Is it safe? Answering that question for therapies based on living cells is not simple.
Therapies based on living cells hold great promise as potential cures for life-threatening diseases. These therapies can repair, restore, replace, or regenerate the affected body system in the patient. One example is the use of ex vivo manipulated peripheral-blood or bone marrow progenitor stem cells as immunotherapies. Researchers are trying to create engineered tissues or biohybrid organs by combining cells with various medical-device biomaterials and other biologically active molecules.
A lingering concern is always the very simple question: "Is it safe?" However, answering that question for therapies based on living cells is not simple because biological safety (biosafety) is largely relative; for cellular-based therapies (CBTs) it is perhaps best discussed in terms of biosafety risk. Such risks can be assessed only after considering numerous factors, such as the source of the cells or tissue, preparation methods, the intended recipient, where in the body the cell-based therapy will be implanted, and the method of cell delivery. This article provides an overview of biosafety issues facing cell- and tissue-based products and discusses a systematic, practical approach for addressing them.
ASSESSINGRISKSINLIVINGCELLS
CBTs are prepared from living sources, which raises clear biosafety concerns regarding the potential presence of infectious disease agents transmitted from the donors. Highly specialized manufacturing processes are often used, commonly necessitating the use of non-FDA approved ancillary materials (for example, media, cytokines, or growth factors) of uncertain safety and quality. Frequently, the final cell-containing product is intended for implantation or delivery into a specific site in the body and interacts with the cellular milieu in a specialized manner to yield the therapeutic effect. This is why biosafety concerns cover the delivery systems used and the post-implantation fate of the cells (Table 1).
Therapies based on living cells or tissues are unique (Table 2). This is important since many physiochemical techniques such as viral inactivation, filtration, and terminal sterilization are incompatible with therapies dependent upon living cells since such methods would render the cells nonviable. Using such techniques to make a CBT safe would eliminate the possibility of it also being efficacious.
We face the inability to completely assess risks that may be lurking in a product derived from living sources and the inability to inactivate or eliminate unknown threats inherent in living cells. It is obvious that providing an absolute assurance of biosafety is not possible. Consequently, a systematic, risk-based approach to biosafety must be employed that considers the unique characteristics of the product in relation to the condition of the recipient.1 Such an approach requires an ongoing biosafety risk assessment throughout all stages of development - before, during, and after administration of the product.
ELEMENTS OF A BIOSAFETY PROGRAM
A comprehensive program for assessing, minimizing, and monitoring risks to biosafety includes the following elements, which are also illustrated in Figure 1:
1. Qualification of materials used in product manufacturing
A. Biosafety and qualification of cells and tissues
B. Biosafety and qualification of key ancillary materials
2. Final product release testing
3. Preclinical pharmacology and toxicology
A. General preclinical design
B. Preclinical biosafety animal studies
4. Delivery system testing
5. Clinical monitoring
6. Post-marketing pharmacovigilance
Qualification of materials used in product manufacturing
Federal rules have mandated establishment of a quality control unit as part of good manufacturing practices (GMP).2 This is particularly important for cell- and tissue-based products, since the manufacturing process entails the use of source and ancillary materials of differing complexity, variability, and risk for introduction of adventitious agents. FDA has made it clear via conference presentations, mass mailings to IND sponsors and guidance documents that developers of gene therapies and cell- or tissue-based products must have a qualification program in place prior to initiating clinical studies.3'7
A critical function of a manufacturing quality-assurance and qualitycontrol program (QA/QC) is the development-qualification program for the source of cells and tissues that will be used as part of the final product, and all the critical reagents (ancillary materials) such as ascytokines and growth factors needed to prepare the final product but are not intended to be part of the final product. An important focus of this qualification program should be biosafety, so we present a sample program in Table 3.
QA/QC is your first line of defense in evaluating and, where possible, eliminating known risks to biosafety presented by a source material or key ancillary material. There may be unforeseen risks to biosafety that only emerge during preclinical safety testing in relevant animal models. Some risks can only be evaluated when this stage has been reached.
Biosafety and qualification of cells and tissues, A core set of documents available from FDA regarding donor screening as well as direct testing of cells and tissues for pathogens of concern, is applicable to the diversity of sources of cells and tissues used to create cellular- and tissue-based products (autologous, allogeneic, xenogeneic, banked vs. non-banked).6'9 For developers of therapies based on human cells and tissues, PDA's draftt guidance contains helpful information on testing autologous and allogeneic cell and tissue sources as well as testing of cell banks.6 The guidance also cites key documents, such as those from the International Conference on Harmonization (ICH).
Similarly, for animal-derived cells and tissues, or cells and tissues that are co-cultured with animal cells (for example, murine feeder cells), valuable information can be found in PDA's guidance on xenotransplantation.7 It is important to recognize that these guidance documents describe only general expectations for biosafety assessments and in the case of ICH guidance documents, often specifically exclude CBTs from their scope. However, in the absence of more specific guidance for cell containing products, the principles described in these documents are applicable.
You may need to make a scientific judgment. In some cases, it may be prudent to perform additional biosafety testing, while in other instances it may not be feasible to perform all described testing. There are additional biosafety concerns to be addressed for cell-based therapies involving ex vivo genetic modification of cells (regulated as gene therapy). Specific biosafety testing or information to be gathered depends on the vector system used and can include product testing and long-term patient follow-Up.8,10
Biosafety and qualification of key ancillary materials. Often, in order for a cell- or tissue-based therapy to possess the desired therapeutic characteristics, it must undergo a variety of manufacturing steps involving enzymes, cell selection or depletion with antibodies, and ex vivo culture in serumcontaining media with a cocktail of cytokines and growth factors. Typically, these processing materials are only transient and are not intended to be part of the final product. The biosafety of these ancillary materials must be assessed since they come in contact with cells that will be subsequently administered to patients. The degree of biosafety necessary to satisfy regulatory authorities depends largely on its origin (synthetic vs. animal- or human-derived) and whether the vendor supplied the reagent for "in vitro research only" versus a product previously approved by FDA for human use.11
Regardless of the regulatory status of an ancillary material, the manufacturer of the cell- or tissue-based product is ultimately responsible for developing procedures to evaluate risks to biosafety and performing additional testing. Demonstrating removal or absence of an ancillary material from the final product is one example where additional testing is needed regardless of whether or not the ancillary material is PDA-approved.
Final product release testing
21 CFR 610 requires that the final product is tested prior to release for distribution and administration in patients.12 These release tests include a number of specifications that must be met for microbiological safety, including bacterial and fungal sterility, pyrogenicity (endotoxin), and mycoplasma (for cultured products). Also included are a number of release specifications relating to product quality and characterization, such as identity, purity, potency, and cellular viability. There are many legitimate issues regarding the need for some types of testing, or the length of time it takes to obtain test results in relation to the limited stability of many CBTs.
Fortunately mechanisms exist for alternative test methods.6 Overall, confirming that the final product meets specifications for release is a key component of ensuring product quality and biosafety for biological products. For example, a variety of cellbased therapies are being investigated for various types of cardiac disease. One approach involves the use of bone marrow to stimulate angiogenesis in ischemie heart tissue. Unfractionated bone marrow contains a complex mixture of cells including T cells, B cells, NK cells, monocytes, macrophages, stromal cells, and neutrophils, which makes it difficult to determine which cells may contain therapeutic vs. non-therapeutic contaminating elements. This raises biosafety concerns since these cells produce a variety of cytokines that could lead to deleterious effects such as inflammation, fever, acute phase response, lymphocyte activation, and proliferation. In vitro and in vivo studies are needed to determine the significance of these concerns. Once information becomes available regarding these issues, it may be necessary to establish release specifications for the purity of the final product to avoid the possibility of adverse clinical events. Thus, the ability to characterize the final product by the purity-impurity profile can have important biosafety implications.
Preclinical pharmacology and toxicology
Undesirable pharmacological activity or unrecognized toxicities are unlikely to be detected through in vitro testing, and generally must be evaluated in a relevant animal model. This is, of course, due to the unique aspects of CBTs.
General preclinical design. Because of the diversity of cell sources and disease targets for CBTs, it is clear that no single type of preclinical study design will be appropriate for all products. The information gained from preclinical studies will be used to design clinical studies. The cellular construct, dose, site of implantation, and duration of treatment should mimic those planned for clinical studies.
Design preclinical animal safety studies to address issues specific to the cellular construct being used, including issues related to the site of implantation, and the post-implantation fate of the cells. For example, if the cells being implanted have been genetically modified, studies evaluating the level and persistence of gene expression, as well as the potential for inappropriate expression of the gene product, will likely be necessary. Similarly, if the cells are implanted as part of a construct containing a biomaterial or seeded within a device component, additional preclinical safety assessments will be needed (Table 4).
