A wide variety of raw materials may be used in manufacturing. They may include relatively simple materials or complex substances, such as cells, tissues, biological fluids, polymeric matrices, mechanical supports, hydrogels, culture media, buffers, growth factors, cytokines, cultivation and processing components, monoclonal antibodies, and cell separation devices. These materials may remain in the final therapeutic product as active substances or as excipients. They may also be used in the manufacturing process as ancillary products. Ancillary products are components or substances that exert an effect on a therapeutic substance (for example, a cytokine may activate a population of cells). However, the mode of action of the ancillary product is limited to the interaction with the therapeutic entity, and the ancillary product is not intended to be present in the final therapeutic product. “Feeder cells,” which are used to provide nutrients or growth factors for the product cell, are an example of an ancillary product. The quality of raw materials used in the production of a cell or gene therapy product can affect the safety, potency, and purity of the product. Therefore, qualification of raw materials is necessary to ensure the consistency and quality of all cell and gene therapy products.

It is the responsibility of the manufacturer of the product to ensure that all raw materials used in manufacturing are appropriately qualified. Qualification is the process of acquiring and evaluating data to establish the source, identity, purity, biological safety, and overall suitability of a specific raw material so as to ensure the quality of all raw materials used in the manufacturing process. The broad natures of the cell and gene therapy products and of the materials used to produce these products make it difficult to recommend specific tests or protocols for a qualification program. Therefore, rational and scientifically sound programs must be developed for each raw material.

Activities involved with raw material qualification will change as products move through various stages from clinical trials to licensure and commercialization. A well-designed qualification program becomes more comprehensive as product development progresses. In the early stages of product development, safety concerns are a focus in a raw material qualification plan. In the later stages, raw material qualification activities should be completely developed and should comply with CGMP. Ultimately, each raw material employed in the manufacture of a cell or gene therapy product should be produced under conditions that are in compliance with CGMP. On rare occasions, complex or unique substances that have been shown to be essential for process control or production may not be available from suppliers that produce them in compliance with CGMP. In these situations, the cell or gene therapy product manufacturer will have to develop a scientifically sound strategy for qualifying the raw material.

A qualification program for raw materials used in cell and gene therapy manufacturing should address each of the following areas: (1) identification and selection, (2) suitability for use in manufacturing, (3) characterization, (4) fetal bovine serum, and (5) quality assurance.

Human-derived tissues may be sourced from normal healthy donors, cadaveric donors, or diseased patients, such as those with cancer. Applicable guidelines and standards for the procurement of human tissue are available from the American Association of Tissue Banks (AATB) and the FDA. Additionally, the federal policy in 45 CFR Part 46 is applicable to all federal or federally supported research. This policy requires that a certified institutional review board review and approve use of any tissue taken from a live human donor. The policy also includes special considerations for research on prisoners, children, and pregnant women or research in other areas involving gestational tissue. In all cases, appropriate written consent must be obtained from the donor or the donor's next of kin, describing which tissue is being procured and for what use it is intended. The donor must meet established guidelines for donor suitability and be tested for the infectious diseases listed in Table 4. The medical history of the donor must be reviewed to ensure the absence of signs and symptoms of these diseases and to rule out issues and behaviors that increase the risk of exposure to such diseases.

Human tissue should be obtained under environmental conditions and controls that provide a high degree of assurance for aseptic recovery. Standard hospital operating room practices are applicable for tissues requiring dissection and surgical procurement. The air quality provided in a typical limited-access operating room is adequate for such procedures. Procurement personnel must be appropriately trained in all aspects of tissue recovery, such as surgical scrubbing, gowning, operating room behavior, anatomy, surgical site preparation, and antisepsis. Special care is required when tissue or organ procurement requires extensive manipulation of the bowel and when sharp dissection may result in the inadvertent puncture of the bowel. Tissue that contains microbial flora (for instance, skin) at the time of procurement can be adequately disinfected by using antimicrobial or bactericidal agents and extensive scrubbing.

Table 4.Infectious Disease Testing for Human Cells and Tissues Used in Cell Therapy Products

Hematopoietic progenitor cells represent one of the most extensively used cell sources in the field of human transplantation. These cells can be collected from the bone marrow, peripheral blood, placental umbilical cord blood, or fetal liver. The source of cells is somewhat dependent upon the patient, the disease, and the clinical protocol. Regardless of the cell source, methods for processing the cells are similar.

Human-derived blood cells and bone marrow cells may be sourced from normal, healthy donors or patients with hematological disorders. Applicable guidelines and standards for the collection and processing of these materials have been published by the American Association of Blood Banks (AABB), the Foundation for the Accreditation of Hematopoietic Cell Therapy, the National Marrow Donor Registry (NMDR), and the FDA. Similar issues regarding consent, infectious disease testing, and donor medical history apply in the sourcing of blood- or bone marrow-derived cells for allogeneic transplants. In cases where these cells will be subjected to selection, expansion, genetic manipulation, or other complex processing procedures, the testing outlined in Table 4 should be followed.

Bone marrow for clinical use is harvested predominantly by percutaneous needle aspiration of the anterior or posterior iliac crests or the sternum. Standard hospital operating room practices are employed by specially trained personnel. Plastic syringes and commercially available aspiration needles are used to draw 3- to 5-mL volumes of marrow from each site of penetration. The material is transferred to a sterile, balanced salt solution or tissue culture medium containing sufficient anticoagulant, such as heparin, to prevent clotting. Removal of bone spicules may be accomplished by passing the material through stainless steel mesh screens or collection kits consisting of sterile, plastic collection bags with in-line filters having about a 200-µm porosity. The volume of marrow collected is dependent upon the body weights and other characteristics of both the donor and the recipient. The maximum volume to be harvested from a donor is about 10 to 15 mL per kg of body weight.

Circulating hematopoietic, peripheral blood progenitor cells (PBPCs) comprise a small population of peripheral blood mononuclear cells that can be utilized in place of or in addition to bone marrow. PBPCs are collected by apheresis, a procedure by which donor blood is withdrawn from a vein and separated ex vivo into some or all of its component parts. One or more of the components are retained as the harvest and the remaining parts are returned to the donor. Conditioning of the donor may enrich the number of circulating PBPCs in the harvest. Examples of such conditioning include collection during recovery from myelosuppressive chemotherapy and administration of hematopoietic growth factors, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF), or steroids. Collections are also improved by increasing the frequency or volume of apheresis. Apheresis requires one or two large-bore peripheral venous catheters in the upper extremities or a single large-bore, thick-walled, central venous double or triple lumen catheter (Mahurkur type). Two types of apheresis technology are available: the discontinuous-flow cell separators (Haemonetics) and the continuous-flow systems (COBE or Fenwall). Anticoagulation for normal to high flow rates is with a citrate-based material. In a closed system, the risk of contamination is low. The procedure is generally performed by trained, dedicated staff in a blood bank or in a donor center associated with a blood bank.

Placental and umbilical cord blood provides a third source of hematopoietic progenitor cells. Compared to bone marrow and PBPCs, the stem cells of placental and umbilical cord blood have a higher proliferative and self-renewal capacity. Volume of collection and thus cell number are limited and depend upon timing and the presence of a dedicated team of personnel. Collections are made during the third stage of labor. Typically, a closed method of collection is employed and involves cannulation or puncture of the umbilical vein with subsequent collection into plastic syringes or blood collection bags containing citrate-based anticoagulant. The procedure is performed in a controlled-access room away from the site of birth. Cellular content of the collection includes large numbers of erythrocytes, leukocytes, platelets, and target mononuclear cells. An open collection technique, which involves drainage of the blood by gravity from the cut end of the cord into sterile tubes containing anticoagulant, does not afford the same aseptic assurance level as the above-mentioned technique.

A major area of concern with the use of placental and umbilical cord blood relates to potential risks of unknown genetic disorders that may be transmitted to the recipient. Donor suitability is established by the usual infectious disease screening of the mother and the completion of a medical questionnaire. The donation remains anonymous and without any long-term follow-up of the child.

The major area of concern with the use of animal tissue relates to the known and unknown risks of potential infectious disease transmission to humans, and as such, the transplantation of animal cells raises unique public health concerns. Introduction of xenogeneic infectious agents into and propagation through the general human population is a risk that must be addressed. Draft Public Health Service (PHS) Guideline on Infectious Disease Issues in Xenotransplantation (August 1996), and any other related regulatory documents that are generated as this field advances, must be consulted when developing xenotransplant cell therapy products. Developers of such products should understand that the product recipients will be subjected to a high level of scrutiny (for instance, clinical and laboratory surveillance or registry in xenotransplantation databases) because of the above-mentioned public health concerns.

The use of animal tissue in the manufacture of cell therapy products requires that the tissue be sourced in a controlled and documented manner and from animals bred and raised in captivity in countries or geographic regions that have appropriate national health status, disease prevention, and control systems. In addition, the care and use of animals should be approved by a certified institutional animal care and use committee. Donor animals must have documented lineage, be obtained from closed herds or colonies, and be under health maintenance and monitoring programs. The facility for housing these animals should be USDA certified (large vertebrate animals) or Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) certified (small vertebrate animals) and should meet the recommendations stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), which can be obtained from the AAALAC. Such facility should be staffed with veterinarians and other trained personnel who will ensure animal health and disease prevention. The procedures employed in the facility should be documented and records should be kept. Health maintenance and monitoring programs are based on standard veterinary care for the species and include physical examinations, monitoring, laboratory diagnostic tests, and vaccinations. Use of a stepwise batch or all-in–all-out method of movement of source animal through the facility, rather than the continuous replacement movement, is recommended. It allows the decontamination of the facility prior to the introduction of the new set of animals, thereby reducing the chance of disease transmission. Feed components should be documented and should exclude, whenever possible, recycled or rendered materials that may have been associated with the transmission of prior-associated diseases.

To provide a high degree of assurance of product safety, screening of donors and of tissues derived from these donors should be performed at several stages throughout the process to rule out the presence of microbial agents. These control tests should utilize assays that are sufficiently sensitive and specific to detect bacteria, mycoplasma, fungi, or viruses of interest. Donor animals can be screened for certain diseases prior to donation of tissue by applying a variety of serological monitoring tests. Tissues can be subjected to a panel of tests including, but not limited to, the following:

Most of these tests are addressed under Analytical Methodologies or under Biotechnology-Derived Articles 1045 and Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin 1050. Post-tissue retrieval necropsies, sentinel animal programs, and archival storage of donor organs, tissues, blood, and other specimens are additional components of the overall program to ensure the safety of animal tissue for use in cellular therapeutic applications.

