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Director, Center for Biologics Evaluation and Research

DEPARTMENT OF HEALTH HUMAN SERVICES Public Health Service Memorandum 1 DATE: Jut FROM: Director, Center for Biologics Evaluation and Research SUBJECT: Points to Consider in the Characterization
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DEPARTMENT OF HEALTH HUMAN SERVICES Public Health Service Memorandum 1 DATE: Jut FROM: Director, Center for Biologics Evaluation and Research SUBJECT: Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993) TO: Manufacturers Utilizing Cell Lines for the Production of Biologics This Points to Consider Document on the Characterization of Cell Lines Used to Produce Biologicals (revised May is intended to replace the document of the same title issued in These Points are neither regulations nor guidelines, but serve to represent the current consensus of the Center for Biologics Evaluation and Research (CBER) staff. We intend to continually revise and update this document as necessary in order to improve it s usefulness. You are requested to review and comment on this document. All comments should be addressed to: Dockets Management Branch Food and Drug Administration Drive, Room l-23 Rockville, MD Note: Comments should be identified by the docket number Two copies of any comments should be submitted. HFM-630 [DOCKET NO. DRAFT OF POINTS TO CONSIDER IN THE CHARACTERIZATION OF CELL LINES USED TO PRODUCE BIOLOGICALS (1993) Submit written comments on this draft to: Dockets Management Branch (HFA-305) Food and Drug Administration rm. l-23, Dr. Rockville, MD Submit written requests for single copies of this draft to: Congressional and Consumer Affairs Branch (HFM-12) Food and Drug Administration 1401 Rockville Pike, Suite 200N Rockville, MD For further information about this contact: Center for Evaluation and Research (HFM-20) Food and Drug Administration 1401 Rockville Pike, Suite 200N Rockville, MD Comments and requests should be identified with the docket number found in brackets in the heading of document. 1 POINTS TO CONSIDER IN THECHARACTERIZATION OF CELL LINES USED TO PRODUCE (1993) CENTER FORBIOLOGICS EVALUATlON AND RESEARCH FOOD AND DRUG ADMINISTRATION Table of Contents INTRODUCTION Page 3 II. HISTORY AND GENERAL CHARACTERISTICS OF CELL LINE 7 A. History of the Cell Line 7 B. General Characteristics of Cell Line THE CELL BANK SYSTEM 8 A. Generation of Cell Banks 8 B. Storage of the Cell Banks 9 IV. Cell Bank Qualification CULTURES AND PRODUCT TESTING A. Cell Culture Media 12 B. Management of Cell Cultures 14 V. CONTROL TESTING 17 A. Tests for the Presence of Bacteria and Fungi B. Tests for the Presence Mycoplasmas C. Tests for the Presence of Viruses 17 D. Tumorigenicity Testing 22 2 VI. VALIDATION OF VIRAL ELIMINATION 24 A. Design. 26 B. Interpretation. 30 C. Statistics 32 D. Revalidation 32 VII. VIII. IX. ATTACHMENT : Letter to Manufacturers Requesting Identification of Bovine/Ovine Components and Sources x Recommended Test Procedures for Mycoplasmas 38 3 INTRODUCTION This document is concerned with the characterization of cell lines used to produce biological products which are subject to under the U.S. Public Health Service Act and also with the identification of possible adventitious infectious agents from the cell lines which might contaminate the final product. The existing general regulations in 21 CFR 200 et seq. and et seq. especially 21 CFR et seq. embody requirements with objectives that the final product be uniform, consistent from lot-to-lot and free from adventitious infectious agents. This document provides information that may be useful to manufacturers in achieving these objectives, but does not create new requirements or rights. Advances in biotechnology are occurring rapidly. Each new product should be evaluated in light of its own particular characteristics and the cell line and manufacturing process being used. Therefore, information in this document is subject to change as new and significant findings become available. Accordingly, this discussion should be interpreted as raising scientific issues that manufacturers who produce biological products from cell lines should consider both during product development under investigational new drug applications and before submitting product license applications Existing general regulations, 21 CFR 200 series and 600 series, are also broadly relevant and should be consulted. 