U.S. PHARMACOPEIA

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MANUFACTURING OVERVIEW
Introduction
The manufacturing of cell and gene therapy products has been divided into three sections. This section, Manufacturing Overview, discusses five topics that apply to manufacturing of all cell and gene therapy products: (1) raw materials, (2) characterization of banked materials, (3) in-process controls, (4) specifications, and (5) validation considerations. Manufacturing of Cell Therapy Products addresses the manufacturing of cell therapy products including cell products into which genetic material has been introduced. Manufacturing of Gene Therapy Products addresses the manufacturing of gene therapy vectors, both viral and nonviral, discussing the design of gene vectors in detail.
All the general principles of current good manufacturing practice (CGMP) as outlined by the FDA in 21 CFR 210, 21 CFR 211, 21 CFR 600s (especially 21 CFR 610), 21 CFR 820, and discussed in USP informational chapters, such as Biotechnology-Derived Articles 1045, apply to the manufacturing of cell and gene therapy products. The manufacturing facility, equipment and process, raw materials, quality systems, and trained personnel are some of the key elements of CGMP. CGMPs apply throughout the clinical development to both the manufacturing process and facility. The extent of control increases as clinical development progresses, with full CGMP compliance expected by initiation of Phase III of the pivotal clinical trial(s). The facility should be carefully designed, built, and validated to accommodate the unique features of the product's manufacturing process. Equipment should be robust, provide consistent product, and allow periodic calibration. Critical equipment, such as incubators and freezers, needs to be fitted with alarm systems that can remotely signal failure. Quality control (QC) and quality assurance (QA) programs should exert control over the manufacturing facilities, the manufacturing process, the validation efforts, and all testing of the raw materials, in-process material, bulk product, and final formulated product. Training and certification programs are central to maintaining a technically competent manufacturing staff. A documentation program should be implemented to support all manufacturing, training, and quality operations. Changes to processes and procedures should follow a formal program based on well-established CGMP and ISO change control principles.
Raw Materials
TYPES OF RAW MATERIALS
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.
QUALIFICATION
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.
Identification and Selection— In the early stages of product development, important decisions regarding the types of raw materials to be employed in the manufacture must be made. As manufacturing progresses and products mature, certain materials that are deemed necessary at this point may turn out to be impossible or prohibitively expensive to qualify. Attention must be paid to issues such as suitability, toxicity, availability, consistency, contamination, and traceability. Raw materials that could be difficult to qualify may have to be investigated and identified in the early stages of product development.
Every material employed in the manufacturing process should be accounted for. The source and intended use for each material should be established, and the necessary quantity or concentration of each material used should be determined. Primary sources, and when possible secondary sources, for each material should be identified. In all cases, suppliers should provide information regarding the traceability of each material, especially for human- and animal-derived raw materials. For instance, human serum albumin and processed allogeneic non-A,B human serum require donor infectious disease status information prior to use, and a material such as fetal bovine serum (FBS) requires herd qualification and country of origin certification before being used in a manufacturing process (see Fetal Bovine Serum).
Suitability— An assessment of the suitability of each raw material used in manufacturing is necessary in order to ascertain the risk that the raw material may pose to the safety, potency, and purity of the final therapeutic product. Knowing the source and the processes employed in the manufacture of each raw material will help determine the relative level of risk for each item. The quantity of the material and its point of introduction in the manufacturing process also affect the risk profile of a raw material. Materials that may have toxic effects or raise biological safety concerns receive special attention. Such materials should be subjected to extensive testing prior to use or should be monitored in the final product. Validation studies that demonstrate that such materials are effectively and consistently removed or rendered inactive in the course of manufacturing will also be necessary for eventual licensure of each product. The biocompatibility of natural or synthetic biomaterials used in cell therapy products may be assessed by subjecting the material to the testing protocols outlined in the FDA's Blue Book Memorandum (May 1, 1995), which is a modification of the ISO document 10993-1:1997 entitled “Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing”. USP chapters Biological Reactivity Tests, In Vitro 87 and Biological Reactivity Tests, In Vivo 88 should also be consulted.
