Gene therapy: Using size exclusion chromatography to evaluate AAV product titers

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Gene therapy: Using size exclusion chromatography to evaluate AAV product titers

Team TFS
Team TFS

Production of recombinant adeno-associated viruses (AAVs) for the purpose of gene therapy is challenging and requires various analytical assays to monitor product quality attributes (PQAs) such as product titer. Due to low production yields, it’s important to develop highly sensitive methods while maintaining good recovery.


Fluorescence AAV Titer 74.pngOne important consideration is the use of the right HPLC sample vials to obtain consistent and accurate results. Another important step is determining AAV product titers even at low sample concentrations. In a recent experiment, a team at Thermo Fisher Scientific demonstrated a simple yet precise way to do just that.


AAV analysis strategy: Use size exclusion chromatography with fluorescence detection

The team used size exclusion chromatography (SEC) to provide a cost-efficient and precise method to rapidly determine AAV product titers following a single-step affinity purification.

In the past, several absorbance-based methods for viral vector titer determination have been proposed, but they have been heavily affected by remaining contaminants such as host cell proteins or DNA. SEC using Thermo Scientific™ MAvPac™ SEC-1 columns efficiently separate AAV capsids from lower molecular weight compounds, which enables precise analysis of the viral capsid concentration. To overcome the common issue of limited sample availability, we employ highly sensitive fluorescence detection, which allows analysis of samples at low concentrations. Therefore, the proposed workflow successfully enables product characterization at various production steps.


Conclusions of the study


  • SEC using MabPac SEC-1 columns is highly suitable for titer analysis of viral vector products during downstream processing.
  • While the obtained results are from AAV5, the proposed workflow is also suitable for analysis of other serotypes.
  • The use of FLD yielded excellent sensitivity.
  • The use of low-binding glass vials aided reproducibility with an observed RSD of below 5%.
  • Bottom line: The presented workflow demonstrated a rapid yet precise and reproducible way of establishing AAV titers during recombinant AAV manufacturing.

Read the customer note to learn more about the workflow.



Version history
Last update:
‎04-17-2023 06:30 PM
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