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JUN 28, 2017 7:00 AM PDT

Analytical methods to measure empty and full AAV particles

Speaker

Abstract

DATE: June 28, 2017
TIME: 7:00am PT, 10am ET

As gene therapy approaches usually require large amounts of AAV vectors for clinical use, few manufacturing processes have been reported to provide high titer and potent quantities of products.
The dose control of AAV vectors is commonly established on titration methods relying either on the quantitation of DNA (viral genome) or transgene expression following cell transduction (infectious genome). However, AAV vector lots are generally a heterogeneous mixture of empty particles (i.e. do not contain DNA) and full particles (i.e. contain DNA). Therefore, it could be considered that empty particles are product related impurities, which can impact on the immunogenicity profile of the product when high doses are administered to patients.

Different indirect methods can be used to establish the ratio between full and empty AAV particles. The quantification of full capsids is performed by using qPCR based technology. The total particles can be evaluated by an ELISA assay, spectrometric analysis, ion-exchange chromatography or SDS-PAGE. However, these methods have limitations and are not applicable to all serotypes without performing a new development.

Using analytical ultracentrifugation approach, we have developed a method to quantify simultaneously empty and full AAV particles as well as intermediate species containing fragmented or incomplete vector genome. We have applied this technique to AAV lots of different serotypes, several sizes of transgene and different process of production. Several examples of AAV vectors analysis will be presented showing that AUC could be implemented as a routine test to monitor AAV product quality and manufacturing consistency.


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JUN 28, 2017 7:00 AM PDT

Analytical methods to measure empty and full AAV particles


Specialty

Biotechnology

Molecular Biology

Cancer Research

Cell Biology

Cell Culture

Biochemistry

Gene Therapy

Cancer Therapeutics

Biomarkers

Laboratory Testing

Molecular Diagnostics

Immunology

Pcr/rt-Pcr/real-Time Pcr

Assay Development

Gene Expression

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