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GENERAL TESTS

• WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE

Y.J H , .. ·

BLG/UNDP/82.1

ORIGINAL: ENGLISH

I

MANUAL OF DETAILS OF TESTS REQUIRED ON FINAL VACCINES

USED IN THE WHO EXPANDED PROGRAMME OF Il~NIZATION

The issue of this document does not constitute

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MANUAL OF TESTS FOR BIOLOGICAL SUBSTANCES

BLG/UNDP/8.21 Page 1

This manual has been prepared, in the first instance, to give details of the tests applied to vaccines in the final form. Such tests may be applied by control authorities to vaccines as they are given to children in order to check that they have adequate potency and do not have undue toxicity.

The tests are identified by the vaccine to which they are applicable or as a general test and are classified according to the complexity of the equipment and facilities required for their execution.

Class A: is a test requiring only the laboratory equipment expected to be present in any microbiological laboratory.

Class B: is a test requ~r~ng special items of equipment but does not require the use of animals.

Class C: is a test requiring the use of animals and, thereby, demanding the facilities of an animal holding unit, an animal breeding station or a reliable source of good quality animals.

BLG/UNDP/82.1 Page 1 bis

GENERAL TESTS

BLG/UNDP/82.1 Page 1 ter


TESTS APPLIED TO VACCINES

General Tests Test Page

1. Sterility GEN-1 6-18

2. Innocuity GEN-2 19

3. Aluminium content GEN-3 20-21

4. Preservatives GEN-4 22-23

5. Hydrogen ion concentration(pH) GEN-5 24

6. Free formaldehyde GEN-6 25-26

7. Opacity GEN-7 27-28

8. Moisture content GEN-8 29-32

Vaccine

B.C.G. Vaccine

1. Identity BCG-1 33-36

2. Sterility See GEN-1

3. Absence of contaminating BCG-2 37 microorganisms

4. Absence of virulent myco-BCG-3 38-40 bacteria

5. Total bacterial content BCG-4 41 or See GEN-7

6. Number of culturable particles BCG-5 42-49

7. Test for stability BCG-6 50-52


Vaccine

Diphtheria Vaccine Test Page

1. Potency DIP-1 53-68


3. Innocuity " GEN-2

4. Adjuvant content " GEN-3

5. Preservative content " GEN-4

6. Test for pH " GEN-5

7. Identity DIP-2 69-70

See also test for free formaldehyde GEN-6 See also stability and

test reveral

DIP-3 71-73

Pertussis Vaccine

1. Potency PER-l 74-90


3. Innocuity GEN-2

4. Adjuvant content II GEN-3

5. Preservative content II GEN-4

6. Test for pH II GEN-5

7. Identity PER-3 93

See also test for opacity GEN-7 See also test for toxicity PER-2 91-92


Vaccine

Tetanus Vaccine

1. Potency

2. Sterility

3.

4.

5.

6.

Innocuity

Adjuvant content

Preservative content

Test for pH

7. Identity

See also test for free formaldehyde

See also test for irreversibility and test for specific toxicity

Poliomyelitis Vaccine (oral)

1. Virus titration (potency)

2, Test for bacteria and fungi

3, Innocuity

4. Identity

Measles Vaccine (Live)

l, Virus concentration (potency)

2. Sterility

3. Innocuity

4. Identity

5. Stability

Test

TET-1

See GEN-1

"

"

"

"

GEN-2

GEN-3

GEN-4

GEN-5

TET-2

GEN-6

TET-3

POL-l

GEN-1

GEN-2

POL-2

MEA-l

See GEN-1

" GEN-2

MEA-2

MEA-3

94-107

108-109

110-111

112-116

117

118-121

122

123


SUMMARY PROTOCOLS FOR THE REPORTING OF RESULTS OF TESTS

Protocol

Diphtheria, pertussis, tetanus

Diphtheria toxoid

Pertussis Vaccine

Tetanus toxoid

BCG Vaccine

Poliomyelitis Vaccine (Oral) Sabin strains

Measles Vaccine (Live)

124-131

132-136

137-140

141-144

145-149

150-161

162-167


VACCINE: ALL VACCINES

CLASS OF TEST: A

GEN-1

TEST: Test for Sterility

GENERAL REQUIREMENTS FOR THE STERILITY OF BIOLOGICAL SUBSTANCES

(Requirements for Biological Substances No. 6) (Revised 1973)

Part A Section 5

THE TEST

1. Sampling from final lot

Samples of final containers from each final lot to be tested shall be taken in such a manner as to be representative of the lot to be tested. Appropriate periodic samples shall be taken including samples at the beginning and the end of the filling operation.

If a product lot is filled through several outlets from a single bulk, samples should be taken from each outlet (filling lot) so as to be representative of the filling assembly.

The number of samples taken shall be at least that approved by the national control authority, provided that for final lots containing 500 or more containers, at least 20 samples shall be taken, including samples at the beginning and the end of the filling operation.


