Cell Expansion: The Rate Limiting Step for Cell Therapies

Emerging cell therapies—the next generation in biopharmaceuticals—bring new hope to patients but also new (and in some instances unanticipated) problems for developers to solve, particularly in achieving economies of scale, or manufacturability. The challenges presented by this ongoing paradigm shift are different from, but equivalent in magnitude to, those faced in the early days of therapeutic biotechnology.

“As newer manufacturing technologies come online, production methods must be industrialized to bring costs down and make these potentially curative therapies readily available to patients,” says Matthew Hewitt, Ph.D., Executive Director, Scientific Services Cell and Gene Therapy at Charles River.

Improvements will require optimizing the many steps in cell therapy production, which include cell preparation (harvesting, purification, transfection), and cell expansion.

First steps

Cells are harvested through whole blood collection, semi-processed buffy coat (with the plasma removed), or, most commonly, through leukapheresis (leukopaks) from peripheral blood.

“Whole blood is the least efficient method because it typically results in the fewest leukocytes,” Hewitt explains. “Buffy coats provide simpler processing, but you end up with the same number of leukocytes as with whole blood collection. Leukopaks still require some processing but typically yield the most cells with the least amount of upfront pre-processing.”

Many kit-based options exist for cell isolation, including magnetic beads, which allow isolation of specific cell populations from a larger, heterogeneous cell population, for example T cells. Since advanced therapies involve extensive manipulation of rare, delicate, living cells, there is no one-size-fits-all approach to obtaining suitable cells at appropriate yields and purities.

Most gene-modified cell therapies for cancer harvest and modify T cells, which exist in high numbers in peripheral blood. Other indications use hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs).

“HSCs are quite rare in peripheral circulation—comprising about 1% of cells collected—while MSCs and iPSCs are derived from progenitor cells,” Hewitt tells Biocompare. Again, no single source can be considered best, even for similar indications or gene transfer methods. Charles River, for example, has experience with a range of immune cell types, including dendritic cells, macrophages, and natural killer cells. “While T cells do make up the majority of work in the cellular immunotherapy space, they are not the only cell types being developed to treat cancer.”

Transfection and expansion—the “killer apps”

Most cell therapy programs use viral vectors to modify cells genetically through transduction. Transfection and electroporation, which do not require viral vectors, are also utilized. Electroporation uses an electrical current to disrupt the cell membrane, creating pores that allow the introduction of genetic material into cells without viral vectors, Hewitt says. “But due to the amount of current required to open these pores we see a drop in cell viability, which can be significant, depending on how well the electroporation transfection protocol was optimized.”

Transduced or transfected cells move into the expansion phase of the process, arguably the core unit operation in manufacture of cell therapies. A course of autologous cell therapy requires between several hundred million cells (e.g., in autologous CAR-T) to 50+ billion cells (autologous tumor infiltrating lymphocyte therapy). Allogeneic cell therapy manufacturing processes can yield tens of billions of cells, while numbers may be even higher for iPSC-derived therapies.

That’s a lot of cells, and the main reason why Hewitt advises simplicity, particularly in early-stage studies.

“The specific issue with cell expansion across different cell therapeutic modalities is that no single platform meets the needs of all therapeutic developers. Some developers may prefer to enter clinical trials with an open, manual manufacturing process. A key component of phase 1 trials is dose-escalation, and the dosing can be quite large, which makes automated manufacturing challenging. Once the dosing regimen is set, after phase 1, developers have a better understanding of the clinical dose going forward. They may then decide whether to switch to a closed, automated manufacturing platform.”

Industrialization

From a commercial perspective as well, cell therapies find themselves in a position not unlike that of therapeutic proteins 30 years ago: dozens of targets, platforms, and molecules, hundreds of preclinical and clinical investigations, and potentially billions of patients, but no clear path to industrialization.

”If cell-based treatments are to become widely available across multiple indications, improvements in automation, process simplification, and supply chain management will be needed to meet demand,” says George White, GM Product Management, Cell and Gene Therapy at Cytiva. “To scale up, manufacturing processes must move from being open and manual to closed and automated, reducing contamination and risk, increasing product consistency and efficiency, and adding traceability throughout the process to ensure chain of custody.”

Automation allows operators to quantify product attributes during critical process steps to tame the inherent variability of starting materials—fragile, living cells. Industrial and academic groups have already begun to adopt technologies that incrementally move the state of the art toward more automated, closed, and scalable systems that minimize risk and anticipate future increases in demand.

“Automation is particularly critical for producing single doses of autologous cell therapies because there is already so much risk of variability,” White adds. “That is why manufacturers can reap tremendous benefits from taking the time, during process development, to automate key steps and reduce the number of manual operations.”

At early adoption stages the main benefit of automation will not be throughput but consistency. Later, as developers gain more experience with autologous cell therapies, it will enable scaling out limited-scale therapies to treat more patients. And some day, when the holy grail of allogeneic therapies becomes feasible and scalable, you will read an article in Biocompare describing single-use 1000-liter bioreactors for expanding immune system cells.