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3.2 Cell Processing and Manufacture

In most jurisdictions, the use of cellular products for medical therapy is regulated by governmental agencies to ensure the protection of patients. Although some stem cell-based products have now been approved for use in humans, a growing number of novel cellular products are being tested for a wide range of disease indications and present new challenges in their processing, manufacture, and pathways for regulatory approval. Given the variety of potential stem cell-based interventions, these guidelines emphasize that cell processing and manufacture of any product be conducted with scrupulous, expert, and independent review and oversight, to ensure the integrity, function, and safety of cells destined for use in patients. Manufacture of cells outside the human body introduces an additional risk of contamination with pathogens, and prolonged passage in cell culture carries the potential for accumulating mutations and genomic and epigenetic instabilities that could lead to altered cell function or malignancy, especially as such cells may outgrow others in the cultures. While many countries have established regulations that govern the culture, genetic alteration, and transfer of cells into patients, optimized standard operating procedures for cell processing, protocols for characterization, and criteria for release remain to be refined for the products of emerging technologies such as genome editing and novel derivatives of pluripotent cells and many attendant cell therapies.

Given the unique proliferative and regenerative nature of stem cells and their progeny and the uncertainties inherent in the use of this therapeutic modality, stem cell-based therapies present regulatory authorities with unique challenges that may not have been anticipated within existing regulations. The following recommendations involve general considerations for cell processing and manufacture.

3.2.1 Sourcing Material

Donor Consent
Recommendation Donors of cells for allogeneic use should give written and legally valid informed consent that covers, where applicable, terms for potential research and therapeutic uses, disclosure of incidental findings, potential for commercial application, and issues specific to the type of intervention under development.

Researchers should ensure that potential donors or their legally authorized representatives adequately understand the stem cell-specific aspects of their research participation. For a list of donor informed consent discussion points, see Section 2.3.2 and Appendix 3.

The initial procurement of tissue from a human donor may or may not require good manufacturing practice (GMP) certification depending on the jurisdiction, but this should always follow regulatory guidelines related to human tissue procurement and maintain universal precautions to minimize the risks of contamination, infection, and pathogen transmission.

Donor Screening
Recommendation Donors and/or the resulting cell banks developed for allogeneic stem cell-based interventions should be screened and/or tested as applicable for infectious diseases and other risk factors, in compliance with applicable regulatory guidelines (see Recommendation 2.4.3).

Tissue procurement for generating stem cell-based interventions is similar to procurement of cells/tissues for other potential clinical purposes and should be governed by the same policies. However, an important distinction between tissue donation and stem cell generation that increases the importance of screening is that, while tissues and organs other than blood are usually distributed to a limited number of recipients, somatic or pluripotent cells derived from allogeneic cells or tissues can potentially be implanted into a large number of patients. Donor screening should include medical examination, collection of donor history and blood testing. This process mitigates the risk of potential transmission of adventitious agents from the donor to patients receiving the stem cell products.  Regulatory agencies such as the (FDA; and the European Medicines Agency (EMA; have issued guidance regarding donor testing and screening. If high-specificity tests are available for adventitious agents, direct testing of the donated cells and tissue can mitigate the need to screen for such agents. However, this type of testing strategy should be prospectively discussed with regulatory authorities to ensure appropriate risk mitigation.

In some cases, in may not be possible to screen donors. For example, the donation of human embryos for the derivation of hESCs often occurs years after the harvesting of gametes and generation of the embryos, consistent with ethical and regulatory standards.  Therefore, screening of the donors at the time of gamete harvest is not appropriate. In these cases, the hESC cell bank can be thoroughly tested to ensure the absence of adventitious agents. However, there still remains the risk of pathogens for which validated tests are not available.

3.2.2 Manufacture

Cellular derivatives generated from stem cells and tissues are considered manufactured products and are subject to various regulations to ensure their quality (consistency, purity, and potency) and safety. 

Quality Control in Manufacture
Recommendation All reagents and processes should be subject to quality control systems and standard operating procedures to ensure the quality of the reagents and consistency of protocols used in manufacturing. Manufacturing should be performed under GMP conditions when possible or mandated by regulation.  However, in early-stage clinical trials it is understood that GMPs may be introduced in a phase appropriate manner in some regions.

The variety of distinct cell types, tissue sources, and modes of manufacture and use necessitate individualized approaches to cell processing and manufacture. The maintenance of cells in culture for any period of time places selective pressures on the cells that are different from those in vivo. Cells in culture age and may accumulate both genetic and epigenetic changes, as well as changes in differentiation behavior and function. Scientific understanding of genomic stability during cell culture and assays of genetic and epigenetic status of cultured cells are still evolving. Guidance documents from the FDA and EMA, as well as other documents, provide a roadmap for manufacture and quality control of cellular products. However, given that many cellular products developed in the future will represent entirely novel entities with difficult-to-predict behaviors, scientists must work with regulators to ensure that the latest information is available to inform the regulatory process. An important goal is the development of universal standards to enable comparisons of cellular identity, purity and potency, which are critical for comparing studies and ensuring reliability of dose-response relationships and assessments of mechanisms of toxicity.

Processing and Manufacture Oversight
Recommendation The oversight and review of cell processing and manufacturing protocols should be rigorous, and consider the manipulation of the cells, their source and intended use, the nature of the clinical trial, and the research subjects who will be exposed to them.