For final lots containing fewer than 500 containers, not less than 10 containers from the lot should be taken for a sample, except that for lots of less than 100 containers, only 10% of the lot need be tested.

1 A suggested rule for

calculating the number of samples is to take 0.4 ./'Ff samples, where N is the number of final containers in the lot.

2. Sterility test for bacteria and fungi

2.1 Culture media

The culture media used for sterility tests for bacteria and fungi shall be those approved by the national control authority. Such media shall have been shown to be capable of supporting the growth of a wide variety of micro-organisms, with both aerobic and anaerobic growth characteristics, including the types found in the environment of the manufacturing operations.

More than one culture medium, generally will be needed to fulfil these criteria. The media used in many countries for sterility tests are fluid thioglycolate medium

2and soybean-casein digest.

medium • Any other media that are used, however, should have been demonstrated to have at least equivalent growth supporting properties.

1 2 Wld Hlth Org. techn. Rep. Ser. 1960 No 200, p. 31

The formulae of these culture media are given in Appendix 1


For testing the growth supporting qualities of each culture medium, strains of microorganisms should be used with exacting nutritive and aerobicanaerobic requirements, in an inoculum of only a small number of the organisms (less than 100). The media should be incubated at the temperatures at which they will be used in the sterility test.

Each lot of dehydrated medium or each lot of medium prepared from basic ingredients should be tested for its growth supporting properties, since every lot may not support the growth of microorganisms as well as desirable. It is desirable that lots so tested should be kept separate and reserved for use in sterility testing. This is a safeguard against occasional unsatisfactory components in a particular lot, or destruction of certain components by over-heating or over-sterilization, which may cause differences in growth response.

In some countries an additional test is made to verify the growth supporting properties of the medium, after the completion of a sterility test in which no growth has occurred in the medium, by inoculation with a small number of suitable organisms (less than 100).


2.2 Performance of the test

Prior to conducting a sterility test on any product, it shall have been determined whether or not the material to be tested itself has the property of killing or inhibiting the growth of micro-organisms, or contains preservatives or other substances that have this effect. If such an effect is shown, the sterility test shall be made using a suitable procedure to counteract the effect.

2.3 Inoculum

The inhibitory effect in a preparation to be tested for sterility may be overcome by increasing the volume of culture medium used, so that the inhibitors are rendered ineffective.

The volume of medium required to overcome the inhibitory effect should be determined. Once established, these quantities may be used for subsequent sterility tests unless a change is made in the composition of the product.

For sterility testing of bulk material, at least 5 ml must be used for inoculation into each culture medium and for each temperature of incubation.

For sterility testing of a final lot from each of the final containers that contain more than 20 ml, an amount of at least 1.0 ml shall be inoculated into each culture medium and for each temperature of incubation. If the volume in each final container is less than 1.0 ml the amount to be inoculated shall be the entire content of the container for each culture medium and for each temperature of incubation. If the volume in each final container is greater than 20 ml, but not more than 100 ml, the amount to be inoculated shall be at least 5 ml for each culture medium and for each temperature of incubation,


2.4 Medium

The quantityof medium put into each vessel for conducting the sterility test shall be sufficiently large to ensure that the volume of material inoculated does not impair the growth supporting properties of the medium by dilution.

All vessels of inoculated media shall be clearly identified by labelling that is adequate to identify the product being tested, each medium used, and each temperature of incubation.

2.5 Incubation

All vessels shall be incubated at the appropriate temperatures for the media. The temperatures selected shall be those approved by the national control authority and shall include 20°-25°C and 30°-36°C.

If only one temperature is selected for a particular medium, generally fluid thioglyco6ate medium is incubated at 30 -36°C and casein diRest medium at 20°-25°C.

In some countries additional temperatures are used for psychrophilic and thermophilic organisms.

It is desirable that the incubation apparatus should have a continuous temperature recording device.

All vessels shall be incubated for a period of at least 14 days and shall be examined at regular intervals and on the last day of incubation for evidence of microbial growth.


If the preparation inoculated into the test vessels has clouded the medium to such an extent that it is difficult to recognize whether or not growth has taken place, subcultures of not less than 1.0 ml should be made from the cloudy medium between the third and seventh days after the start of the test. The original and transfer vessels should each be incubated and observed for a total of not less than 14 days.

3. Interpretation of test results

If no evidence of growth is found in any of the vessels inoculated for the test for sterility, the bulk material or final lot, whichever is applicable, meets the requirements for this test. If evidence of growth is found, the preparation tested fails to meet the requirements for the test for sterility, unless it can be demonstrated to the satisfaction of the national control authority, by retests or by other means, that the test was invalid.

The decision between failure of the product to pass the test and invalidity of the test procedure requires competent judgement of a trained individual.

