Rabies Vaccine

(Ph Eur monograph 0216)

 

The label may state 'Rab/Vac'.

 

Ph Eur


 

 

Definition

 

Rabies vaccine for human use prepared in cell cultures is a freeze-dried preparation of a suitable strain of fixed rabies virus grown in cell cultures and inactivated by a validated method.

 

The vaccine is reconstituted immediately before use as stated on the label to give a clear  liquid that may be coloured owing to the presence of a pH indicator.

 

Production

 

The production of the vaccine is based on a virus seed-lot system and, if a cell line is used for virus propagation, a cell-bank system. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy; even with authorised exceptions, the number of passages beyond the level used for clinical studies shall not exceed five.

 

The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9).

 

SUBSTRATE FOR VIRUS PROPAGATION

 

The virus is propagated in a human diploid cell line (5.2.3), in a continuous cell line approved by the competent authority or in cultures of chick-embryo cells derived from a flock free from specified pathogens (5.2.2).

 

SEED LOTS

 

The strain of rabies virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation.

 

Working seed lots are prepared by not more than five passages from the master seed lot.

 

Only a working seed lot that complies with the following tests may be used for virus propagation.

 

Identification

 

Each working seed lot is identified as rabies virus using specific antibodies.

 

Virus concentration

 

The virus concentration of each working seed lot is determined by a cell culture method using immunofluorescence, to ensure consistency of production.

 

Extraneous agents (2.6.16)

 

The working seed lot complies with the requirements for virus seed lots. If the virus has been passaged in mouse brain, specific tests for murine viruses are carried out.

 

VIRUS PROPAGATION AND HARVEST

 

All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin complying with the monograph on Human albumin solution (0255). Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from infectious extraneous agents; trypsin complies with the monograph on Trypsin (0694). The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest.

 

Only a single harvest that complies with the following requirements may be used in the preparation of the inactivated viral harvest.

 

Identification

 

The single harvest contains virus that is identified as rabies virus using specific antibodies.

 

Virus concentration

 

Titrate for infective virus in cell cultures; the titre is used to monitor consistency of production.

 

Control cells

 

The control cells of the production cell culture from which the single harvest is derived comply with a test for identification and with the requirements for extraneous agents (2.6.16).

 

PURIFICATION AND INACTIVATION

 

The virus harvest may be concentrated and/or purified by suitable methods; the virus harvest is inactivated by a validated method at a fixed, well defined stage of the process which may be before, during or after any concentration or purification. The method shall have been shown to be capable of inactivating rabies virus without destruction of the immunogenic activity. If betapropiolactone is used, the concentration shall at no time exceed 1:3500.

 

Only an inactivated viral suspension that complies with the following requirements may be used in the preparation of the final bulk vaccine.

 

Inactivation

 

Carry out an amplification test for residual infectious rabies virus immediately after inactivation or using a sample frozen immediately after inactivation and stored at -70C. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 doses of vaccine into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further 14 days and then examine the cell cultures for rabies virus using an immunofluorescence test. No rabies virus is detected.

 

Residual host-cell DNA

 

If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method as described in Products of recombinant DNA technology (0784), is not greater than 100 pg per single human dose.

 

FINAL BULK VACCINE

 

The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approved stabiliser may be added to maintain the activity of the product during and after freeze-drying.

 

Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.

 

Glycoprotein content

 

Determine the glycoprotein content by a suitable immunochemical method (2.7.1), for example, single-radial immunodiffusion, enzyme-linked immunosorbent assay or an antibody-binding test. The content is within the limits approved for the particular product.

 

Sterility (2.6.1)

 

The final bulk vaccine complies with the test for sterility, carried out using 10 ml for each medium.

 

FINAL LOT

 

The final bulk vaccine is distributed aseptically into sterile containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to avoid contamination and the introduction of moisture.

 

Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated viral suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.

 

Identification

 

The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method (2.7.1) using specific antibodies, preferably monoclonal; alternatively, the assay serves also to identify the vaccine.

 

Tests

 

Inactivation

 

Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further 14 days and then examine the cell cultures for rabies virus using an immunofluorescence test. No rabies virus is detected.

 

Bovine serum albumin

 

Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.7.1).

