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Nanyang Technological University

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

9.1K Views

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10:31 min

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January 13th, 2022

DOI :

10.3791/62829-v

January 13th, 2022

•

Md Nasimuzzaman¹^,², Sophia Villaveces¹, Johannes C. M. van der Loo¹^,²^,³, Sivani Alla¹

¹Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, ²University of Cincinnati College of Medicine, ³Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia

Summary

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Explore More Videos

Adeno Associated Virus AAV

Insect Cell Culture

Cell Propagation

Virus Production

Purification Protocol

Good Manufacturing Practice GMP

Cryopreservation

Fetal Bovine Serum FBS

Dimethyl Sulfoxide DMSO

Liquid Nitrogen Storage

Chapters in this video

0:05

Introduction

0:44

Generation of Baculovirus-infected TIPS Cells

3:05

Production of AAV Vector

5:08

Lysis of Cells and Release of AAV

6:07

Purification of AAV Vector Using Affinity Column Chromatography System

8:03

Concentration and Diafiltration of AAV Vector Using Tangential Flow Filtration (TFF)

8:54

Results: Purification of Adeno-Associated Virus (AAV)2 Vector Using Baculovirus-Insect Cell Culture System

9:48

Conclusion

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