4. Sterility test for mycoplasmas

For viral vaccines made by growing the virus in animal tissues or cell cultures, sterility test for mycoplasmas of virus culture fluid and control fluid specified for the particular product shall be made by a


h d d b h . 1 1 h . 1 a met o approve y t e nat~ona contro aut or~ty.

The samples taken may include material prior to clarification or filtration in the case of live vaccines produced from in vitro living cell cultures or an~mal tissues. In the case of inactivated virus vaccines produced from such cell cultures or tissues, the samples may be taken prior to inactivation, from each virus harvest pool and from control pooled fluid.

Control cultures should be included in each test.

5. Sterility test for viruses

The sterility tests for the detection of extraneous viruses in products where their presence is unacceptable shall be performed as specified in the requirements for the individual products.

6. Tests for specific micro-organisms

The tests to ensure the non-survival of microorganisms in inactivated vaccines and additional tests for the absence of specific extraneous micro-organisms in such products shall be performed as specified in the requirements for the individual products.

7. Records

Records shall be permanent and clearly indicate all steps in the sterility testing of the product, including temperatures and incubation times, as well as the relation-

1 A description of tests for mycoplasmas is given

in Appendix 2


ship of the samples under test to the manufacturing operations followed in the production of the lot under test. The records shall be of a type approved by the national control authority. Written records shall be kept of all tests performed, irrespective of their results. They shall be retained throughout the dating period of the product, and be available for inspection by the national control authority. Records shall be maintained of all cultures kept in the establishment. Such records shall include clear labelling for the identification of the cultures and the purposes for which the cultures are used.

For each sterility test performed a record shall be kept of the name and identifying numbers of the product under test, the quantities inoculated, the batch, type and tests of culture media used, the temperatures of incubation, the dates of inoculation and the results.

8. Additional samples

For each final lot for which sterility tests are made, additional samples shall be retained as reference material throughout the dating period of the product, in a manner that ensures the identity of the lot or batch of the product.

It is desirable, that where possible, manufacturers should retain sufficient additional samples to permit repetition of the sterility tests.


Appendix 1

MEDIA FOR THE DETECTION OF AEROBIC & ANAEROBIC BACTERIA & FUNGI*

1. Fluid thioglycolate medium

L-cystine 0.5 sodium chloride 2.5 glucose (C6Hl206H20) 5.5

agar 0.75 yeast extract, water soluble 5.0 pancreatic digest of casein 15.0 distilled water 1000 thioglycolic acid 1 0.3 (or sodium thioglycolate 0.5 resazurin sodium (0.10% fresh solution) 1.0

Final pH • 7.0- 7.2

Preparation:

g g g

g g g ml ml g) ml

Thoroughly grind the first six ingredients, in the order given above, in a mortar. Stir in some heated water, transfer to a suitable container, add the remainder of the water and complete the solution by_ heating in a boiling water bath, taking special care to ensure complete solution of the L-cystine. Add the thioglycolic compound, then 1 N sodium hydroxide so that the pH of the completed and sterilized medium will be 7.0-7.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. Distribute into suitable vessels and sterilize by autoclaving for 18-20 minutes at 121°C.

*

1

The quality of the components should be in accordance with Specifications for Reagents mentioned in the International Pharmacopoeia, Geneva, World Health Organization 1967, unless otherwise stated. Reagent quality sodium thioglycolate is more stable than thioglycolic acid.


0 0 0 Cool promptly to 25 C and store at 20 C-30 C, avoiding excessive light. If the uppermost portion of the medium has changed to a pink colour and this exceeds one-third of the depth of the medium, it is unsuitable for use, but may be restored once by heating in steam. Medium more than 3 weeks old should not be used.

2. Soybean-casein digest medium

pancreatic digest of casein papaic digest of soybean meal sodium chloride dipotassium hydrogen phosphate glucose (C

6H

12o6H20)

dist~lled water

Final pH- 7.1 - 7.5

Preparation:

17.0 3.0 5.0 2.5 2.5

1000

g g g g g

ml

Dissolve the solids in the water, warming slightly, then cool the solution to room temperature. Adjust the reaction with 1 N sodium hydroxide if necessary, so that the pH of the completed and sterilized medium will be 7.1-7.5. Filter, if necessary, to clarify, distribute into suitable vessels and sterilize in an autoclave for 18-20 minutes at 121°C.


Appedix 2

TESTS FOR MYCOPLASMAS

Allvarieties of mycoplasma are not capable of growth in the same medium. Some strains require special culture media. Suitable media should therefore be selected having regard to particular conditions, e.g. the tissue in which a virus is grown or source of animal serum, and the growth requirements of the potential contaminants, e.g., the need for yeast extract. Solid and semi-solid or liquid media coytaining serum fractions rich in certain lipids (sterols and 'phospholipids) are used to provide the nutritive requirements. Control cultures on solid and semi-solid or liquid media of known fastidious strains of mycoplasma, both sterol-requiring and non-sterolrequiring, are included in each test to show that the media are capable of supporting the growth of mycoplasmas.

