Implementing a robust platform analytical procedure for measuring adeno-associated virus vector genome titer

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.