A. Introduction

Development and manufacturing a cellular therapy based product under current Good Manufacturing Practices (cGMP) while adhering to all FDA regulations may seem a daunting task for any researcher or clinician. The following chapter has been designed to assist the reader in understanding the necessities of developing a safe effective cellular therapy product using cardiac repair as a prime example where cell-based therapy can potentially help treat numerous individuals suffering from a complex disease.

B. Testing and Characterizing the Cellular Therapy Product

Once the cell type has been chosen, donors identified and processing protocols established, several assays need to be performed in order to garner as much information about the cell before moving forward in the development of a cellular therapy product. The first assays that need to be performed are:

Cellular Counts

Generally most cellular therapies will depend upon a particular cell dosage, i.e. for an effective umbilical cord blood transplant a minimum cell dose of 2.3 x 107 cells/kg are required (1). The most effective and reproducible method would be an automated cell counter. Based on these analyses, if the cell counts are insufficient then a different processing, collection methodology, tissue source or even cell type must be selected in order to obtain the predetermined cell numbers required for cell therapy-based applications.

Purity of the Cell Population/Characterization by Flow Cytometry.

In order to determine the purity of the cell population and the viability at numerous stages (pre-process, post-process, during cell culture etc.) flow cytometry may be employed. There are numerous established cell surface antigens commercially available so characterization should be simple and quickly performed.

>Differentiation studies.

After the purity of the cellular therapy product has been determined, in vitro differentiation assays should be performed in order to determine the potential of a given cell type. It is imperative that the given cell type can easily and effectively differentiate into the tissue of interest, in this case cardiac tissue.

Molecular/Proteomic Characterization of the Cells

becomes particularly important if the cells that are going to be used in cardiac therapy are expanded ex vivo. It has been demonstrated with different clones of bone marrow-derived MSCs that have the same cell surface antigens have different proliferative capabilities and different protein expression patterns (2). Characterization of human MSCs using different molecular techniques also revealed that the cells show changes during expansion which is accompanied by changes in cell marker expression over the culture period (3). In addition, protein expression maps, created using two-dimensional polyacrylamide gel electrophoresis for continuous subcultures of clonal bone marrow MSCs up to 10 passages, also revealed variation in the proteome during cell expansion (4). These proteomic profile changes could represent very serious biochemical alterations to the cells, therefore concerted effort must be put into determining what biochemical changes are occurring to the cells particularly during expansion. Currently there are no robust approaches for verifying the status of the cells during long-term culture in vitro (5-7). Recently, several new technologies including the analysis of gene expression or proteomic profiling have demonstrated the potential to better define MSC preparations (8-10). One very promising technology is the use of the SELDI-ToF (Surface Enhanced Laser Desorption/Ionization – Time of Flight Mass Spectroscopy). SELDI-ToF technology can be used to evaluate the proteomic profile of stem cells in culture and the proteomic profile can be used to assay changes that occur during cell growth (11).

C. Production of the Cellular Therapy Product According to cGMP

Producing cellular therapy products according to current Good Manufacturing Practice (cGMP) is a global challenge for the production of all cells for use in humans. GMP is a quality assurance system used in the pharmaceutical industry, to ensure the final product meets preset specifications of both manufacturing and testing of the final product. cGMP requires traceability of all raw materials and a standard operating procedures (SOPs) for all manufacturing processes. (see Fig. 1). The FDA has established standards for cellular therapy and these standards regulate quality and safety for donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells (12).

Figure 1. Overview of a Quality Management System

Briefly, Standard Operating Procedures (SOPs) will need to be generated that cover all the processes involved in manufacturing the cellular therapy product. Additionally forms will also need to be generated to cover these processes and equipment used in the production. Documentation for all training activities, audits, and supplier approvals will need to be generated. A fully documented quarantine and release procedure will need to be in place for all materials used in the production of a cellular therapy product. A complaints procedure with the associated Corrective Action and Preventative Action (CAPA) will also need to be implemented. These are the minimal requirements for a Quality Management System.

A current misconception is that clinical-grade cellular therapy products may be produced by transfer of current methodology into clean-room facilities. Such facilities are needed and important in avoiding microbial contamination of the product, but equally and perhaps even more important in implementing GMP will be the development of validated SOPs for the entire process, from cell isolation to freezing and storage of the cell products (13). In addition, another key aspect of the transfer of cell production to GMP standards is the establishment of quality control methodology and release criteria of the final cell therapy product. It is also important to emphasize that implementing cGMP manufacturing will not necessarily ensure that the produced cells are the highest possible quality or are the most efficient cells for a particular type of therapy. However, it does ensure that the cells are produced in a reproducible manner and meet preset specifications that will ensure the safety of the patient.

The translation of research-based procedures into cGMP-compliant procedures for large-scale production of clinical-grade cellular therapy products requires detailed analysis of all the risks and benefits in order to identify and control all critical aspects of the manufacturing process (14). When establishing a cellular therapy product the following parameters described below need to be considered.

Different collection methods of stem cells result in variable cell yield and viability. An excellent example would be MSCs from cord blood. With this source of MSCs the success rate in isolating and further expanding MSCs is influenced greatly by the time between collection and processing, the volume of cord blood collected and the cell content (15).

Age and viral testing falls under the same criteria as for all other cell- and tissue-based products (16). Age can be an extremely important criterion; children may have a higher level or progenitors and age can be directly linked to decreased proliferation and multipotency in vitro (15).

If the cells can be harvested from the donor tissue in sufficient numbers to use therapeutically, then expansion ex vivo may not be necessary, since the local microenvironment is sufficient to facilitate proliferation and differentiation in the target tissue. In addition, the lack of expansion ex vivo keeps the cells in the realm of minimal manipulation, which will keep these cells under a cellular therapy product regulatory designation as opposed to culturing the cells, which now makes them a drug product.