Samples of the material of not less than 6 ml for a single test (i.e. using one solid and one semi-solid or liquid medium) are stored either between 2° and 8°C for no longer than 24 hours or at -20°C or lower if stored for longer than 24 hours.

Not less than 2.0 ml of each sample are inoculated and evenly distributed over the surface of at least 10 plates of each solid medium used, and not less than 1.0 ml of each sample is inoculated into each of at least 4 tubes of each semi-solid or liquid medium used. Each tube should contain 10 ml of medium. Larger volumes of inoculum, e.g. making a total of 50 ml of material in 400 ml of medium, have been used advantageously. Ultracentrifugation before testing such samples has also been used.

0 0 The plates and tubes are incubated at 36 C + 1.0 C

for not less than 14 days. One half of each set-is

1 Non-sterol-requiring mycoplasmas will grow on media containing sterols.


incubated aerobically in an environment of adequate humidity, and the other half is incubated anaerobically in an environment of 5-10% carbon dioxide in nitrogen, also of adequate humidity. At the end of 3 days and again at 14 days, 0.5 ml from each of 2 tubes being incubated aerobically are combined and inoculated on to not less than 4 plates. The 2 tubes being incubated anaerobically are treated"likewise. · Since some mycoplasmas may not grow on media in Petri dishes, sucf subcultures on solid media may also be made in tubes.

All material seeded originally on solid media is subcultured on the day after the primary inoculation, on the third day and on the sixth day, on other suitable solid and liquid media, and these subcultures are incubated similarly, half aerobically and the other half anaerobically as described, and examined after 10 days, while the original cultures are examined at the end of the 14-day period. All plates (and tubes) after incubation for not less than 14 days are observed for the growth of mycoplasmas by appropriate procedures.

If no evidence is found of the presence of mycoplasmas the material, e.g. virus pool, meets the requirements of these tests. If evidence of growth of mycoplasmas is found, the mycoplasmas should be identified and the source traced.

For detection and identification of mycoplasmas the following procedures are suggested but other methods are available:

1. For detection of mycoplasmas

(a) typical colonies or specific coloration (blue) by Diene's stain of colonies spread on blocks of agar in Petri dishes, and viewed under the microscope;

1 In order to induce mycoplasmas to grow in liquid media, similar subculturing could be done, but three or four passages may be needed.


1. For detection of mycoplasmas Cont ...

(b) phase contrast microscopy of colonies (strips or plaques) for globular bodies, filaments and granules;

(c) examination under the electron microscope of the pellet centrifuged from a semisolid culture;

(d) examination under the light microscope (x 1000) of a film stained by Giemsa.

2. For separation of mycoplasmas into classes

(a) enzymatic effects; fermentation of glucose, breakdown of arginine and reduction of triphenyltetrazole, using decoloration of phenol red as indicator for the first two effects and red colour of the tetrazole derivative for the last;

(b) haemolysis in a 4% suspension of washed guineapig cells, added and incubated for 18-48 hours aerobically at 37°C;

(c) haemadsorption of a 4% suspension of guineapig cells poured over the growth surface or fixation of red blood corpuscles by the colonies after incubation at 37°C for 30 minutes.

3. For specific identification

Growth inhibition by specific anti-mycoplasma serum (prepared in the rabbit); such inhibition might be demonstrated by depositing paper discs impregnated with the serum on 2-4 hour growth cultures and observing them from the third day.

Immunofluorescence techniques for this purpose are being developed.

VACCINE: Diphtheria Toxoid Pertussis vaccine and Tetanus Toxoid

TEST: Test for innocuity


CLASS OF TEST C

GEN-2

REQUIREMENTS FOR DIPHTHERIA TOXOID, PERTUSSIS VACCINE TETANUS TOXOID AND COMBINED VACCINES

(Requirements for Biological Substances Nos. 8 and 10) (Revised 1978)

Addendum 1980

Part A Section 19

THE TEST

Each final lot shall be tested for abnormal toxicity by the injection of one human dose, but not more than 1 ml, into each of 5 mice (weighing 17-22 g) and at least one human dose, but not more than 1 ml, into each of 2 guineapigs (weighing 250-350 g) by the intraperitoneal route. The final product shall be considered as innocuous if the animals survive for at least 7 days without showing significant signs of toxicity.

The animals used in this test shall be from a healthy stock. If there is any doubt about the health of the stock similar groups of animals should be injected with 1 ml of saline and the results compared with those animals given the vaccine. If any animals die within 7 days of being given saline the health of the animals should be investigated.


