Artist’s rendition of AAV virus particles

AAV manufacturing solutions for gene therapy commercialization

The rapidly growing interest in gene therapy has led to the need for more cost-effective and scalable viral vector manufacturing platforms to deliver these therapies. Adeno-associated virus has become the vector of choice as it stands out for its safety profile since infection with the vector is not pathogenic. Also, AAV cannot replicate on its own and is not directly integrated into the host genome.

Thermo Fisher Scientific offers innovative products and services that are designed to accelerate your therapy projects through AAV vector development. With solutions that span the entire AAV production workflow, our aim is to provide superior product depth and expertise to help companies develop breakthrough therapies.

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AAV workflow

There are multiple strategies to develop AAV vectors, but typical steps include plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, and fill and finish. Transient expression of AAV in either adherent or suspension cell lines are most common in pre-clinical and early phase development. An alternative option for AAV production is the development of a stable producer cell line which helps ensure long-term stable supply of the viral vector.

The use of an adherent cell line for production, such as HEK293 cells, requires the transfection of multiple plasmids into the cells with a transfection reagent. When Sf9 cells are used for production, the genes of interest are introduced through the Baculovirus expression vector system. Both options require medium- to large-scale plasmid production.

Workflow graphic depicting the 6 steps of AAV production from plasmid development to fill and finish

We offer solutions and expertise for every phase of your AAV manufacturing workflow

Plasmid development & production

When large amounts of plasmid DNA are needed to transfect the host cells and before transfection can take place, plasmid DNA needs to get produced. After bacterial expression, the plasmid DNA is harvested and purified from the fermentation culture. In order to be released for clinical use, safety tests are conducted to help ensure the plasmid DNA is free from any process- or product-related impurities.

Plasmid production products

From vector selection to large-scale production and purification, we have solutions to meet your needs.

Gene synthesis and cloning services

Invitrogen GeneArt Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence. You’ll receive a bacterial stab and/or purified plasmid containing your synthesized gene—ready for downstream applications.

Cell expansion, plasmid transfection & viral vector production

Before transfection is performed, a suitable mammalian or insect cell line is selected and expanded. In large-scale production, cells which can grow in suspension such as suspension adapted HEK293 or Sf9 insect cells, have an advantage over adherent cells as they provide easier scalability. Nevertheless, adherent HEK293 cultures are easily set up and often used in process development phase or when scalability is not an issue.

Once the cells are expanded and the genes of interest are introduced, viral vector production takes place. No matter the production scale or cell systems used, we can supply you with state-of-the-art products across your upstream AAV production workflow.

Cell culture platforms—adherent cells

Cell factory in a laboratory incubator

Cell culture platforms—suspension cells

Scientist using HyPerforma Single-Use Bioreactor in the lab

Bioprocessing liquids and cell culture media preparation

Automation

In addition to standard cell lines, we offer custom cell line engineering services through our Viral Vector Services CDMO group.

Purification

After several days of production the AAV vectors are ready for harvest and purification. When all regulatory requirements are met, the product is ready for final fill and finish.

Downstream processing can be a laborious and costly process, therefore effective and scalable methods of generating high-purity and high yield of AAV particles is critical.

For manufacturing at scale, cGMP-compliant purification techniques to separate the viral vectors from the various process and product related impurities produced during the upstream process are key. Affinity chromatography, providing high purity and yield in a single step, can reduce the number of steps during AAV purification, thereby improving yield and reducing process lead times.

A polish chromatography step is needed to enrich for full capsids. There is only a slight difference in isoelectric point (pI) between the empty and full particles. Ion exchange chromatography performed with a high-resolution resin is desired to ensure separation of empty and full capsids.

POROS CaptureSelect AAV affinity resins, are specifically designed for the purification of a large subset of AAV serotypes, thereby offering a scalable platform for the industry. In addition, the high-resolution POROS resins provide an excellent solution for polishing.

Chromatography resins

Mixers, buffers and single-use solutions

Fill & finish

Once the final product has been purified, successfully passed the contaminant and impurity testing and received regulatory approval for clinical use, it is ready for formulation, final fill and finish. Thermo Fisher has the capabilities to provide drug product fill, finish, storage, and delivery services. If clients prefer to complete fill and finish independently, we provide a portfolio of sterile container systems for therapy delivery, including bioprocess containers and bags. We offer the solutions you need to fit your unique AAV application and workflow.

Plasmid development & production

When large amounts of plasmid DNA are needed to transfect the host cells and before transfection can take place, plasmid DNA needs to get produced. After bacterial expression, the plasmid DNA is harvested and purified from the fermentation culture. In order to be released for clinical use, safety tests are conducted to help ensure the plasmid DNA is free from any process- or product-related impurities.