Characterization— Once the suitability of each raw material employed in manufacture has been assessed, specific QC characterization tests need to be developed or implemented for each material. The test panel for each raw material should assess a variety of quality attributes, including identity, purity, functionality, freedom from adventitious or microbial contaminants, and suitability for intended use. The level of testing for each component is a product of its risk assessment profile and the knowledge gained about each component during development. Test specifications should be developed for each raw material to ensure consistency and performance of the manufacturing process. Acceptance criteria should be established and justified on the basis of the data obtained from lots used in preclinical and early clinical studies, lots used for demonstration of manufacturing consistency, and relevant development data, such as those arising from analytical procedures and stability studies.
Many raw materials, such as cells isolated from blood, serum-derived fluids and extracts, or growth factors, are biological in nature; therefore, they are highly complex and difficult to fully characterize. Tests for sterility, pyrogenicity, mycoplasma, and adventitious or infectious viral agents, including RCV, should be performed on these materials. The specific adventitious viral agent test panel is dependent on the source of the component and how that component is manufactured or prepared. Because these materials exert their effects through complex biological activities—and biochemical testing may not be predictive of process performance—functional or performance testing may be necessary. Performance variability of such materials may have a detrimental impact on the eventual potency of the final therapeutic product. Examples of complex functionality testing for raw materials are growth-promotion testing of individual lots of FBS on the cell line used in manufacturing, performance testing of digestive enzyme preparations, and in vitro tissue culture cytotoxicity assays.
Fetal Bovine Serum— One commonly employed animal-derived material in manufacturing is fetal bovine serum (FBS). FBS is often added to the cultivation medium to promote cellular proliferation of a wide variety of cell types, including cell cultures that are derived from primary tissue explants and biopsy specimens. Growth factors, hormones, and other nutritive components present in FBS, many of which are undefined or present in trace quantities, provide the necessary components required by many cells to survive and undergo cellular division in vitro. The production of high-titer gene therapy vectors from cell lines can also require rich culture medium that includes FBS at levels between 10% and 15%. Defined, serum-free media have been developed for a number of cell types. Although some cell lines may be gradually adapted to serum-free or low-serum culture conditions, this may not be possible for certain fastidious cells, thereby necessitating FBS use.
Although FBS use may be required, development and assessment of serum-free or reduced-serum culture media should be considered. A number of bovine-derived bacteria, mycoplasma, and viruses are known to be associated with FBS. These organisms could potentially enter the process stream and contaminate the final product. The potential risk of BSE, the bovine form of TSE (transmissible spongiform encephalopathy), transmission with this material has been the subject of international discussion. Although FBS has been categorized as a low-risk material, adequate testing and sourcing of FBS lots must be managed appropriately in a qualification program. Reduction or elimination of FBS in the manufacturing process can reduce the risks associated with contamination by adventitious agents.
FBS must be obtained from herds that are monitored for specific diseases relevant in agricultural settings (for example, tuberculosis or brucellosis) and that are from regions known to be free from BSE. Each lot must meet established guidelines for sterility and endotoxin content as well as freedom from specific bovine viruses. To increase the level of safety assurance, consideration should be given to employing methods, such as irradiation or nanofiltration, that remove or inactivate viral entities known to be associated with FBS.
It should be noted that defined media formulations typically include components, such as albumin and transferrin, that are purified from animal or human plasma. The purification, processing, and extensive testing of such components further minimize, but do not eliminate, the risk of viral or microbial contamination. Apart from the risks associated with adventitious agents, residual FBS in the final product may trigger an immune response in patients. The level of residual FBS in the final product does not necessarily correlate with the starting amount of FBS, and it can depend upon the nature of the product and the purification process. Even if FBS is not included in the manufacture of the product, residual amounts of other components used in the manufacturing process, including recombinant proteins or other defined media components, may be potentially antigenic.
Quality Assurance— The components of this part of the qualification program are multifaceted and should reflect those found in a typical manufacturing environment for a pharmaceutical product produced in compliance with CGMP. These activities should include the following systems or programs: (1) incoming receipt, segregation, inspection, and release of materials prior to use in manufacturing, (2) vendor auditing and certification, (3) certificate of analysis verification testing, (4) formal procedures and policies for out-of-specification materials, (5) stability testing, and (6) archival sample storage.
