Quantification of Linear Polyethylenimines in Recombinant Adeno-Associated Virus by High-Performance Liquid Chromatography with Charged Aerosol Detection
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View abstract on PubMed
Summary
This summary is machine-generated.Quantifying residual polyethylenimine (PEI) in gene therapy products is crucial. A new HPLC method accurately measures PEI in adeno-associated virus (AAV) manufacturing, ensuring product safety and quality.
Area Of Science
- Biotechnology
- Analytical Chemistry
- Gene Therapy Manufacturing
Background
- Polyethylenimine (PEI) is a common transfection reagent in adeno-associated virus (AAV) production.
- Residual PEI poses toxicity risks and requires accurate quantification in final gene therapy products.
- Quantifying PEI in complex biological samples like AAV is challenging due to PEI-nucleic acid interactions.
Purpose Of The Study
- To develop and validate a robust analytical method for quantifying residual linear PEI in purified AAV samples.
- To overcome challenges associated with PEI quantification in complex biological matrices.
- To provide a reliable tool for quality control in gene therapy manufacturing.
Main Methods
- High-performance liquid chromatography (HPLC) coupled with charged aerosol detection (CAD).
- Sample preparation involving acid treatment (trifluoroacetic acid and hydrochloric acid) at 60°C to denature proteins and hydrolyze nucleic acids.
- Validation using spike-and-recovery studies to determine method sensitivity and accuracy.
Main Results
- The developed HPLC-CAD method demonstrated high sensitivity and specificity for PEI quantification.
- Achieved a limit of detection (LOD) of 5 µg/mL and a limit of quantitation (LOQ) of 10 µg/mL.
- Quantification was confirmed through visual peak identification, ensuring reliability.
Conclusions
- The validated HPLC-CAD method offers a sensitive, specific, and reproducible approach for measuring residual PEI in rAAV products.
- This method is valuable for process monitoring and ensuring the safety and quality of gene therapy manufacturing.
- The technique effectively addresses the challenges of quantifying PEI in complex biological samples.
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