VIRUS PRODUCTION SERVICES

Adeno-associated virus (AAV)

 

CUSTOM ADENO-ASSOCIATED VIRUS (AAV) PRODUCTION (Serotypes available for customized production service: AAV2, AAV5, AAV6, AAV8, AAV9)

 

Upstream process development and downstream processing for production at medium/large scale

 

ADENO-ASSOCIATED VIRUS VECTOR PRODUCTION STEPS

• Cloning the gene of interest (GOI) into the corresponding p-AAV-ITR-GOI plasmid.
• Preparation of the p-AAV-ITR-GOI plasmid and complementary plasmids.
• Co-transfection in a complementing cell line for virus production.
• Harvest and clarification.
• Downstream processing (Scalable two-step chromatography process: Affinity Chromatography followed by Anion Exchange chromatography for separation of empty and full AAV capsids).
• Analytical quantification by ddPCR and functionality assays.

 

Versatile AAV manufacturing platform

Production system based on a highly reliable cell line for viral packaging, which is cultured in suspension in serum-free media, using methodologies and trade secrets with exclusive know-how generated in our laboratories. Protocols are easily adaptable to a variety of product requirements for academic and industry.

 

Custom vector design and optimization

Supported by an extensive knowledge in molecular biology, it includes the design and production of the vector containing the gene of interest (GOI), focused on the tropism and specific application of the AAV (gene cloning, tissue-specific selection of promoter, Rep/Cap promoter optimization, subcellular localization of the recombinant product).

 

Medium/large-scale production under GLP

Flexible capabilities for process optimization and production in shake flask cultures and bioreactors of 1L, 3L and 20L capacity. The process can be easily scaled up to larger-sized bioreactors.

 

Upstream process development

Our scientists have extensive expertise in the upstream processing and scale-up of viral vectors. Our process development for production and optimization of transfection has consisted of media screening; optimization of the cell density at the time of transfection; transfer vector, Rep/Cap and helper plasmid ratio optimization; and plasmid DNA:transfection reagent ratio optimization. Consistent with our expression system in use, we guarantee an antibiotic-free process, serum-free and animal components-free, chemically defined media.

 

Downstream processing

Downstream Process Development (DSP) fully developed at VVector Bio. Our optimization of the DSP includes harvest time, lysis method and nuclease treatment optimization, purification optimization by affinity and ion exchange chromatography for efficient separation of empty and full particles.

 

Analytics

For Quality Control, we developed a standardized protocol for the measurement of the virus titer (as genome copies/mL) for all the AAV being produced. Tittering is performed using digital droplet PCR (ddPCR) with primers targeting the ITRs, WPRE, CAG promoter present in the viral genome. Quantification based on specific detection of the gene of interest is also conducted. AAV capsid VP1, VP2 and VP3 are also detected and purity assessed by polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. Additional quality control services required for your AAV order can be added or developed upon request.