VACCINES: All Vaccines containing adjuvants

TEST: Test for Aluminium content

CLASS OF TEST A

GEN-3

REQUIREMENTS FOR DIPHTHERIA TOXOID, PERTUSSIS VACCINE TETANUS TOXOID AND CO}ffiiNED VACCINES


Part A Section 3.4.3

Procedure

To duplicate 3 ml samples of the product in boiling tubes are added 1 ml of concentrated sulfuric acid and six drops of concentrated nitric acid. The tubes are heated until dense white fumes evolve. Heating is continued, with dropwise addition of nitric acid, until the contents of the tubes are colourless. The tubes are cooled and 10 ml of distilled water is carefully added. If the solution is cloudy the tubes are boiled until clear. Sodium hydroxide (50% w/v) is added to each tube until a pink-yellow endpoint is obtained with methyl orange , indicator. Any precipate forming is dissolved by the addition of dilute sulfuric acid. The contents of the tubes are transferred to separate 250 ml Erlemeyer flasks, the tubes washed out with 25 ml of distilled water and the washings pooled with the contents of the flasks. To each flask are added 25 ml of M/100 disodium ethylenediamine tetra acetate, and 10 ml of acetate buffer (containing 68 g of sodium acetate, 38.5 g of ammonium acetate, 125 ml of glacial acetic acid in a total volume of 500 ml). The flasks are boiled gently for three minutes and 1 ml of pyridylazonaphthol indicator (0.1% solution in 95% ethanol) is added. Each hot


solution is titrated with M/100 copper sulfate solution until a purple-brown endpoint is reached. Simultaneously a blank determination is also carried out using distilled water in the digestion procedure instead of sample. Each ml of M/100 Cuso

4.5H

20 = 0.2698 mg Al+++.

Calculation of the results:

mg of Al per ml of sample = Blank titre-Sample titre Volume of sample (ml)

X 0.2698 X 4.52

Criteria for acceptance

The aluminium content in the final bulk product should not exceed 1.25 mg Al per single human dose.


VACCINE: All Vaccines containing Thiomersal

TEST: Test for Thiomersal

CLASS OF TEST A

GEN-4




TEST FOR THIOMERSAL CONTENT

By chemical means

Procedure

The thiomersal content (merthiolate, thimerosal, ethylmercuri-thiosalicylate) is determined spectrophotometrically using diphenylthiocarbazone (dithizone).

Separatory funnels are washed with concentrated nitric acid and rinsed with tap and distilled_ water. Two aliquots of a standard 0.01% acueous solution of thiomersal (0.5 and 1.0 ml) and two samples of the test solution (1 ml) are added to individual separatory funnels and the volume adjusted to 10 ml with a 1% solution of ammonium acetate at pH 6.0. Ten milli-litres of a 1 in 10 dilution of a fresh solution of 0.01% dithizone in chloroform is added to each funnel and the contents shaken vigorously for 45 seconds. The chloroform layer is carefully separated.


The 0 and 100% transmission points on the spectrophotometer are set at 490 nm using the diluted dithizone solution and a scan is taken of the test solutions from 470 to 520 nm. The transmission at 520 nm is plotted against the thiomersal concentration of the standards on semi-logarithmic paper and the concentration of thiomersal in the sample determined.

By microbiological means

Nutrient agar is poured to a depth of 4 mm on a suitable rectangular glass plate set in a metal frame (a large Petri dish may be used but can accommodate only a few samples). It is essential that the plate is absolutely horizontal when the agar is being poured. Circular wells of approximately 8 mm diameter are cut in the agar so that adjacent wells are at leas-t 30 mm apart. The plate is seeded with sufficient Staphylococcus aureus organisms to give a confluent growth at 48 hours. Into each well 100 microlitre samples of 1:2.5, 1:5, 1:10, 1:20, 1:40 and 1:80 dilutions of a standard solution of 1 g/litre sodium thiomersalate are randomly dispersed as well as samples of the test vaccines. The location of each preparation is carefully recorded.

The plates are incubated for 48 hours. The diameter of the zones of inhibition, produced by each of the test vaccines, is measured and compared with the zones produced by the reference dilutions of thiomersal. The effective concentration is expressed in terms of the dilution of reference thiomersal solution to which it most closely approximates.


The thiomersal content should be between 0.005 and 0.02%.


VACCINES: Diphtheria, Pertussis, Tetanus and Poliomyelitis

CLASS OF TEST A

GEN-5

TEST: Test for Hydrogen Ion concentration pH

TEST FOR pH

Procedure

A pH meter with glass and calomel reference electrodes is standardized with standard buffer solutions at pH 5.0 and 7.0. The electrodes are rinsed with distilled water, dried and the pH of the test solution recorded.


The pH value of DPT vaccine shall be 6.7-7.0, of Diphtheria, Tetanus and DT vaccines between 6.0-6.7, and for Pertussis vaccine alone 7.0 + 0.3. Poliomyelitis vaccine (ora~) should be 6.8 but should never be more than 7.0. Poliomyelitis vaccine (oral) contains an indicator (phenol red) and the vaccine should be an orange colour. If the colour is bright red or purple the pH is too high and should be checked. Vaccine containing magnesium chloride as a stabilizer but at a high pH will not be stable.