Characterization of Cell and Virus Banks
Cell Banks— A cell bank is a collection of vials containing cells stored under defined conditions, with uniform composition, and obtained from pooled cells derived from a single cell clone. The cell bank system usually consists of a master cell bank (MCB) and a working cell bank (WCB), although more tiers are possible. The MCB is produced in accordance with CGMP and preferably obtained from a qualified repository source (source free from adventitious agents) whose history is known and documented. The WCB is produced or derived by expanding one or more vials of the MCB. The WCB, or MCB in early trials, becomes the source of cells for every batch produced for human use. Cell bank systems contribute greatly to consistency of production of clinical or licensed product batches, because the starting cell material is always the same. Mammalian and bacterial cell sources are used for establishing cell bank systems.
Virus Banks— The master virus bank (MVB) is similar to the MCB in that it is derived from a single production run and is uniform in composition. The working virus bank (WVB) is derived directly from the MVB. As with the cell banks, the focus of virus bank usage is to have a consistent source of virus, shown to be free of adventitious agents, for use in production of clinical or product batches. In keeping with CGMP guidelines, testing of the cell bank to be used for production of the virus banks, including quality assurance testing, should be completed prior to the use of this cell bank for production of virus banks.
Qualification— Cell and viral bank characterization is an important step toward obtaining a uniform final product with lot-to-lot consistency and freedom from adventitious agents. Testing to qualify the MCB or MVB is performed once and can be done on an aliquot of the banked material or on cell cultures derived from the cell bank. Specifications for qualification of the MCB or MVB should be established. It is important to document the MCB and MVB history, the methods and reagents used to produce the bank, and the storage conditions. All the raw materials required for production of the banks, namely, media, sera, trypsin, and the like, must also be tested for adventitious agents.
Qualifying Master Cell Bank— Testing to qualify the MCB includes the following: (1) testing to demonstrate freedom from adventitious agents and endogenous viruses and (2) identity testing. The testing for adventitious agents may include tests for nonhost microbes, mycoplasma, bacteriophage, and viruses. Freedom from adventitious viruses should be demonstrated using both in vitro and in vivo virus tests, and appropriate species-specific tests such as the mouse antibody production (MAP) test. Identity testing of the cell bank should establish the properties of the cells and the stability of these properties during manufacture. Cell banks should be characterized with respect to cellular isoenzyme expression and cellular phenotype and genotype, which could include expression of a gene insert or presence of a gene-transfer vector. Suitable techniques, including restriction endonuclease mapping or nucleic acid sequencing, should be used to analyze the cell bank for vector copy number and the physical state of the vector (vector integrity and integration). The cell bank should also be characterized for the quality and quantity of the gene product produced.
Qualifying Master Virus Bank— Testing of the MVB is similar to that of the MCB and should include testing for freedom from adventitious agents in general (such as, bacteria, fungi, mycoplasma, or viruses) and for organisms specific to the production cell line, including RCV. Identity testing of the MVB should establish the properties of the virus and the stability of these properties during manufacture.
Qualifying Working Cell or Virus Bank— Characterization of the WCB or WVB is generally less extensive, requiring the following: (1) testing for freedom from adventitious agents that may have been introduced from the culture medium, (2) testing for RCV, if relevant, (3) routine identity tests to check for cell line cross-contamination, and (4) demonstration that aliquots can consistently be used for final product production.
In-Process Control
Manufacturing processes should have well-defined go–no go decision point criteria that are applied to key in-process intermediates and are used to pool material that has been processed through a step in several sublots. Quality must be built into the product, rather than tested during batch release. In-process controls are the assays or tests that are performed to ensure that the in-process intermediate is of sufficient quality and quantity to ensure manufacture of a quality final product. Examples of in-process controls are listed in Table 3. The main reason for performing the in-process control is to ensure that the correct product with anticipated quality and yield is obtained. Intermediate in-process material that fails to satisfy the in-process control criteria should not be used for further manufacturing. This material may be reprocessed if there are procedures in place for such activities. The reprocessed material must satisfy the original in-process specifications before it can undergo further manufacturing. If several sublots are to be pooled for further processing, sublots that fail to satisfy the criteria should not be included in the pool, even if the pool containing these failed sublots would pass the in-process assay criteria.
