Gene and gene-modified cell therapies are one of the fastest growing segments of the biopharmaceutical market. These novel medicines have the potential to treat if not cure what were once thought to be untreatable diseases. With several having achieved success in the clinic and on the market, developers large and small are attracting significant investment designed to accelerate their advance towards approval and commercialization.

Indeed, despite the COVID-19 pandemic, financing for regenerative medicines and advanced therapies reached a record $19.9 billion in 2020, with gene therapy financing up 73% that year, according to the Alliance for Regenerative Medicine.¹ At the end of 2020, there were nearly 1,100 developers of cell, gene, and tissue-based therapies active worldwide (approximately 100 more than the previous year) conducting over 1200 clinical trials, with 31.5% in Phase 1, 56% in Phase 2, and 12.5% in Phase 3.

Two additional approvals were awarded to gene therapies in 2020 as well: Orchard Therapeutics’ gene therapy Libmeldy by the European Medicines Agency (EMA) and Kite Pharma’s Tecartus chimeric antigen receptor T-cell (CAR-T) therapy by the U.S. Food & Drug Administration (FDA). Eight additional regulatory decisions are expected in 2021 for eight new regenerative medicine products.¹

As early as 2019, FDA predicted it would receive more than 200 investigational new drug applications per year for cell and gene therapies and that by 2025 10 to 20 cell and gene therapy products would receive approvals each year.²

The Process is the Product
Regulatory authorities, are, however, watching gene and cell therapy manufacturing closely because, as is often noted, “the process is the product.” Gene and gene-modified cell therapies are produced using viral vectors. For gene therapies, the vector is the therapy, serving as the delivery vehicle for the gene of interest that will replace missing or dysfunctional genes. In gene-modified cell therapies, such as CAR T-cell treatments, viral vectors are used to deliver genetic material to cells to engineer them in some manner that provides enhanced or additional bioactivity.

The most used viral vectors are adeno-associated virus (AAV) and lentivirus (LV), but adenovirus and some retroviruses are also being employed for gene and gene-modified cell therapy candidates in the clinical pipeline.

These viral vectors are produced via cell culture, most often transfection of mammalian cells (typically HEK293) with plasmid DNA, but in some cases (such as for AAV) via coinfection of insect-based baculovirus cells or using human-derived herpes simplex virus (HSV) Type 1 systems.

More stable packaging and producer cell lines have been developed and used in some cases, but these solutions have much longer development timelines and require significant additional improvements before they will be widely employed.

With gene therapies, minimizing the time it takes to get into the clinic and more importantly reach the market is essential, as the first to launch will dominate the market; unlike with monoclonal antibodies (mAbs), there is no opportunity to gain market share for later entrants with curative products.

Consequently, the focus is initially on demonstrating drug efficacy and less so on process efficiency, robustness, and scalability. Indeed, many gene therapy processes initially developed at lab scale in static plasticware were sufficient for discovery work and even the production of early-phase clinical trial materials. However, they involve numerous manual manipulations and are not practical for large-scale.

Initial Focus on Suspension Cell Culture
Most gene therapies, including those in development today, begin as adherent cell-culture processes because adherent cells are complex and costly at the pre-clinical stage. They provide a means for rapid discovery and development and entry into the clinic.

Conventional biologic manufacturing processes for recombinant proteins and monoclonal antibodies (mAbs), however, are performed in suspension in stirred-tank reactors (STRs). These reactors were first developed for use in the chemical industry and then adapted for brewing and ultimately biologics production. For mAbs, they offer many advantages, including scalability and well-known fluid dynamics behavior, which allows for automated control and a high level of reproducibility. Over several decades, Chinese hamster ovary (CHO) cells have been adapted to perform well in suspension cell culture, affording robust and highly productive cell lines capable of yielding dramatically higher titers than initial cell lines.

Scaling of suspension cell culture processes in STRs, which increasingly are single-use systems, can be achieved readily from the milliliter to the 6000 L or greater scale. An inoculum is first prepared in a shake flask and then the volume is increased, typically in 10-fold steps in smaller bioreactors, until the necessary seeding quantity is obtained.

Limitations of Stirred Tank Bioreactors
Suspension cell culture in STRs has limitations for viral vector manufacturing, however. Cell culture of any type requires suitable concentrations to enable communication between the cells via secretion and absorption of various cofactors. Because of concentration effects in STRs, production bioreactors must be seeded at high concentrations—much higher than for the initial adherent processes—bringing added time and cost.

The suspension cells are then adapted from adherent cells. Since they are not in their natural state, they do not behave in the same manner and tend to afford lower yields than their adherent counterparts. Work on the adaptation of HEK293 cells for suspension has only proceeded for a few years, not decades, so they tend to be less robust at large scale, and thus many challenges with scaling also remain.

Transfection of HEK293 cells with plasmid DNA requires homogeneous mixing to be effective but achieving homogeneity within large STRs is difficult as mixing efficiency decreases with scale. Agitation and gas flow rates (sparging of oxygen, leading to the presence of large, dispersed bubbles) must be increased non-linearly to account for increases in the ratio of the total vessel volume (m3) to the surface available for heat and gas transfer (m2). Greater agitation means higher mechanical forces and greater risk for local oxidative stresses.

The mammalian cells used for viral vector production are sensitive to these shear and oxidative stresses prevalent in large STRs. Shear stress can also damage the plasmid DNA and plasmid DNA-transfection agent complexes required for transfection, creating the need for precautions during pumping as well as during the actual process.