CLASS OF TEST A

GEN-6

VACCINES: Diphtheria, Pertussis and Tetanus

TEST: Test for free Formaldehyde




TEST FOR FREE FORMALDEHYDE

Procedure

1 ml of the vaccine or toxoid is diluted with distilled water to 10 ml. 2 ml of the diluted sample is transferred into 50 ml Nessler tube (with about 65 ml capacity) and diluted with distilled water to about 35 ml. 10 ml diluted sulfuric acid (one vol. H2so

4 of sp. gr.

1.84: one vol. water) is added and the Ilu1d is diluted with distilled water to the 50 ml mark. Similarly a series of tubes with known amounts of formaldehyde are prepared to serve as formaldehyde standards for comparison, using a 0.01% dilution of Analar solution of formaldehyde (containing not less than 37% of CH 0) and selecting quantities close to that expected in t5e sample. To the sample tube and to each of the standard tubes 10 ml of fuchsin-sulfurous acid test solution is added. The mixtures are allowed to stand one hour at room temperature and a colorimetric comparison of the sample tube with the standards is made.

Fuchsin-sulfurous acid test solution

0.2 g of basic fuchsin is dissolved in 120 ml of hot


distilled water and the solution allowed to cool. A solution of 2 g of anhydrous sodium sulfite in 20 ml of distilled water is added and then 2 ml of hydrochloric acid. The solution is diluted with distilled' water to 200 ml and allowed to stand at least one hour. This solution must be freshly prepared.


The vaccine shall be shown to have not more than 0.02% free formaldehyde.

Note: If the B. pertussis organisms in the pertussis vaccine are killed with chemicals other than formaldehyde or by heat then there is no purpose in applying this test to pertussis vaccine not combined with toxoids.

VACCINE: Pertussis and BCG

TEST: Test for opacity


CLASS OF TEST A

GEN-7




TEST FOR OPACITY

This test is applied as an "in process" control to the pertussis component before any admixture takes place and before the addition of adjuvant; it is applied also to final BCG vaccine. The test is included in this manual because control laboratories should be aware of the test even though it cannot be applied to the final vaccine.

The opacity of each single harvest shall be determined not later than two weeks after harvesting and before the bacterial suspension has been subjected to a process capable of altering its opacity. The opacity shall be determined in comparison with the International Reference Preparation of Opacity or an equivalent reference preparation approved by the national control authority.

A suspension of pertussis organisms that on visual inspection has an opacity approximately equal to 10 IU of opacity may be considered for practical purposes to


1 have a concentration of 10 000 million organisms per ml .

Procedure

The sample shall be diluted until the opacity is identical with the Fifzh International Reference Preparation of Opacity when compared by EYE. The opacity of such a suspension is 10 Opacity Units.

Taking the dilution factor into account, the opacity of the sample is calculated.

If the final bulk, when diluted with an equal volume of saline has the same opacity as the Fifth International Reference Preparation of Opacity, the bulk has an opacity of 20 Opacity Units.

1

2

The concentration determined by direct count, however, would probably be less than 10 000 million per ml. The question of relationship of the international unit of opacity to concentration of organisms has been considered by the Expert Committee on Biological Standardization (WHO Technical Report Series, No. 96, 1955, page 8, and No. 259, 1963, page 26).

Available from Chief, Biologicals, WHO, Geneva

VACCINE: BCG and Measles

TEST: Test for moisture content

TEST FOR MOISTURE CONTENT


CLASS OF TEST A

GEN-8

The moisture content is determined by measuring the loss of weight of the test sample after drying under detiined conditions.

The acceptance is assessed according to the criterion specified for each vaccine.

Test Procedure

A weighing bottle with a stopper provided with a capillary of 0.20 to 0.25 mm inside diameter is dried and accurately weighed at a relative humidity of no higher than 45%.

A 30-50 mg portion of the test sample is transferred into the weighing bottle and the bottle is accurately weighed to obtain the net weight of the test sample.

The loaded bottle is dried over anhydrous P2o~ at a pressure of no higher than 5 mmHg and at 60°C for 3hours, then placed in a dessicator containing anhydrous P

2o

5 or

silica gel, cooled to room temperature and weighed accurately.


The moisture content is calculated by the following formula:

Moisture content

%

the weight lost of the test sample by drying the weight lost of the test sample before drying

X 100

The moisture shall be less than 3% for BCG vaccine and less than 2% for measles vaccine.

VACCINE: BCG

General Remarks


The system for the control of BCG vaccine is different from that of other vaccines. In view of the special problems in standardizing production and control of this vaccine it is a WHO-centralized system based on the principle that the number of BCG production and control centres should be kept to a minimum.