 

Sterility (2.6.1)

 

The vaccine complies with the test for sterility.

 

Bacterial endotoxins (2.6.14)

 

Less than 25 IU per single human dose.

 

Pyrogens (2.6.8)

 

The vaccine complies with the test for pyrogens. Unless otherwise justified and authorised, inject into each rabbit a single human dose of the vaccine diluted to ten times its volume.

 

Water (2.5.12)

 

Not more than 3.0 per cent, determined by the semi-micro determination of water.

 

Assay

 

The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against the effects of a lethal dose of rabies virus, administered intracerebrally, with the quantity of a reference preparation of rabies vaccine necessary to provide the same protection. For this comparison a reference preparation of rabies vaccine, calibrated in International Units, and a suitable preparation of rabies virus for use as the challenge preparation are necessary.

 

The International Unit is the activity contained in a stated quantity of the International Standard. The equivalence in International Units of the International Standard is stated by the World Health Organisation.

 

The test described below uses a parallel-line model with at least three points for the vaccine to be examined and the reference preparation. Once the analyst has experience with the method for a given vaccine, it is possible to carry out a simplified test using a single dilution of the vaccine to be examined. Such a test enables the analyst to determine that the vaccine has a potency significantly higher than the required minimum but will not give full information on the validity of each individual potency determination. The use of a single dilution allows a considerable reduction in the number of animals required for the test and must be considered by each laboratory in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.

 

Selection and distribution of the test animals

 

Use in the test healthy female mice about 4 weeks old, each weighing 11 g to 15 g, and from the same stock. Distribute the mice into six groups of a size suitable to meet the requirements for validity of the test and, for titration of the challenge suspension, four groups of five.

 

Preparation of the challenge suspension

 

Inoculate mice intracerebrally with the CVS strain of rabies virus and when the mice show signs of rabies, but before they die, sacrifice them, remove the brains and prepare a homogenate of the brain tissue in a suitable diluent. Separate gross particulate matter by centrifugation and use the supernatant liquid as the challenge suspension. Distribute the suspension in small volumes in ampoules, seal and store at a temperature below -60C. Thaw one ampoule of the suspension and make serial dilutions in a suitable diluent. Allocate each dilution to a group of five mice and inject intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe the mice for 14 days. Calculate the LD50 of the undiluted suspension using the number in each group that, between the fifth and fourteenth days, die or develop signs of rabies.

 

Determination of potency of the vaccine to be examined

 

Prepare three five-fold serial dilutions of the vaccine to be examined and three fivefold serial dilutions of the reference preparation. Prepare the dilutions such that the most concentrated suspensions may be expected to protect more than 50 per cent of the animals to which they are administered and the least concentrated suspensions may be expected to protect less than 50 per cent of the animals to which they are administered. Allocate the six dilutions one to each of the six groups of mice and inject intraperitoneally into each mouse 0.5 ml of the dilution allocated to its group. After 7 days, prepare three identical dilutions of the vaccine to be examined and of the reference preparation and repeat the injections. Seven days after the second injection, prepare a suspension of the challenge virus such that, on the basis of the preliminary titration, 0.03 ml contains about 50 LD50. Inject intracerebrally into each vaccinated mouse 0.03 ml of this suspension. Prepare three suitable serial dilutions of the challenge suspension. Allocate the challenge suspension and the three dilutions one to each of the four groups of five control mice and inject intracerebrally into each mouse 0.03 ml of the suspension or one of the dilutions allocated to its group. Observe the animals in each group for 14 days and record the number in each group that die or show signs of rabies in the period 5 days to 14 days after challenge.

 

The test is not valid unless: for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses given to the mice; the titration of the challenge suspension shows that 0.03 ml of the suspension contained not less than 10 LD50; the statistical analysis shows a significant slope and no significant deviations from linearity or parallelism of the dose-response lines; the fiducial limits of error (P = 0.95) are not less than 25 per cent and not more than 400 per cent of the estimated potency.

 

The vaccine complies with the test if the estimated potency is not less than 2.5 IU per human dose.

 

Labelling

 

The label states the biological origin of the cells used for the preparation of the vaccine.

 

 

 


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