Stem cells can proliferate in culture for extended periods of time. This proliferative capacity carries risks. When maintained in culture for prolonged periods of time, cells may acquire mutations, grow and differentiate into inappropriate cellular phenotypes, form benign or malignant outgrowths, and fail to mature. Appropriate tests must be devised to maximize safety of stem cell derived products. 

Factors that are common to many stem cell products include the cells’ proliferation and differentiation potentials, source (autologous, allogeneic), type of genetic manipulation, if any, homologous versus non-homologous or ectopic use, their persistence in the recipient, and the anticipated integration of cells into tissues or organs (versus, for example, encapsulation). Culture composition and purity of desired phenotype vs. extent of residual undifferentiated progenitors should be carefully evaluated. In order to mitigate the risk associated with these factors, stem cell-based interventions should be thoroughly tested in safety and efficacy preclinical studies. Given that each stem cell therapy is a unique product, the evaluation of each product should be informed by the characteristics of the cell product and the risk/benefit associated with the clinical indication. 

Assays available for genetic and epigenetic assessment of stem cell-based products are evolving. Researchers should be aware of the limitations of these assays in predicting clinical outcomes. For cryopreserved or otherwise stored products, any impact of short- or long-term storage on product potency and stability must be determined. Human or xenogeneic materials associated with elevated risk (for example, human allogeneic and pooled source materials or xenogeneic reagents such as fetal bovine serum) should be stringently tested for safety and quality.

Components in Culture or Preservation of Cells
Recommendation Human or chemically defined components should be used in the culture or preservation of cells whenever possible. 

Cells are likely to be expanded in culture and might be exposed to xenogeneic materials before transplantation. Components of non-human animal origin present risks of transferring pathogens or unwanted biological material and can be quite variable in composition and bioactivity. As such the risk of transmission of viruses and other infectious agents is proportionately greater when using xenogeneic materials. Researchers can mitigate this risk by properly sourcing xenogeneic reagents from regions reasonably assumed to be clear of known pathogens.  If xenogeneic components cannot be reasonably removed, researchers should demonstrate the lack of feasible alternatives and document favorable risk/benefit in using animal-based components. These risks can be mitigated by using reagents in which the manufacturers have included pathogen reduction steps which remove pathogens (such as viral inactivation), or testing cell lines (such as CHO lines), used in the manufacture of these reagents. In addition, adventitious agent testing of the cells should include testing for appropriate xenogeneic pathogens; these requirements are specified in guidance documents published by the FDA, EMA and other regulatory agencies. Careful adherence to regulations and tracking of cells and reagents and the development of a risk mitigation plan is crucial to translation and uptake of cell-based therapies. 

Recommendation All reagents used in manufacturing stem cell-derived therapeutics should be of the highest quality available. 

In order to ensure the safety of stem cell products, the reagents and raw materials used in manufacturing should be manufactured under GMPs, whenever possible.  It should be noted that while manufacturing under GMPs ensures product consistency and purity, it does not necessarily assure absence of adventitious agents.  Therefore, a risk mitigation assessment and adventitious agent testing plan should be performed that address risks associated with all reagents used in manufacturing. 

In some cases, GMP grade reagents may not be available. In these cases, it is recommended to use reagents that comply with compendial requirements (e.g., USP, British Pharmacopoeia) and which were manufactured using the highest level of controls possible. It may also be necessary to clarify the appropriateness of reagents and raw materials with regulatory agencies if there are doubts about whether reagents that are not made under GMPs are of a sufficiently high enough quality for human use. It is essential that documentation including lot numbers and certificates of analysis and certificates of origin be retained for every reagent used in the isolation, expansion, and manipulation of stem cells that will eventually be used in the generation of therapeutic products.

Release Criteria
Recommendation Criteria for in process and release specifications should be developed during the regulatory review process. Culture-acquired genetic abnormalities may be a significant risk and should be part of in process and/or final product testing for stem cell products that have undergone extensive expansion in vitro.

The genetic and epigenetic stability of pluripotent stem cell-derived products warrants careful scrutiny. During manufacturing, it will be important to test cytogenetic abnormalities, as well as additional genetic and epigenetic parameters as defined by the protocol review process. The limitations of any such tests will be assessed and weighed against the risk /benefit and patient population for any given case. 

Recommendation Criteria for release of cells should include the assessment of off-target cells, using the most sensitive assays possible. 

Release criteria for stem cell-based interventions should utilize qualified or validated assays that assess the identity, purity, sterility, activity, and potency of the product. Because stem cell products may consist of heterogeneous populations of cells, it is important to include assays that detect and quantify the target cells responsible for the bioactivity of the product as well as other “off-target” cell populations. 

The off-target cells may be cells from different lineages, cells from the same lineage, partially differentiated cells, or undesirable cells such as undifferentiated stem cells.  For pluripotent stem cell-derived products, there is concern about residual undifferentiated pluripotent stem cells that may remain in the product. Given the nature of pluripotent stem cells and their innate capacity to form teratomas, there is a particular concern for the potential tumorigenicity of stem cell-based interventions. Therefore, the development of sensitive assays to detect contaminating undifferentiated pluripotent stem cells in the final product is critical, especially when delivering large cell doses. Further, the sensitivity of these assays should be documented in regulatory submissions. Some techniques (such as FACS analysis) may be suitable for doses of millions of cells but for doses in the 108-109 range more sensitive assays may need to be developed.

In summary, all stem cell-based interventions should be defined with their constituents as completely as possible, including at a minimum the proportion of therapeutic (on target) cells within the final cell product as well as minimizing the cells capable of causing major side effects, including tumor formation.