As far as the quality control is concerned, this is coordinated by the BCG Dept. of the State Serum Institute, Copenhagen. The data from the most important test (estimation of the number of culturable particles - see below) are collected in Copenhagen, punched on cards and calculated in Geneva by computer. In this way at least the vaccines available internationally are standardized in order to ensure their efficacy.

The production and control of BCG vaccine is possibly only by specially trained staff. As a rule such training takes place at the Copenhagen laboratory, where there is an ongoing training programme for this purpose.

Control tests of BCG vaccine are now performed in a number of control laboratories (Budapest, Prague, Bucharest and Madras for vaccines supplied to UNICEF) as well as in other national control laboratories and also production laboratories. The results from most of these laboratories are analysed in the coordination programme and it is clear that this system functions efficiently.

The backgrounds and philosophy of the present situation in the field of BCG vaccine control has been fully described in WHO/TB/Technical Guide/77.8, "WHOsponsored International Quality Control of BCG Va~e". The details of the in-vitro test methods are to be found in


detail in WHO/TB/Technical Guide/77.9, "In vitro Assays of BCG Products".

The most recent WHO Requirements were published in 1979: "Revised Requirements for dried BCG Vaccine", WHO Technical Report Series, No. 630 1979 Annex 2, in which the test methods are found on pages 128-135.

BCG VACCINE

VACCINE: BCG

TEST: Test for Identity


CLASS OF TEST B

BCG-1

REQUIREMENTS FOR DRIED BCG VACCINE (Requirements for Biological Substances No. 11)

(Revised 1978)

Part A Section 5.1

THE TEST

Identity Test

An identity test shall be performed on samples of vaccine from each final lot.

The identity of each filling lot of vaccine shall be verified by the morphological apperance of the bacilli in stained smears and by the characteristic appearance of the colonies grown on solid media.

The medium used is generally the Lowenstein-Jensen or a similar medium. The smears are stained by the Ziehl- Neelsen method.

Lowenstein-Jensen Medium

Lowenstein's medium with 0.75% Glycerin (usually called Lowenstein-Jensen medium) (Zentralblatt fur Bakteriologie, Parasitenkunde u. Infektionskrankheiten I. Abt. Orig. 125, 222, 932 and Advances in the Control of Zoonoses, p. 11 and 35, World Health Organization, Geneva 1953).


Lowenstein-Jensen medium Cont ...

For the culture medium, the following mixture is made up:

Monopotassium phosphate Magnesium sulfate Magnesium citrate 1-Asparagin Glycerine (twice distilled)

0.4% 0.04% 0.1% 0.6% 2.0%

A flask containing 100 ml of this solution is boiled for 2 hours; after cooling, 30g of potato flour are added and the mixture is then boiled in a water-bath with continual and thorough stirring to complete pastiness. The mixture is then cooled to about 56°C before the eggs are added. 20-22 fresh eggs with large yellow yolks are cleaned carefully, broken into a large flask, shaken vigorously and filtered through sterile gauze. One litre of egg and 600 ml of the salt-potato flour solution are mixed; after shaking 20 ml of 2% aqueous malachite-green solution is added. The medium is tubed, slanted, and coagulated at 85°C for 40 minutes (Fig. 2). The following day the tubes are heated to 70°C for 30 minutes. The cotton plugs are then parafined or closed in another way, and stored in a refrigerator at 4°C.

(From: Jensen, Bulletin International Union against Tuberculosis 24: 102-103)

Ziehl-Neelsen Method:

Concentrated Alcoholic Solution of basic Fuchsin

Basic fuchsin (certified) 95 per cent ethyl alcohol

90 g 945 ml

1. Add 300 ml alcohol to the 90 g fuchsin. 2. Heat in water bath to boiling for 2 minutes. 3. Allow to stand 1 minute and decant off; save

the supernatant.


4. Add a second 300 ml portion of the alcohol to the sediment, boil and decant as before.

5. Add the remaining 345 ml alcohol to the sediment, boil as before and add to portions collected previously.

5 per cent Phenol:

Distilled water Phenol crystals

950 ml 50 g

Carbol Fuchsin Solution for Staining Acid-fast Bacilli:

Concentrated alcoholic solution of basic fuchsin

5 per cent phenol

Filter stain just before using to remove

105 ml

895 ml

any acid-fast organisms which may be present.

Acid Alcohol for Decolorizing:

Hydrochloric acid 95 per cent ethyl alcohol Mix thoroughly

Staining Procedure

3 ml 97 ml

It is difficult for stains to penetrate cells of the Mycobacteria because of their high lipoid content. Only after prolonged treatment with carbol fuchsin, one of the most penetrating stains, or the application of heat during the staining process, can these bacteria be coloured. Once stained, however, the waxy material of the cells holds the carbol fuchsin even in the presence of acid alcohol. The steps of the Ziehl-Neelsen method which demonstrate the


property of acid-fastness are:

1. Smear out material (cultures or specimens) dry and fix as usual.

2. Cover smear with carbol fuchsin and steam over a low flame or a water bath for 5 minutes. The stain should never boil nor should it be allowed to dry.

