Appendix XV J. Cell Substrates for the Production of Vaccines for Human Use

This general chapter deals with diploid cell lines and continuous cell lines used for the production of vaccines for human use; specific issues relating to vaccines prepared by recombinant DNA technology are covered by the monograph on Products of recombinant DNA technology (0784). Testing to be carried out at various stages (cell seed, master cell bank, working cell bank, cells at or beyond the maximum population doubling level used for production) is indicated in Table 5.2.3.-1. General provisions for the use of cell lines and test methods are given below. Where primary cells or cells that have undergone a few passages without constitution of a cell bank are used for vaccine production, requirements are given in the individual monograph for the vaccine concerned.

Diploid cell lines A diploid cell line has a high but finite capacity for multiplication in vitro.

Continuous cell lines A continuous cell line has the capacity to multiply indefinitely in vitro; the cells often have differences in karyotype compared to the original cells; they may be obtained from healthy or tumoral tissue.

For injectable vaccines produced in continuous cell lines, the purification process is validated to demonstrate removal of substrate-cell DNA to a level equivalent to not more than 10 ng per single human dose, unless otherwise prescribed.

Cell-bank system Production of vaccines in diploid or continuous cell lines is based on a cell-bank system. The in vitro age of the cells is counted from the master cell bank. Each working cell bank is prepared from one or more containers of the master cell bank. The use, identity and inventory control of the containers is carefully documented.

Media and substances of animal and human origin The composition of media used for isolation and all subsequent culture is recorded in detail and if substances of human or animal origin are used they must be free from extraneous agents.

If human albumin is used, it complies with the monograph on Human albumin solution (0255).

Bovine serum used for the preparation and maintenance of cell cultures is tested and shown to be sterile and free from bovine viruses, notably bovine diarrhoea virus and mycoplasmas.

Trypsin used for the preparation of cell cultures is examined by suitable methods and shown to be sterile and free from mycoplasmas and viruses, notably pestiviruses and parvoviruses.

Cell seed The data used to assess the suitability of the cell seed comprise information, where available, on source, history and characterisation.

Source of the cell seed For human cell lines, the following information concerning the donor is recorded: ethnic and geographical origin, age, sex, general physiological condition, tissue or organ used, results of any tests for pathogens.

For animal cell lines, the following information is recorded concerning the source of the cells: species, strain, breeding conditions, geographical origin, age, sex, general physiological condition, tissue or organ used, results of any tests for pathogens.

Cells of neural origin, such as neuroblastoma and P12 cell lines, may contain substances that concentrate agents of spongiform encephalopathies and such cells are not used for vaccine production.

History of the cell seed The following information is recorded: the method used to isolate the cell seed, culture methods and any other procedures used to establish the master cell bank, notably any that might expose the cells to extraneous agents.

Full information may not be available on the ingredients of media used in the past for cultivation of cells, for example on the source of substances of animal origin; where justified and authorised, cell banks already established using such media may be used for vaccine production.

Characterisation of the cell seed The following properties are investigated:

(1)  the identity of the cells (for example, isoenzymes, serology, nucleic acid fingerprinting);

(2)  the growth characteristics of the cells and their morphological properties (optical and electron microscopes);

(3)  for diploid cell lines, karyotype;

(4)  for diploid cell lines, the in vitro life span in terms of population doubling level.

Cell substrate stability Suitable viability of the cell line in the intended storage conditions must be demonstrated. For a given product to be prepared in the cell line, it is necessary to demonstrate that consistent production can be obtained with cells at passage levels at the beginning and end of the intended span of use.

Infectious extraneous agents Cell lines for vaccine production shall be free from infectious extraneous agents. Tests for extraneous agents are carried out as shown in Table 5.2.3.-1.

Depending on the origin and culture history of the cell line, it may be necessary to carry out tests for selected, specific potential contaminants, particularly those that are known to infect latently the species of origin, for example simian virus 40 in rhesus monkeys. For cell lines of rodent origin, antibody-production tests are carried out in mice, rats and hamsters to detect species-specific viruses.

Cell lines are examined for the presence of retroviruses as described below. Cell lines that show the presence of retroviruses capable of replication are not acceptable for production of vaccines.

Tumorigenicity For the preparation of live vaccines, the cell line must not be tumorigenic at any population doubling level used for vaccine production. Where a tumorigenic cell line is used for the production of other types of vaccine, the purification process is validated to demonstrate that residual substrate-cell DNA is reduced to a level equivalent to not more than 10 ng per single human dose of the vaccine, unless otherwise prescribed, and that substrate-cell protein is reduced to an acceptable level.

A cell line which is known to have tumorigenic potential does not have to be tested further. If a cell line is of unknown tumorigenic potential, it is either regarded as being tumorigenic or it is tested for tumorigenicity using an in vitro test as described below; if the result of the in vitro test is negative or not clearly positive, an in vivo test as described below is carried out. The tests are carried out using cells at or beyond the maximum population doubling level that will be used for vaccine production.

The MRC-5, WI-38 and FRhL-2 cell lines are recognised as being non-tumorigenic and further testing is not necessary.

Chromosomal characterisation Diploid cell lines shall be shown to be diploid. More extensive characterisation of a diploid cell line by karyotype analysis is required if the removal of intact cells during processing after harvest has not been validated. Samples from four passage levels evenly spaced over the life-span of the cell line are examined. A minimum of 200 cells in metaphase are examined for exact count of chromosomes and for frequency of hyperploidy, hypoploidy, polyploidy, breaks and structural abnormalities.

The MRC-5, the WI-38 and the FRhL-2 cell lines are recognised as being diploid and well characterised; where they are not genetically modified, further characterisation is not necessary.

Identification Nucleic acid fingerprint analysis and a relevant selection of the following are used to establish the identity of the cells:

(1)  biochemical characteristics (isoenzyme analysis);

(2)  immunological characteristics (histocompatibility antigens);

(3)  cytogenetic markers.

Contaminating cells The nucleic acid fingerprint analysis carried out for identification also serves to demonstrate freedom from contaminating cells.

Bacterial and fungal contamination The master cell bank and each working cell bank comply with the test for sterility (2.6.1), carried out using for each medium 10 ml of supernatant fluid from cell cultures. Carry out the test on 1 per cent of the containers with a minimum of 2 containers.

Mycoplasmas (2.6.7) The master cell bank and each working cell bank comply with the test for mycoplasmas by the culture method and the indicator cell culture method. Use one or more containers for the test.

Test for extraneous agents in cell cultures The cells comply with the test for haemadsorbing viruses and with the tests in cell cultures for other extraneous agents given in chapter 2.6.16 under Production cell culture: control cells. If the cells are of simian origin, they are also inoculated into rabbit kidney cell cultures to test for herpesvirus B (cercopithecid herpesvirus 1).

Co-cultivation Co-cultivate intact and disrupted cells separately with other cell systems including human cells and simian cells. Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses. The cells comply with the test if no evidence of any extraneous agent is found.

Retroviruses Examine for the presence of retroviruses using:

(1)  product-enhanced reverse transcriptase (PERT) assay (2.6.21) carried out for cell bank supernatants using cells at or beyond the maximum population doubling level that will be used for production;

(2)  transmission electron microscopy.

If test (1) and/or test (2) gives a positive result, test (3) is carried out;

(3)  infectivity assays carried out on human cells with an endpoint PERT assay on the supernatant.

Since the sensitivity of PERT assays is very high, interpretation of a positive signal may be equivocal and a decision on the acceptability of a cell substrate is based on all available data.

Tests in animals Inject intramuscularly (or, for suckling mice, subcutaneously) into each of the following groups of animals 10⁷ viable cells divided equally between the animals in each group:

(1)  2 litters of suckling mice less than 24 h old, comprising not fewer than 10 animals;

(2)  10 adult mice.

Inject intracerebrally into each of 10 adult mice 10⁶ viable cells to detect the possible presence of lymphocytic choriomeningitis virus.

Observe the animals for at least 4 weeks. Investigate animals that become sick or show any abnormality to establish the cause of illness. The cells comply with the test if no evidence of any extraneous agent is found. The test is invalid if fewer than 80 per cent of the animals in each group remain healthy and survive to the end of the observation period.

For cells obtained from a rodent species (for example, Chinese hamster ovary cells or baby hamster kidney cells), tests for antibodies against likely viral contaminants of the species in question are carried out on animals that have received injections of the cells.

Tests in eggs Using an inoculum of 10⁶ viable cells per egg, inoculate the cells into the allantoic cavity of 10 SPF embryonated hens' eggs (5.2.2) 9-11 days old and into the yolk sac of 10 SPF embryonated hens' eggs 5-6 days old. Incubate for not less than 5 days. Test the allantoic fluids for the presence of haemagglutinins using mammalian and avian red blood cells; carry out the test at 5  ±  3  °C and 20-25  °C and read the results after 30-60 min. The cells comply with the test if no evidence of any extraneous agent is found. The test is invalid if fewer than 80 per cent of the embryos remain healthy and survive to the end of the observation period.

Tests for tumorigenicity in vitro The following test systems may be used:

(1)  colony formation in soft agar gels;

(2)  production of invasive cell growth following inoculation into organ cultures;

(3)  study of transformation activity using, for example, the 3T3 assay system for active oncogenes.

Tests for tumorigenicity in vivo The test consists in establishing a comparison between the continuous cell line and a suitable positive control (for example, HeLa or Hep2 cells).

Animal systems that have been shown to be suitable for this test include:

(1)  athymic mice (Nu/Nu genotype);

(2)  newborn mice, rats or hamsters that have been treated with antithymocyte serum or globulin;

(3)  thymectomised and irradiated mice that have been reconstituted (T^–, B⁺) with bone marrow from healthy mice.

Whichever animal system is selected, the cell line and the reference cells are injected into separate groups of 10 animals each. In both cases, the inoculum for each animal is 10⁷ cells suspended in a volume of 0.2 ml, and the injection may be by either the intramuscular or subcutaneous route. Newborn animals are treated with 0.1 ml of antithymocyte serum or globulin on days 0, 2, 7 and 14 after birth. A potent serum or globulin is one that suppresses the immune mechanisms of growing animals to the extent that the subsequent inoculum of 10⁷ positive reference cells regularly produces tumours and metastases. Severely affected animals showing evident progressively growing tumours are killed before the end of the test to avoid unnecessary suffering.

At the end of the observation period all animals, including the reference group(s), are killed and examined for gross and microscopic evidence of the proliferation of inoculated cells at the site of injection and in other organs (for example lymph nodes, lungs, kidneys and liver).

In all test systems, the animals are observed and palpated at regular intervals for the formation of nodules at the sites of injection. Any nodules formed are measured in 2 perpendicular directions, the measurements being recorded regularly to determine whether there is progressive growth of the nodule. Animals showing nodules which begin to regress during the period of observation are killed before the nodules are no longer palpable, and processed for histological examination. Animals with progressively growing nodules are observed for 1-2 weeks. Among those without nodule formation, half are observed for 3 weeks and half for 12 weeks before they are killed and processed for histological examination. A necropsy is performed on each animal and includes examination for gross evidence of tumour formation at the site of injection and in other organs such as lymph nodes, lungs, brain, spleen, kidneys and liver. All tumour-like lesions and the site of injection are examined histologically. In addition, since some cell lines may give rise to metastases without evidence of local tumour growth, any detectable regional lymph nodes and the lungs of all animals are examined histologically.

The test is invalid if fewer than 9 of 10 animals injected with the positive reference cells show progressively growing tumours.