Upstream Bioprocessing for Manufacturing Viral Vectors

Viral vectors are fascinating tools that the biopharmaceutical industry is using to deliver genetic material for therapeutic application. In recent years, there has been huge increase in the gene therapy pre-clinical development and applications for clinical research in order to commercialize novel therapeutics. To meet the industries requirement for large quantities of GMP-compliant therapeutic viral vectors for various applications, high quality robust products must be manufactured that can be effectively processed and purified for clinical use.

In this blog, we will focus on key elements and technologies involved in the upstream bioprocessing for vector manufacturing processes.

Upstream viral vector manufacturing is comprised of multiple unit operations such as cell expansion, transfection or infection of the vector producing cells and viral vector production. Within individual unit operations there are numerous variables such as the cell/virus cultivation platform, cell line, medium formulation and bioprocess parameters (temperature, pH, dissolved oxygen) that impact final viral vector productivity.

As there is no universal platform or process suitable for all therapeutic applications, the choice of virus, cell line and bioprocess system should be made early in the development process for each drug product. In addition, factors like development timeline, cost of goods, product yield, time to market and process robustness are equally important to consider during drug development.

There are currently two preferred modes of viral vector production that are widely used. Most investigators employ either transient transfection or they generate stable producer cell lines in mammalian or insect cells.

The majority of transient transfection vector production is being carried out using attachment dependent cell lines where the chosen cell type requires favourable surfaces to which they attach, grow and produce viral vectors. On the other hand, stable-producing cell lines are generally suspension adapted cell lines which do not require a substrate to attach, grow and produce viral vectors.

There are advantages and disadvantages associated with the individual modes of vector production. As such, one needs to identify the target dose and patient population very early in clinical development, to guide selection of the optimal vector manufacturing platform which is capable of meeting future commercial demand.

Transient Transfection

Transient transfection involves the insertion of genetic material into cells without needing to integrate genetic material in the host cell genome. This process is relatively simple and straightforward operationally. It also benefits from the fact that the transfected genetic material resides in the cell for a limited period of time and does not transfer to newer dividing cells. The viral vectors produced using this process can be harvested from cells in a short window of time post transfection.

The process development timeline associated with transient transfection based vector production is relatively short, compared to the stable producer cell line. The biopharmaceutical industry is well established with multiple suppliers and various tools required for efficient transient transfection based vector production. Whereas, the vector production approach using stable producer cell line is relatively young and the work is ongoing to support industry’s growing demand.

Transient transfection is a very flexible and efficient mode of viral vector production. It can decrease development time and expedite time to market strategy. However, it requires using costly GMP grade plasmids for the transfection steps, as well as additional purification steps to remove helper plasmid, cellular DNA and transfection reagents.

Scale-Up of Adherent Cells for Vector Manufacturing

Industrial scale production of viral vectors is achieved by either increasing the number of culture system units (scale-out) or sequential use of larger devices (scale-up).

Historically, much of the work to date for vector manufacturing is carried out in cell factories, roller bottles, HyperFlasks^♦ and HyperStacks^♦ etc. Each of these manufacturing platforms requires an increase in the number of culture units for large scale production. This is time consuming, labor intensive, costly and requires a large footprint.

Bioreactors facilitate scale-up of adherent cell manufacturing for viral vector production, with many advantages over scale-out processes. These manufacturing advantages include:

• Ease of monitoring
  
• Reduced number of unit operations
• Fewer open steps to prevent contamination risk
•  Lower operational costs
• Controlled environment within the bioreactor – which can improve productivity as well as quality of the final product

Hollow fiber bioreactors (e.g. Quantum^♦) and fixed-bed bioreactors (e.g. Pall’s iCELLis® bioreactor) offer increased scale, with multiple scalability options within units for manufacturing scale production. In addition to added scalability, the iCELLis bioreactor platform can support higher cell density per unit volume and produces similar cell-specific titers compared to traditional adherent culture. Although both fixed-bed and hollow fiber bioreactors provide significant scale-up advantage compared to historic scale-out options, the scale is currently restricted to 500 m². If a therapeutic product dose titer or commercial demand exceeds the current manufactured amount per batch, the process would require scale-up to a larger surface area.

Microcarrier platforms can also provide various scale-up options for adherent cell expansion which can meet vector manufacturing quantities for research, clinical and manufacturing scale. The vaccine industry has successfully scaled-up virus production to ≥ 2000 L using microcarriers in stirred tank bioreactors. For example, a 2000 L stirred tank bioreactor operating at 10 cm²/mL microcarrier density provides 2000 m² of surface area. A potential downside of incorporating microcarrier platforms for vector manufacturing is that developmental studies to optimize cell attachment, growth and transfection efficiency can be lengthy for some systems.

Read more in next week’s blog where we continue the discussion of Upstream Bioprocessing for Manufacturing Viral Vectors (part 2 of 2).

References:

1. https://labiotech.eu/sponsored/producing-adeno-associated-virus-vectors-cevec/
2. https://www.pharmasalmanac.com/articles/transient-transfection-at-a-large-scale-for-flexibility-in-clinical-and-commercial-vector-manufacturing

HyperFlasks and HyperStacks are trademarks of Corning Inc., Quantum is a trademark of Terumo Corporation.