Researchers who introduce laboratory-scale production processes for emerging therapeutic modalities are justifiably proud. However, these researchers might, if pressed, admit that these newly developed processes tend to be long on promise and short on commercial manufacturing substance. All too often, these processes fail to address matters such as scale-up parameters and regulatory requirements.

Unresolved scale-up and regulatory issues are certainly evident in the laboratory-scale platforms for cell and gene therapies. Also unresolved is the matter of which platform types will fade into the background, and which will advance. Perhaps producers of cell and gene therapies will come to focus on a few platform types, following the example set by producers of conventional biologics. (During a decades-long evolution, the producers of monoclonal antibodies and recombinant biologics came to rely on CHO cells. It was hardly a coincidence that during this time, conventional biologics went mainstream.)

Young fields have growing pains, especially fields evolving as rapidly as the one for cell and gene therapies. To help this field mature, manufacturers are introducing more sophisticated tools, and regulatory authorities are preparing more detailed guidance documents. These developments will facilitate the cell and gene therapy market’s delivery of life-saving therapies to those in dire need.

Communication and consolidation

According to Jose Vidal, PhD, COO of CytoImmune Therapeutics, companies in the cell and gene therapy field are engaged in extensive communications with each other and with regulators. As a result, some processes are beginning to be standardized. At some point, one or two platform types will prevail.

Whatever platform types come into favor, they will have to be capable of producing therapeutics that meet regulatory requirements. Unfortunately, the path to regulatory fitness is unclear. Yes, dozens of FDA and EMA documents for gene and cell therapy have been issued, but they have been adapted from documents for biologics. As a result, they are not so helpful. “We are building the plane with regulators as we fly,” Vidal remarks.

Generalized regulations are open to interpretation and challenging for process development. “The majority of the time,” Vidal admits, “we are not in complete alignment with the regulators.”

Timing plays an important role in managing process development. When research-grade processes are introduced, they are relatively crude. And due to the young age of the industry, the companies that supply raw materials have yet to reach the GMP level.

CytoImmune Therapeutics Team
CytoImmune Therapeutics operates a facility in Puerto Rico that manufactures chimeric antigen receptor (CAR)-engineered natural killer (NK) cells. The facility, which covers 37,000 square feet, employs more than 30 people, including the 10 people shown in this photograph. The team is helping to advance CytoImmune’s differentiated pipeline of off-the-shelf tumor-reactive CAR NK cell therapies. Indications include non-small cell lung cancer and other solid tumors, as well as acute myeloid leukemia and multiple myeloma.

Companies are allowed to start with processes that require development. But if they wait too long and bring changes to the industrial process late in clinical trials, the changes may call for comparability studies or add-ons to clinical trials. If resources are available, research-grade and industrial processes can be developed in parallel. Alternatively, even a suboptimal industrial process can be developed, and post-approval commitments can be proposed during the approval process.

“As soon as we move into Phase II, we try to get the industrial process in place,” Vidal says. “[We try to ensure that] we do not have to adjust it much.” CytoImmune is aware that regulations are promulgated by multiple jurisdictions. For each jurisdiction, it stays current with every relevant Common Technical Document module, every Good Manufacturing Practice guidance, and every Chemistry, Manufacturing, and Controls guidance. Then the company complies with the regulations that are most stringent, whatever jurisdiction they may have come from.

“In the long run, critical mass will speed up the process,” Vidal predicts. “So many programs are going on simultaneously that the regulators are exposed to a lot of information and modalities. This will help them expedite the process of issuing more defined guidance.

“It is a two-way street. Consolidation and communication between all stakeholders will accelerate improved regulations. The benefit is so big that it is all worth it. There is no lack of passion for making this happen.”

Investing in future cell therapies

Cell therapies leverage the sophisticated capabilities of the immune system. “The complexity of chimeric antigen receptor [CAR] T-cell therapies [exceeds that of] traditional biologics or small-molecule medicines,” says Henrik Andersen, PhD, senior vice president, Cell Therapy Development, Bristol Myers Squibb. “[Producers of CAR T-cell therapies] use a patient’s own cells to start a highly sophisticated and personalized manufacturing process.”

Significant challenges come from the complexity, cost, and availability of the many investigational materials, which include plasmids and viral vectors. Analytically, many of the vector tests require the use of cells as indicators of the vector characteristics, thus adding further variability and requiring considerable time and resources.

Bristol Myers Squibb cell manufacturing facility in Devens, MA. Soon, the facility will provide 244,000 square feet of floor space and house equipment tha
Bristol Myers Squibb indicates that construction is nearly complete on its new cell manufacturing facility in Devens, MA. Soon, the facility will provide 244,000 square feet of floor space and house equipment that will enable quick capacity expansions for clinical and commercial cell therapy products. These products will include Breyanzi, a cell-based gene therapy for large B-cell lymphoma.