Most of the same aseptic procurement issues apply to animal tissue and to human tissue. Again, the tissue should be obtained under environmental conditions and controls that provide a high degree of assurance of aseptic recovery. Specifically designed procurement facilities, usually closely associated with the animal holding facility, are typically employed. These facilities have specific attributes and design features that may not be available or applicable in the hospital operating-room setting. Such features include the following: (1) staging of various events, such as shaving, sedation, and operating-room preparation, in different rooms that are often separated with air locks for environmental control; (2) high-efficiency particulate air (HEPA) filtration; (3) adjacent but separate facilities for further tissue processing; and (4) dedicated areas for carcass removal. The issues regarding the training of personnel, bowel manipulation and puncture, and disinfection that are applicable to human tissues apply to the surgical procurement of tissue from animals as well (see Human Tissue).

The general principles for processing human and animal tissues following aseptic procurement are independent of the tissue source. The manufacture of cell products may occur at a clinical site or at a central cell-processing facility. Sites involved in cell processing should ensure reproducibility and safety of the manufactured products through appropriate QC and QA programs.

Regardless of the location, processing should occur in a dedicated area physically separated from the site of procurement. To the greatest extent possible, the facility design and processing procedures should be consistent with those provided by the FDA's Guidelines on Sterile Drug Products Produced by Aseptic Processing (June 1987), or provided under Pharmaceutical Compounding—Sterile Preparations 797 for processes involving open manipulation. Generally, this requires that properly trained and outfitted processing staff handle blood or tissue samples in a critical zone supplied with class 100 HEPA-filtered air, which is provided by a biological safety cabinet located in a controlled clean room supplied with class 10,000 HEPA-filtered air. The facility and processing areas should be monitored for air quality in a manner that provides a high level of process asepsis. For guidance in this area, see Microbiological Evaluation of Clean Rooms and Other Controlled Environments 1116. The material should be packaged in sterile, leak-proof containers and transported from the procurement area to the processing area under controlled conditions that maintain cell viability. The fluid medium in which the specimens are bathed during transportation should be optimized to maintain cell and tissue viability. This transport medium can be supplemented with antibiotics. If so, the antibiotic levels in process buffers are decreased and eventually eliminated during subsequent processing steps, so that antibiotics are not present in the final cellular product. In the case of blood products or tissues containing substantial amounts of blood, the transport media or buffered electrolyte solution should contain an anticoagulant such as heparin or a citrate-based material.

Solid organs or tissues are usually dissected to expose a desired region. This material may be used as is for transplantation or it may be processed further. If multicellular organoids (for instance, islets of Langerhans) or single-cell suspensions are desired, the tissue may be subjected to mechanical or enzymatic disaggregation. Physical disaggregation may be accomplished through the use of instruments that impart high shear forces on the material (namely, to homogenize) or break the tissue into smaller pieces. Alternatively, the material can be pressed or passed through screens of defined mesh sizes.

Enzymatic digestion of the extracellular connective tissue, which holds cells together within the tissue, is another common method for dissociating solid tissue. Typically, the tissue is minced into small cubes, usually larger than 1 mm3, and incubated in a buffered solution containing a digestive enzyme. Alternatively, the intact organ is infused with a solution to rinse the blood from the tissue followed by the enzymatic solution that aids the digestion. Various enzymes are used to accomplish this. Examples include collagenase, trypsin, elastase, hyaluronidase, papain, and chymotrypsin. Enzymes with nuclease activity, such as deoxyribonuclease, may be added to digest nucleic acids released from damaged cells, preventing excessive cell clumping. At the end of the incubation process, the cell suspension may be subjected to a mild pumping action to further break up multicellular clusters into those of desired size or composition. Enzymatic and physical disaggregation methods are often combined to achieve the desired result.

Because cells isolated from blood and bone marrow products are inherently cell suspensions, mechanical manipulation is limited to plasma removal, which is accomplished by centrifugation and physical removal of clots that occurred during transport via 200-µm filtration.

Cell suspensions at this stage may be transferred directly to culture vessels as described for Propagation under Cell Propagation and Differentiation, genetically manipulated as described under Introduction of Genetic Material into Cells, or formulated by various techniques as described under Formulation of Cell Therapy Products. Cell suspensions often consist of a mixture of cell types that may require further processing to isolate a cell population of interest or to decrease the level of an undesirable cell type such as potentially contaminating tumor cells. Various cell isolation and separation techniques exist that provide high yields of pure cell populations.

Each cell type typically possesses specific size and density; therefore, different cell types will sediment at different rates in a centrifugal field or at unit gravity. Cell populations can be selectively sedimented to yield pure fractions by varying the centrifugation forces and the duration of centrifugation. Separation can also be achieved by isopycnic centrifugation, where the cell suspension is centrifuged in a gradient medium that encompasses all of the densities of cells in the sample. In this procedure, the various cell populations sediment to an equilibrium position at the gradient density equal to the density of the cell population. Specifically designed continuous-flow elutriation centrifuges separate cell populations by subjecting a cell suspension to opposite centrifugal and fluid stream forces in a special chamber within the centrifuge rotor mechanism. Cell populations separate within the rotor on the basis of their various sizes and densities, and they are selectively eluted out of the rotor chamber by increasing the fluid stream force. Finally, methods that do not require centrifugation but instead involve the addition of high-density agents, such as hydroxyethyl starch, to the cell suspension will result in cell separation. The mixture is allowed to settle in a tube at unit gravity, resulting in the separation of different cell types based on buoyant density. Concentration and separation procedures such as these frequently result in cell loss due to clumping and aggregation.

Cell separation can also be achieved by applying techniques that take advantage of unique cytological or biochemical characteristics of different cell populations. Soybean agglutinin binds to and agglutinates cells that bear a particular carbohydrate moiety expressed on mature blood cells, but not stem cells, allowing for purification of the stem cells. Lymphocytes possess the CD2 antigen that acts as a receptor for sheep red blood cells. The lymphocytes form rosettes, which then can be separated via differential centrifugation.

Some applications take advantage of the ability of certain cell populations to adhere to the surface of specific solid substrates such as tissue culture plastic, collagen-coated materials, and natural and synthetic polymeric scaffolds. The specifically bound cell type is selectively recovered onto the surface and removed from the initial cell suspension. When placed under the appropriate culture conditions, these cells will multiply and eventually occupy the available surface or void volume of the substrate.

Monoclonal antibodies directed against specific cell surface antigens or receptors can be used for both positive and negative cell selection. For example, a monoclonal antibody–labeled cell population can be removed from the cell suspension immunomagnetically, after exposure to magnetic particles coated with antimonoclonal antibody. The magnetic particles and their bound cells are removed from the cell suspension magnetically. Cells are released from the complex following incubation with reagents, such as specific peptides, that dissociate the monoclonal antibody from the cell. Unlabeled cell suspensions can be poured over or incubated on surfaces such as plastic flasks or microspheres coated with monoclonal antibodies as a means of isolating particular cell populations. In addition, a fluorescence-activated cell sorter (FACS) can be used to separate different cell types by binding antibodies tagged with fluorescent markers to a particular cell type.

Various other techniques purify particular cell populations by destroying unwanted cells present in the mixture. For example, certain cell-bound monoclonal antibodies are able to fix and activate complement, which is added to the cell suspension, resulting in lysis of the cell. Some procedures use cytotoxic agents or mitotic inhibitors to selectively impede or kill unwanted cells in a cell product. These methods typically target an unwanted cell subpopulation with a high growth rate, such as tumor cells. Finally, an antibody can be conjugated to a toxic moiety, such as ricin, allowing delivery of the cytotoxic agent to the targeted cell population. Most of these procedures require several washing steps after the exposure of the cells to the cytotoxic agents to ensure the removal of the dead cells, cell fragments, and cytotoxic agents from the final cell product.

A key issue for cell therapy products is the ability to manufacture and deliver a therapeutically relevant dose of the required cell population to the patient. Depending on the application, the product may be a pure, homogeneous cell type or it may be a mixture of different functional cell types. Many target cell populations are present at low level or low purity in complex primary source tissues. In such cases, production of a therapeutic dose may be achieved only by specific enrichment and propagation of the required cells.

Propagation of cells may occur in suspension culture (for example, T cells or hematopoietic stem and progenitor cells), adherent culture (for example, mesenchymal stem cells, embryonic stem cells, neuronal stem cells, or dermal fibroblasts), or a mixture of both (for example, bone marrow stroma expansion). Numerous devices of varying degrees of sophistication and automation exist for cell culture.

In the simplest iteration, cells can be propagated in tissue culture flasks (T flasks), roller bottles, on polymeric scaffolds, or nonrigid, gas-permeable bags inside regular incubator units that are controlled for temperature, humidity, and gas composition. Multilayered plastic cell factories, cell cubes, and multi-bag systems have been developed that enable expansion, harvesting, and formulation to be carried out in a closed system.

Traditional small-scale fermenter units can be used for expansion of cells in suspension culture. It is also possible to expand adherent cells in such units either by providing a surface for attachment (coated beads or disks) or by adapting the cells to propagate in suspension culture. Some culture systems are specifically designed for the propagation of cells for therapeutic applications. These systems tend to be closed systems that use disposable bioreactor cartridges, such as those made of hollow fiber or molded plastic, in automated processing units with direct control of parameters such as temperature, gas composition, and media perfusion rate. These units can provide a completely automated, closed system for expansion and harvesting. In some cases the automated software is set up for patient–donor tracking and will document culture conditions and manipulations for the entire processing run. These features are useful in the design and implementation of QC product-release testing programs and for the QA documentation of processing runs.

In the case of adherent culture, the cells are usually released from the surface upon which they have expanded. Methods of release include physical agitation, enzymatic cleavage with enzymes such as porcine or bovine trypsin, collagenase, or dextranase, chelation of metal ions (for example, with edetate disodium), and competitive inhibition of adhesion or matrix molecules. As described above, consideration must be given to the source, safety, toxicology, and residual testing for any reagent used to release adherent cells during manufacturing.