The principles involved in designing and developing appropriate preclinical testing to determine the safety of cell-based products are similar to those encountered for other biological products. Key components of protocol design for preclinical studies include:
* discerning activity and toxicity of the product (mechanism of action)
* recommendation of an initial safe dose and dose escalation scheme in humans
* identification of potential toxicity or activity target organs
* identification of parameters that should be monitored clinically
* identification of patient eligibility criteria.13
When available, relevant animal species and animal models of disease should be utilized in preclinical studies. We all recognize that no single species will be representative or predictive for all patients. Ideally, the, animal disease model chosen should have a similar pathophysiology and anatomy to humans, which should improve predictability of human risks as well as facilitate modeling of route of administration and dose exploration. Standard animal models of disease are frequently modified to generate the preclinical toxicity data. For example, immunological reactions in animal models often require that preclinical toxicology studies be performed with autologous animal cellular products (animal products that are analogous to the intended clinical product), rather than the actual human product.14 Follow good laboratory practices (GLP) for preclinical safety studies. However, given the unique design requirements of preclinical assessments of CBTs, we recognize that this is not always possible. In these cases the "spirit of GLP" should be followed.
Performing preclinical safety studies in an animal model of the disease depends onthe existence of such a model. Where models do exist, recognize their limitations, such as whether the model is relevant to the pathophysiology of the disease in humans, the physical size of the animal, and potential technical limits on feasibility. Other important considerations are the availability of the animal model, including age, gender, and numbers needed for statistical considerations; the need for specialized housing; animal welfare concerns; costs; and the availability of historical data to support the animal model.
In some cases, more than one animal model may be needed. For example, a small-animal model could provide important preclinical safety and proof or principle information. However, a study in larger animals may still be necessary if there are issues relating to the size of the cellular implant or if specialized tools are needed to deliver the therapy that cannot be effectively modeled in the small animal. Limit unnecessary use of animals, and discuss specific concerns with knowledgeable consultants or directly with regulatory authorities prior to initiating preclinical pharmacology and toxicology studies.
Preclinical biosafety animal studies. What do you do when no relevant preclinical animal model exists? In those situations, obtaining preclinical safety data in normal animals is necessary. At a minimum, treated animals should be monitored for general health status, serum biochemistry, and hematologie profiles. Microscopically examine target tissues for histopathological changes. Pharmacological studies in normal animals may also provide useful information regarding the in vivo function, survival time, and appropriate trafficking of the modified cells.8
One consequence of not being able to obtain relevant information concerning the pharmacological and toxicological effects of a CBT in an animal model is the need to design a resource-intensive clinical program. This includes heavy patient monitoring for adverse effects, since limited information is available as to which clinical safety parameters are important. Additional patients may be necessary, since information on the effects of the product in relation to the specific stage of disease may be unclear. It may be necessary to include long-term patient follow-up since information on long-term consequences of the therapy, such as patient morbidity or mortality, cannot be anticipated in the absence of animal studies.
The goal of a scientifically sound pre-clinical study design is the ability to identify potential biosafety concerns that need to be examined in the clinical trial. The data from preclinical studies serve as the basis for risk-benefit decisions that are made in the context of the patient population, severity of disease, and the availability of alternative therapies.
Delivery system
CBTs are delivered to patients in a number of ways, including infusion or injection, via syringes or catheters and, in some cases, surgical implantation as part of construct-containing cells and medical-device biomaterials. Since there are no systems specifically approved for the delivery of CBTs, some preclinical assessment is usually necessary. For example, catheters may present issues with clogging of lumens by the cell suspension, loss of viability of the cells, and uncertainty regarding the dose of cells delivered due to dead space in the catheter system. For catheters with needles there may be additional safety concerns regarding the ability to precisely deliver cells to a localized injection site versus inadvertent injection into the systemic circulation.15
Biosafety issues related to the post-implantation fate of the cells include a variety of risks that often overlap (Table 5). For example, a product derived from stem cells may be injected into a specific region of the brain. Ideally, safety studies in an animal model would address the ability of those cells to migrate away from the implantation site. Such migration might be acceptable if the cells are expected to integrate into appropriate anatomic structures within the brain that are distant from the implantation site. Conversely, this may be undesirable if the cells migrate to regions where they normally do not exist and aberrantly secrete excitotoxic substances. Similarly, it would be important to determine the influence of the local microenvironment at the implantation site on cellular differentiation and expression of the desired phenotype.
