Cell-based therapy is progressing quickly, at least in terms of research and development. Important trends include hematopoietic progenitor cell transplantation, autologous therapies for wrinkles and cartilage defects, autologous cellular immunotherapies, and chimeric antigen receptor (CAR) T-cell therapies. In addition, many novel cell therapies are in the development pipeline.

It must be emphasized, however, that cell-based therapies are complex. They represent living products. Accordingly, they present unique manufacturing challenges. These include challenges in process scale-up, process analytics, and product characterization. Until these challenges are overcome, cell-based therapies won’t receive widespread rollouts.

Initiatives to improve the manufacture of cell-based therapies were highlighted at PepTalk: The Protein Science Week. This event, which was held January 20–24 in San Diego, included a “Cell and Gene Therapies” pipeline that delivered world-class presentations about emerging opportunities and persistent hazards in manufacturing and analytics.

Shorten time to therapy

One of the major challenges for cell therapy is that the manufacture of autologous products cannot be scaled up. Unlike traditional biopharmaceutical products, where larger reactors often represent a convenient solution to the scale-up problem, autologous cell therapies are single-batch products from individual patients. One strategy for getting around this difficulty is using therapies based on natural killer (NK) cells rather than T cells.

At the PepTalk event, this strategy was discussed by Sandro Matosevic, PhD, assistant professor, department of industrial and physical pharmacy, Purdue University. He presented his work on the developing CAR NK-cell immunotherapies against solid tumors.

Matosevic said that NK cells have a higher potential to be used allogeneically. That’s because they work when there is a human leukocyte antigen (HLA) or major histocompatibility complex (MHC) mismatch. Matosevic says that NK cells look for cells that are a mismatch and kill them, which means the bigger the mismatch, the better it works as a cell therapy. In contrast, T-cell therapies will not work when mismatched. In addition, CAR NK-cell therapies do not lead to graft-versus-host disease (GVHD), which is a major adverse event associated with CAR T-cell therapies in clinical studies.

Typically for autologous T-cell therapies, it takes 3–4 weeks to manufacture the therapy. That’s because blood has to be taken from the patient and taken to a facility so that T cells from the blood can be engineered into a therapy, and then shipped back to the hospital where the patient receives it.

“For NK cells, though, this can be shrunk a little bit because we can have off-the-shelf cells. If the cells are already waiting and the patient doesn’t have to give blood, they can be ready a little quicker,” asserted Matosevic. That’s accomplished by using stem cells to create synthetic blood cells in the laboratory.

NK cells engineered to target CD73 have been shown to kill glioblastoma cells, lung cancer cells, and prostate cancer cells in vitro. For glioblastoma, a notoriously immunosuppressive tumor type, targeting multiple pathways with NK cells instead of a single antigen has shown promise.

“Targeting multiple pathways at the same time through genetic engineering is really the way to go for these treatments,” Matosevic emphasized. “Targeting just one antigen at the same time like CAR T cells do for leukemia does not work for these tumors.”

One manufacturing challenge for NK cells is persistence. T-cell therapies can be persistent in the body for months, leading to a long-lasting immune response. NK cells typically last no more than two weeks. Persistence can be prolonged by infusing the cells into patients with cytokines. According to Matosevic, in a clinical trial, NK cells given to patients with IL-15 had a presence in the body up to or over 12 months.

“ It’s really an important consideration in terms of their activity when they’re given as drugs,” Matosevic pointed out. “You do want a higher response than what they’re able to sustain, but you don’t want them to be active forever.” Exosomes as alternatives to cells As promising as cell therapies are, they have some disadvantages. Those include issues with efficacy and cytotoxicity, high cost, long wait times, and adverse pharmacokinetics in the body. Exosomes are a potential alternative that can be adapted to deliver many different types of therapies. In nature, exosomes play a role in communicating signals in the cell as well as transmitting disease. As a therapeutic, exosomes have an advantage over cell therapies in that they do not need to be genetically engineered. Also, exosomes are made of cell membranes rather than synthetic polymers or modified cells. So, exosomes are well tolerated by the host.

Exosomes, however, also present some unique manufacturing difficulties. Their very small size makes them difficult to separate, and they overlap in size and surface chemistry with microvesicles, apoptotic bodies, viruses, and other objects in the cell. They don’t tolerate extreme chemical conditions, and their surface chemistry is not uniform, limiting the type of purification processes that can be used.

Wasfi AlAzzam, PhD, CSO at TechnoPharmaSphere, presented a novel process for manufacturing exosome therapies. “In cell therapy, you need to separate the cells from patients, and then you need to genetically modify the cells,” he said. “Accordingly, the degree to which these immune cells are accepted will vary. They may be attacked by the patient’s immune system. Exosomes don’t have these side effects, so they can be very good therapeutic agents.”

