Adsorbed Diphtheria Vaccine
Ph Eur
DEFINITION
Diphtheria vaccine (adsorbed) is a preparation of diphtheria formol toxoid with a mineral adsorbent. The formol toxoid is prepared from the toxin produced by the growth of Corynebacterium diphtheriae.
PRODUCTION
Specific toxicity
The production method is validated to demonstrate that the product, if tested, would comply with the following test: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing 250-350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test.
For the production of diphtheria toxin, from which toxoid is prepared, seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored by deliberate reselection. A highly toxinogenic strain of Corynebacterium diphtheriae with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is separated aseptically from the bacterial mass as soon as possible. The toxin content (Lf per millilitre) is checked (2.7.27) to monitor consistency of production. Single harvests may be pooled to prepare the bulk purified toxoid. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxification.
Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
Absence of toxin and irreversibility of toxoid
Using the same buffer solution as for the final vaccine, without adsorbent, prepare a solution of bulk purified toxoid at 100 Lf/mL. Divide the solution into 2 equal parts. Maintain 1 part at 5 ± 3 °C and the other at 37 °C for 6 weeks. Carry out a test in Vero cells for active diphtheria toxin using 50 µL/well of both samples. The sample should not contain antimicrobial preservatives and detoxifying agents should be determined to be below the concentration toxic to Vero cells. Non-specific toxicity may be eliminated by dialysis.
Use freshly trypsinised Vero cells at a suitable concentration, for example 2.5 × 105 mL-1 and a reference diphtheria toxin diluted in 100 Lf/mL diphtheria toxoid. A suitable reference diphtheria toxin will contain either not less than 100 LD50/mL or 67 to 133 lr/100 in 1 Lf and 25 000 to 50 000 minimal reacting doses for guinea-pig skin in 1 Lf (diphtheria toxin BRP is suitable for use as the reference toxin). Dilute the toxin in 100 Lf/mL diphtheria toxoid to a suitable concentration, for example 2 × 10-4 Lf/mL. Prepare serial twofold dilutions of the diluted diphtheria toxin and use undiluted test samples (50 µL/well). Distribute them in the wells of a sterile tissue culture plate containing a medium suitable for Vero cells. To ascertain that any cytotoxic effect noted is specific to diphtheria toxin, prepare in parallel dilutions where the toxin is neutralised by a suitable concentration of diphtheria antitoxin, for example 100 IU/mL. Include control wells without toxoid or toxin and with non-toxic toxoid at 100 Lf/mL on each plate to verify normal cell growth. Add cell suspension to each well, seal the plates and incubate at 37 °C for 5-6 days. Cytotoxic effect is judged to be present where there is complete metabolic inhibition of the Vero cells, indicated by the pH indicator of the medium. Confirm cytopathic effect by microscopic examination or suitable staining such as MTT dye. The test is invalid if 5 × 10-5 Lf/mL of reference diphtheria toxin in 100 Lf/mL toxoid has no cytotoxic effect on Vero cells or if the cytotoxic effect of this amount of toxin is not neutralised in the wells containing diphtheria antitoxin. The bulk purified toxoid complies with the test if no toxicity neutralisable by antitoxin is found in either sample.
Antigenic purity (2.7.27)
Not less than 1500 Lf per milligram of protein nitrogen.
The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used.
Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 per cent of the intended amount.
Sterility (2.6.1)
Carry out the test for sterility using 10 mL for each medium.
The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination.
Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/L, the test for free formaldehyde may be omitted on the final lot.
IDENTIFICATION
Diphtheria toxoid is identified by a suitable immunochemical method (2.7.1). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/L solution. Maintain at 37 °C for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate.
TESTS
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the absorbent.
Free formaldehyde (2.4.18)
Maximum 0.2 g/L.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115 per cent of the quantity stated on the label.
Sterility (2.6.1)
The vaccine complies with the test for sterility.
ASSAY
Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose.
LABELLING
Ph Eur