“We need to consider the pace of the evolution of platforms and technologies,” Andersen emphasizes. “The technologies we employ today must still be relevant when products are in the commercial stage.” To cope with technological change, producers are simplifying process development and reducing cost of goods for therapies based on autologous cells, allogeneic cells, and induced pluripotent stem cells.

“We also need to ensure that platforms enable ‘speed to clinic’ while meeting quality objectives,” Andersen adds. “A large part of the cost is labor and the operation of the facility. We continue to explore technologies that decrease manual manipulations and manufacturing footprints.”

The company is heavily investing in high-throughput capabilities that will allow it to rapidly increase its understanding of the process and identify the critical process parameters to improve yield and drive vector quality. According to Andersen, one exciting area is the use of nonviral delivery technologies, which could drastically reduce the complexity, duration, and cost of the overall cell therapy manufacturing process.

Technology needs to evolve with the field to better define quality as a function of enhanced analytics. As the attributes of viral vectors and drug products become better understood, the introduction of improved processes for viral vectors and drug products should proceed more seamlessly.

“With our numerous strategic partnerships,” Andersen declares, “we are reimagining the future of cell therapy.”

Process design considerations for CAR T cells

Inherently, allogeneic CAR T cells have certain challenges when it comes to scale-up. “For the allogeneic model to work, unit operations must accommodate cell numbers that are often an order of magnitude (or more) greater than corresponding operations in autologous CAR T-cell processes,” says James Bolling, senior director, Cell Therapy Process Development, Precision BioSciences. While cGMP-compliant processing equipment options remain limited for cell therapy as a whole, equipment capable of handling the high cell numbers and volumes generated in allogeneic CAR T-cell processes are quite scarce.

Bolling recognizes that a number of process design considerations are important. He also believes that certain considerations can reinforce each other and result in the most impactful approaches. These considerations include controlled cell expansion and limited process duration, highly precise gene editing with targeted CAR integration, limited manipulation and hold times, and optimized cryopreservation procedures.

The two main drivers of the process scale are the number of T cells at the start of the process, and the number of cell doublings allowed during the expansion phases. Precision BioSciences has driven scale by focusing on the input cell number, while actually shortening the process and controlling cell expansion.

To increase the input cell number, the company has invested in deep understanding and optimization of the apheresis collection and T-cell enrichment processes. With this combination of high starting cell number and short process duration, the cell phenotype and functionality is optimized without significantly compromising process yield.

According to Bolling, cryopreservation can be one of the most impactful unit operations on final drug product quality, especially if it is not well controlled. “We have invested tremendous effort in optimizing our drug product formulation and each step of the controlled rate freeze protocol,” Bolling details.

Besides having conducted freezer mapping and vial load studies, the company has developed a freeze protocol that is capable of precisely controlling prefreeze cooling, ice nucleation, and latent heat of fusion to cool the cells consistently at the target rate.

Ramping up AAV production

Plasmid manufacturing was a natural addition to Forge Biologics’ suite of scalable manufacturing services, which already included adeno-associated virus (AAV) process development, analytical development, cGMP manufacturing, and automated final fill capabilities. Together, these services offer a way to streamline the entire development process.

Forge Biologics is a hybrid gene therapy contract manufacturing and clinical-stage therapeutics development company. Besides being poised to become the largest global manufacturer of dedicated AAV vectors, Forge Biologics has several new offerings that include research-grade and GMP-pathway plasmids for use in early-phase clinical trials. Full GMP-grade plasmids are scheduled to become available in 2023. Manufacturing processes currently range from 1 to 1,000 L and are on track to scale up to 5,000 L.

“Our proprietary technologies are the basis of our platform,” says John Maslowski, COO of Forge Biologics. “They focus on a commercially oriented cell line and an adenovirus-derived (Ad) helper plasmid, enabling developers to go from research through commercial stages in a complete end-to-end process.

“Proprietary technologies include our suspension HEK293 Ignition Cell Line, which has a well-documented history allowing for commercial use. Our Ignition cells were clonally selected in-house. One of the selection criteria was packaging of full AAV capsids. The selected clone generated 40% full capsids at harvest and >90% after downstream purification.”

The company’s pEMBR Ad helper plasmid contains a simplified backbone with an improved safety profile. The reduced size of pEMBR allows for increased efficiency in manufacturing while maintaining AAV vector yield and quality.

“Our hybrid business model has allowed us to expedite the development and manufacture of FBX-101 for infantile Krabbe disease due to direct access to development and manufacturing services,” Maslowski asserts. Krabbe disease, a devastating neurodegenerative disease inherited in an autosomal recessive manner, affects about 1–2.5 in 100,000 people in the United States. The infantile form usually results in death by age 2.

According to Forge Biologics, FBX-101 has been shown to functionally correct the central and peripheral neuropathy associated with Krabbe disease, improve gross motor outcomes, and significantly prolong lifespan in animal models. The company asserts that its approach has the potential to overcome some of the immunological safety challenges observed in traditional AAV gene therapies and extend the duration of gene transfer.

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