Some product-specific systems that do not require the release of adherent cells have been developed. In these systems, the cells are expanded upon a synthetic or natural matrix that is then applied topically (for example, in dermal repair products) or the cells are grown inside or outside of fibers for ex vivo perfusion (for example, hepatocytes in hollow-fiber devices to treat liver disease). In these applications, the matrix and device composition must be biocompatible and, in some cases, biodegradable.

In all of the above systems, standard cell culture parameters must be optimized for maximum process efficiency. Such parameters include composition of cellular source material, initial seeding density, media composition, rate of media exchange, temperature, gas composition, and rate of delivery. Depending on the nature of the product, the potential effect of process parameters on the potency and function of the target cells should be defined.

In closed bioreactor systems, it can be difficult to observe or sample cells so as to determine and control the rate of proliferation and thereby the point of harvest. Measurement of traditional fermentation parameters, such as rate of nutrient usage or production of metabolic products, can provide a surrogate method, amenable to validation, with which to evaluate the rate of proliferation and predict when to harvest the cell product. The relationship of such parameters to the viability, potency, and function of the cell product should be well defined. Postexpansion purification and enrichment of target cells by using methods such as those described above may be required.

Some cell therapies require lineage or functional differentiation of the source cells. For example, hematopoietic stem cell expansion processes normally result in products containing a mixture of multipotent stem cells, lineage-committed progenitor cells, and lineage-differentiated cells. The composition of these products can be manipulated by using different combinations of growth factors and cytokines during the expansion process. The inverse is true for processes in which mature cells are de-differentiated to enable them to then be recommitted to a lineage pathway (for example, chondrocytes in cartilage repair).

Specific examples of ex vivo manipulation are the programming of professional antigen-presenting cells, such as dendritic cells and monocytes or macrophages, and the production of antigen-specific T cells to target various specific disease indications. In these applications, the manipulated cells may be engineered to target and attack a specific tumor or tumor cell type, to induce a specific antibody or other cellular response, or to potentially vaccinate a patient. The processes for production of such products can involve one or more exposures of the relevant cells to disease-specific synthetic immunogens (for example, peptides) or natural immunogens (for example, dead tumor cells, viruses, cell membrane fractions, or purified natural molecules) before, after, or during culture expansion. Alternatively, the target cells may be genetically engineered with a specific gene product, such as an HIV-specific receptor. In some applications, relevant cells are cocultured with tumor cells, other diseased cells, or cells producing a transduceable or transfectable gene construct to generate a specifically targeted product.

Again, prior to delivery, the manipulated target cells may require further purification and enrichment by applying the methods described throughout this section. In the case of certain T-cell products, the desired antigen-specific cells can be cloned and then further expanded to provide the therapeutic dose.

Formulations for cell therapy products depend upon the desired length of storage and whether the cells are administered as a suspension or in combination with a matrix. Regardless of the route of administration, cells that will be administered as a suspension can be frozen or not frozen. The most common formulation for cells that are cryopreserved is a 5% to 10% solution of dimethyl sulfoxide (DMSO), with or without hydroxyethyl starch (generally 6%), and a plasma protein, such as 4% to 10% human serum albumin, in a balanced salt solution. DMSO prevents dehydration by altering the increased concentration of nonpenetrating extracellular solutions during ice formation at the time of freezing. The high molecular weight polymeric hydroxyethyl solution protects the cells from dehydration as water is incorporated into the extracellular ice crystals. The use of protein often results in maximum recovery and viability of cells after thawing. Serum (5% to 90%) has been used in place of specific proteins. Some cryopreservation formulations are completely free of protein. If the solution contains a buffer, the pH of the buffer should not be affected by changes in temperature. The optimal concentration of cells for cryopreservation depends on the cell type, but it generally ranges from 106 to 107 cells per mL. The purity of the cell population can also affect recovery. For instance, granulocytes can be damaged by the cryopreservative and the cell viability can decrease. These effects are dependent upon the concentration of cryopreservative. Both effects subject the patient to an increased level of infusion-related toxicity, although this is related to the volume administered and the final concentration of the cryopreservative.

Formulations for cell suspensions stored without freezing generally contain cell culture media, often without any protein. Because cells continue to metabolize their media even at the reduced temperatures used for storage, the medium supplies the amino acids and other nutrients that help in maintaining cell viability.

Many cell therapy products are administered in combination with a biocompatible matrix. For instance, wound healing or skin substitute products contain cells seeded on a matrix. The biochemical and physical structure of the matrix and the method for combining cells with the matrix are specific to the application. Some common examples include the following:

When manufacturing such products, the primary consideration is the sourcing of a quality matrix material. The matrix material should be biocompatible, should not interfere with cell function, and should not trigger an immune response in the patient. If it is intended that the cells proliferate after loading onto or into the matrix, the matrix and the supporting culture system must allow exchange of nutrients and waste products. Cells may form tissue-like structures under favorable conditions and for those applications where this is required. A thick, impermeable matrix will lead to forming regions of necrotic tissue. Many of these devices are designed so that they can be removed from the patient after a certain period of time.

In all cases where cells are combined with biocompatible matrices, the use of closed systems for the manufacture and the delivery of product is preferable. As cell therapy products of this type can be quite intricate, the manufacturing details for such products are outside the scope of this chapter.

A typical gene therapy vector is composed of (1) the vector backbone, viral or plasmid, (2) a promoter, (3) the therapeutic gene of interest, including introns, and (4) a polyadenylation signal. Murine and human retroviruses, adenoviruses, parvoviruses such as adeno-associated virus (AAV), herpes viruses, poxviruses, toga viruses, nonviral plasmid therapy systems, and synthetic antisense-oligonucleotide therapy systems are being developed for gene therapy applications. The properties of these vectors (see Table 5) differ greatly in terms of their capacity to deliver genes to cells. Some viral vectors preferentially target dividing cells while others are capable of transducing both dividing and nondividing cells. There are significant variations in transgene capacity, meaning that there are limitations on the size of the foreign DNA fragment that can be incorporated into the recombinant genome. The ideal gene therapy vector has often been described as one capable of efficient transduction, targeted delivery, and controlled gene expression. The level, timing, and duration of gene expression required will depend on the clinical indication. Low-level, long-term gene expression is thought to be required for some diseases including adenosine deaminase (ADA) deficiency or type A and type B hemophilia. High-level, short-term expression may be more appropriate for cancer when genes that induce apoptosis are used, or for cardiovascular disease where preventing hyperproliferation of smooth-muscle cells may impede restenosis of saphenous vein grafts.

Table 5. Types of Gene Vectors

Vectors are designed and selected for disease states on the basis of the following criteria:

Selection of the route of administration and manipulation of the total dose of vector are strategies that can be used to compensate for some features of specific vector systems. The design and selection of a vector system include the evaluation of the disease of interest.

Additionally, there are advantages and disadvantages for the manufacture of each of the different vector systems. Production consistency favors those systems with well-defined fermentation or culture systems, such as plasmid, retroviral, or adenoviral vectors, or chemically defined systems, such as synthetic antisense-oligonucleotide systems. For those viral vector systems that require helper functions (see below), a rationally engineered cell line can overcome the scalability and consistency limitations of cotransfections. Engineered cell lines can also eliminate the possibility of replication-competent recombinant virus appearing in viral culture. Use of a cell line that is adapted to suspension culture can affect scalability and cost efficiency.

To be effective, a vector must first find and transduce its target cell. Viruses have a natural host range that is strongly influenced by the expression levels of specific cell-surface receptors in target tissues, the cell cycle status of the target cells, and the route of administration. Integrins are a class of cell-adhesion receptors known to interact with either the penton base or the fiber protein of adenoviruses. The Coxsackie and adenovirus receptor (CAR) is also known to interact with adenoviruses. However, the expression levels of integrins and of CAR vary according to tissue type, affecting the transduction efficiency of adenoviral vectors. Amphotropic retroviruses infect cells via a sodium-dependent phosphate transporter molecule that is expressed at a detectable level in every human cell type.

The host and tissue range can be modified or targeted by a variety of approaches. Retroviruses, and lentiviruses, in particular, encode an envelope protein that mediates virus binding and entry via a specific host-cell receptor. Envelope proteins from one retrovirus may be interchanged with a protein from another retrovirus or a protein, such as the vesicular stomatitis virus glycoprotein from an entirely different virus. This process is referred to as pseudotyping. Viral protein coats may be modified in several ways. By engineering the fiber and knob of adenovirus, it is possible to change the intrinsic integrin specificity. Similarly, viral coat proteins can be chemically modified for ligand-mediated receptor targeting. It is feasible to create ligand–plasmid fusion molecules for receptor-mediated targeting of nonviral vectors. Some lipid formulations for nonviral vectors incorporate antibody Fab fragments or ligands to target plasmid delivery.

With respect to cell cycling, adenoviruses easily infect both quiescent and rapidly dividing cells, while murine leukemia virus-based retroviral vectors are efficient only when transducing rapidly dividing cells. Lentiviral vectors can infect quiescent cells, including cells of neuronal origin. In general, nonviral vectors have lower transduction efficiencies than viral vectors. Transduction efficiencies of nonviral vectors are strongly influenced by the formulation used and route of administration.

Regardless of the route of administration, the intended target cell, and the dose, the vector is likely to encounter some component of the immune system as it moves toward the target cell. For viral vectors, the humoral (antibody-based) immune system cannot readily distinguish between wild-type viral infections and recombinant viral vectors because the humoral response is directed against proteins contained in the viral coat or package. Protein-containing formulations of nonviral vectors can also elicit a humoral immune response. Either specific or cross-reacting humoral responses may pre-exist or they may be elicited during dosing, and the antibody response may vary in its capacity to diminish gene transduction in individual patients. It is possible to compensate for the neutralizing activity of the antibodies by increasing the vector dose or by altering the dosing interval to coincide with periods of low antibody titer. Because neutralizing capacity is frequently enhanced upon multiple dosing, effective dosing by repeated administration may be problematic. This issue is generally avoided by the use of nonviral systems. Alternatively, viral vectors can be engineered to evade the immune system. For adenoviral vectors, one approach involves increasing expression of specific viral genes that allow the virus to evade the host's humoral response.