Table 3.Examples of In-Process Control Applications
Type of Product Attribute to Control
Cell therapy Quantity and viability of cells after a
key processing step
Cell phenotype after a culture step
Viral gene therapy Quantity and viability of cells during
bioreactor culture
Quantity of virus after virus culture
Specific activity of virus in fractions
after column chromatography
Quantity of host-cell DNA in frac-
tions after column chromatogra-
phy
Nonviral gene therapy Optical density or change in oxygen
consumption during culture
Amount and form of plasmid prior to
culture harvesting
Amount and form of plasmid after
extraction steps
Amount of pyrogen or endotoxin
after extraction steps in plasmid
pool
Antisense-oligonucleo-
tide therapy		 Purity from strand side-products after
key extension steps
Quantity in fractions after chroma-
tography
During clinical development, assays for product quality and yield should be performed after most processing steps to determine which steps are critical and which assays are most sensitive to deviations in the process. The information from these runs is also used to set the criteria for the selected assays. In-process controls are performed for fully validated processes to ensure that the process continues to be under control. The results of these assays should be trended and actions should be taken to correct problems as they arise.
Specifications
Specifications for cell and gene therapy products should be chosen to confirm the quality of the product by testing to ensure the safety and efficacy of the product. Selected tests should be product-specific and should have appropriate acceptance criteria established to ensure that the product exhibits consistent quality parameters within acceptable levels of biological variation, loss of activity, physicochemical changes, or degradation throughout the product's shelf life. The development and setting of specifications for cell and gene products should follow the principles outlined in the International Conference on Harmonization (ICH) guidance entitled “Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products”.
Establishing specifications for a drug product is part of an overall manufacturing control strategy that includes control of raw materials, excipients, and cell and virus banks; in-process testing; process evaluation and validation; stability testing; and testing for consistency of lots. When combined, these elements provide assurance that the appropriate quality is maintained throughout the manufacture of the product.
Appropriate specifications are established on the basis of thorough characterization of the product during the development phase and an understanding of the process and its capability. Characterization should include measurements of the physicochemical properties, safety, purity, process and product-related impurities, potency, viability, sterility, and quantity. Specifications for each product should be developed from this information by applying appropriate statistical methods. The data should include lots used in preclinical and clinical studies and should also include assay and process validation data that can be correlated to safety and efficacy assessments. Specifications should allow for the inherent variabilities exhibited by the production process and by the assay. The traditional lot-release specifications that apply to biologics may have to be re-examined for these product types. For example, the general safety test stated in 21 CFR 610.11 is a lot-release requirement that has been deleted for cell therapies, because it exhibits little relevance for these products.
Specifications for the product are anchored by an appropriate reference standard for the product. The reference standard for the product ensures that the process, as measured by the release assays, does not change significantly over time. The reference standard is made from a lot that is produced under CGMP and passes all in-process and final release testing. In addition, this reference standard is subjected to an additional level of characterization that includes tests not normally performed for product release. The reference standard need not be stored at the same dose, formulation, or temperature as the product. However, the stability of this reference standard needs to be determined. The reference standard verifies that a test produces acceptable results (passes its system suitability tests). Alternatively, a specific assay standard (working standard) can be used. If so, in the test it should behave similarly to the reference standard. Changing to a new reference standard (lot) should include many tests, all of which are run side by side with the existing reference standard. The impact of any change in the properties of the new reference standard should be carefully evaluated before it is adopted. One option for a reference standard for a cell product with a short shelf life or for a patient-specific application can be a bank of normal donor cells of the appropriate cell type. This cell bank can be used to ensure that the manufacturing process is capable of making a consistent product.