If the goal is to manipulate the cells by culturing them before administration, cell plating density is another critical parameter to ensure expansion of the cell therapy product and preserve their differentiation potential. A lower cell density has been associated with the development of early progenitors/stem cells (17).

Stem cells that are adherent, i.e. MSCs, have normal growth inhibition at confluence. Therefore, for large-scale production of these cell types, many sequential passages may affect the quality of the product. For most stem cells, the proliferation rate slows and the cells lack multipotency after 3 weeks in culture and ~ 12-15 doublings (18). Any cell type that is expanded ex vivo with a high number of population doublings, can undergo senescence and some can undergo genetic instability. Important concerns have been raised regarding the risk of transformation after long-term cultures that are related to cells escaping the senescence program. Senescence and transformation are tightly linked; 19 cells becoming senescent could transform after a transient senescence crisis or abrogation of senescence mechanisms by a re-increase of telomere length or repression of p16ink4a and p53 activity (20). During culture, senescence of stem cells induces growth arrest with normal telomere shortening, decreased differentiation, down-regulation of genes related to tumorigenesis such as ras and an upregulation of some gene clusters and some microRNAs (21). One study recently demonstrated that clinical-grade MSCs could undergo senescence and never transform, with or without aneuploidy, mainly duplications of chromosomes 5 and 16 (18) However, regardless of all these data, since the first clinical trial in humans, tumors have never been reported with adult stem cells, particularly with MSCs.

To avoid the main risks during the use of expanded cells, the genetic stability of the GMP-expanded cells must be tested. Different assays can be used to test for genetic stability. Recently experts from the European Regulatory Authorities proposed a pragmatic idea: the number of population doublings should be minimal, and conventional karyotype combined with CGH array and/or FISH (fluorescence in situ hybridization) are only necessary when recurrent abnormalities are found.

Controls for cell identity are straightforward and are based on FACS (Fluorescent Activated Cell Sorting) analysis of cell surface antigens. Some of these have been well established, depending on the particular cell type, for example the antigens that are used to characterize MSCs have been established by the International Society for Cellular Therapy (22). The efficacy will be largely based on previous data generated in animal models and in vitro during the preclinical phase of process development.

To maintain phenotypic and genotypic stability of stem cells during multiple passages, optimization of the media conditions is absolutely essential for cGMP production. Even though consensus is lacking on the ideal method for culturing most of these stem cell types, basically Dulbecco’s modified Eagle’s medium (DMEM) or alpha-minimal essential media is commonly used, with the addition of animal serum (FCS) or human serum or plasma and growth factors (23,24). The choice of the right FCS is crucial because large batch-to-batch variability may negatively the production efficiency (25). Since these media are based on a xenogenic source of growth factors (FCS) they could transmit unexpected or emerging disease. Therefore new developments are based on supplementation with human blood-derived products secured by serological and DNA testing of blood-transmitted viruses (i.e. HIV and hepatitis C) with eventually a supplemental step of virus inactivation. The most reliable and used human supplementation product is platelet lysate, consisting of plasma enriched by platelet growth factors released by freezing/thawing cycles (143). The reduced efficiency of animal serum-free culture conditions could potentially be overcome with human AB serum and with cytokines like fibroblast growth factor 2 (26).

The production of any cellular therapy product requires aseptic conditions and the validation of those aseptic conditions. Therefore, the development and use of closed automated devices are an important step facilitating cell therapy product expansion under GMP. A first step can be the use of multi-layer systems that could be used in stacked incubators. This allows for the safe use of wide surface areas and the easy production of hundreds of millions to a billion cells in 2 to 3 weeks time. However, in terms of GMP requirements, these processes are not fully closed systems and would require a class A cabinet for each manipulation. For a simpler and safer process, a fully closed and automated bioreactor may be used. The main criteria for growing a cellular therapy product in a bioreactor are a large ratio of surface area to volume, a closed system, automated inoculation and harvesting, and automated control of the culture parameters. There are several different designs of bioreactors – parallel plate, hollow fiber, or micro fluidic could improve these criteria. Multi-layered systems could be coupled with fully automated systems allowing media circulation and assisting in media replacement, i.e. Terumo’s fully automated bioreactor (27). Lined with animal-or serum-free media, these advancements can provide the optimal tools for delivering cellular therapy products of clinical grade to the clinical arena. Moreover, bioreactors could help manage environmental conditions that change or improve the behavior of the cell therapy product, i.e. supplying continuous low oxygen improves the growth and genetic stability of MSCs (28). A full validation of aseptic conditions must be considered to be a crucial part of any process and closed systems instead of the classic, plastic flask-based cell manipulations are preferred.

One of the main requirements for cell therapy product production is that controls give relevant information on the safety and efficacy of the released cell therapy product. These controls should permit the determination of the identity and purity of the cell product and if applied to the release of product, should be reproducible and fast.

D. Concluding Remarks

Regenerative medicine is a new and rapidly developing area for cardiac repair. Several cell types have been identified and investigated in both pre-clinical and clinical research, generally with positive results. There is a wealth of pre-clinical and early clinical data demonstrating the feasibility, and early efficacy of cardiac cell therapy.

Given the worldwide prevalence of cardiac dysfunction and the limited availability of tissue for cardiac transplantation, cellular therapy, could ultimately fulfill a large-scale unmet clinical need and improve the quality of life for millions of people suffering from cardiovascular disease. However, the use of these cells in a clinical setting is still in its infancy. There is still much that needs to be elucidated bout the mechanisms by which stem cells repair and regenerate myocardium, the optimal cell types and modes of their delivery and the safety issues that will accompany their use.

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