4 These points are not all-inclusive. Alternative approaches may well be suitable in specific situations, and certain aspects may not be applicable to all situations. Furthermore the scientific basis for determining the appropriateness of the points specified for consideration here is developing rapidly and more appropriate approaches may be developed in the future. Therefore, the Center for Evaluation, and Research (CBER) will review the adequacy of testing of any cell line on a case basis. This document supersedes the Points to Consider (PTC) in the Characterization of Cell Lines Used to Produce (1987) and reflects a number of changes emanating several international workshops held since that time As stated in the 1987 PTC, the current approach to working with cell lines to produce biological products focuses on: 1. production, identification and characterization of the cell substrate; 2. validation of the manufacturing process for removal and/or inactivation of adventitious agents; and 3. testing of the bulk and final product to assure safety. However, it should be noted that a number of tests previously recommended have been revised or eliminated. Specifically: Karyology 5 In 1978 an ad hoc committee met to revise the recommendations on karyology control. The Committee s report was published in 1979 (3). The detailed characterization and monitoring procedures described in 1979 applied specifically and are still applied to diploid cell lines used for the production of, for example, live virus vaccines. However, the utility of katyology for the characterization of continuous cell lines is probably minimal; therefore, routine karyology is not recommended in these circumstances Tumorigenicity testing Experience has shown that virtually all cell lines of rodent origin are tumorigenic; therefore, rodent cells need not be tested for tumorigenicity. Human epithelial cells and all cells used for live virus vaccine production should, however, be tested for tumorigenicity. In addition, some special cases regarding somatic cell or gene therapy may require tumorigenicity testing. 3. Oncogene testing Recent studies indicating that oncogenes may be involved in normal cell growth suggest that testing for endogenous oncogenes is not necessary. The results of tests described in this document may be submitted to support the acceptability of a cell line to produce a biological product: In general, testing should be performed in compliance with Good Laboratory 6 Practice requirements (21 CFR 58). These tests should not be interpreted as checklists. Rather, the selection of tests depends on many variables such as the nature of the cell line, the manufacturing situation and the product indication. In addition, the amount of testing that is needed may be greater to support approval of a PLA than that for an IND application. The testing required for initiating clinical trials depends on the product and its use. The points discussed here do not generally address the basis for test selection due to the variety of issues each manufacturer must. consider when making these decisions. The characterization of a cell line intended for use in the manufacture of includes: 1. history and general characteristics of the cell line; 2. the cell bank system; and 3. quality control testing. In addition, in many cases there is need for validation studies removal and inactivation by the manufacturing process. virus Additional information concerning the testing of cell lines used to produce antibodies and recombinant DNA technology products for in vivo and select in vitro use may be found in the Points to Consider in the Manufacture and Testing of Antibody Products for Human Use (June (now under revision), Points to Consider in the Production and Testing of New Drugs and Produced by Recombinant DNA Technology (April 1985) and the supplement to the recombinant DNA 7 Points to Consider, Nucleic Acid Characterization and Genetic Stability If a cell line or cells are to be returned into humans to produce its biological product(s) in then the Points to Consider in Human Somatic Cell Therapy and Gene Therapy (1991) and also the Points to Consider in the Collection, Processing and Testing of Ex-Vivo-Activated Mononuclear Leukocytes for Administration to Humans (1989) should be consulted. II. HISTORY AND GENERAL CHARACTERISTICS OF THE CELL LINE A. History of the Cell Line The history of any cell line used for the production of biological products should include, when possible: 1. age, sex and species of the donor; 2. for human cell lines, the donor s medical history and if available, the results of tests performed on donor for the detection of adventitious agents: 3. culture history of the cell line including methods used for the isolation of the tissues from which the line was derived, passage history, media used and history of passage in animals, etc.; 4. previous identity testing and the results of all available adventitious agent testing. General Characteristics of the Cell Line The growth pattern and morphological appearance of the cell line should be determined and should be stable from the master cell bank to the end-of-production ceils [Points to Consider: Nucleic Acid Characterization and Genetic Stability If there are specific markers that may be useful in characterizing the ceil line (such as marker chromosomes, specific surface markers), these should be characterized for stability. If the cells have an identified finite life expectancy, the total number of population doubling levels through senescence should be determined. THE CELL BANK SYSTEM A. Generation of Cell Banks Once a cell line is chosen as the biological source of a product, a cell bank system should be generated to assure that an adequate supply of equivalent cells exist for use over the entire life span of the product. In addition to providing a constant supply of starting material, the advantages of a cell bank system include allowing for a detailed characterization of the cell line and decreasing the likelihood and increasing the detection of both cell line contamination and adventitious agent contamination. Ordinarily, the cell bank system would consist of two tiers: a master cell bank (MCB) and a manufacturer s working cell bank (MWCB). 