Production of a safe and efficacious product involves establishing not only lot-release specifications but also specifications designed to maintain control of the manufacturing process and the final product. This includes in-process specifications (see In-Process Controls), raw material and excipient specifications (see Raw Materials), product-release specifications, and shelf-life specifications. Specifications should be established for acceptance of raw materials and excipients used in the final formulation of the product. In addition, tests should be performed at critical decision steps during manufacture or at points where data serve to confirm consistency of the process. In-process release specifications should be established for each control step. Heterogeneity can result from the manufacturing process or storage of the product. Therefore, the manufacturer should define the pattern of heterogeneity within the product and establish limits that will maintain the therapeutic efficacy and safety of the product.
In some cases, specifications may be established for lot release as well as for shelf life. As discussed in ICH guideline Q5C, presented under Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products 1049, the use of different specifications should be supported by sufficient data to demonstrate that the clinical performance is not affected. Acceptance criteria should be established and justified on the basis of data obtained from lots used in preclinical and clinical studies and lots used for demonstration of manufacturing consistency and on the basis of relevant development data, such as those arising from validated analytical procedures and stability studies. Acceptance criteria should also be correlated with safety and efficacy assessments.
Once specifications have been established, test results should be trended. Results that are out of specification (OOS), or even those that are out of trend, need to be investigated prior to dispositioning of the material. The purpose of an investigation is to determine the cause of the discordant result. The FDA's Draft Guidance for Industry: Investigating Out of Specification (OOS) Test Results for Pharmaceutical Production provides a systematic approach for conducting an investigation. An assay result can be rejected if it can be confirmed that an error, such as an analyst error, calculation error, or equipment failure, has taken place. If the investigation concludes that the product is not within the specification, the lot should be rejected. In unique situations, a product that does not meet all specifications may have to be administered to a patient. However, procedures must be in place to govern the communication of the OOS results to the physician or to the person responsible for making the decision to use the product and to provide instruction for any follow-up testing, patient monitoring, and communication of those results.
Considerations for Validation
The potential for wide biological variation in cell and gene therapy products, particularly for patient-specific treatments, affects the validation effort. Nevertheless, the basic principles of process validation for any biological product, including those recommended by the ICH and FDA guidance documents and recommended under Validation of Compendial Methods 1225 and Validation of Microbial Recovery from Pharmacopeial Articles 1227, apply to the validation of most cell and gene therapy products. Guidelines for validating viral vaccines can be relevant to gene therapy processes that produce viral vectors. The hold steps in a manufacturing process should be validated to ensure that in-process intermediates are within specification and that the final product can be formulated successfully. Any assay used during the process validation must itself be validated before the process validation is commenced.
Process validation for patient-specific products, such as autologous cell therapy products or custom gene therapy products, presents some unique issues. First, the starting materials for patient-specific products typically arise from patient-derived materials, such as biopsy material or apheresis cell products. The process should be designed to accept a wide range in the quality and quantity of starting material. Sometimes use of alternative procedures with additional steps are required when the starting material is of poor quality or below specified amounts. Validation should confirm that these alternative procedures still result in a final product that satisfies release specifications. Procedures should also be in place to deal with receipt of substantially more of the starting material than normally expected. Such procedures should address the disposition of the extra material. Second, manual processing of cells and tissues will exhibit a degree of inherent variability. It is essential to develop processing steps that will successfully and consistently result in appropriate process components and final product, even if the process is confronted with nonstandard or variable tissue materials, such as a T-cell suspension contaminated with red blood cells or low-weight biopsy material. Process validation should take this variability into consideration and ensure that critical manufacturing and testing endpoints consistently meet specifications. The process validation shows that the procedures can produce a product free of microbial contamination. It should also show that there is no cross-contamination among different patient product lots. If possible, the process should be validated for virus clearance as discussed in ICH Q5A: Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. If this is not possible, cells used for production of the product should be evaluated for their ability to propagate viruses that are known to contaminate these cells or source materials. This should include raw materials used as ancillary products.
As a result of the variability discussed above, the consistency and the robustness of the manufacturing process need to be assessed by testing more than three lots. It is not expected that every manufacturing effort will be successful for patient-specific therapies. However, the success rate should be established and tracked so as to discover any decrease in that rate and to take actions to correct the problem. Well-characterized banked primary cells may be used in the validation of the process if the donors have a range of profiles expected for the patient population to which the therapy will ultimately be directed. Trending of a number of statistically acceptable product administrations can also be appropriate.