Once protein expression is under way, cellular immune responses can lead to a rapid removal of both viral and nonviral vector-transduced cells from the body and a decrease in therapeutic effectiveness. Although protein synthesis is not required for cellular immune responses to viral vector envelope proteins, de novo synthesis of viral genes can exacerbate host-cellular responses. To reduce potential cellular responses, viral vectors have been designed with specific backbone deletions to eliminate the expression of viral structural genes. Examples of such vectors include the E1- and E4-deleted adenoviruses, the adenoviruses and herpesviruses in which all viral genes have been deleted (gutless) or are helper dependent, and the recombinant adeno-associated viral vectors. Certain plasmid sequences expecially those with the CpG motif, can elicit a strong cellular immune response.

The therapeutic gene product may also be antigenic. When proteins that are retained in the target cell are used, cellular responses may eliminate the target cell. In some cases this is the desired therapeutic effect, particularly in the antigen-based immunotherapy for cancer or a viral disease. However, if sustained protein expression is required, the cellular immune response may decrease the effectiveness of the therapy or eliminate it entirely. The antigenicity of the therapeutic gene may reflect a variety of experimental conditions. If a gene such as the cystic fibrosis transmembrane conductance regulator (CFTR) is truncated to fit within a chosen vector, this modification may result in creation of a distinct antigen. By using the gene that encodes thymidine kinase derived from the herpes simplex virus (HSV), a foreign protein is introduced into a human subject and thus it can function as an antigen. Any patient with a monogenic deficiency disorder is at risk for lack of tolerance to the normal protein that is defective or absent in the disease state (for example, dystrophin in Duchenne muscular dystrophy).

Retroviral vectors are also subject to another host-defense mechanism—the complement component of the immune system. Retroviral vectors are reported to be rapidly inactivated by complement in sera from primates, but not from lower mammals. In considering the replication cycle of retroviruses, it is known that glycosylation epitopes are derived from the host cell during the budding process. Because many retroviral vectors used in gene therapy are murine in origin and have been grown in mouse packaging cell lines, they will have envelopes containing mouse glycoproteins. When retroviral vectors are made in human cells, they are substantially more resistant to human complement. It is reasonable to assume that the mechanism of resistance involves incorporation of natural human cell-membrane complement control proteins that have been incorporated into the vector envelope during the budding stage of particle assembly.

Once the vector reaches the target cell, several factors can affect the level and duration of therapeutic gene expression, and these factors dictate the choice of an appropriate vector system for a specific clinical indication. The localization of the vector genome within the cell, the strength of the gene expression control elements, the stability of the message, and the stability of the translated protein will all affect the therapeutic impact. Alphavirus-based vectors, such as those derived from Sindbis or Semliki Forest virus, reside in the cytoplasm and typically exhibit a very high level of gene expression. Retroviral, adenoviral, and other viral vectors have advantages in gene delivery with their natural mechanisms for nuclear delivery of the therapeutic gene and reasonable levels of gene expression from viral or other promoters. Nonviral plasmid vectors are episomal and are often susceptible to DNA degradation when they are shunted into cell endosomes. However, some nonviral systems incorporate nuclear targeting signals as a means of increasing therapeutic gene-transcription efficiency.

Another means of controlling gene expression is to incorporate tissue-specific promoters to stimulate or to restrict expression of the therapeutic gene. Drug-responsive promoters are being used to control gene expression. Rapamycin, mifepristone, or the tetracycline on systems have been used to repress gene expression. This type of regulation may be required for certain proteins, such as erythropoietin, where constitutive expression may produce toxicity.

Replication status is another important consideration for vector design and selection. Viral vectors are most frequently constructed to be incompetent or replication-defective in order to limit uncontrolled vector spread and pathogenicity. However, when effective therapy requires infection of virtually all the target cells, replication can be engineered to be conditional when specific viral gene interactions are matched with intracellular pathway targets. When these targets are defective or missing, such as in cancer cells, the virus can replicate, but when the target cell is functioning normally, viral replication is repressed. One of the risks inherent in the use of conditionally replicating viral vectors is that the growth of the virus is not absolutely restricted to a single cell type, that is, the system may be leaky. As compensation, the susceptible target cells may be efficiently transduced at a dose that is significantly lower than that necessary for nontarget cells.

Nonviral vectors are normally designed as nonreplicating systems, but some groups are experimenting with replicating nonviral plasmids to increase gene expression levels given the low transduction efficiency of most nonviral systems and to increase the duration of gene expression. Additional preclinical studies are needed to establish the safety of these systems. Artificial chromosomes have also been designed to take advantage of normal mechanisms for retaining gene expression in rapidly dividing target cells.

The duration of gene expression is also a function of the stability of the vector genome. Retroviral vectors can stably integrate into the host-cell genome. Adenoviruses do not integrate because their DNA remains episomal. Recombinant adeno-associated virus (AAV) vectors integrate, but because the rep genes responsible for site-specific integration are normally excluded from the construct in order to increase the vector-packaging capacity, integration is not site-specific as it is for wild-type AAV. Nonviral plasmid DNA does not integrate efficiently. However, stable episomes have been observed in certain cell types, such as muscle cells. Site-specific integration can be a desirable feature for vectors intended to correct genetic disorders. Although it is not currently possible, the control of the site of integration is desirable in order to prevent insertional mutagenesis. Insertional mutagenesis has the potential to kill a cell, if a critically functioning gene is inactivated, or to predispose a cell to malignant transformation, if a tumor-suppressor gene is inactivated.

The success of any gene therapy product is dependent on the relationship between the vector-delivery system and the requirements of the disease application in terms of the site, level, and duration of therapeutic gene expression. It is unlikely that there will ever be a universal vector, and the challenge is in fitting the vector to the disease.

Viral and nonviral gene-transfer vectors are constructed by using standard molecular biology protocols. For viral vectors, the vector backbone consists of viral RNA or DNA sequences from which the regions encoding viral structural genes or the regions required for replication have been deleted. The deleted region of the vector is usually modified with specific restriction endonuclease sites used to allow insertion of the gene of interest. For nonviral vectors, the plasmid DNA backbone contains multiple restriction sites for cloning and the bacterial elements necessary for plasmid production. Vector backbones can accommodate single or multiple gene inserts depending on the maximum amount of sequence they can carry. The promoter that facilitates transcription of the gene insert can be a related viral promoter, such as murine leukemia virus long terminal repeat (MuLV LTR), or a heterologous promoter that is either tissue-specific, such as alpha crystalline promoter (of the eye), or constitutive, such as cytomegalovirus (CMV). For example, in a retroviral vector construct containing two gene inserts, transcription of one is regulated from the 5¢-LTR-promoter sequence, while a second gene insert can be linked to an internal heterologous promoter from Simian virus 40 (SV40). The complementary DNA (cDNA) containing the therapeutic gene of interest, including its introns, is excised from its source by using restriction enzymes and is inserted at the multiple cloning site of the gene-transfer vector. The polyadenylation signal can be derived from multiple sources such as the SV40 virus or human growth hormone. Characterization and testing of gene therapy vectors are described under Analytical Methodologies.

Recombinant viral vectors are most often modified to be replication-defective, a condition created by deletion or modification of the viral genes needed for replication and production of infectious virus. As a result, viral vectors require help to produce infectious vector particles. Helper functions are often provided by packaging cell lines to deliver the necessary viral element from a source outside of the gene of interest (in trans). Packaging cell lines should be designed to minimize the risk of production of RCV through recombination between the vector and the packaging elements.

Plasmids encoding the necessary elements are introduced into the packaging cell by standard methods such as calcium phosphate–mediated transfection or electroporation. If multiple trans-acting elements are needed, these elements are introduced on separate plasmids in order to increase the number of recombination events needed to form a wild-type viral genome, thus decreasing the frequency of the event. An additional approach to eliminate production of RCV is the elimination of common sequences between the packaging cell plasmids and the gene therapy vector.

Stable packaging cell lines should be selected and clonal MCBs prepared. In retroviral vector production systems, typically the pro-viral form of the retroviral vector is stably incorporated into the packaging cell, resulting in what is referred to as the producer cell line. A stable, banked packaging cell–producer line will lead to consistency in production and control of adventitious agent contamination. Alternatively, the system can be transient, with the packaging plasmids transfected along with the gene therapy vector for each round of vector production. However, a transient transfection system is less efficient and limited in scalability.

Typical helper function systems are as follows:

Retrovirus and adenovirus have classically been produced on the laboratory scale by using traditional cultivation methods for anchorage- and serum-dependent cell lines, employing flasks, trays, and roller bottles. Initially, gene therapy vectors were produced by using these exact methods because large volumes of product were not required for early clinical studies. Cell bank systems are used as the source of cells and virus banks as the source of virus for clinical production. In most cases, supernatant is collected, clarified, and stored frozen in bags at 70. In many early clinical trials unpurified supernatant has been used for ex vivo gene transfer.

More recently, larger-scale upstream production methods have been reported including suspension, bioreactor, and fixed-bed or microcarrier culture methods. Some groups have reported adapting their process cells to serum-free culture conditions. Cells are harvested and lysed or supernatant collected. The harvest is clarified and purified to remove host-cell debris, host-cell DNA, and other process-derived contaminants.

Traditionally, viruses are purified by gradient ultracentrifugation, but this is time-consuming and unsuitable for larger-scale production purposes. The selection of downstream process steps and their sequence is determined by the nature of the virus itself and the upstream process used for manufacturing the virus. As processes are being developed for the manufacture of gene therapy vectors, many different purification steps have been reported. These include ion-exchange and sulfonated-cellulose chromatography, zinc ion affinity chromatography, size-exclusion chromatography, and DNase or other nuclease treatments. AAV production and lentiviral production are complicated by a need for transient transfection or cotransfection of plasmid or helper virus. These processes have so far required anchorage-dependent cell lines that are difficult to scale up. The development of stably transfected cell lines would allow large-scale production.

Plasmids are double-stranded, circular DNA molecules that exist in bacteria as extrachromosomal, self-replicating molecules. They have been modified to serve as cloning systems, to contain multiple restriction endonuclease recognition sites for insertion of the cloned transgene, and to contain selectable genetic markers for identification of cells that carry the recombinant vector. Plasmid-based nonviral vectors are frequently used as gene delivery systems for both in vivo and ex vivo gene therapies. They are in the form of naked DNA or complexed with lipids or other agents that facilitate transfer across the cell membrane and delivery to the cell nucleus without degradation. An advantage of a plasmid-vector system is the efficient production of large quantities of the vector that is easily characterized and involves no risk of contamination with the RCV.

