The aim of upstream processing in viral vector development and manufacturing is to generate a suitable quantity of vectors while ensuring that purification in downstream processing can be achieved efficiently. One key decision that must be made prior to upstream processing during lentiviral and gammaretroviral vectors is whether to rely on transient transfection or stable producer cell lines.

In this blog, we examine the advantages and disadvantages of transient transfection and explore the reasons why many viral vector developers are keen to move towards the use of stable producer cell lines for lentiviral vector development.

Viral vector upstream processing

Successful upstream processing will rely on achieving scalable expansion of producer cells as well as suitable cell viability and density to ensure optimal generation of viral vectors. The methods used will ultimately depend on the initial plasmid design, the pseudotype of the vector, and the chosen cell line. The choices at this stage will also impact downstream processing as well as all other processes downstream. Consequently, viral vector developers must take their time to carefully consider the platform that will be used and build a thorough understanding of the options available.

Introducing the genetic material encoding the viral vector – be it an lentiviral or a gammaretroviral vector – to the producer cell line will always require an initial transfection step. However, this can either be with transient transfection in mind or as part of developing a stable producer cell line. With advantages and disadvantages to both, viral vector developers need to choose between transient transfection and stable producer cell lines on a project-by-project basis.

Transient transfection

In transient transfection, the viral vector plasmids will be introduced to 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 genes encoded will generally only be detectable for a short period (typically days) post-transfection, with the viral vectors being harvested within this time.

Choosing this method to deliver viral genes to producer cell lines means that the production of every viral vector batch will require this transfection step. At clinical levels of manufacturing and beyond, this will necessitate the use of expensive clinical-grade plasmids and transduction reagents. Transient transfection is also associated with a certain degree of variability between batches.

Despite these challenges, transient transfection can achieve high expression levels of viral vector due to the high copy number of transfected genetic material. Additionally, transient transfection can be a simpler alternative to the often arduous development of stable cell lines.

Stable cell line generation

To produce some types of viral vectors, developers will choose to generate a stable cell line 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.

The generally recommended method of developing stable producer cell lines is to use a cell line where a packaging plasmid (encoding gag and pol) and an envelope plasmid (encoding env) have already been transfected and the genes integrated. A transfer plasmid with the transgene of interest can then be introduced. 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 (MCB) for future batches.

Using stable producer cell lines for vector production has a number of advantages:

• Reduced batch-to-batch variability
• Decreased cost involved in using clinical grade transfection agents and plasmids
• Simplified upstream culture and harvest
• Can generate greater quality particles, being nearly homologous

Weighing up the benefits and drawbacks

Despite the many advantages that stable cell lines offer, there are some circumstances where their generation may not be suitable.

Even for retroviral vectors, where using a stable producer cell line for development and manufacturing is the norm, transient transfection methods might be preferable in some situations. For example, if a self-inactivating (SIN) transfer plasmid is being used, stable cell lines will be more difficult to produce due to the self-inactivating U3 in the 3’ long terminal repeat (LTR).

Transient transfection is also generally the preferred method for large-scale batch production of lentiviral vectors for clinical trials due to the difficulties involved in developing a suitable stable cell line. The cytotoxicity of certain lentiviruses envelope proteins means that cell death can occur before suitable cell densities are achieved when using a stable cell line for production.

This issue can be overcome by genetically modifying the cell line so that the expression of the envelope protein is controlled and inducible under certain conditions. However, developers must carefully assess whether this is more beneficial than relying on transient transfection.

It is also important to remember that stable producer cell lines can be arduous to develop, requiring isolation and evaluation of individual clones, which can take a long time. Also, it is common that lower titers will be achieved as compared to transient transduction methods (predominantly due to the cytotoxic effects that viral protein expression can have on cells).

Key lessons

When considering the advantages and disadvantages of stable producer cell lines and transient transfection, viral vector developers will need to make the choice on a case-by-case basis with the project’s needs in mind.

As a contract development and manufacturing organization (CDMO) with both lentiviral and gammaretroviral expertise, Genezen can help navigate the challenges of choosing between these two options. For more information, get in touch.