Manufacturing Biotherapeutics with Living Cells

Most medicines on the market today are small molecules, which made up around 82% of new drug applications in 2021.¹ However, there’s also a growing market for biologics,² therapies based on proteins, nucleic acids, living cells, and other biological materials.³ Most famously, these include the Pfizer-BioNTech and Moderna’s COVID-19 vaccines based on messenger RNA.⁴

A typical biological manufacturing process begins by engineering cells to produce the desired product. Monoclonal antibody production, for example, begins by creating a cell bank of manufacturing cells that encode for the therapeutic protein.⁵ For cutting-edge CAR-T cell therapies, a patient’s own T cells are modified so they express a surface protein designed to fight cancer.⁶

In all cases, the next stage is to culture the engineered cells to produce millions of identical copies. These copies might be returned to a patient’s body, in the case of CAR-T. Alternatively, these cells might be used to produce huge volumes of protein or RNA, which can be packaged up for administration to patients.

Whatever the application, the cells can be cultured in a flask, cell factory, or bioreactor.

Flasks and cell factory systems

The first decision to make depends on whether you’re using adherent or suspended cell culture. Adherent cultures grow the cells on a surface whereas suspended cultures have the cells free-floating in the growth media.⁷ Most cells derived from vertebrates need to be cultured on a surface, but some have been adapted to work in suspension.

“During early testing and research stages, scientists can use a bioprocess container, like the Nunc™ Flask, to perform small-batch adherent cell culture,” explains Brian Nowlin, Vice President, General Manager for Thermo Fisher Scientific’s bioproduction business.

As scientists scale up their processes, they can choose to move to a cell stack or factory, such as the Thermo Fisher Nunc™ Cell Factory System. The Nunc™ is a stack of up to 40 trays for adherent cell culture. Cells attach themselves to the treated polystyrene surface of the trays and there is an option to automate the filling, harvesting, and dissociating of cells from the trays.

According to Nowlin, cell factory or stack systems are a simple, cost effective, and proven technology. They are good for adherent cells because the stack has a large surface area for the cells to grow on.

Turning to bioreactors

Adherent cell factory systems, however, are limited by the surface area of the stack. As such, bioreactors are typically used to produce the larger volumes of cells or proteins required for clinical trials or commercial-scale manufacturing.

A bioreactor is simply a vessel that provides the right conditions for cells to grow, including mixing the culture media to ensure the cells are at a comfortable temperature, pH, and have access to nutrients. According to Nowlin, a bioreactor like Thermo Fisher’s DynaDrive Single-Use Bioreactor, performs suspension cell culture, where the cells do not adhere to the side of the chamber.

Bioreactors “not only have measuring capabilities, but [you] can also adjust parameters to keep a stable environment,” explains Saskia Schoenen, Bioprocess Sales Specialist at Eppendorf. By controlling parameters like pH or oxygen “you gather a lot of knowledge about your process, which can then be transferred to a larger scale.”

Eppendorf’s main customer base works in R&D and on small-scale production and, as such, their bioreactors start at a 100 mL volume—about equivalent in size, she says, to a jar of marmalade. Many customers move to a small-scale bioreactor after culturing cells in a dish or flask, and then have the option to scale up to 1, 3, 10, or even 50 liters volume and beyond.

Different types of bioreactors

There are several types of bioreactors, with a crucial initial decision being between reusable and single-use. Single-use bioreactors can be used for multiple different products as they don’t need to be cleaned between batches, explains Fiona Bellot, Sales Director of Cellexus. However, for volumes of 2,000 liters or above, she says stainless steel bioreactors remain quite common.

The most common type of bioreactor is stirred tank.⁸ These have an impeller to move the cell culture to maintain constant temperature, pH, and nutrient concentrations. However, Bellot adds, they exert harmful shear forces on the cells. Alternatives include airlift bioreactors, such as those offered by Cellexus, which bubble gas through the cell culture media.

“The advantage of airlift is there’s good gas transfer, efficient mixing, and no dead spots within the bioreactor. There are also no shear forces on the cells, so they’re not broken up [as with] an impeller system,” she says. Popular applications among her customers include growing bacteriophages, which have tails that can be damaged by an impeller.

Another technology is the rocking or wave-motion bioreactor. These consist of a bag on a moving platform, which helps to mix and keep the cells in suspension. According to George White, Product Manager for Cell Therapies at Cytiva, whether a company should adopt a rocking or stirred-tank bioreactor, can come down to the cell type and its characteristics.

Speaking about the emerging field of cell therapies, he says, Cytiva is building out datasets to help its customers select the preferred bioreactor solution. “We’ve got a lot of proof-of-concept work being done to help customers make those decisions,” he says.

Growing adherent cells

For customers who want to grow adherent cells in a bioreactor, Schoenen explains that options include microcarrier beads that float around in the culture media. The cells grow on the surface of the beads.

An emerging solution are bioreactors that contain macro-beads or meshes to which cells can adhere. These solutions have the benefits of a bioreactor, such as the circulating flow of culture media, combined with the ability to grow adherent cells on a large surface area. The latter is a major benefit of cell factory systems.^9,10

Eppendorf offers a packed-bed bioreactor, which circulates culture media through a bed of Fibra-Cel^® disk macro-carriers. These disks are made of a three-dimensional fiber-mesh material that can be packed into the center of the bioreactor, which does not have a typical impeller. According to the company, a packed-bed bioreactor with a working volume of 10.5 liters is equivalent to 706 roller bottles in terms of the surface area for growing cells.

References

1. (Accessed 2023) Small Molecule API Market Overview (2022 to 2032) 

2. (Accessed 2023) Salib, V. Comparing Small Molecule and Biologics Drug Development Challenges, PharmaNewsIntelligence 

3. (Accessed 2023) What Are "Biologics" Questions and Answers, U.S. Food and Drug Administration, modified 02/06/2018 

4. Accessed 2023) Katella, K. Comparing the COVID-19 Vaccines: How Are They Different? Yale Medicine, 05/22/2023 n

5. Hawkins, S. Biomanufacturing: How Biologics are Made, biotechPrimer, 06/16/2022 

6. (Accessed 2023) CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers, National Cancer Institute 

7. Baghrizade, R. (2021) Adherent versus suspension based platforms: what is the near future of viral vector manufacturing? Cell & Gene Therapy Insights Vol 7(11), pp. 1365–1371

8. Garcia-Ochoa, F, et al (2011) 2.15 Stirred Tank Bioreactors in Comprehensive Biotechnology, 2nd edition Vol. 2, pp. 179-198

9. Portner, R. and Fashian, R. (2018) Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture, in Growing and Handling of Bacterial Cultures, 

10. Upton, T. and Goral, V. (2021) How fixed bed bioreactors are changing the game for adherent cell culture-based vector production, Cell & Gene Therapy Insights, Vol. 7 (3), pp. 337-343