Selecting the right producer cell line for the development and manufacturing of oncolytic virus (OV) therapeutics can be challenging. The inherent highly heterogeneous nature of viruses means that there is no “magic bullet” cell line suitable for all types of live viruses and vector projects. As a result, it is of critical importance to determine the cell line that is optimal for the application. Moving from pre-clinical stages to phase I trials and beyond, the implications of the choice of cell line will become increasingly apparent as the project scales.
A key decision that must be made early on the path to IND is between the use of adherent and suspension cell culture platforms. Offering distinct advantages depending on circumstance, the two culture platforms must be weighed to assess the best option on a project-by-project basis.
Cell line selection
The choice of cell line will predominantly depend on the virus type being produced and its destined application. Cell lines most commonly used in oncolytic virus manufacturing currently include:
- A549 – a cell line isolated from human lung tissue
- VERO – derived from kidney cells of African green monkey
- Chicken embryo fibroblasts (CEFs)
- HeLa S3 – a clonal derivative of the parent HeLa immortal cervical cancer cell line
- HEK293 – derived from human embryonic kidney cells
As well as those listed, there has been a constant influx of new cell lines entering the virus manufacture market in the last decade, mainly comprising proprietary designer cell lines. This has been driven by the specific needs of different virus types and the lack of an “all-inclusive” producer cell line. Depending on the type of OV being produced and its characteristics, cell lines with specific attributes could offer significant benefits as compared with others.
- Cell lines for enveloped viruses
Certain cell lines may not be suitable for the production of enveloped viruses, as the presentation of producer cell-specific immunological markers could potentially impact oncolytic viruses in vivo efficacy.
- Cell lines for replication-deficient oncolytic virus production
Often, it will be important for patient safety that the chosen OV cannot replicate within patient cells, only in the producer cell line. This can be achieved with certain cell lines; HEK293/HEK293T cell lines, for example, were originally designed to produce replication-deficient adenoviruses. Engineered to supplement the E1 adenoviral genes needed for replication in the cell genome, adenoviruses deficient in E1 can replicate in HEK293/HEK293T but not in cells lacking these genes.
- Cell lines approved for use
Not all cell lines are regulatory approved for live virus/vaccine manufacture and licenses may be required for the use of cell cultures derived from protected animals (e.g., VERO). Oncolytic virus developers must be aware of relevant CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) regulations if cells are to be transported between different countries, such as shipping to manufacturing partners.
Suspension and adherent cell culture
One of the most important factors that will need to be considered when selecting a producer cell line for oncolytic virus production is their ability to grow as adherent or suspension cell cultures. The ability to scale upstream processes to produce the smaller yields needed at pre-clinical stages to the large volumes required at commercial scale will depend on this decision. Although most cell lines commonly used for oncolytic virus development are capable of adherent cell culture, not all can be adapted to suspension culture (Table 1).
| Cell line | Description/origin | Adherent or suspension? |
|---|---|---|
| VERO | Established from the kidney of normal adult African green monkey | Mainly adherent, although suspension has been possible |
| A549 | Derived from explanted human lung adenocarcinoma | Mainly adherent, as adaptation to suspension is not simple |
| HEK293 / HEK293T | Immortalized/transformed through transfection of Human Embryonic Kidney cells | Adherent and suspension |
| HeLa S3 | Derived from cervix carcinoma patient | Suspension-adapted |
| BHK21 | Derived from immortalized, Baby Syrian Hamster Kidney cells | Adherent and suspension |
| MDCK | Madin-Darby-Canine (dog)-Kidney tubules | Adherent and suspension |
| HER (HER.96, HER.911) | Proprietary cell line; derived from human embryonic retinoblast cells | Adherent and suspension |
| MRC-5 | Medical Research Council cell strain 5, UK; derived from male human fetal lung | Adherent only |
| WI-38 | Wistar Institute fetus 38; derived from female human fetal lung | Adherent only |
| CEFs | Chicken embryo fibroblasts | Adherent only |
Table 1. The ability of cell lines to grow as adherent or suspension cultures
Adherent cell culture platforms
With the majority of cell lines used for oncolytic virus development and manufacturing relying on adherent culture methods, a wide array of culture vessels is available for use at various scales.
2D cell vessels such as Corning CellStacks®, Hyperflasks®, and HyperStacks® can be scaled with increasing units (“scaling-out”). However, the time requirements for operator handling of these vessels can be extensive at large scale and potentiate contamination risk. Additionally, scale-out in this manner can necessitate significant facility floor space due to the vast surface area needed for cell growth.
Alternatively, fixed-bed bioreactors are a promising alternative to 2D adherent culture methods, offering a large growth surface area in a compact space. The iCELLis® (PALL) fixed-bed bioreactors are robust, supporting a wide range of cell lines for production of a diverse panel of viruses, and come in two versions for seamless scale-up (the iCELLis Nano and the GMP-compliant iCELLis500+). Fixed-bed bioreactors are particularly well suited for collection of viruses from the supernatant, allowing relatively simple bulk harvest operations.
Suspension cell culture platforms
Although not all cell lines can be adapted to suspension culture, these platforms can provide vast benefits as compared with adherent alternatives, especially when OV projects require scaling. Offering relatively linear scale-up, bioreactor systems are available from many companies (including Sartorius, Cytiva, Thermo Fisher Scientific, Abec, etc.) and can have capacities of up to 20,000 L if stainless steel. These systems are commonly seen in protein production, though not readily utilized in viral production at this scale.
With minimal manual handling required (except for cleaning and decontamination procedures), suspension cell platforms often offer improved simplicity, less operating time, and a lower risk of contamination as compared with adherent platforms.
In the past five years, single-use bioreactors have also become commercially available up to working volumes commonly of 50 L, 200 L, 500 L, and 2000 L systems, with the more recent introduction of large-scale 5000 L (ThermoFisher, HyPerforma DynaDrive) and 6000 L (ABEC Inc.), further reducing contamination risk and time between project transfer. These bioreactors are typically designed to be cylindrical in shape with constant ratios for height and diameter, enabling the simple transfer of process between all available scales.
Key lessons
From the onset of an oncolytic virus project, developers should be looking ahead to determine how their initial decisions could impact success as the therapy moves toward the clinic and eventually to a commercial scale. Being proactive and taking time to assess how the choice of cell line and culture platforms could impact milestones like IND application will ultimately simplify this often difficult journey.
Specializing in live virus and viral vector development and manufacturing, Vibalogics can help to navigate these challenges in order to deliver vital transformational oncolytic viruses to patients. Speak to our team to find out how we can help to simplify your path to IND, contact us today.
