Cell therapy using embryonic or adult stem cells for regenerative medicine is generating high interest in the global medical community and in the general population.Physicians and patients are looking to cell therapies as potentially curative treatments for diseases such as diabetes, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Graft versus Host disease (GvHD), and cancer. Cell-based therapeutic products have been administered in clinics for nearly 90 years in the form of blood transfusions and for 50 years in the form of bone marrow transplants. With vast improvements over the past two decades in cell characterization, isolation, and in vitro manipulation, cell therapy has grown to include FDA-approved products such as

• cell-infused support matrices to regenerate damaged or injured skin

• investigative products for clinical trials such as cell-based vaccines for autoimmunity and cancer

• cell-based therapeutics for the treatment of cardiovascular, inflammatory, autoimmune, and neurodegenerative diseases and cancer. Furthermore, an increasing number of novel cell-based products for such indications have progressed closer to regulatory approval within the past three years.

PRODUCT FOCUS:CELL THERAPIES

PROCESS FOCUS:MANUFACTURING

WHO SHOULD READ: R&D, PROCESS DEVELOPMENT, QA/QC, MANUFACTURING

KEYWORDS: CELL ISOLATION, CELL PROCESSING, CLINICAL TRIALS, FACS (fluorescence-activated cell sorting), flow cytometery, CGMP

LEVEL:INTERMEDIATE

Cell therapy can be defined as the treatment or prevention of disease by administering cells that have been selected, manipulated, or altered outside the body. As more cell-based therapeutic products progress into clinical trials and commercialization, developing bioprocesses compliant with current good manufacturing practices (CGMP) has been challenging. This is because the final products are not traditional biological (secreted) molecules such as monoclonal antibodies but rather the cells themselves.

Industry focus has turned to one element of the cell-based product manufacturing process, cell isolation, because of the demonstrated importance of cell purity and the special considerations related to protocol compliance to CGMP regulations. Here, we compare technical aspects of fluorescence-activated cell sorting (FACS) and other cell isolation methods, summarize regulatory guidance for CGMP-compliant cell isolation, and provide points to consider when using FACS within a CGMP compliant manufacturing environment. This is not intended to be a comprehensive review of the field, but provides a basis for further reading and understanding.

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General Steps in Cell-Based Product Manufacturing

The general steps for manufacturing a cell-based product are harvesting, debulking and isolation, ex vivo manipulation (e.g., activation, expansion, and/or genetic modification), and cryopreservation. Some cell-based products might be used after isolation without further manipulation or cryopreservation. Others might involve cell isolation after ex vivo manipulation. However, nearly all cell-based therapy products require some level of cell isolation or purification as part of the overall manufacturing process. These products are often sourced from heterogeneous cell populations with rare cells that need to be enriched or with contamination from unwanted cells that must be removed.

The production of regulatory T (Treg) cells, which are under study as a potential treatment for GvHD, offers an example of a selection strategy playing a key role in determining the activity and safety of a therapeutic product (1,2,3). Treg cells, which promote tolerance after allogeneic (donor) organ transplant or prevent GvHD after stem cell transplantation, share several cell surface antigens with alloreactive T cells, which are capable of causing GvHD in recipients. Isolation of Treg cells from peripheral blood using only a single antigen parameter results in contaminating alloreactive T cells. Furthermore, if the expansion of Treg cells is part of the manufacturing process, alloreactive T cells might be preferentially expanded under certain culture conditions, thereby resulting in lower therapeutic activity of the final product or unintended effects after patient administration (4,5,6).

Other examples in which cell purity is a factor include treatments based on human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cell-derived products. Final products contaminated with undifferentiated hESC or iPS cells might form teratomas after injection (7,8). For some such issues, purification of specific therapeutic cell populations or removal of unwanted cell types during manufacturing might be critical in reducing some potential risks.