Nonviral vectors are typically produced by using an Escherichia coli bacterial system. Plasmids are transfected into Escherichia coli, and an appropriate single bacterial colony is selected and expanded to create an MCB. After reselection of a colony from a bacterial plate inoculated from the MCB, plasmid DNA is isolated from cultures that can range in size from 1 L on a laboratory scale to hundreds of L in bacterial fermenters. Plasmid DNA can be purified by several methods including affinity or ion-exchange chromatography and cesium chloride–ethidium bromide density gradients. Cesium chloride–ethidium bromide density gradients are not recommended for production of clinical-grade material.

Antisense oligonucleotides are manufactured by synthetic chemistry procedures. Currently, the method of choice is solid-phase phosphoramidite chemistry. Synthesis is linear, rather than convergent, and a high level of efficiency must be maintained at each synthesis step. This is accomplished by using molar excesses of highly pure raw materials to drive the reaction kinetics towards completion. Synthetic oligonucleotide manufacturing may require metric-ton quantities of nucleoside phosphoramidites and other compounds such as activator and sulfur-transfer reagents for commercial-scale manufacturing. An issue for oligonucleotide manufacturing is that during the preparation of raw materials and the synthesis of oligonucleotides, precaution must be taken regarding moisture, because moisture is detrimental to both yield and purity. Purification of the single-strand oligonucleotide product requires removal of residual solvents and synthetic strand by-products. Nevertheless, current oligonucleotide-manufacturing technology is readily scalable and cost-efficient, and it results in products with purity levels similar to those of classical small-molecule pharmaceuticals.

Final formulations for vector products are still in early development. So far, mannitol, sucrose, lipids, polymers, and serum albumin have been utilized as stabilizers. Aseptic filling of large numbers of vials, using classical manufacturing processes, may be problematic. For example, some viral vectors are thermally sensitive and storage at ultra-low temperatures is often required. Progress is being made in both viral and nonviral vector lyophilization and in the use of stabilizers for liquid formulations.

Prior to administration, on-site preparation of the cell or gene therapy product may involve one or more manipulations. These manipulations include the following:

Facility requirements for performing on-site preparative steps or administration of cell and gene therapy products depend on the nature of the products, their applications, and the manipulations required. The most important determinant of facility features is the level of risk for microbial contamination associated with each step. Definition of low-risk and high-risk conditions can be made according to a framework similar to that defined for Low-Risk Level CSPs and High-Risk Level CSPs in the CSP Microbial Contamination Risk Levels section under Pharmaceutical Compounding—Sterile Preparations 797.

Cell and gene therapy products that undergo on-site preparative steps or manipulations must be subjected to appropriate checks or tests to ensure that all quality specifications are met prior to release for patient administration. The nature and extent of manipulations will determine whether release requirements or critical specifications must be added to those required immediately after initial manufacture. Pre-release requirements usually include the following:

In addition, products considered to be high-risk products according to the description under Pharmaceutical Compounding—Sterile Preparations 797 should undergo additional product testing. For all high-risk products, quality assays for the identity, potency, and purity of the active ingredients should be defined and performed. For high-risk products in Category II, sterility and endotoxin testing should be performed.

Depending on the specific cell or gene therapy application, steps may need to be taken by trained patient-care staff to prepare the patient for product administration. These steps are aimed at ensuring that the product will provide the intended therapeutic outcome and at minimizing the risk of adverse effects.

In cases where autologous, selected allogeneic, or xenogeneic tissue is the source of the cell or gene therapy product, determination of patient suitability for the therapy, including the evaluation of histocompatibility between the donor and the recipient, typically occurs prior to the product preparation. However, because of the possibility of changes in clinical status of the patient after the time of tissue collection, such as fever, infection, recurrence or spread of tumors, or organ dysfunction, a thorough re-evaluation of the patient's general condition and suitability for therapy must be performed in close proximity to product administration. This evaluation usually includes a patient history, physical examination, and laboratory studies such as blood counts and chemistries. In addition, baseline physical or functional measurements, laboratory tests, or imaging studies relevant to the specific application may be obtained. Examples include pulmonary function tests for a therapy aimed at improving lung function, measurement of blood levels of an enzyme that is the gene product in a gene therapy application, and nuclear imaging of organs prior to anticancer therapies.

A variety of patient interventions related to route of product administration may be required before product administration. For cellular therapies requiring intravenous administration, patients with poor peripheral venous access may require placement of a central venous catheter. In applications where cells or matrices combined with cells are implanted into the patient, the site of implantation may require preparation in the operating room. This may involve surgically opening the site, removing the degenerated or damaged tissue, trimming of the adjacent tissue to accommodate the implant, and excising the tissue from a second site to be used as an anchor or support for the implant. For instance, in the case of cell products for wound healing, it is critical that the site for grafting be free from infection and that it demonstrates a well-prepared wound bed. In cells intended to repair cartilage defects, the site of damage needs to be prepared so that the cells can be applied to a watertight compartment. For applications involving direct administration of the product into an organ system (for example, bronchioalveolar system) or vascular network (for example, coronary arteries), the patient may require endoscopic or surgical access to these sites.

In all cases, the need for adequate anesthesia and premedication must be carefully evaluated in conjunction with these steps prior to product administration. For example, if it is anticipated that DMSO will remain in a thawed, cryopreserved cellular product, the patient is given an antihistamine prior to administration of the cells to block adverse effects associated with histamine release induced by the DMSO. Pre-administration patient evaluation must also include assessment of concurrent therapies that may interact with the cell or gene therapy product to modify its effects. Some therapies may be considered adjunctive to the cell or gene therapy, such as cytokines that promote proliferation or differentiation of the infused or implanted tissue. Other commonly used drugs such as antibiotics, antineoplastics, anticoagulants, and anti-inflammatory agents must be evaluated for possible effects on the efficacy of the cell or gene therapy product.

Some cell or gene therapy products are patient-specific, in that they are manufactured from a selected tissue source, such as autologous, selected allogeneic, or xenogeneic tissue. Certain patient-specific products have a defined potential for benefit or adverse immunoreactivity. Systems must be in place to prevent administration of such a product to the wrong patient. Recommended systems include procedures similar to those used for administration of human blood products, with special attention given to the correct identification of the patient and patient-specific product by at least two people immediately prior to administration.

Cell and gene therapy products can be administered by a variety of routes. These include the intravenous route, the parenteral routes (subcutaneous, intramuscular, and intra-arterial), and the respiratory or gastrointestinal tract route. Other possibilities include direct application of cell or gene therapy products into regional vasculature, organs, tissues, or body cavities by means of needles or catheters or following surgical exposure of the tissue. While parenteral administration can be accomplished in routine outpatient or inpatient facilities, the other means of administration may require specialized facilities, such as an aseptic operating theater or endoscopic suite. In all cases, standard operating procedures and a quality program must be in place to ensure that the product is administered in the intended manner.

There should be written policies and procedures for monitoring patient outcomes and managing reports of adverse events. Patient-outcome assessment should include indicators that are likely to detect errors or problems related to the entire manufacturing process, with special attention given to manipulations, storage, or transportation after the initial manufacture of the product. Management of adverse reactions should include procedures for ensuring prompt medical evaluation and treatment of patients with suspected adverse effects and a system for reporting and evaluating adverse effects that may point to a potential defect in the administered product. Reporting procedures include providing details required for federal, state, or USP adverse-event reporting programs.

Follow-up and monitoring procedures should be implemented for patients who have received gene therapy vectors or ex vivo gene therapies. To the extent that it is relevant and that it can be assessed, vector or modified cell biodistribution and persistence in vivo should be monitored. With direct administration of vectors, localization to the germ line may be an issue. Although preclinical studies can be used to address this issue, useful information may be gained through patient monitoring. In the case where a retroviral vector has been administered, patients should be monitored for replication-competent retrovirus (RCR) according to the FDA's Draft Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-Up of Patients in Clinical Trials Using Retroviral Vectors (October 2000). This involves active monitoring during the first year and archiving of patient samples thereafter if RCR is not detected initially.

Database systems to collate and track patient-monitoring results are essential to management of this information. National registries or publication of data should be considered for establishing the collective safety of gene therapy.

Safety testing for cell and gene therapy products focuses on three issues: (1) preventing the unwitting use of contaminated cells, tissues, or gene therapy agents with the potential for transmitting infectious diseases, (2) preventing the use of improperly handled or processed, and consequently contaminated, products, and (3) ensuring safety when cellular and gene therapies are adapted for use other than in their normal functions or setting.

The primary means of assessing safety are the performance of biological assays to measure adventitious agents directly. Molecular biology-based assays that measure adventitious agent DNA or RNA are also used.

Direct transmission of infectious disease is a major concern for cell therapy products. The degree of risk is dependent upon various factors such as whether the cells or tissues are to be used in a person different from the one they were obtained from; whether they are banked, shipped, or processed in a facility that handles cells and tissues from multiple donors; and how extensively they are processed. Improper handling can alter the integrity and function of cell therapy products by introducing microorganisms or by contaminating the therapeutic cell products with other donor or patient cells during collection, processing, or storage.

In addition to transmittable-disease screening and testing of allogeneic donors of all viable and nonviable tissues intended for use as cell therapy products, appropriate labeling and tracking are required. These requirements are not only based on the potential risk of disease transmission from donor to recipient but also on the following: (1) the unusual, but documented, possibility of product-to-product transmission (for example, viral contamination may occur among disrupted bags in the liquid phase of a liquid nitrogen freezer) or (2) the possibility of erroneous administration of a product to the wrong recipient. Specific donor screening and testing requirements for allogeneic cell products are based on those currently required for human blood products. These include (1) specific donor testing for HIV Type 1, HIV Type 2, hepatitis B, hepatitis C, and syphilis and (2) medical history screening for high risk for HIV, hepatitis B, Creutzfeldt-Jakob disease, and tuberculosis. Some of these tests and screening measures are also recommended, but not required, for autologous tissues.

The risk of cross-species infectivity during xenotransplantation is still unknown. Assessing the risk of infection from a new transmissible agent is difficult. In vitro coculture assays involving sensitive human indicator cell lines for the donor species should be developed. In particular, assay of endogenous retrovirus (ERV) present in the xenogeneic cell or tissue is required. In the case of porcine cells and tissues, both PCR and RT-PCR assays for porcine endogenous retrovirus (PERV) have been described and are applied to donor cells and tissues. These tests are also being used for patient monitoring. Assays for PERV antibody have also been developed for patient monitoring. Published studies indicate that the risk of PERV transmission to patients may be low.

