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Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production

Pete Gagnon¹, Blaz Goricar¹, Nina Mencin¹

¹BIA Separations, Sartorius Company, Mirce 21, 5270 Ajdovscina, Slovenia.

|January 22, 2021

Summary

This summary is machine-generated.

Enhanced high-performance liquid chromatography (HPLC) methods improve adeno-associated virus (AAV) characterization. Combining UV, fluorescence, and light scattering offers more accurate empty and full capsid ratio determination for therapeutic drug development.

Keywords:

AAV HPLC adeno-associated virus empty capsids extrinsic fluorescence full capsids in-process analysis intrinsic fluorescence light scattering process development validation

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Area of Science:

Biochemistry
Analytical Chemistry
Biotechnology

Background:

High-performance liquid chromatography (HPLC) is crucial for characterizing adeno-associated virus (AAV) empty and full capsids.
UV-based monitoring in HPLC often overestimates full capsids and is limited for unpurified samples.

Purpose of the Study:

To enhance HPLC utility for characterizing therapeutic adeno-associated virus (AAV) drug substances.
To improve the accuracy of empty and full capsid ratio determination.
To enable better monitoring of DNA contamination during AAV purification.

Main Methods:

Combined dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence (Picogreen), and light scattering.
Applied enhanced HPLC methods to anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC).
Utilized intrinsic fluorescence for sensitive AAV detection and objective capsid ratio estimation.
Employed light scattering for AAV identification in complex samples and semiquantitative capsid ratio analysis.
Used extrinsic Picogreen fluorescence for semiquantitative tracking of DNA, including host DNA contamination.

Main Results:

Intrinsic fluorescence enhanced AAV detection sensitivity and improved empty/full capsid ratio accuracy.
Light scattering allowed identification of AAV in complex matrices and semiquantitative ratio determination.
Extrinsic Picogreen fluorescence enabled DNA tracking throughout purification, detecting external DNA and host contamination.
The combined methods provide comprehensive characterization of AAV samples.

Conclusions:

Enhanced HPLC methods significantly improve the characterization of adeno-associated virus (AAV) products.
These advanced techniques offer more accurate assessment of empty and full capsid ratios.
The developed methods support accelerated process development, in-process monitoring, and validation for therapeutic AAV drugs.