Monday, August 2, 2010



1. Cell Banks Qualification of cell banks

2. Pre-Production Cells Visual inspection or an identity test may be performed on cells directly prior to production.

3.Pre-Filtered Harvest or Post-Production Cells

4.Control Cells

Use of Control-Cell Cultures

5.Post-Filtered Harvest (Final Bulk)

Cell banking assures that an adequate supply of equivalent, well-characterized cells exist for production over the expected lifetime of the product. In addition to providing a constant supply of biological starting material, cell banking provides you with the opportunity to undertake a comprehensive characterization of the cell substrate and to minimize the chance of adventitious agent contamination and/or to maximize the chance of detection of a contaminant.

Cell Banking Strategies and Methods Ordinarily, the cell bank system consists of two tiers: a master cell bank (MCB); and a working cell bank (WCB), sometimes called a manufacturer’s working cell bank (MWCB). The MCB represents a collection of cells of uniform composition derived from a single source prepared under defined culture conditions. Sometimes, a parent cell bank, comprising vials of the progenitor cells to the MCB, is also maintained. The WCB is derived from one or more vials of cells from the MCB, which are expanded by serial subculture. The pooled cells are dispensed into individual vials and cryopreserved to form the WCB. One or more vials from the WCB are generally used for the production of a lot of a vaccine. If cells from more than one WCB vial are used, the cell suspensions should be pooled at the time of thawing. The population doubling level or passage level of cells used for production should not exceed an upper limit based on your cell substrate characterization, including at end-of-production level

When using a two-tiered banking system, we generally recommend that you perform an extensive characterization of the MCB. Because all WCBs would be derived from the well-characterized MCB, your WCBs may be tested in a more limited manner, focusing on adventitious agents to which the WCB could have been exposed during expansion from the MCB. The use of a cell banking system might allow you to perform more limited testing of individual vaccine lots, focusing on adventitious agents to which the cells could have been exposed during manufacturing from the WCB. Alternatively, some manufacturers might choose to extensively characterize each WCB in lieu of a one-time thorough characterization of the MCB. The testing strategy chosen will depend on your own circumstances, but you should characterize at least one cell bank (MCB or WCB) extensively. However, in the absence of thorough characterization of the MCB, we may request a thorough characterization on the WCB used to produce each lot.

Vaccine manufacturer may use other cell banking systems, for example, when the original source of your cell substrate is limited and/or the need to maintain a low passage level restricts expansion of cell numbers. Regardless of what cell banking system you use, cells at some level of expansion should be characterized completely, with a justification for the choice of the passage level tested. In either case, the derivation and cell bank designations should be thoroughly described and explained.

The requirements set out in 21 CFR 610.18 relate to cultures, including cell banks. Cell banks should be stored under conditions shown to be suitable for long-term stability (e.g., liquid nitrogen, ultra-low temperature freezer) (21 CFR 610.18(a)). Storage of cell banks in the vapor phase (as compared to the liquid phase) of liquid nitrogen might reduce the potential for cross-contamination. Cell stability under the freezing and storage conditions should be validated using cell recovery or viability data. You should store the MCB and WCB in two or more separate locations within the facility or at a distant site in order to avoid loss of the cell substrate due to local disaster or equipment malfunction. Access to these cell banks by your staff should be limited and controlled. Under 21 CFR 610.18(d), appropriate records must be maintained. You should maintain a record of the location, identity, and inventory of individual ampoules of cells.

Qualification of Cell Banks and Primary Cell Cultures
The purpose of qualifying cell banks is to demonstrate their suitability for use in vaccine production. This section includes recommendations on testing for different types of cells and stages of cell banking.
The same basic principles apply to the qualification of cell banks and to the qualification of primary cell cultures. Characterization of a cell bank is dependent on its use, and your particular use should guide your determination of which recommended testing is necessary and whether additional testing might be necessary. Consideration of the choice of tests will be based on the assessment of the risks that the cell substrate might represent for the product type and its clinical indications.

Testing to qualify the MCB should be performed directly on the cell bank, except when it is more appropriate to test cell cultures derived from the cell bank.
Tests that one might perform on his MCB include tests for bacteria, fungi, mycoplasma, and viruses (e.g., in vitro and in vivo testing, specific tests for retroviruses, and specific tests for viruses known to exist in the species of origin or that could be acquired during serial passage in cell culture). Other specific tests might be warranted, depending on the passage history of the cell substrate.

If the species from which your cells were derived is susceptible to infection with Mycobacterium tuberculosis, an appropriate test should be performed for this agent as well. Depending on the source species, you may consider specific tests such as the test for avian leukosis virus for products produced in avian cells. If you are using primary monkey kidney cell cultures, for example, you should test for species-specific simian agents, such as SV40 and herpes B virus, and other simian agents, such as simian polyomaviruses and simian cytomegalovirus.

Testing either the MCB or the reagents to which it was exposed may be done to provide assurance that these reagents were not a source of adventitious agents, an issue of particular concern when reagents are animal-derived.