Often the shelf life of cell therapy products is shorter than the time required to test for sterility and adventitious agents using traditional cell-based methods. However, as already discussed, development of validated rapid PCR-based methods allows both assessment and timely release. Presence of mycoplasma and a range of specific adventitious viruses and bacteria can be tested by using PCR or DNA- or RNA-hybridization dot blot analysis. Fourteen-day sterility testing is not always a viable alternative for final release of cell therapy products; in such cases, automated methods that rely on colorimetric detection or on continuous monitoring may be acceptable if they are validated. Facility and process validation are necessary adjuncts to ensure safety with regard to sterility and mycoplasma, particularly when rapid-release strategies are employed.

Additional testing for safety may be required when cell therapy products are used in the patient for a purpose other than that which the cells or tissue fulfills in its native state or when placed in a location of the body where such structural function does not normally occur. Testing should be designed to predict product behavior under these settings and should be designed based on the context of use. For example, in the case of a cell therapy product for cancer patients, where the cells are activated by culture on a feeder layer of cells during processing, it may be necessary to test the product cells for the presence of feeder cells. Residual feeder cells in the final product may cause an inflammatory response. In addition, products using non-human feeder cells are considered xenogeneic products. Cell therapies are exempted from general safety testing.

If the cells were modified by a viral gene vector during manufacturing, presence of RCV must be tested. Typically, RCV testing (see Viral Gene Therapy Products under Safety) is limited when rapid release is required by shelf life. Again, molecular biology–based methods such as PCR can be used in rapid screening situations. In such cases, during product development, testing that employs cell-based assays (for example, detection of cytopathic effect on indicator cell lines) is performed after release to validate the molecular biology–based test result.

One of the primary safety concerns associated with viral vectors used for gene therapy is RCV. Regardless of the virus, these concerns are based on the potential lack of predictability for the pathogenicity of a contaminating virus for a specific route of administration, particularly if it is not the normal route of infection or if humans are not a natural host for the virus.

The pathogenesis of a wild-type adenovirus infection is known but may not be predictive for the routes of administration employed with recombinant adenoviral vectors. For adenoviral vectors, a limit of one RCA per dose is currently considered acceptable; other limits have been established for dose levels greater than 1 × 109 particles, specific indications, and routes of administration based on preclinical safety studies and patient-monitoring studies during clinical development. Limits as high as several thousand RCAs per dose have been reported. Typically, RCA levels are determined by using a cell-based assay that allows amplification of the RCA while preventing replication of the product. The cell line recommended for amplification and detection of RCA is the A549 cell line. However, some recombinant adenoviral vectors express therapeutic genes that interfere with analysis on A549 cells. In such cases, a bioassay utilizing two cell lines is used, with the first cell line chosen on the basis of resistance to the effects of expression of the therapeutic gene of interest and with subsequent passage of cell lysate onto A549 cells for amplification and detection of the RCA. RCA is most often detected by visual observation of the cytopathic effect but it may also be detected in the A549 cell culture by using immuno- or PCR-based methods. Quantitation of the RCA level is based on the quantity of sample tested and the detection limit of the assay. Typically, RCA bioassays are validated as being able to detect 1 plaque-forming unit or infectious unit of RCA in the test sample over a wide range of test-sample sizes. Test-sample sizes can range from 1 × 108 to 1 × 1012 particles but they are typically based on clinical-dose size. To verify detection limits, spike controls should be included as part of the test, even with validated assays. For recombinant adenoviruses produced using 293 cells, RCA detection by PCR methods can be confounded by detection of residual 293 host-cell DNA (detection of the E1 region). PCR assays, however, can be designed to specifically quantitate host cell DNA contamination and can be made specific to particular forms of slow-growing RCA. Quantitative PCR assays can be used in conjunction with a cell-based method for precise quantitation of RCA levels. When a tested sample is found to be positive, the identity of the RCA is usually confirmed by conducting PCR analysis. This rules out the possibility that contamination of the assay by exogenous wild-type adenovirus or other adventitious agents is responsible for the positive result.

For retroviral vectors, testing for RCR is required for cell banks, viral vector production lots, and any resultant ex vivo product lots (see the FDA's Draft Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-Up of Patients in Clinical Trials Using Retroviral Vectors, October 2000). Standard assays have been designed to detect replication-competent murine leukemia virus (MLV). The pathogenesis and potential long-term toxicity of low-level amphotropic MLV in human beings is not known. Methods commonly used to detect RCR include an amplification of virus titer by application of product to a replication-permissive cell line such as Mus dunni. Because infection is limited by the ability of a virus to reach the cells through Brownian motion, procedures (e.g., centrifugation and filtration) that physically bring the virus into contact with the cells may be used to enhance detection. However, high-titer recombinant vector can interfere with the detection of low-level RCR and this interference may be enhanced through such methods. Infected cells are passaged several times to allow viral replication. Culture medium is harvested at the end of the culture period and RCR detected by using an indicator cell line. If the product is an amphotropic MLV, RCR may be detected by using a feline cell-based PG4 S+L– assay, a mink cell-based MiCl S+L– assay, or a marker rescue assay. In S+L– assays, the RCR expresses proteins that lead to transformation and subsequent plaque formation on the monolayer. In a marker rescue assay, RCR infects a cell line that expresses a retroviral vector encoding a marker gene such as -galactosidase, drug resistance, or a fluorescent protein. The vector is packaged by the proteins supplied to it in trans by the RCR. The potentially vector-laden supernatant is transferred to naive target cells that are then screened for expression of the marker vector.

Testing for RCR is performed by cocultivation of the cell line or amplification of vector supernatant with an RCR replication-permissive cell line, typically Mus dunni, for several passages. Culture medium is harvested at the end of this cocultivation process and applied to an appropriate indicator cell line as described above. It is important to note that artifacts may be generated during the cocultivation assay by expression of an endogenous virus in the permissive cell line or through fusion if the vector-producing cell line is cultured directly with a marker rescue cell line. In addition, cocultivation may not be possible for ex vivo cell products that have specific culture requirements or limited culture life spans.

Methodologies for testing the presence of RCR in crude, purified bulk or final vector products are not specified. The FDA has deposited a reference standard of an amphotropic hybrid MLV with the ATCC. This viral stock has been assigned a label titer and should be used in assay validation. Method validation should demonstrate the ability to reproducibly detect a single RCR particle in individual product types because the product and its related impurities can interfere with the detection of RCR. Currently there are no acceptable limits for RCR contamination in products. Any product lot found to contain RCR cannot be used for human use.

Reference standards for assessing RCV in other viral vectors including ecotropic, xenotropic or pseudotyped MLV, adenovirus, and lentivirus have not been developed. Amplification and detection of replication-competent HIV, especially its pseudotyped variants, may warrant special containment and handling procedures.

Additional safety testing usually focuses on methods similar to those described under Biotechnology-Derived Articles 1045, in Safety Tests—Biologicals under Biological Reactivity Tests, In Vivo 88, and under Sterility Tests 71. For viral gene therapies produced using a human cell line, performance of the in vitro adventitious agent bioassays using 3 cell lines is recommended on either the bulk or final product. For adenoviral vectors, specific tests for adeno-associated virus are also recommended on either the bulk or final product. For adeno-associated virus, specific tests for adenovirus and herpesvirus are recommended on either the bulk or final product.

Safety testing usually focuses on methods similar to those described under Biotechnology-Derived Articles 1045, in Safety Tests—Biologicals under Biological Reactivity Tests, In Vivo 88, and under Sterility Tests 71. However, the General Safety test is not required for therapeutic DNA plasmid products (even if formulated). Safety testing should be performed on nonviral formulated material. If the shelf life of the formulated nonviral therapy is very short, then the components should be tested individually.

An assay that precisely measures the amount of the product is referred to as a dose-defining assay, and it is selected on the basis of its precision and accuracy. An assay that measures therapeutic activity of the product is referred to as a potency assay and it is designed to measure product function. The design of the assay is dependent upon the type of product. In the case of drugs, the assays measuring the amount of active ingredient (dose) are referred to as strength assays.

Product dose can be defined as the concentration or amount of the drug product administered to the patient and it is typically measured as product mass. For cell and gene therapy products, attributes such as viable cell number, milligrams of plasmid or antisense oligonucleotide, or the viral particle number are often used to define the dose of the product.

Cell therapy products may be dosed on the basis of enumeration of one or more cell populations. For ex vivo gene therapy, dose may be based on cell number as well as level of expression of the gene product. For products in the form of a homogeneous, single-cell suspension, viable cell number is the most frequently used assay. Such assays may include enumeration of all cells, total nucleated cells, or another subset of cells. Viability assays are usually based on a cell's ability to exclude a supravital dye, such as trypan blue. Results are expressed as the number of cells that exclude the dye and are therefore considered viable. The compound 7-AAD, a red-fluorescing compound that binds to nuclear proteins and is also excluded by viable cells, may be incorporated into flow-cytometric methods for simultaneous determination of viability and cell-identity markers.

Cell counting may be performed rapidly by manual or automated methods. Manual cell counting by visual enumeration of cells in a hemacytometer chamber is a readily available technique with acceptable accuracy, but a lower degree of precision than most automated methods. Typical instruments for automated cell counting provide reproducible enumeration of nonnucleated cells (e.g., erythrocytes and platelets) and nucleated cells and differential counting of the nucleated cells into mononuclear and polymorphonuclear leukocyte populations. Further discrimination of specific cell populations usually requires cell-surface phenotype analysis by flow-cytometric or other methods (see Cell Therapy Products under Identity). The proportion of a specific subpopulation of cells may be determined by FACS analysis or by flow cytometry.

An example of a cell enumeration assay is the enumeration of CD34-positive (CD34+) hematopoietic progenitor cells, the number being expressed as the number of cells per recipient's body weight. In numerous studies this measurement has been shown to predict hematologic reconstitution following myelosuppressive or ablative therapy in autologous or allogeneic hematopoietic transplantation.

For products that contain cells in a nonhomogeneous suspension, such as cells that form a two- or three-dimensional structure, alternative measures for cell enumeration, such as total area of a cell sheet, wet weight, total protein, and total DNA, have been used. If such measures are used to determine product dose, then supplemental tests must be performed to demonstrate therapeutic activity.