Both the shear and oxidative stresses negatively impact the process. This often requires additional development work to move suspension viral vector processes from the lab to the pilot plant to commercial production—with yields generally remaining suboptimal.

Additionally, the typical lab-scale transfection process involves repeated washing of the transfection mixture. This is not feasible for large-scale suspension processes in STRs because it would involve hundreds to thousands of liters of liquid. To avoid this issue, the cells are either concentrated via filtration or the transfection mix is added directly to the cell culture without washing—both approaches add time and cost.

Interest in perfusion cell culture is increasing as a means for sustaining cell culture for longer periods to achieve higher productivity. In STRs, perfusion requires use of an external cell retention device, however, low-shear options do exist for this technology, which helps minimize cell stress.

Adherent Cell Culture in Fixed-Bed Bioreactors 
Adherent cell culture in fixed-bed reactors (FBRs) provides an alternative to suspension culture in STRs. Immobilized on the bed, the cells are decoupled from the liquid medium, allowing for optimization of process parameters without impacting the cells. Specifically, FBRs contain a solid but permeable support matrix that encourages homogeneous distribution of the adherent cells and through which media is gently circulated or perfused with minimal mechanical or oxidative stresses, leading to higher growth and productivity than is possible in FBRs.

The design of the fixed bed supports approximately the same quantity and throughput of cells with a volume reduction of 100-fold compared to STRs. The lower volume translates to a more compact footprint with a 50 L FBR having throughput equivalent to an STR of 1000 L or more.

The much higher cell densities in FBRs also dramatically reduce the required seeding quantities and thus a lower operational burden for seed generation. Since the cells are naturally retained by the bed, FBRs are advantageous for perfusion processes as well because there is no need for complex cell retention devices, which lowers process complexity and cost while also reducing the potential for exposure of the cells to shear forces.

Furthermore, the ratio of vessel volume to surface for gas and heat exchange does not change as much when scaling-up or down, making it easier to maintain homogeneous conditions inside the vessel. This property also simplifies the transfection process, which can be directly transferred from small to large scale without the need for cell concentration, dilution, or redevelopment of the transfection mixture.

This feature of FBRs enables linear scalability from the lab to the industrial scale leveraging single-use technologies and a high level of automation and control, enabling more rapid development and commercialization with reduced risk.

It should be noted that in adherent FBR processes, the inoculum is prepared by growing the cells first in flatware and then in a multi-tray dish(es). When enough has been produced, the cells are enzymatically detached to seed the FBR. To promote even cell distribution throughout the bed, it is essential to ensure that the cells are present as single units. Care should be taken if shear-sensitive viral vectors are produced outside of the cells, as they will circulate in the FBR.

Applying FBRs to Vector Manufacturing
Univercells Technologies developed the scale-X FBR portfolio for easy transfer and scaling of existing adherent processes. The scale-X hydro (2.4 m2), scale-X carbo (10–30 m2), and scale-x nitro, 200–600 m2) support process development, pilot scale, medium-to-large-scale industrial, and larger scale industrial production of gene and gene-modified cell therapies, respectively. The scale-X bioreactor portfolio has also demonstrated to be applicable for suspension-based processes. In this instance, the suspension cells are entraped in the fixed-bed to enable a direct process transfer from suspension-based processes to the scale-X FBR without impacting process quality or productivity.

The design results in conditions like those observed in static plasticware, with gentle circulation of the media across and through the cells. A tightly packed support matrix consists of spiral-wound, non-woven polyethylene terephthalate (PET) fabric layers that allow for high cell densities in a small-footprint bioreactor. A magnetic centrifugal impeller provides good mixing to ensure even availability of nutrients throughout the fixed bed and aeration via the creation of a “falling film” in the vessel headspace to increase the surface area available for gas exchange and realize a homogeneous environment in both the vertical and horizontal directions.

The linear velocity of liquid media travelling through the fixed bed remains constant across scales and, as a result, the cells experience similar low-shear conditions. A high level of automation, meanwhile, ensures control of key process parameters.

Meeting Large-Scale Viral Vector Manufacturing Needs
The basic mechanics and operational constraints of stirred tanks bioreactors can lead to extended process development timelines and impact manufacturing productivity, efficiency, and cost. And while suspension cell culture is widely employed to produce viral vectors, more robust and scalable solutions are needed to achieve true industrialization. As advances continue to be made in both adherent and suspension-based processes, a platform that supports both approaches will optimize productivity and response time by decoupling the process from technology.

Cell culture in fixed-bed bioreactors offers a solution for the large-scale production of viral vectors and will play a crucial role in facilitating the manufacture of large quantities of affordable gene and gene-modified cell therapies. With the scale-X FBR, early development processes can be directly transferred and scaled in smaller footprint single-use equipment using simpler processes that afford greater productivity and higher yields.
In addition, recent and ongoing investigation of the applicability of the scale-X FBR for suspension cell culture for viral vector manufacturing has suggested that this single-use bioreactor technology also offers benefits for gene therapy developers leveraging suspension-based processes. Specific data will be forthcoming soon. 

References

1. Alliance for Regenerative Medicine, “Alliance for Regenerative Medicine Annual Report Highlights Record Sector Growth and Resilience in 2020,” Press Release, March 16, 2021. https://alliancerm.org/press-release/alliance-for-regenerative-medicine-annual-report-highlights-record-sector-growth-and-resilience-in-2020.
2. Statement from FDA Commissioner Scott Gottlieb, MD, and Peter Marks, MD, PhD, director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. News release. FDA website. January 15, 2019. https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics.