Table 1 shows examples of virus tests to be performed once only at various cell levels, including MCB, WCB, and cells at the limit of in vitro cell age used for production.

Table 1. Examples of Virus Tests to be Performed Once At Various Cell Levels

1. Master Cell Bank

Extensive screening for both endogenous and nonendogenous viral contamination should be performed on the MCB. For heterohybrid cell lines in which one or more partners are human or nonhuman primate in origin, tests should be performed in order to detect viruses of human or nonhuman primate origin because viral contamination arising from these cells may pose a particular hazard.

Testing for nonendogenous viruses should include in vitro and in vivo inoculation tests and any other specific tests, including species-specific tests such as the mouse antibody production (MAP) test, that are appropriate, based on the passage history of the cell line, to detect possible contaminating viruses.

2. Working Cell Bank

Each WCB as a starting cell substrate for drug production should be tested for adventitious virus either by direct testing or by analysis of cells at the limit of in vitro cell age, initiated from the WCB. When appropriate nonendogenous virus tests have been performed on the MCB and cells cultured up to or beyond the limit of in vitro cell age have been derived from the WCB and used for testing for the presence of adventitious viruses, similar tests need not be performed on the initial WCB. Antibody production tests are usually not necessary for the WCB. An alternative approach in which full tests are carried out on the WCB rather than on the MCB would also be considered acceptable.

3. Cells at the Limit of In Vitro Cell Age Used for Production

The limit of in vitro cell age used for production should be based on data derived from production cells expanded under pilot-plant scale or commercial-scale conditions to the proposed in vitro cell age or beyond. Generally, the production cells are obtained by expansion of the WCB; the MCB could also be used to prepare the production cells. Cells at the limit of in vitro cell age should be evaluated once for those endogenous viruses that may have been undetected in the MCB and WCB. The performance of suitable tests (e.g., in vitro and in vivo) at least once on cells at the limit of in vitro cell age used for production would provide further assurance that the production process is not prone to contamination by adventitious virus. If any adventitious viruses are detected at this level, the process should be carefully checked in order to determine the cause of the contamination, and should be completely redesigned if necessary.

Numerous assays can be used for the detection of endogenous and adventitious viruses. Table 2 outlines examples for these assays. They should be regarded as assay protocols recommended for the present, but the list is not all-inclusive or definitive. Since the most appropriate techniques may change with scientific progress, proposals for alternative techniques, when accompanied by adequate supporting data, may be acceptable. Manufacturers are encouraged to discuss these alternatives with the regulatory authorities. Other tests may be necessary depending on the individual case. Assays should include appropriate controls to ensure adequate sensitivity and specificity. Wherever a relatively high possibility of the presence of a specific virus can be predicted from the species of origin of the cell substrate, specific tests and/or approaches may be necessary. If the cell line used for production is of human or nonhuman primate origin, additional tests for human viruses, such as those causing immunodeficiency diseases and hepatitis, should be performed unless otherwise justified. The polymerase chain reaction (PCR) may be appropriate for detection of sequences of other human viruses as well as for other specific viruses. The following is a brief description of a general framework and philosophical background within which the manufacturer should justify what was done.

Table 2. Examples of the Use and Limitations of Assays Which May be Used to Test for Virus

1. Tests for Retroviruses

For the MCB and for cells cultured up to or beyond the limit of in vitro cell age used for production, tests for retroviruses, including infectivity assays in sensitive cell cultures and electron microscopy (EM) studies, should be carried out. If infectivity is not detected and no retrovirus or retrovirus-like particles have been observed by EM, reverse transcriptase (RT) or other appropriate assays should be performed to detect retroviruses that may be noninfectious. Induction studies have not been found to be useful.

2. In Vitro Assays

In vitro tests are carried out by the inoculation of a test article (see Table 2) into various susceptible indicator cell cultures capable of detecting a wide range of human and relevant animal viruses. The choice of cells used in the test is governed by the species of origin of the cell bank to be tested, but should include a human and/or a nonhuman primate cell line susceptible to human viruses. The nature of the assay and the sample to be tested are governed by the type of virus which may possibly be present based on the origin or handling of the cells. Both cytopathic and hemadsorbing viruses should be sought.

3. In Vivo Assays

A test article (see Table 2) should be inoculated into animals, including suckling and adult mice, and in embryonated eggs to reveal viruses that cannot grow in cell cultures. Additional animal species may be used, depending on the nature and source of the cell lines being tested. The health of the animals should be monitored and any abnormality should be investigated to establish the cause of the illness.

4. Antibody Production Tests

Species-specific viruses present in rodent cell lines may be detected by inoculating test article (see Table 2) into virus-free animals and examining the serum antibody level or enzyme activity after a specified period. Examples of such tests are the mouse antibody production (MAP) test, rat antibody production (RAP) test, and hamster antibody production (HAP) test. The viruses currently screened for in the antibody production assays are discussed in Table 3.

Table 3. Virus Detected in Antibody Production Tests

It is recognized that some cell lines used for the manufacture of product will contain endogenous retroviruses, other viruses, or viral sequences. In such circumstances, the action plan recommended for manufacture is described in section V. of this document. The acceptability of cell lines containing viruses other than endogenous retroviruses will be considered on an individual basis by the regulatory authorities, by taking into account a risk/benefit analysis based on the benefit of the product and its intended clinical use, the nature of the contaminating viruses, their potential for infecting humans or for causing disease in humans, the purification process for the product (e.g., viral clearance evaluation data), and the extent of the virus tests conducted on the purified bulk.