Particle concentration is a commonly used measure for viral vector product dose. Particle concentration may be measured by physical, biophysical, or in vitro cell-based assays. For example, quantitation of purified adenoviral particles may be determined by using the optical density of a solution of virus in 0.1% (w/v) sodium dodecyl sulfate (SDS) solution, at 260 nm, because a relationship between absorption and particle concentration has been published for adenovirus. The particle number concentration is equivalent to the product of the absorbance at 260 nm in a 1-cm cell, the dilution factor, and 1.1 × 1012 particles.2 Other methods to determine particle concentration include particle counting by electron microscopy and integration of viral peak area against an authenticated reference standard in an anion-exchange resin-based high-pressure liquid chromatographic (HPLC) assay.

Virus concentration can also be assessed through the measurement of selected structural proteins with known molecular masses and known copy numbers within the virion. For this method, the virus has to be lysed, and the structural proteins have to be separated by using an appropriate, high-recovery chromatographic procedure (e.g., reverse-phase HPLC). The chromatographic separation and the identity and the purity of the selected structural protein has to be verified during assay validation by methods such as SDS polyacrylamide gel electrophoresis (SDS-PAGE), peptide sequencing, and mass spectroscopy. The selected structural proteins have to be quantified, for example, by integrating chromatographic peaks at 214 nm and comparing the area to an authenticated reference standard. The virus concentration can then be calculated based on the molecular mass, the copy number, and the measured mass of the protein. Very importantly, the virus concentration can be estimated simultaneously for multiple structural proteins, allowing the use of this assay in relatively impure virus preparations. This method has been applied to adenovirus and should be applicable to other viral vector types.

Biophysical methods of determining particle number include direct quantitation of vector nucleic acid by radiolabeled-probe hybridization and indirect quantitation by amplification of template nucleic acid (e.g., PCR and RT-PCR) or by signal amplification (e.g., branched-chain DNA using multiple-probe hybridization).

In cases where biophysical methods are not available, bioassays that measure gene-vector titer have been used. These involve infection, transfection, or transduction of a susceptible cell line in vitro, followed by some measure of the product uptake. Methods for quantitation or estimation of the number of infection, transfection, or transduction events include plaque-forming unit assays, tissue culture infectious dose, 50% (TCID50 ) assays based on cytopathic effect or immunofluorescent detection of an expressed vector protein, or a quantitative DNA-hybridization assay. Examples are as follows.

For retroviral or lentiviral gene therapy products or AAVs that carry a selectable marker (e.g., that for neomycin resistance) or a reporter gene (e.g., -galactosidase) in addition to the therapeutic gene, the infectious titer is commonly determined by measuring the number of transduced or infected cells expressing these nontherapeutic proteins. Vector titer is typically reported as the number of colony-forming units (cfu) per mL for cells transduced with viral vectors containing drug-resistance markers and selected for growth in drug-containing medium. Titer based on -galactosidase can be expressed in terms of blue (cfu) per mL after staining and counting the cells that convert the -galactosidase substrate X-Gal into a blue chromophore. For vectors without a marker gene, quantitation of transduction has been measured precisely by using quantitative PCR.

Most nonviral gene therapy products contain plasmid DNA and their usual measure of dose is the DNA mass. The DNA mass may be determined in the formulated state, and, if recombinant protein is included in the formulation, the total combined mass of all formulation components based on a specific ratio can be used. DNA concentrations greater than 500 ng per mL are most simply determined by using optical density measurement at 260 nm. This method is not generally applicable to lipid-formulated DNA. Because RNA and proteins also have significant absorbances at 260 nm, other analyses must be performed to demonstrate that there is minimal contamination with RNA, protein, or residual host-cell chromosomal DNA. Dyes that specifically bind to double-stranded DNA allow the DNA concentrations of less than 500 ng per mL to be measured accurately when calculated against an authenticated DNA standard curve. PicoGreen is one such fluorescent dye and it is minimally affected by single-stranded DNA, RNA, proteins, salts, and detergents. The fluorescent dye Hoechst 33258 also binds to both double-stranded and single-stranded DNA and it can be used to determine DNA concentrations as low as 0.3 ng per mL. The Hoechst 33258 does not bind to protein or RNA and it can accurately determine the DNA concentrations in crude samples.

Methods, such as capillary electrophoresis, employing an authenticated reference material, can also be used to determine the strength of nonviral and antisense-oligonucleotide products.

Potency is defined as the therapeutic activity of the drug product. Together with dose, potency defines the biological activity of each lot (see General Considerations under Dose-Defining Assays). Potency may be assessed through in vitro or in vivo bioassays. It is not uncommon for these assays to have coefficients of variation between 30% and 50%. These assays require a well-defined, representative reference material that can be used as a positive control for the assay. The positive control serves to qualify the performance of an individual assay. Potency assay development should focus on characterizing and controlling variability. The high-precision assays are more effective tools in monitoring product quality. Information about potency-assay variability should be incorporated into the stability study design and the proposed statistical approach to assignment of expiration date (see Stability).

Functional assays that can be performed on cellular products are application-related and include viable cell number and a wide range of colony-forming assays, proliferative assays, cell-to-target killing assays, and assays that quantitate gene expression following gene transduction. For hematopoietic progenitor cells prepared from marrow, peripheral blood, or cord blood, traditional colony-forming assay quantitates committed progenitor cells such as colony-forming unit-granulocyte–macrophage (CFU-GM); this assay has been correlated with clinical engraftment outcomes in some studies. More recently, process-monitoring programs incorporate assays such as the long-term culture-initiating cell (LTCIC) assay or the in vivo animal models such as competitive repopulation in immunodeficient mice to monitor the activity of the most primitive hematopoietic stem cells. In the case of cells intended for structural repair, proliferation under a set of defined ex vivo conditions may be used as the potency assay. If the cells release an enzyme or active molecule, a potency assay could be based on the units of enzymatic activity or on the total of active molecules released. For instance, the production of insulin in response to changes in glucose levels could be the basis of a potency assay for cells intended to treat diabetes.

Patient-specific products, such as autologous cancer vaccines that elicit an in vivo immune response, present a challenge in demonstrating therapeutic activity in an in vitro or in vivo assay system. Assessment of potency in these circumstances is currently the subject of public-policy debate. Novel approaches to measuring potency, such as the correlation of clinical outcome to other characterization tests such as identity tests, may be appropriate and should be discussed with regulatory authorities early in development. For example, the ability to determine specific cell-surface identity markers by employing flow-cytometric techniques or vital stains may be an acceptable measurement of potency if properly validated and correlated with clinical outcome.

Bioassays employed to measure the potency of viral and nonviral gene therapy products generally involve infection, transfection, or transduction of a susceptible cell line in vitro, followed by some functional measure of the expressed gene of interest. Functional assays for the therapeutic gene (e.g., those measuring enzyme activity and cytokine activity) should generally be used instead of analytical methods such as enzyme-linked immunosorbent assay (ELISA), HPLC, or FACS, which provide information about the level of expression but only infer function. In addition, for viral vectors, infectious titer measurements by themselves are generally not considered an adequate measure of product potency. The design and ultimate suitability of an assay system for determining product potency will depend on the relationship between the intended human target cell in vivo and the following: (1) the transduction or transfection efficiency of the cell line used in vitro (2) the protein expression levels, and (3) the duration of expression required for the therapeutic effect.

In vivo tests may also be used to measure vector-product potency. Readouts may be based on a response per animal (e.g., blood levels of therapeutic protein 24 hours after treatment) or a group response rate (e.g., percentage of animals that elicited an immune response or survived virus challenge). The availability of an appropriate in vivo test system will depend on the vector-host range (for viral vectors), the pharmacokinetics and biodistribution of the vector and its resultant gene product relative to its human counterpart, and the time frame required to observe the therapeutic effect or surrogate. Issues of cost, facilities, validation, and ethics will determine the practicality of an in vivo potency test.

Analytical methods that separate, isolate, and specifically quantify the intended active product components determine product purity. Impurities are either product- or process-related components that can be carried through to the final product. The manufacturing and purification process should be optimized to consistently remove impurities while retaining product activity. The requirement to test for a particular impurity for product lot release will depend on the following: (1) the demonstrated capability of the manufacture and purification process to remove or inactivate the impurity through process validation, and (2) the toxicity potential associated with the impurity.

Examples of process-related impurities associated with cell and gene therapy products include residual production-medium components (e.g., FBS, antibiotics, cytokines, and Escherichia coli chromosomal DNA in a plasmid product), ancillary products used in downstream processing (e.g., nucleases such as DNase I), and residual moisture for lyophilized vector products. Impurities may be bioactive (e.g., cytokines and hormones) or immunogenic (e.g., product aggregates, degradation products, plasmid-selection markers, and nonhuman-derived proteins) or they may have other deleterious effects (e.g., they may compete with the product) if administered at a dose equal to that of the product. Product-related impurities are specific to each product type. Examples include differentiated cells in a stem-cell therapy product, nicked plasmid forms in nonviral products, and defective or immature virus particles in retroviral or adenoviral vector products. Analytical methodologies to assess purity require quantitation or physical separation of intended product from its impurities. Common sense should drive the need to quantify specific impurities. It may be possible to validate the manufacturing process to the extent that specific lot-release testing for impurities will be very limited. An emphasis may be placed on demonstrating the consistency of the product-impurity profile.

Testing for impurities is often extensive during product characterization and process validation when the consistency of the manufacturing and purification process is being demonstrated. Testing for impurities as part of lot-release testing should reflect the safety risks associated with the impurity and the ability of the process to consistently remove that impurity.

One measure of purity is the percentage of viable cells in the total cell population. Another measure is the percentage of transduced cells or the percentage of cells with a specific marker. If an entire population of cells is the therapeutic agent, methods should determine the relative amounts of each subpopulation. Limits should be defined for each cell subpopulation. These assays may be based on immunological methods utilizing flow cytometry or DNA-hybridization dot blot analysis. Additional tests for process contaminants are performed depending on the specifics of the manufacturing process. For example, a quantitative ELISA for residual serum proteins may be required. If the cells are genetically modified during manufacture, then testing for residual vector may need to be performed.

For gene-modified cell therapies, determining the purity of the cell therapy product depends on the availability of reagents and methods to distinguish therapeutic cells from the other cells present in the product. As in the case of gene therapy products, gene-modified cells can be distinguished from the unmodified cells on the basis of expression of the transgene. FACS using an antibody that detects the therapeutic protein or fluorescent probes that detect expressed RNA allow the separation and quantitation of the transgene expressing and nonexpressing cells. By adding an antibody to a cell-specific phenotypic marker and by using a double-sorting technique, FACS can be used to further identify the subpopulation of cells that are modified.

