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Related Experiment Video

Updated: Jun 22, 2025

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

Published on: January 13, 2022

Recombinant AAV Production.

Pranav R H Joshi¹, Alina Venereo-Sanchez²

¹Department of Bioengineering, McGill University, Montreal, QC, Canada.

Methods in Molecular Biology (Clifton, N.J.)

|July 1, 2024

Summary

This summary is machine-generated.

Insect cell-baculovirus expression vectors (IC-BEV) offer a scalable platform for producing recombinant proteins and gene therapy vectors like adeno-associated virus (AAV). This chapter details a One-Bac protocol for efficient AAV production in insect cells.

Keywords:

Baculovirus IC-BEVS Insect cell culture Plaque assay Recombinant adeno-associated virus rAAV production rBEV

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Last Updated: Jun 22, 2025

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

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Area of Science:

Biotechnology
Molecular Biology
Virology

Background:

Insect cell-baculovirus expression vector (IC-BEV) platforms are widely adopted for producing recombinant proteins and biologics.
These platforms are comparable to mammalian cell-based systems due to their simplicity, high yield, and robust bioprocessing.
Recombinant adeno-associated virus (rAAV) vectors are critical gene delivery tools produced using these systems.

Purpose of the Study:

To describe a modified One-Bac protocol for efficient rAAV production in insect cells.
To present a method adaptable to various baculovirus expression vector platforms.
To detail the key steps involved in rAAV production and clarification.

Main Methods:

Utilized a stable transformed Sf9 cell line containing AAV Rep2/Cap5 genes.
Employed a single recombinant baculovirus expression vector (rBEV) encoding the transgene.
Included steps for rBEV stock preparation, rAAV production, and centrifugation-based clarification.

Main Results:

The described protocol enables efficient production of rAAV vectors in insect cells.
The protocol is versatile and can be applied to traditional Three-Bac, Two-Bac, and Mono-Bac systems with minimal modifications.
Demonstrated the feasibility of using a modified One-Bac system for rAAV production.

Conclusions:

The modified One-Bac system provides a robust and adaptable method for rAAV production.
Insect cell-based expression systems are a valuable alternative for manufacturing gene therapy vectors.
This protocol facilitates scalable and high-quality production of rAAV for research and therapeutic applications.