Upstream Manufacturing for Cell and Cell-based Gene Therapies

Cell and cell-based therapies have seen explosive growth since 2017 when Kymriah, the first receptor-T cell (CAR-T) therapy, was approved for use in the United States as a treatment for lymphoma.^1,2 Today, CAR-T is regarded as mainstream within the emerging advanced therapy industry, with up to 12 new cell (and gene) therapies expected to be approved in the U.S. in 2023 alone.¹

However, despite its early successes, cell therapies like CAR-T still face limitations and challenges, sometimes due to the quality of the starting cells or difficulties during processing. In this article, we discuss what to consider during upstream manufacturing of cell and cell-based therapies, including during cell harvesting, preservation, and growth.

The limitations of cell therapies

Cell therapies use cells to fight cancers, deliver drugs, or repair tissue damage. Immune, stem, and skin cells are among the cell types used. In CAR-T, an autologous therapy, a patient’s own immune cells are removed and reengineered to fight cancer. In other types of cell therapy, called allogeneic therapies, cells are collected from a donor and engineered to treat a range of patients, as with more traditional therapies.³

CAR-T is the most established cell therapy but, according to Rob Tressler, Co-founder and Chief Scientific Officer of Excellos, it’s not yet completely effective for all patients. “Right now, in clinic, there are a lot of partial responses and not enough complete responses.” A partial response to a cell-based therapy might give a patient with Stage Four malignant melanoma 22 months of additional life, he says, but six percent of patients who have a complete response survive for ten years.⁴

Improving starting materials

According to Tressler, a major cause of the differences in response rate for adoptive immunotherapies is that as little as 5% to 20% of the cells returned to the patient may be effective at treating cancer. The rest may be irrelevant, or even harmful.

He believes this is partly due to the quality of the cells collected from the patient for autologous therapies or the donor for allogeneic therapies, as patients frequently are older and extremely unwell with compromised immune function, and allogeneic donors are not well characterized. This is critical for CAR-based therapies, as these cells to make them more efficient, he says, is like “putting a turbo charger onto a 1950s Volkswagen four-cylinder [as opposed] to a Maserati. It’ll perform better, but it won’t win the race.”

Therefore, the starting point for improving cell quality and health during manufacturing is ensuring the starting cells are high quality. “The freshness of the material is very important,” says Wini Luty, Senior Director of Biologic Operations at BioIVT, a cell donor center. Cell therapy manufacturers working with immune cells often use leukopaks³, mononuclear white blood cells preferentially separated out of a patient’s blood during a multi-hour collection.

According to Luty, leukopaks are more expensive than doing a whole blood draw but are a more concentrated and higher quality product. “It’s a clean premium product to start with, which is why customers prefer it [in terms of ensuring good] cell quality and yield,” she says. Leukopak donors also tend to be healthier, as they must sit through a multi-hour cell collection procedure. They also tend to have undergone more detailed pre-screening for health conditions, she says, adding that it’s important to screen for any medications, as these can affect cell viability.

There are multiple methods to get a pure population of cells from blood, but it’s critical to ensure the method is standardized, explains Fang Tian, Director of Biological Content at ATCC. “By using commercially validated reagents in a [well]-designed experiment, people can get consistent results.”

Careful cell thawing

Most therapy manufacturers use cryopreserved (frozen) cells instead of those freshly collected from donors, explains Luty. It’s easier to plan laboratory time and frozen cells can be transported longer distances from the clinic, where they are collected, to a manufacturing site.

To keep the cells viable, it’s important to avoid shocking them during thaw. “Even if cells are highly viable when you freeze them, you need to thaw them properly,” adds Luty. The cells need to be thawed gradually, drop by drop, she says. As the dimethyl sulfoxide (DSMO) in freezing media can be toxic, the cells should be transferred as quickly as possible into a warm culture media.

Maintaining viability in processing

The next stage in cell-based manufacturing is activating and modifying the cells to express a therapeutic protein and to optimize them for survival in patients.⁵ Companies use a variety of protocols, including using lentivirus to deliver genetic material. Modifying the cells by, for example, infecting them with a virus or punching a hole in them with electroporation, will temporarily reduce their viability.

Subsequently, the modified cells are encouraged to replicate during an expansion step. This larger population of cells can be returned to a single patient, or frozen ready to treat a larger group. During this stage, it’s important to keep cells healthy by understanding the relationship between cell density in the culture media, nutrition, and waste removal. “If you maintain a higher density of cells […] the stress will be much higher than at a lower density, so it’s essential to design and validate a feeding strategy for example, to balance these things,” says Dr. Tian.

Testing and monitoring

Tian explains that, to maintain cell quality, it’s important to use a standard set of validated methods for engineering cells, and to use Quality by Design (QbD)⁶ to specify how viable and potent each cell should be during processing. According to Tressler, it can be helpful to monitor the durability, metabolic fitness, and potency of the cells before, and after, the engineering step. This can be done with live-cell analysis systems, such as Axion BioSystems Maestro Z platform, which is designed to quantify cell growth and death in real-time with small electrical currents delivered through electrodes to the surface of a well plate.

“Monitoring viability in real time […] can help detect issues early so they can be addressed quickly,” writes Danielle Califano, Ph.D., Product Manager at Axion Biosystems. “This is especially important when the growth and viability, or ability to kill, is a critical quality attribute of [the product].”

Contamination of the cells also needs monitoring during upstream manufacturing, says Tian. Host cells can become infected by microbes or viruses, the reagent can become contaminated, and it’s even possible for host cells to become contaminated by cells of the same species. “I can’t stress enough that contamination [of host cells] isn’t always cross-contamination [from an earlier/different process],” she says. A short-read repeat assay can help to identify the contaminant cells within a culture. For example, mouse cells that have contaminated a human cell line, which—if left unidentified—might outcompete it.

References

1. (Accessed 2023) Hewitt, M. (2023) The Age of Cell and Gene Therapy is Here, Charles River Laboratories

2. U.S. Food & Drug Administration (2017) FDA approval brings first gene therapy to the United States 

3. (Accessed 2023) Charles River Laboratories Leukopak product information 

4. Seitter S.J, et al. (2021) Impact of Prior Treatment on the Efficacy of Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma. Clinical Cancer Research. Vol 27(19), pp. 5289-5298. 

5. STEMCELL Technologies (2020) CAR-T Workflow: Isolation, Activation and Expansion

6. Yu, L. X et al (2014) Understanding Pharmaceutical Quality by Design, AAPS Journal Vol 16 (4), pp. 771-783, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070262/