9 The Master Bank is defined as a collection of of uniform composition derived from a single tissue or cell. cryopreserved in stored in the liquid or vapor phase of liquid nitrogen. The MCB for a diploid cell line should be prepared from cells at a low population doubling level. The Manufacturer s Working Cell Bank (MWCB) is derived from one or more the MCB. The.MCB source cells are expanded by, serial subculture up to a passage number selected by the manufacturer and approved by CBER. At that point the cells are combined into one pool, dispensed into individual ampules and cryopreserved to form the MWCB. One such ampules from the MWCB would be used for the production of a lot of a biological product. If cells from more than one MWCB ampule are used, the cell suspensions should be pooled at the time of thawing. The population doubling level of cells used for production should not exceed an upper limit based on written. criteria established by the manufacturer. B. Storage of the Cell Banks Both the MCB and the MWCB should be stored in either the liquid vapor phase of liquid nitrogen. The location, identity and inventory of individual ampoules of cells should be thoroughly documented. is recommended that the MCB and MWCB should each be stored in or more widely separate areas within the production facility well as at a site in order to avoid loss of the cell line. 10 C. Cell Bank Qualification 1. The Master Cell Bank Testing to qualify cell banks should be done either on an aliquot of the cell bank or on cell cultures derived from the cell bank, as appropriate. Testing to qualify the MCB includes testing to demonstrate freedom from adventitious agents and identity testing. The testing for adventitious agents should include tests for bacteria, fungi, mycoplasmas and for viruses. Testing for adventitious viruses should include routine in and cell culture inoculation tests and any other specific tests that are warranted, based on the passage history of the cell line, to detect possible contaminating viruses. Some of the tests which are relevant in selected circumstances are described in part V. Finally, testing should be performed in most circumstances to determine if the cells produce retroviruses or retrovirus particles. This testing is also described in part V. Extensive identity testing of the MCB should be done once and should include all tests needed to establish all significant properties of the cells and the stability of these properties throughout the manufacturing process. Such characteristics should include: 11 a. morphology, as determined by light and electron microscopy; b. species of origin (and sex, if human); c. split ratio; d. data demonstrating that the ceils can be used for their intended purpose. If the ceils contain an expression system to produce a recombinant derived protein, data should be obtained to demonstrate the copy number and physical state of the expression system and the quality and quantity of the protein it produces ( see Points to Consider on products) ; e. a meaningful test should be performed which will also be performed for routine identity testing production cultures used for each lot of product; and f. other such tests which may be useful for demonstrating that the cell bank is comprised of cells with the intended characteristics. 2. Manufacturers Working Cell Bank I I 12 The MWCB being derived from the MCB and propagated for an approved number of passages in tissue culture, only needs to be spot checked for contaminants that may have been introduced from the culture medium, Recommended tests include sterility, mycoplasma, routine virus (in vitro and in vivo) tests and cell line authenticity to check for cell cross-contamination. When a manufacturer moves from a serum containing to a serum free defined growth medium,it is suggested that the cells which are weaned into the serum free medium should be to establish a new MCB MWCB of cells for optimal growth in the defined medium. IV. PRODUCTION CULTURES AND PRODUCT TESTING Quality control of cell substrates used for production is an important part of product quality control. Specific areas to be addressed include cell culture media, management of cell cultures, and specific testing. A. Cell Culture Media Accurate records should be kept of the composition and source of the cell culture medium. In cases where the manufacturer of a biological product uses a proprietary medium or medium supplement, the manufacturer of the medium or medium supplement may be 13 required