Characterization of Cell and Virus 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.
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.
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.
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.