Plasmid production products

From vector selection to large-scale production and purification, we have solutions to meet your needs.

Gene synthesis and cloning services

Invitrogen GeneArt Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence. You’ll receive a bacterial stab and/or purified plasmid containing your synthesized gene—ready for downstream applications.

Cell expansion, plasmid transfection & viral vector production

Before transfection is performed, a suitable mammalian or insect cell line is selected and expanded. In large-scale production, cells which can grow in suspension such as suspension adapted HEK293 or Sf9 insect cells, have an advantage over adherent cells as they provide easier scalability. Nevertheless, adherent HEK293 cultures are easily set up and often used in process development phase or when scalability is not an issue.

Once the cells are expanded and the genes of interest are introduced, viral vector production takes place. No matter the production scale or cell systems used, we can supply you with state-of-the-art products across your upstream AAV production workflow.

Cell culture platforms—adherent cells

Cell factory in a laboratory incubator

Cell culture platforms—suspension cells

Scientist using HyPerforma Single-Use Bioreactor in the lab

Bioprocessing liquids and cell culture media preparation

Automation

In addition to standard cell lines, we offer custom cell line engineering services through our Viral Vector Services CDMO group.

Purification

After several days of production the AAV vectors are ready for harvest and purification. When all regulatory requirements are met, the product is ready for final fill and finish.

Downstream processing can be a laborious and costly process, therefore effective and scalable methods of generating high-purity and high yield of AAV particles is critical.

For manufacturing at scale, cGMP-compliant purification techniques to separate the viral vectors from the various process and product related impurities produced during the upstream process are key. Affinity chromatography, providing high purity and yield in a single step, can reduce the number of steps during AAV purification, thereby improving yield and reducing process lead times.

A polish chromatography step is needed to enrich for full capsids. There is only a slight difference in isoelectric point (pI) between the empty and full particles. Ion exchange chromatography performed with a high-resolution resin is desired to ensure separation of empty and full capsids.

POROS CaptureSelect AAV affinity resins, are specifically designed for the purification of a large subset of AAV serotypes, thereby offering a scalable platform for the industry. In addition, the high-resolution POROS resins provide an excellent solution for polishing.

Chromatography resins

Mixers, buffers and single-use solutions

Fill & finish

Once the final product has been purified, successfully passed the contaminant and impurity testing and received regulatory approval for clinical use, it is ready for formulation, final fill and finish. Thermo Fisher has the capabilities to provide drug product fill, finish, storage, and delivery services. If clients prefer to complete fill and finish independently, we provide a portfolio of sterile container systems for therapy delivery, including bioprocess containers and bags. We offer the solutions you need to fit your unique AAV application and workflow.

Analytics: contaminant and impurity QC testing

To help ensure regulatory approval of the final AAV product, QC testing must be implemented. We offer rapid molecular methods for contaminant and impurity testing across the AAV production workflow, such as mycoplasma screening during cell culture and residual DNA testing following virus harvest and purification.

Mycoplasma testing of cell culture–based therapies

Per regulatory requirements, cell culture–based therapies must be free of mycoplasma. Manufacturers have traditionally outsourced testing to labs that specialize in the 28-day culture-based test method. For manufacturers of gene and cell therapy products, as well as other low-dose and short shelf-life therapeutics, it is not feasible to wait 28 days for test results. Real-time PCR–based assays provide a viable alternative to the culture-based method and provide results in hours while meeting the required sensitivity. 

Residual host cell DNA testing of cell culture–based therapies

Quantification of residual host cell DNA from the producing cell line is part of the purity tests for Thermo Fisher Scientific gene therapy products. Regulatory agencies provide guidance on the maximum acceptable level of DNA per dose of vector. Therefore, rapid and accurate quantification is crucial to manufacturers. Gene therapy products and other low-dose, short shelf-life therapies are not suitable for testing that requires a long time to receive results and uses large sample volumes. Real-time PCR based assays provide accurate, actionable results in hours, while meeting sensitivity requirements.


Viral vector contract development

4-step workflow graphic including process development, clinical supply, analysis and validation, and commercial launch

Our Viral Vector Contract Manufacturing Services, supports clients from drug development through clinical trials to full-scale manufacturing. With expertise in cGMP manufacturing practices, our team has a keen understanding of the challenges scientists and clients face in this field. We provide the technical expertise and operational capabilities required to bring novel gene therapies to market.

In addition to product release, stability testing, process and analytical qualification, we provide global quality compliance and regulatory support.

Learn more about our CDMO services

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