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Human cells form the source material for cell therapies and they require a variety of assays to detect bacterial, fungal, and viral contaminants.
Though cell therapy is a recent innovation, with the first therapies approved by FDA in 2017, the use of human cells has been a standard of care for decades in hematology and oncology (1). A cell therapy can be derived from a variety of sources, including hematopoietic, skeletal muscle, neural, and mesenchymal stem cells (i.e., adult stem cells that differentiate into structures such as connective tissues, blood, lymphatics, bone, and cartilage). Lymphocytes, dendritic cells, and pancreatic islet cells can also function as source cells. Many cell-based therapies currently in development are based on induced pluripotent stem cells (iPSCs), adult cells that have been genetically reprogrammed back into a pluripotent state (i.e., so that they can differentiate into one of many types of cells in vivo) (2).
There are two types of cell therapy: autologous and allogeneic. In autologous cell therapy, the source cells come from the patient to whom the therapy is administered. In allogeneic cell therapy, the source materials are cells from an independent donor, and the therapy can be administered to a number of patients.
Given the urgency of ensuring good patient outcomes, it is essential to have a strategy in place for preventing contamination of source cells, as well as the resulting cell therapies. Any biocontaminants found in critical zones of the cleanroom environment can endanger patients’ lives, and have a devastating impact on finances and reputation (3).
Maintaining an aseptic environment is critical to minimizing the risk of contamination from extrinsic sources. Intrinsic contamination risks also exist in the cell manufacturing processes, however, primarily due to the fact that the source cells cannot be sterilized. This can raise concerns over the risks of contamination as well as cross-contamination.
Contamination control over the manufacturing of a cell-based product requires mapping out the entire process (4). This approach helps developers and manufacturers understand the risks involved at each step of the manufacturing process. Having a proactive contamination-control mindset is key to mitigating risks in general.
“There are unique challenges in both allogeneic and autologous cell therapy production, given the human origin of the cells,” says Andrew Bulpin, head of process solutions, MilliporeSigma. In allogeneic therapies, these cells typically come from healthy donors, Bulpin points out, but there is a limit to how many doses can be grown from a single donation, given the limited number of times a cell can double. This introduces the need to address donor-to-donor variability in incoming material for therapeutic uses.
“Autologous therapies have the other extreme-each dose starts with the cells of the sick patient. This introduces supply chain challenges (i.e., harvesting, shipping from the patient, manipulating, shipping to the patient, and administering cells),” Bulpin adds. Care must be taken to ensure the chain of custody so that each patient receives his or her own cells. Adding to the complications is the variability in cell health from the patient, which demands that processing be extra robust.
To assess cell health and the presence of any contamination in autologous or allogeneic therapies, a range of assays is required to test the source materials. According to Bulpin, this includes assays that are highly sensitive and specific to a particular target contaminant (e.g., polymerase chain reaction (PCR) assays), as well as assays that offer broad detection capabilities, such as next generation sequencing (NGS), in-vitrocell-based assays, and in-vivoassays.
“The choice of assay is influenced by a number of factors, including the ability of the contaminant to be cultivated in vitroor tested in vivo, but also the expectations of the regulatory agencies that will be reviewing the assay data,” Bulpin states.
PCR assays are used to detect mycoplasma and disease-causing viruses in both autologous and allogeneic source cells, Bulpin says, and they typically generate results in one to three days. A broader cultivation assay-based on a rapid, semi-automated method-is often employed for the detection of bacteria and fungi, and usually generates results in five to 10 days, he says. “Turnaround of these assays is time critical in delivering successful treatment to the patient from whom the cells have been isolated. For allogeneic cells, broad detection assays are also used to address the risk of ‘unknown’ and known contaminants being passed from donor to recipient. Tests include the use of transmission electron microscopy, NGS, and in-vitrocell-based assays,” he says.
For commercial-scale manufacturing in the case of allogeneic cell therapies, it is important to perform cell banking and cell characterization to support scale up. This includes establishing and testing the working cell bank (WCB) and cells at the limit of in-vitrocell age (LIVCA) separate from the original master cell bank (MCB).
Bulpin points out that the specific requirements for the detection of contaminants at the WCB and LIVCA level are laid out in relevant regulations and guidelines for advanced therapy medicinal products (ATMPs). These requirements are similar to the requirements for the MCB, with less testing required at the WCB level. “In particular, the risk of introducing contaminants during scale-up must be addressed through testing, including the detection of human viruses, bacteria, fungi, mycoplasma, and pyrogens,” he says.
The requirements are similar for controlling the vector material and associated production cells. Rigorous testing should be performed on all starting and in-process materials, such as plasmids, vector production cell banks, vector bulk harvests, and purified bulk, says Bulpin. It is at this stage that specific risks, such as the presence of replication-competent viral vectors in bulk harvest and transduced cells must be addressed, he explains.
When moving from clinical to commercial-scale manufacturing for a cell therapy, other considerations must be taken into account, and phase-appropriate validations used to assess the materials. During early clinical phases, for example, assays used for the release of clinical material should be suitably validated and qualified in the presence of a test matrix that uses a single batch of material. For late-stage or commercial-scale development, assays should be fully validated and qualified in the presence of a test matrix using a minimum of three batches under a formal product-specific qualification, Bulpin says.
A battery of tests is generally used to assess and validate the purity of the cell therapy end-product. Tests include PCR assays for viral pathogens and mycoplasma and a sterility assay for bacteria and fungi. For cell therapies that have been transduced using a viral vector, the risk of replication-competent virus being present must be addressed, Bulpin asserts. This is typically done using a detector cell-based in-vitrocultivation assay with a vector-specific endpoint test. “Furthermore, purity of the cell therapy must be authenticated to the level of the individual. Finally, assays used for lot release of clinical or commercial material should be suitably validated and qualified in the presence of test matrix,” he concludes.
The complex nature of cells and cell-to-cell interactions calls for advanced assays to help detect even minute levels of contamination and to validate their purity. The most advanced analytical assays that are used for detecting contamination in human cell sources include novel molecular sensitive techniques with broad molecular detection capabilities. Examples include deep sequencing/next-generation sequencing/high-throughput sequencing, degenerate polymerase chain reaction for whole virus families or random priming methods, hybridization to oligonucleotide arrays, and mass spectrometry, according to Bulpin.
1. D. Clarke, et al., Cytotherapy 18 (9) 1063–1076 (2016).
2. ARM, “Cell Therapy,” alliancerm.org/technologies/cell-therapy, accessed March 20, 2020.
3. R. Hansen, “Reducing the Risk of Bio-contamination in Gene, Cell and CAR-T Therapy,” Bioquell.com/news, August 2018.
4. Microrite, “Mitigating Contamination Challenges in Cell Based Regenerative Therapies,” microrite.com, accessed March 13, 2020.
Vol. 33, No. 4
When referring to this article, please cite it as F. Mirasol, “Contamination Control for Cell Therapy,” BioPharm International 33 (4) 2020.
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