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Related Experiment Video

Updated: Aug 20, 2025

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System
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AAV process intensification by perfusion bioreaction and integrated clarification.

João P Mendes1,2, Bárbara Fernandes1,2, Earl Pineda3

  • 1iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.

Frontiers in Bioengineering and Biotechnology
|November 24, 2022
PubMed
Summary

This study shows that alternating tangential flow (ATF) and tangential flow depth filtration (TFDF) can improve adeno-associated virus (AAV) production. These methods enhance AAV titers and enable integrated, efficient harvest and clarification processes.

Keywords:
adeno-associated virusalternating tangential flowgene therapyintegrated manufacturingperfusionprocess intensificationtangential flow depth filtration

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Area of Science:

  • Biotechnology
  • Viral Vector Production
  • Bioprocess Engineering

Background:

  • Increasing demand for adeno-associated virus (AAV) vectors in clinical applications necessitates enhanced production strategies.
  • High doses required for AAV administration challenge current bioprocessing, driving the need for increased volumetric productivity.

Purpose of the Study:

  • To evaluate alternating tangential flow (ATF) and tangential flow depth filtration (TFDF) for high-cell-density perfusion production of AAV8.
  • To assess the integration of AAV harvest and clarification into a single step using these filtration techniques.

Main Methods:

  • Implementation of a high-cell-density perfusion process using HEK293 cells and transient transfection.
  • Comparison of ATF and TFDF for AAV production, cell viability maintenance, and continuous harvest/clarification.
  • Optimization of shear rates within ATF and TFDF systems.

Main Results:

  • AAV expression improved with medium exchange strategies in perfusion cultures.
  • Optimized shear rates in ATF and TFDF enhanced AAV-specific titers by up to 4-fold compared to non-optimized perfusion.
  • TFDF demonstrated superior recovery yields for continuous harvest and clarification.

Conclusions:

  • ATF and TFDF techniques are effective for supporting AAV production and continuous harvest.
  • These methods facilitate integrated clarification, simplifying operations and improving manufacturing efficiency for AAV.
  • The study highlights the potential of perfusion and advanced filtration for scalable AAV manufacturing.