One should characterize cells that are expanded to or beyond the end of production passage level. Such cells are referred to as End-of-Production Cells (EOPC). You should demonstrate the stability of the cell substrate characteristics using your EOPC. Your characterization should include, if applicable, growth characteristics, tumorigenic phenotype, expression of endogenous viruses, stability of expression of the inserted or engineered genes, and genetic stability of the cells (such as karyotyping or DNA fingerprinting).

Special Considerations for Primary Cell Cultures
Primary cells are obtained directly from the tissues of healthy animals. Primary cells are more likely to contain adventitious agents than banked, well-characterized cells. This concern with primary cells is mitigated by rigorous qualification of source animals and primary cell substrates. Animals from which primary cultures are established should be from specific-pathogen-free (SPF) closed flocks, herds, or colonies, when feasible. The term “closed” refers to the maintenance of a group (flock, herd, and colony) free from introduction of new animals (new genetic material). Animals that are not from closed flocks, herds, or colonies should be quarantined and thoroughly evaluated for a period sufficient to detect signs of disease or infection before introducing them into the flock, herd, or colony. Animals should be screened serologically for appropriate adventitious agents to determine their suitability as a source for the primary cell substrate. Animal husbandry practices should be described in the application.

Embryonated chicken eggs used as the source of chick embryo tissue for the propagation of viral vaccines should be derived from flocks certified to be free of Salmonella pullorum, avian tuberculosis, fowl pox, Rous sarcoma virus, avian leukosis virus, reticuloendotheliosis virus, and other adventitious agents pathogenic for chickens. Appropriate records must be retained for documentation (21 CFR 610.18(d)). If eggs are procured from non-SPF flocks, assurance that the vaccine is free of such agents may be provided by testing or by validating the process for their removal.Control cells are often used with primary cell substrates

2. Pre-Production Cells Visual inspection or an identity test may be performed on cells directly prior to production.

3.Pre-Filtered Harvest or Post-Production Cells
In general, the stage at which adventitious agents are most likely to be found is the stage at which the most extensive adventitious agent testing should be performed for each product. For many viral vaccines, the pre-filtered harvest is the stage of manufacture that is most concentrated and at which the least processing has been performed. For these reasons, this might be the best stage of production for testing for adventitious agents.
In addition to testing the viral or vaccine harvest for cultivatable mycoplasmas, as required under 21 CFR 610.30, you should also test the viral or vaccine harvest for non-cultivatable mycoplasmas (and spiroplasmas, if appropriate), and adventitious viruses by in vitro methods. Adventitious virus tests should also include in vivo methods if the cells and vaccine seeds have not already been tested. This might include a test for hemadsorbing viruses. If the production process is capable of inducing expression of retroviruses, PCR-based reverse transcriptase (RT) testing or infectivity studies might also be recommended at this stage. If appropriate, a test for Mycobacterium tuberculosis should be performed. if the assay system used for in vitro or in vivo adventitious virus testing is capable of supporting replication of the vaccine virus, and if the vaccine virus replicates to levels that interfere with the adventitious agent tests, then it might be necessary to neutralize the vaccine virus prior to performing these tests. If neutralization is difficult to achieve, the other considerations in cn be adapted.

If cells survive the production process (e.g., if the vaccine virus does not result in a lytic infection), post-production cells may also be tested for adventitious agents. If multiple harvests are performed for a single vaccine lot, testing should be performed on each individual harvest (rather than the pooled harvest) in order to avoid dilution of a potentially contaminated harvest with uncontaminated harvests

4.Control Cells

Use of Control-Cell Cultures
It is recommended the use of control cells when it is not feasible to directly test cells or product at various stages of manufacture. For example, if you are using primary cell cultures to propagate your vaccine virus, complete testing of the primary culture might not be feasible prior to inoculation of virus. In this situation, when possible, you should produce and test uninfected control-cell cultures that are derived in parallel with, and handled in the same manner as, the production culture.

5.Post-Filtered Harvest (Final Bulk)
The post-filtered harvest (generally referred to as final bulk) should be tested for bacterial and fungal sterility. Depending on the product, other testing might be appropriate to assure the safety, identity, purity, potency, and quality of the final bulk. These may include testing for levels of residual cellular proteins and cellular nucleic acids. If the cell line used to produce the vaccine is known to produce viruses other than the vaccine virus, the clearance of these viruses should be demonstrated in the production processes.
If processes are not validated for removal of residual cells, a test to demonstrate the absence of residual cells is recommended at this stage.

6. Final Filled Product
The regulations for General Biological Products Standards under 21 CFR Part 610 provide that each lot must be tested for potency, general safety, sterility, purity and identity. Sterility must be demonstrated as required under 21 CFR 610.12. Products excepted from these requirements (e.g., under 21 CFR 610.12(g)(4)(ii)) should still be assessed by the microbial limits test for bioburden

Under 21 CFR 610.11a, general safety testing for each lot of inactivated influenza vaccine includes a demonstration that it contains less endotoxin than a reference preparation provided by US FDA. Other final filled products should also be assessed for endotoxin levels. In addition, a general safety test for detecting extraneous toxic contaminants, under 21 CFR 610.11, is required on final filled containers, except as described in 21 CFR 610.11(g). Other tests should be performed to assess the safety, identity, purity, potency, and quality of the final filled product. These tests are determined case-by-case depending on the product and should be described in their IND and BLA submissions.

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