On the analytics side, some useful technologies for detection of exosomes are SEC-MALS (size-exclusion chromatography with multiangle light scattering), SECimmunofluorescence, and SEC-picogreen. These can provide insights that complement conventional monitoring methods. SEC doesn’t discriminate exosomes from non-exosomal vesicles, but it can give a helpful perspective on the overall size distribution of contaminants. Reduction of contaminants can be carried out by tangential flow filtration or commercial kits that precipitate contaminant vesicles.

AlAzzam noted that other size measurements, such as those obtainable via Luminex’ Amnis flow cytometry instrumentation, can enable estimation of exosome size, counts, and conservation of immunological integrity. The overall exosome development process, he added, could be scaled up or scaled down for development, validation, and manufacturing.

Meeting supply demands

Kelly Kemp, PhD, director of process development at ViaCyte, offered an overview of her company’s work scaling up cell-based processes for clinical trials. ViaCyte is developing islet replacement products that are based on pluripotent stem cells as the starting material for manufacturing. She says that ViaCyte is trying to overcome the limitations of islet transplants such as limited supply, high cost of organ procurement, inconsistent quality, and immunosuppression.

ViaCyte has a portfolio of three different product candidates that are based on its core technologies—human pluripotent stem cells, directed differentiation to pancreatic precursors, and a family of subcutaneous delivery devices. Its most advanced program, PEC-Direct, delivers pancreatic precursor cells in an open device that requires the use of immune suppression. PEC-Encap delivers the cells in a closed device designed to allow for nutrient exchange but protect the cells against immune rejection. A third preclinical program, PEC-QT, which ViaCyte is pursuing in partnership with CRISPR Therapeutics, aims to use gene editing to create cells that evade the immune system.

Kemp said that there is increasing demand for a supply of cells not just in the field of cell therapy but also in other fields, creating a need to manufacture a large amount of pluripotent stem cells, particularly as clinical development progresses. ViaCyte’s initial production process used 2D flasks and 3D roller bottles.

“If we were to scale this out, basically replicate that process, the number of lots we would need to produce for the commercial phase becomes unrealistic very quickly,” Kemp noted. “So, we need to develop a scaled-up manufacturing process to efficiently produce high-quality cells.”

Scale-up hurdles include time and resource constraints, difficulties in starting the development process early enough, and the need to develop scale-up technologies that can meet stringent process and forecast requirements. Kemp highlighted the stirred-tank bioreactor as a scale-up technology with many advantages. For example, there are no surface area limitations, and processing may occur in a closed and controlled environment.

“We can consistently create aggregates of a specific size and then also expand them while maintaining pluripotency,” she declared. “Similarly, with the differentiation process, we have a hundred runs under our belt where we’ve been able to demonstrate the ability to make pancreatic precursor cell aggregates efficiently and effectively in this 3D environment.”

ViaCyte, a regenerative medicine company that develops novel cell replacement therapies, is working to grow human pluripotent stem cells (hPSCs) as aggregates in a 3D stirred-tank bioreactor to support scale-up production efforts. Culture conditions are first characterized at a small scale, and then optimal parameters are applied to larger scale bioreactors. (A)hPSCs after aggregation and expansion in a stirred-tank bioreactor. (B) Flow cytometry results show that the hPSC aggregates express proteins for pluripotency (SOX2 and POU5F1/OCT4) and not of proteins that would indicate spontaneous differentiation (FOXA2). (C) Similarly, gene expression analysis indicates comparable expression of genes for pluripotency (POU5F1/OCT4) when hPSCs are grown in 2D in flasks (blue) or as 3D aggregates in a stirredtank bioreactor (green), and no expression of off-target genes (FOXA2).

Playing catch-up

Manufacturing control in cell-based therapy products is sometimes limited due to a relative lack of product knowledge, suggested Mo Heidaran, vice president of technical, regulatory, and technical CMC consulting for cell, gene therapy, and tissue engineering at Parexel International. He noted that in some cases, manufacturing control for cell-based therapies may lag that for biologics by 15–20 years.

One of the biggest hurdles to overcome, he emphasized, is product consistency. “Many of these products have been developed in academic settings and are very complex,” he said. “Characterization of the product is very challenging because of the biological complexity and poorly defined mechanism of action. One issue that has to be overcome is understanding the complex attributes of these types of products—and recognizing that the process really is the product.”

With the addition of genetic modifications, including CAR T-cell therapies and edited cellular products, these technologies bring additional layers of complexity.

Considerations for manufacturing include how to deal with manufacturing changes, product quality assessment, challenges in collecting biological source material, compliance with donor eligibility requirements, donor-to-donor variability of starting materials, and limited shelf life of products. Establishing good manufacture control requires extensive characterization of the drug product by better understanding the relevant critical quality attributes, critical process parameters, and key process parameters.

Because the FDA has a phase-based approach for initiating Investigational New Drug Applications, there is more emphasis on the safety of products and less emphasis on control of manufacturing and consistency. As a result, companies tend to pay less attention to critical aspects of manufacturing control. Heidaran recommended that companies pay special attention to the development of appropriate phase-based manufacturing control— especially if the companies receive expedited program designations and can proceed without having to introduce major manufacturing changes during a licensing trial.

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