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With growing interest to develop treatments that circumvent the drawbacks of traditional first-line treatments for advanced cancers including surgery and chemotherapy, innovative immunotherapy approaches are taking center stage. The oncolytic virus (OV) market has subsequently been projected to reach $962 million by 2030 at a CAGR of 26.28% [1].
Development and manufacturing of OVs is no simple task, and there are many challenges surrounding production, formulation, and product stability that could arise. Careful consideration must go into every stage of production, from cell line selection to analytical development.
In this, the first of our blog series “Oncolytic virus manufacturing: challenges, considerations, and opportunities”, Chief Scientific Officer of Vibalogics, Kai Lipinski, explores the landscape of cell lines commonly used for OV manufacture. Further, he emphasizes the importance of understanding the potential benefits offered by suspension and adherent cell cultures and choosing the most suitable platform for each product.
As viruses are highly heterogeneous, there is no one cell line that is suitable for production of all live viruses and viral vector products. However, there are several cell lines that offer key advantages that make them better suited for certain applications.
Cell lines for varying applications
The Vero cell lineage (isolated kidney epithelial cells extracted from an African green monkey) is a continuous cell line and has an abnormal number of chromosomes. As it has a defective innate anti-viral defense mechanism – being interferon-deficient – they are suitable for production of viruses from various families, making them a popular choice for live virus manufacturing. It is important to remember when using Vero cells that the lowest possible passage number (< p140) should be used for production, as with passage number tumorigenicity increases
Cell lines for oncolytic adenovirus manufacturing
Adenovirus is one of the most frequently used OVs, offering powerful oncolysis and immune response stimulation, as well as promising efficacy and safety in preclinical and clinical trials. Human-derived cell lines that are deficient for genomic adenoviral sequences, including A549 (lung carcinoma epithelial cells) and HeLa S3 cells (an immortal cervical cancer cell line) are commonly used for oncolytic adenovirus manufacturing.
HEK293 (human embryonic kidney 293) cells can also be used for production of oncolytic adenoviruses, but it is essential that contamination of wildtype (WT) adenovirus is prevented and carefully monitored. This is because recombination events between HEK293 cell genomic Ad5 sequences and vector-derived adenovirus sequences can lead to generation of replication-competent adenovirus.
Cell lines for enveloped viruses
Enveloped viruses budding from the host cell form an envelope comprised of lipids and proteins of the host cell’s plasma membrane. These host-cell-specific immunological markers can impact the efficacy of the OV. When selecting a cell line, it is essential that consideration is taken early in the process to determine the impact of these marker molecules on in vivo efficacy and toxicology.
Suspension or Adherent Cell Cultures
Choosing between the use of cell lines that can be grown in suspension or those that require a surface to adhere to is an important decision that can impact all processes further downstream. Each offers distinct advantages and disadvantages, which are further emphasized at the point of scaling up OV projects to commercial scale.
Suspension or Adherent Cell Cultures
Adherent cell cultures
Adherent cultures are ideal at development stages, but traditional 2D multiple-surface cultivation systems like cell stacks are difficult to scale for larger volume needed at Phase II/III and commercial manufacture. The scaling of these systems requires increasing the number of production units (scaling-out) as opposed to increasing unit size (scale-up). Scale-out can be achieved with the use of roller bottles, scalable cell culture vessels, micro-carrier bioreactor processes, and fixed-bed bioreactors.
Fixed-bed bioreactor systems for adherent cell culture are generally preferred over other vessels at large-scale, offering reduced manual operation times, minimized contamination risk, and batch homogeneity. In these systems, the bulk of the medium is always separated from the cells, allowing simplified bulk harvest of viruses in the medium supernatant. They are also well-suited to the harvesting of non-enveloped viruses, offering the option to perform chemical lysis in situ with the addition of detergent to the media volume.
Suspension cell cultures
As compared with adherent platforms, suspension cell culture platforms offer increased scalability and ease-of-use for cell lines capable of growth in suspended media. Bioreactor systems for suspension cell culture come in various sizes, with capacities up to 5000 L. Many single-use options are available for suspension cell cultures, helping to further reduce possible contamination between projects.
Cell expansion for suspension processes typically begins in shake flasks. Rocker-style bioreactors can then be used as the final vessel or as an intermediate before inoculating a larger final vessel, such as a stirred-tank bioreactor. Wave bags are also available and can offer integrated perfusion membranes to allow high-density cultures to be used during cell build (e.g., n-1 passage) or as production vessels.
Additional Considerations
Early in the development process, OV manufacturers must also consider the cell line productivity of the chosen cell type and culture methods.
OV productivity (e.g., infectious particles (IP)/mL bioreactor volume or IP/cm2 growth surface), multiplicity of infection (MOI), and potential drug product (DP) clinical dose must be brought into alignment at this stage. If this is not achieved, clinical efficacy, costs per dose, and market viability could be jeopardized.
Additionally, if the transportation of cells between different countries is required, Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) regulations must be considered to avoid delays when shipping cells to partners.
Key Conclusions
There are many critical considerations that developers and manufacturers must keep in mind to ensure successful delivery of OV products to patients. Choosing the most suitable cell line for the application, deciding between adherent or suspension cell culture processes, and keeping OV productivity and regulations in mind requires a thorough understanding of critical upstream processes and their potential impact downstream. When bringing innovative OV products to market, partnering with a contract service partner with expertise and experience can help developers navigate these difficult decisions.
Unlock our Virotherapy Insider: Deep Dive into GMP Manufacturing of Oncolytic Viruses, to learn the critical steps involved in OV development, manufacturing, testing, and explore the measures that need to be taken at each stage to ensure success.
About Vibalogics
Since 2003, Vibalogics has operated as a specialized global Contract Development and Manufacturing Organization (CDMO) offering process and analytical development, manufacturing, testing, and fill-finish services to innovators developing transformational virotherapy products. From its headquarters and commercial facility in Boxborough, MA, USA and operations in Cuxhaven, Germany, Vibalogics supports its customers in the development and commercialization of oncolytic viruses, gene therapies and viral vaccines.
