online, 2019
- A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy ProductsMolecular Therapy - Methods & Clinical Development, 2019
- The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First TimeTrends in Biotechnology, 2019
- Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)Biomolecular Detection and Quantification, 2017
- A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cellsJournal of Pharmaceutical and Biomedical Analysis, 2015
- Clarification technologies for monoclonal antibody manufacturing processes: Current state and future perspectivesBiotechnology & Bioengineering, 2015
- Residual DNA analysis in biologics development: Review of measurement and quantitation technologies and future directionsBiotechnology & Bioengineering, 2011
- Molecular Dynamics Simulations of DNA/PEI Complexes: Effect of PEI Branching and Protonation StateBiophysical Journal, 2011
- Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity☆Advanced Drug Delivery Reviews, 2006
- Stability of PEI–DNA and DOTAP–DNA complexes: effect of alkaline pH, heparin and serumJournal of Controlled Release, 2001