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Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors
Published on: December 3, 2013
Bioprocess development for canine adenovirus type 2 vectors.
P Fernandes1, C Peixoto, V M Santiago
1Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Oeiras, Portugal.
Gene Therapy
|July 6, 2012
Summary
A new bioprocess using MDCK-E1 cells enables scalable production of canine adenovirus type 2 (CAV-2) vectors. This GMP-compliant method yields high-quality vectors suitable for clinical applications, addressing previous regulatory concerns.
Area of Science:
- Gene therapy vector production
- Adenovirus vector development
- Biotechnology and bioprocessing
Background:
- Canine adenovirus type 2 (CAV-2) vectors offer advantages over human adenovirus (AdV) vectors due to lower immunogenicity.
- CAV-2 vectors efficiently transduce neurons and traffic to afferent brain regions.
- Current CAV-2 vector production in dog kidney (DK) cells poses challenges for clinical-grade material and regulatory approval.
Purpose of the Study:
- To develop a Good Manufacturing Practice (GMP)-compliant bioprocess for scalable CAV-2 vector production.
- To establish a robust downstream purification process for clinical-grade CAV-2 vectors.
- To assess the quality and productivity of CAV-2 vectors produced using the new bioprocess.
Main Methods:
- Development of an MDCK-E1 cell line for CAV-2 vector production.
- Cultivation of MDCK-E1 cells in scalable stirred tank bioreactors using serum-free medium.
- Purification of CAV-2 vectors using column chromatography.
- Characterization of vector identity, particle size, and Zeta potential via SDS-PAGE and dynamic light scattering.
Main Results:
- Achieved productivities of approximately 10^9 infectious particles (IP)/mL and 2 x 10^3 IP/cell.
- Developed a scalable downstream process with 63% global recovery.
- Purified CAV-2 vectors exhibited a particle-to-IP ratio <20:1, meeting regulatory standards for AdV vectors.
- Vectors produced in MDCK-E1 cells were comparable to those from DK cells.
Conclusions:
- A GMP-compliant bioprocess for scalable CAV-2 vector production was successfully developed.
- The new process utilizes MDCK-E1 cells and scalable bioreactor technology.
- This advancement facilitates the production of clinical-grade CAV-2 vectors, supporting their use in gene therapy applications.
