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Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass
Published on: May 1, 2013
A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual
Shu-Min Zhang1, Matthew Roberts1, Marisa Jones1
1Structure & Function Characterization, CMC Analytical, GlaxoSmithKline, Collegeville, PA, USA.
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.
Insights
Polyethyleneimine (PEI) interferes with DNA quantification in antibody purification. Removing PEI using specific agents allows sensitive and accurate residual DNA measurement, meeting regulatory standards.
Area of Science:
- Biopharmaceutical Manufacturing
- Analytical Chemistry
- Molecular Biology
Background:
- Polyethyleneimine (PEI) is a common flocculent in monoclonal antibody purification.
- PEI residuals interfere with sensitive DNA quantification assays like real-time quantitative PCR.
- High sample dilutions to overcome PEI interference compromise DNA assay sensitivity and regulatory compliance.
Purpose of the Study:
- To address the interference of PEI in residual DNA quantitation.
- To develop a method for accurate DNA quantification in the presence of PEI.
- To ensure compliance with regulatory limits for residual genomic DNA.
Main Methods:
- Investigated the inhibitory effect of PEI on DNA quantitation.
- Evaluated high sample dilutions as a method to overcome PEI interference.
- Assessed the efficacy of sodium dodecyl sulfate, heparin, and sarkosyl in removing PEI while preserving DNA.
Main Results:
- PEI significantly inhibits real-time quantitative PCR-based DNA assays.
- Extreme sample dilutions (1:10,000) overcome PEI interference but reduce assay sensitivity.
- Treatment with sodium dodecyl sulfate, heparin, and/or sarkosyl effectively removes PEI, enabling accurate DNA quantitation.
Conclusions:
- PEI interference necessitates alternative strategies for accurate residual DNA measurement.
- Chemical removal of PEI is a viable method to maintain assay sensitivity and meet regulatory requirements.
- Optimized methods for PEI removal enhance the reliability of residual DNA quantitation in biopharmaceutical manufacturing.
