Viral Vector Process Development

Viral vectors are modified viruses used for gene delivery in gene therapy and used as vaccines. Modified viruses lack the potential to reproduce and cause disease but still retain specialized mechanisms to efficiently deliver genetic information into specific cells.There are several types of viral vectors that can be used to deliver genetic information into target cells including Retrovirus, Lentivirus, Adenovirus, Adeno-Associated Virus (AAV), and Baculovirus. The most suitable choice of viral vector will depend on your specific application and needs. Producing viral vectors in cell lines grown in bioreactors instead of conventional 2D cultivation systems allows for close process monitoring and control , simplifies bioprocess scale-up , and as a result opens up new possibilities for bioprocess optimization to increase yield and reproducibility.

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Upstream bioprocess development for viral vector production

Viral vector process development in mammalian cell cultures typically requires several stages. The process starts with plasmid production or selection, followed by cell expansion and plasmid transfection, and finally, viral vector production and purification.Let’s look at some of these stages and discuss potential approaches for optimization to ensure your product has higher yields, is simpler to scale, and is commercially viable.

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Plasmid production

In the context of viral vector production, plasmids can be adapted to contain the instructions for producing viruses with specific genomes. Through standard plasmid cloning, viruses can therefore be engineered to contain important genes for either gene therapy or vaccination purposes. Viral vectors can be used as a powerful tool for the delivery of this genetic information into mammalian cells. During the plasmid production stage, plasmids are either constructed in the laboratory or selected from existing stock. The type and number of plasmids you require will depend on the viral vector you choose to use and whether you intend to use transient or stable cell lines. Learn more about plasmid production in our webinar!

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Cell expansion and virus production

Selecting a suitable cell line platform is a key factor in viral vector process development. During the cell expansion stage, an appropriate cell line must be selected and cultured to a sufficient density in order to prepare for transfection. The method you employ to grow your cell culture will vary depending on the type of cell you use. The majority of the cell lines used to generate viral vectors are naturally adherent. However, the use of suspension-adapted cell lines becomes increasingly popular, as they ease production scale-up and enable higher production capacity.

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Suspension cell culture

In suspension culture cells are free floating in the culture medium. Therefore, they are well suited for cultivation in stirred-tank bioreactors. Suspension cell culture in stirred-tank bioreactors promises higher production capacity, increased yield, and simplified production scale-up compared to attachment-dependent expression systems. Eppendorf offers a range of scalable reusable and single use bioreactors that can be used to optimize your cell growth and viral vector production. Get more information about how Eppendorf bioprocess systems were used to cultivate suspension cells to develop viral vector production processes.

Adeno-associated virus production in suspension HEK293 cell culture using the SciVario^® twin

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HEK293 suspension cell culture using the BioFlo^® 320 with BioBLU^® 3c Single-Use Bioreactors

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Development of a scale-down model for rAAV viral vector production using a Sf9/BEV system

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rAAV production in suspension CAP GT^® cells in BioBLU^® 3c and 10c Single-Use Bioreactors

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Cultivation of adherent cells

Growing adherent cells relies on a growth support matrix. Conventionally, 2D culture systems like flasks and dishes have been used, but as technology has evolved, cultivation in bioreactors provides an alternative option that exploits the benefits they offer in terms of process control and scalability. The use of microcarrier-based culture systems and packed-bed bioreactors filled with Fibra-Cel^® Disks are options for cultivating adherent cells in bioreactors.

Fibra-Cel^® Disks

Fibra-Cel^® is a solid support growth matrix, which can be used in combination with packed-bed bioreactors from Eppendorf. Its three dimensional structure provides a large surface area for cell attachment and growth in both fixed and suspended states and protects cells from shear forces. Fibra-Cel^® Disks eliminates the need for cell filtration to separate cells from the end product and like this enables sustained long-term periods of high-density growth in perfusion.

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Product brochure

Learn more about Fibra-Cel^® Disk

PDF 4.3 MB

Application note

Read, how lentiviruses were produced in adherent cells grown on Fibra-Cel^® Disks

PDF 0.4 MB

Microcarrier-based cell culture systems

Microcarrier-based cell culture systems promote high cell yield and can be used with many bioreactor control systems from Eppendorf including the small scale DASbox^® Mini Bioreactor System and the benchtop BioFlo^® 320 bioprocess controller and SciVario^® twin bioprocess controller. Used in combination with spin filters or cell-lift impellers with decanter columns they enable the setup of perfusion processes.

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Application note

Read about perfusion for adherent cell culture using an Eppendorf bioreactor equipped with microcarrier spin filter

PDF 1.3 MB

Application note

Read about perfusion cell culture on microcarriers using a cell-lift impeller

PDF 2.9 MB

Plasmid transfection, viral vector production, and harvest

A transfection step is necessary to introduce the plasmid into the cultured cell line and subsequently, produce the encoded viral vector. In general, there are two approaches for viral vector production, transient and stable transfection.

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Learn more about transient and stable transfection‌

In transient transfection, viral vector plasmids are inserted into the producer cell but will only exist intracellularly for a limited time before they are broken down. Due to the transient existence of the plasmid and the fact that the genes encoding the viral vector components are not integrated into the cell’s genome, the genetic material will not be passed to the next generation during division. As a result, expression of the viral genes is only detectable for a few days post-transfection, with the viral vectors being harvested within this time. This option requires multiple batches and is linked to batch-to-batch variation; however, transient transfection can achieve high expression levels of viral vector due to the high copy number of transfected plasmids. Additionally, transient transfection can be a simpler alternative to the often-arduous development of stable cell lines.

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Alternatively, stable producer cell lines can be used, where genes for viral vector production are integrated into the host genome. This method is particularly common for the development and manufacturing of retroviral vectors. Since the genes for viral vector generation are stably integrated, transfected clones can be serially diluted to isolate single clones. From these, high titer clones can be selected to produce a stable master cell bank for future batches. This approach reduces batch-to-batch variability and operating costs, and can generate greater quality virus vectors. However, these cell lines are challenging to produce and maintain due to the inherent toxicity of the viral genes when they integrate into cell genome.

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With advantages and disadvantages to both, viral vector developers need to carefully choose between transient transfection and stable producer cell lines on a project-by-project basis.

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Bioprocessing for AAV vector production

Cells will need to be harvested and the viral vectors isolated for characterization and quantification. An important part of this process is determining the ratio of viral vectors with genomes to those without, known as the full/empty ratio. This can be done using methods including ELISA, electron microscopy, analytical ultracentrifugation (AUC), and high-pressure liquid chromatography (HPLC), each of which has benefits and limitations of its own. For information on AAV vector production workflow in a bioreactor, check out this application note!

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Lab scene showing lab equipment needed for viral vector process development

Eppendorf upstream bioprocessing solutions for cell culture

Eppendorf offers a scalable bioreactor portfolio for the cultivation of suspension and adherent cells, which can support you in bioprocess development for viral vector production.

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Small scale bioreactor systems‌

Bench scale bioreactor systems‌

Large scale bioreactor systems‌

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Viral Vector Process Development

Upstream bioprocess development for viral vector production

Plasmid production