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Related Experiment Video

Updated: Jun 5, 2025

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification
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Implementing a robust platform analytical procedure for measuring adeno-associated virus vector genome titer.

Thomas W Powers1, Courtney D K Sloan1, Don Stano1

  • 1Pfizer, Analytical Research and Development, 875 Chesterfield Pkwy. West, Chesterfield, MO 63017, USA.

Molecular Therapy. Methods & Clinical Development
|December 13, 2024
PubMed
Summary

A new size-exclusion chromatography method accurately measures vector genome titers for AAV gene therapies. This precise and reliable technique offers a viable alternative to PCR for manufacturing and development.

Keywords:
AAVAAV dosingSE-HPLCadeno-associated virusgene therapyvector genome titervg titer

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Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Gene Therapy

Background:

  • Accurate vector genome (vg) titer measurement is critical for recombinant adeno-associated virus (AAV) gene therapy development, impacting patient dosing and manufacturing control.
  • Current methods like qPCR and digital PCR are common, but chromatographic techniques offer promising alternatives aligned with biotherapeutic standards.

Purpose of the Study:

  • To introduce and validate a novel method for measuring vg titer using size-exclusion high-performance liquid chromatography (SE-HPLC) with UV detection.
  • To demonstrate the method's precision, linearity, stability-indicating properties, and applicability as a platform technology for AAV.

Main Methods:

  • Development and implementation of a SE-HPLC method with UV detection for vg titer quantification.
  • Evaluation of method precision (relative standard deviation <2%), linearity across concentration ranges, and performance with varied particle content.
  • Assessment of the method's stability-indicating capabilities and its ability to be bridged with existing vg titer assays like qPCR.

Main Results:

  • The SE-HPLC method demonstrated excellent precision (<2% relative SD) and linearity across tested concentrations and particle variations.
  • The method is stability-indicating and showed no significant bias when compared to established vg titer procedures, such as qPCR.
  • The approach proved applicable across different AAV serotypes and transgenes, highlighting its potential as a platform method.

Conclusions:

  • The developed SE-HPLC UV detection method provides a precise, reliable, and versatile approach for measuring vg titers in AAV gene therapy products.
  • This method can be implemented even in late-stage pharmaceutical development due to its performance and bridgeability with existing techniques.
  • The technology represents a significant advancement for AAV therapeutic development and manufacturing.