Endotoxin testing is also required. Biomaterials used with cell therapy products should also be tested for their biocompatibility.

Product-related impurities for viral vectors include aggregates and defective and immature particles that may be produced during the manufacture or purification of the recombinant vector. Aggregates of vector may form if the product is highly concentrated, stored under certain conditions (e.g., under certain pH or temperature), or reconstituted after lyophilization. Assays to detect aggregates include particle size analysis by laser light-scattering and the use of nonreducing, nondenaturing PAGE, followed by staining of the gel or transfer and detection of viral proteins by Western blot analysis. Sedimentation rate analysis also allows separation of aggregates from monomers based on size. Optical density analyses of light-scattering are also used to assess vector aggregation.

Defective particles are viral particles that do not contain the appropriate recombinant genome, that is, they contain some other nucleic acid or contain no genome at all, or the vector has some missing, defective, or otherwise altered structural component that impairs its ability to transduce a cell. For viral vector systems that have capsomeric symmetry, which requires the appropriate nucleic acid incorporation for configuration, empty particles may be readily distinguished from those carrying genomes. For enveloped viruses, empty particles may not be as readily separated from those with encapsidated nucleic acid.

For some viral vector products, active viral particles may be separated from defective particles by using analytical HPLC. Anion-exchange resins have been used to separate active adenovirus from defective virus particles. However, this method might not be useful for an adenoviral vector purified by anion-exchange chromatography unless the resin for the assay is different from that used during manufacture. Depending on the nature of the viral vector and its nonactive or defective forms, other methods of separation, such as equilibrium centrifugation in a cesium chloride density gradient, may need to precede the quantitation of the active particle. Ideally, the method of separation will allow quantitation.

Defective particles that carry a noncell-derived oncogenic gene or other undesirable genes may pose a special concern. For example, in murine-based retroviral packaging cell lines, small viral elements called VL30 sequences can be packaged in about one third of all particles. Assays may need to be developed to quantify specific defective particles if they are known to be present in quantities sufficient to pose a safety concern.

Virus quality and the comparability of preparations can also be assessed by measuring selected structural proteins with known molecular masses and known copy numbers within the virion. For this method, the virus is lysed, and the structural proteins are separated by using reverse-phase HPLC or some other high-recovery chromatographic procedure. The chromatographic separation should be validated and the identity of the selected structural proteins verified by methods such as SDS-PAGE, peptide sequencing, or mass spectroscopy. One can fingerprint the batch based on quantification of the selected structural proteins and comparison to a reference standard. When the method incorporates the use of mass spectroscopy, impurities such as structural variants can also be identified. For adenovirus preparations, some precursor and most mature virion proteins can be monitored, thus allowing monitoring of the product and of the immature virion forms.

Host cell-derived proteins may be considered impurities for some viral vector products and may be separated and quantified by PAGE or HPLC or detected by amino acid analysis, Western blot, or immunoassay-based methods. However, for enveloped viruses such as retroviruses, host cell-derived membrane proteins are an integral part of the product. In those vector systems, it may be difficult to determine the presence of contaminating exogenous host-derived proteins.

Presence of specific process-related impurities depends on the manufacture and purification process of each vector or product type. However, most products will need to be tested for residual endotoxin (see Bacterial Endotoxins Test 85). Acceptable limits of endotoxins have been determined and can be directly applied to viral vector products.

Although genomic DNA derived from continuous cell substrates used to manufacture biological product has been considered historically as potentially tumorigenic, recent studies suggest that the risks are very low. However, every attempt should be made during process development to reduce contaminating DNA levels. The need to test for residual DNA as part of product lot release should be evaluated on a case-by-case basis and may be dependent upon the size distribution of the DNA, its association with the product or its formulation components, and the route of administration of the product. Quantitative PCR assays have been developed to analyze the amount of residual host-cell DNA by using primers designed to amplify evolutionarily conserved and abundant target sequences, such as 18S for 293 cells.

Quantitation of residual serum components such as bovine serum albumin (BSA) can be achieved by using ELISA and a BSA reference standard. Specific functional or immunological methods may need to be developed for other ancillary products including other culture media or purification process components such as cytokines or enzymes (e.g., nucleases such as DNase I or benzon nuclease).

Testing is usually performed on the individual components, the plasmid DNA, the lipid or lipoplex components and (recombinant) protein components if any are present in the formulation. Plasmid DNA is characterized for a variety of impurities including residual host-cell DNA, residual RNA, and residual protein. Residual protein testing is frequently included in lot-release testing. Optical density ratios, usually the ratio of the measurement at 260 nm to that at 280 nm, are frequently used in purity specifications for plasmid DNA.

In addition, the plasmid DNA should also be characterized with regard to its form. Plasmid DNA forms include monomeric supercoiled, relaxed monomer, and linear forms. The profile of forms needs to be monitored for product consistency. Additionally, it may be possible to correlate form with in vivo transfection behavior. While monomeric supercoiled plasmid has been shown to be more efficient than relaxed monomer, linear, or multimeric forms of the plasmid in transfecting cell lines in vitro, in vitro transfection has been shown to not always predict in vivo behavior. Formulation, delivery method, and route may impact in vivo transfection. Agarose gel electrophoresis can resolve these forms of plasmid, which are then detected by UV after ethidium bromide staining. This method provides information about the relative levels of the plasmid forms, but it is not highly quantitative for the individual species. Analytical anion-exchange HPLC can be used as a quantitative assay for monomeric supercoil and percentage of other forms, including concatamers. Other sophisticated methods, such as capillary zone electrophoresis, linear-flow dichroism, and atomic-force microscopy have been proposed as replacements for agarose gel analysis. Until they are validated, these analytical methods may be more appropriate for characterization studies in support of process development and validation rather than for lot-release testing. The appropriate methods for lot release will depend on what effect these alternate plasmid forms have on the product potency. Many of these methods, such as HPLC, are also applicable to the assessment of the purity of antisense-oligonucleotide products and the determination of the level of by-products.

Tests for process-related impurities, such as cesium chloride, must also be conducted. In the case of antisense-oligonucleotide products, residual solvents must be quantified. Lipid and lipoplex formulation components must also be tested for their chemical purity. Testing for specific chemical impurities is commonly performed by using gas chromatography–mass spectroscopy (GC–MS), high-pressure liquid chromatography (HPLC), or thin-layer chromatography (TLC) methods.

If protein is part of the formulated complex, then the protein must also be tested for purity. The methods outlined under Biotechnology-Derived Articles 1045 or under Biotechnology-Derived Articles—Tests 1047 are relevant.

Bacterial protein, DNA, RNA, and endotoxins are the major types of host-derived process contaminants. Standard protein assays (e.g., Lowry, Bradford, or Coomassie), PAGE followed by silver staining or Western blot analysis, or ELISA can be used to detect residual host protein in the nanogram range. Host chromosomal DNA may be detected by slot blot hybridization (detection in picogram range) or by PCR using highly conserved target sequences (e.g., 18S for Escherichia coli). However, low background may be unavoidable in PCR-based assays because the recombinant polymerases used for the amplification of target also contain residual bacterial DNA. PAGE or agarose gel electrophoresis followed by fluorescent dye staining may be used to detect residual RNA. Quantitation may not be required given the labile nature of RNA and the low-level toxicity associated with it.

Certain antibiotics, such as kanamycin, that may be used during the fermentation process must be removed during the process, and validation of the process or lot-release testing must be performed to confirm removal during the purification of the plasmid. HPLC is one method that can be used to detect low-level residual antibiotic.

Residual moisture may affect the stability of a lyophilized vector product. The FDA's Guideline for the Determination of Residual Moisture in Dry Biological Products (January 1990) recommends a 1% residual moisture level, although data indicating no adverse effects on product stability at higher levels will be considered acceptable. Residual moisture levels can be determined by using a standard method (see Water Determination 921) that is compatible with the formulated product.

Lot-release testing for cell and gene therapy products must include an identity test. This test serves to specifically identify the product. The complexity of the identity test will depend on the nature of the specific product and the array of products being manufactured. For example, more extensive and rigorous testing may be performed for an autologous gene-modified cell therapy product at a facility where multiple patient products are manufactured than for a viral vector product produced at a site that manufactures a single vector product.

Cell therapy identity tests must be relevant to the cell type and manipulations applied during processing. Differential surface markers (for instance, CD3, CD4, CD34, and CD45) are frequently used to ascertain product identity. Flow-cytometric immunoassay methods are the most common means of detecting and quantifying these markers. In this type of assay, a sample of the cells is stained with fluorescently labeled antibodies directed against specific identity markers and then passed as a single cell suspension in front of a laser source. Identification and quantitation of particular cell subsets is accomplished by multiparameter analysis, usually of size and granularity (measured by forward and side light-scattering) and of one or more identity markers (measured by emitted fluorescence). Simultaneous quantitation of cell viability can be performed by adding 7-amino-actinomycin D (7-AAD) to cell suspensions marked with antibodies conjugated to green (e.g., FITC) or orange (e.g., phycoerythrin) fluorescent compounds.

Analyses, such as isoenzyme analyses, employing biochemical markers are also used. For example, isoenzyme analyses are used to confirm species in the case of xenotransplants. Cell morphology can be used if it can distinguish specific cell types or unique function. Morphology can be combined with doubling-time parameters to better distinguish different cell types.

Restriction enzyme mapping and sequencing of the transcription unit DNA are the most commonly used approaches to establishing the identity of viral vectors for characterization purposes. PCR-based methods, restriction enzyme mapping, and transgene expression–based immunoassays are most commonly used to confirm the identity during lot-release testing.

Restriction enzyme mapping is the most common identity method for plasmid-DNA and antisense-oligonucleotide products. The number of enzymes used to create the vector fingerprint will vary with the complexity of the DNA and the degree of similarity between multiple products. If lipids, lipoplex agents, or proteins are used to formulate the DNA, then their identity must also be tested. Lipids and lipoplex chemicals may be identified by procedures used for traditional pharmaceuticals, such as GC–MS, TLC, and the like. Protein components of the formulation may be identified by peptide mapping or other means outlined under Biotechnology-Derived Articles—Tests 1047.