Clinical biosafety monitoring
It should go without saying that assessing biosafety is an essential element of investigational clinical studies. After all, a fundamental tenet of good clinical practice (GCP) is that a clinical trial of an investigational therapy should be initiated and continued only if the anticipated benefits justify the risks.16 GCP forms the basis of the worldwide clinical trials system. Initiation of a clinical study should be predicated on a risk-benefit assessment largely based on biosafety data accumulated from preclinical safety studies, qualification of components, and testing of the final product. For example, in the US, adequate information about chemistry, manufacturing and control of the product, as well as adequate pharmacological and toxicological data, must be provided prior to initiating investigational studies.17 To ensure the safety of patients, incorporate the information collected regarding possible risks and side effects into the design of the clinical studies.
Post-market pharmacovigilance
Intense clinical monitoring of the human subject is expected after administration of the product. FDA requires sponsors to report serious and unexpected adverse events associated with use of a product as soon as possible and within 15 calendar days. Sponsors must report any unexpected fatal or life-threatening experience associated with the use of the product as soon as possible and within seven days.
The need to have a well-defined clinical monitoring program is something FDA has emphasized for both cell-based and gene therapy products in recent years.4,5 In terms of gene therapy products, which include genetically modified cells, FDA recently announced the "Gene Therapy Patient Tracking System," which includes a provision for the collection of short- and long-term patient safety and outcome data.18 A similar system is under consideration for long-term patient follow-up for recipients of xenotransplantation products.19,20 Upon marketing approval, FDA expects ongoing post-marketing safety data collection and risk assessment based on observational data, as it does for all approved products, as part of pharmacovigilance.22
SUMMARY
Products based on living cells hold significant therapeutic promise for addressing unmet medical needs. In light of the approximately 340 ongoing clinical trials involving cell therapies, perhaps this is an understatement.23 However, such enthusiasm must be tempered by the potential biosafety risks these products present. Many of these risks, such as those presented by infectious agents, are common to all biological products, and well-established procedures such as donor screening and testing, as well as qualification of source and ancillary materials, should greatly minimize those risks.
However, the very characteristics that many cells possess, such as the ability to proliferate, differentiate, and integrate into a diseased or damaged body system, also elicit concerns regarding biosafety. Since there are a number of uncertainties regarding the ability of in vitro and in vivo systems to address these issues, we are left to ponder the notion that "absence of evidence is not evidence of absence."24 An ongoing biosafety vigilance program (before, during, and after administration of the product) should significantly minimize the actual biosafety risks inherent in CBTs.
DEDICATION
This article is dedicated to my former colleagues in the Office of Cellular, Tissues and Gene Therapies (OCTGT), CBER, FDA. The basis of the information presented in this article has been gleaned from conversations with many of you over the years, as well as guidance documents you have written and presentations you have made. all I know, I owe to you.
[Sidebar]
QA/QC is your first line of defense in evaluating and,where possible, eliminating known risks to biosafety presented by a source material or key ancillary material.
[Reference]
REFERENCES
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15. Jensen D. Cardiac catheters for delivery of cell suspensions. Presented at FDA Biologies Response Modifiers Advisory Committee Meeting; 2004 Mar 18; Silver Springs, MD. Available at: www.fda.gov/ ohrms/dockets/ac/04/slides/4018s1 .htm.
16. International Conference on Harmonisation. Good clinical practice, consolidated guideline E6. Available at: www.ich.org.
17. FDA.Codeof Federal Regulations, Title 21 Part312: investigational new drug applications. 2003 April 1.
18. FDA, CBER. Gene therapy patient tracking system. 2002 june 27. Available at: www.fda.gov/cber/ genetherapy/gttrack.htm.
19. FDA, CBER. Availability for public disclosure and submission to FDA for public disclosure of certain data and information related to human gene therapy or xenotransplantation. Available at: www.fda.gov/cber/rules/frgene011801.htm.
20. Public Health Service. PHS guideline on infectious disease issues in xenotransplantation. 2001 Jan 19. Available at: www.fda.gov/cber/gdlns/ xenophs0101.htm.
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23. Weber DJ, Simek S, and Puri RK. FDA educational partnerships to improve the development of cell and gene therapy products. BioProcessing Journal 2003;2(4):23-25.
24. Favorite guote of Philip Noguchi, M.D., former Acting Director Office of Cells, Tissue and Gene Therapies, FDA, CBER. Original source of guotation is unknown.
[Author Affiliation]
Darin J.Weber,Ph.D.
Darin J. Weber, Ph.D. is a senior consultant in cell and gene therapies, Biologies Consulting Group, LLC, 7712 2nd Avenue NE, Seattle,WA 98115, 206.940.3923, fax 206.374.8281, dweber@biologicsconsulting.com. www.biologicsconsulting.com