to the necessary data directly to CBER, in the form, example, of a Master File Application. serum or additives derived from animal sources are added to the cell culture medium, they should be certified to be free from contaminants and adventitious agents, such as the agent responsible for the production of Bovine Spongiform Encephalopathy. Information should be provided with regard to the identity and source of, and testing for adventitious agents carried out on these additives. Acceptance of certified raw materials based on certification provided by the supplier should be based on a determination by the manufacturer accepting the product that process used for certification is sufficient. Since animal serum may produce allergic responses in human subjects, attempts should be made to reduce serum levels required for the propagation of production cell cultures as much as possible. The residual amount of serum or additives in the final should be determined, and shall not exceed (b)). CFR If porcine trypsin is used in passaging cells, it should be free from adventitious agents, including porcine parvovirus (9 CFR and ). Pursuant to 21 CFR , manufacturers of biological products are requested to provide information regarding the source(s) and control of any bovine- or ovine-derived (see attachment 14 Penicillin or other beta lactam antibiotics should not be present in production cell cultures. Minimal concentrations of other antibiotics or inducing agents may be acceptable CFR (c)]. However, the presence of any antibiotic or inducing agent in the product is discouraged. B. Management of Cell Cultures Lot-to-lot characterization of the product and routine monitoring for adventitious agents is part of the quality control of the biological product. It includes testing of production cell cultures and unprocessed and processed cell culture fluids. Appropriate approaches to quality control of cell substrate depend on the nature of the propagation system used. Cell substrates are propagated as monolayer cultures, in suspension cultures, or in bioreactors, and may be held on a short term, long term, or even on a potentially indefinite basis. When short-term cultures are used, the is obtained either from a single harvest of cell culture fluid or from multiple harvests. In some cases the quality control testing may need to be performed on each harvest before pooling into the bulk lot. If the product is an infectious virus, it will usually replicate in one or more of the cell cultures used for routine testing for adventitious viruses. Nonreplicating viruses, used as vectors for gene therapy, may be tested in the usual cell culture tests for the presence of adventitious agents. In such cases a proportion of the vessels 15 containing the cell substrate prepared for production (commonly, about 10% of the vessels) should be held as control cultures. The uninoculated control cell cultures and fluids are tested for adventitious agents. (This should not be confused with the product identity test, which is typically a procedure in which the virus is neutralized and inoculated into a susceptible cell culture.) When long term cultures are used, multiple harvests may be pooled. into bulk lots at intervals. In these cases quality control testing should be performed on each bulk lot, and, if possible, on cells separated from the production harvest pooled into the specific bulk. The management of cell substrates for the purposes of quality control testing should be designed to optimize sensitivity of the testing. Criteria for termination of long-term cultures should be established and followed. Testing for bacterial and sterility is generally performed on the unprocessed bulk lot, the final bulk lot and the final product. The unprocessed bulk is the pooled harvests of cell culture fluids that constitutes a homogeneous mixture for manufacture into a unique lot of product. It is important that testing for adventitious agents be performed prior to further processing such as filtration, clarification or other procedures, unless such testing is made more sensitive by initial partial processing (e.g., unprocessed bulk may be toxic in test cell cultures, whereas filtered bulk may not). Final bulk product is a concentrated, purified product in a homogeneous prepared for with excipients and filling into final 16 containers. The final bulk product is subjected to a variety of lot release tests which often include sterility testing if it is intended to be sterile. Final product should be tested for sterility and endotoxin. Routine testing for mycoplasmas and in and in testing for adventitious viruses should be performed on every lot using production cells and unprocessed bulk fluids. If a cell line is known to produce a virus that is routinely present in the unprocessed bulk, testing on a lot-to-lot basis to demonstrate its absence from the product after purification may be required, unless the virus is the product, as is the case in viral vectors for gene therapy. Lot-to-lot testing
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