3. Let slide cool a minute. Wash thoroughly in water.

4. Decolorize in acid alcohol (95 ml of 95 per cent ethyl alcohol plus 5 ml concentrated HCl) for 5 minutes or until no more color leaves the smear.

5. Wash in water. 6. Counterstain with methylene blue for 30 seconds. 7. Wash, dry and examine with oil immersion objective.

By this method acid-fast bacteria appear red against a blue background.

(From: Kelly and Rite, Microbiology, ed. AppletonCentury-Crofts, New York)

VACCINE: BCG

TEST: Test for absence of contaminating micro-organisms


CLASS OF TEST B

BCG-2


(Revised 1978)

Part A Section 5.2

THE TEST

Samples from each final lot shall be tested for bacterial and mycotic contamination according to the requirements given in Part A, Section 5, of Requirements for Biological Substances No. 6 (Requirements for the Sterility of Biological Substances) (11, page 4).

For details of this test see GEN-1.


VACCINE: BCG

CLASS OF TEST C

BCG-3

TEST: Test for absence of virulent Mycobacteria


(Revised 1978)

Part a Section 5.3.1

THE TEST

"If the test for the absence of virulent mycobacteria applied to the final bulk, is unsatisfactory, it shall be repeated with a sample of a final lot (see Part A, section 3.4.3).

This test may be applied to the final lot and omitted from the final bulk".


A sample of the final bulk intended for this test shall be stored for not more than 72 hours after harvest at 4°C.

At least 6 guinea-pigs, all of the same sex, each weighing 250-350 g are used.

Some countries prefer to use guinea-pigs that have been shown previously to be tuberculinnegative.


A dose of BCG organisms corresponding to at least 50 human doses of vaccine intended for intradermal injection shall be injected into ealh guinea-pig by the subcutaneous or intramuscular route • The animals shall have been maintained on a diet that is free from added substances, such as antibiotics, that might interfere with the test. The guinea-pigs shall be observed for at least 6 weeks. If, during that time, they remain healthy and gain weight, the final bulk shall be considered to be free from virulent mycobacteria.

At the end of the observation period, the animals shall be killed and examined post mortem for macroscopic evidence of progressive tuberculous disease; similarly, any animals that die before the end of the observation period shall be subjected to post mortem examination.

The vaccine lot shall pass the test if none of the guinea-pigs shows evidence of progressive tuberculous disease and if at least two-thirds of them survive the observation period.

Should more than one-third of the guinea-pigs die during the observation period (and freedom from progressive tuberculous disease is verified), the test shall be repeated with another sample of the final lot on at least 6 more guinea-pigs •

If, on the second occasion, more than one-third of the animals fail to survive the observation period, the vaccine lot shall not pass the test.

1 When a more concentrated vaccine, intended for administration by the percutaneous route, is tested, a dilution factor approved by the national control authority shall be applied, so that the mass of BCG injected corresponds to 50 human doses of intradermal vaccine.


Should a vaccine lot fail to satisfy the requirements of this test because animals die from causes other than tuberculosis, the procedure to be followed by the manufacturer should be determined with the approval of the national control authority.

If evidence of progressive tuberculous disease is seen, the vaccine lot shall be rejected, all subsequent vaccine lots shall be withheld, and all current vaccine stocks shall be held pending further investigation. The manufacture of BCG vaccine shall be discontinued and it shall not be resumed until a thorough investigation has been made and the cause or causes of the failure determined, and then only with the approval of the national control authority.

VACCINE: BCG

TEST: Test for total bacterial content


CLASS OF TEST A

BCG-4

REQUIREMENTS FOR DRIED BCG VACCINES (Requirements for B-iological Substances No. 11)

(Revised 1978)

Part A Section 5.4

THE TEST

The total bacterial content of the reconstituted vaccine shall be estimated for each vaccine lot by a method approved by the national control authority, and shall have a value within a range approved by the national control authority.

The estimation of total bacterial content may be made either directly by determining the dry weight of organisms, or indirectly, by an opacity method that has been calibrated in relation to the dry weight of organisms.

It is desirable that one method of estimation should be adhered to for all the vaccine lots produced by a manufacturer.

An International Reference Preparation of opacity is available for the determination of the opacity of a suspension of BCG organisms. The comparison is made by EYE, and the test is described in GEN-7.


VACCINE: BCG

CLASS OF TEST B

BCG-5

TEST: Test for number of culturable particles

REQUIREMENTS FOR DRIED BCG VACCINES (Requirements for Biological Substances No. 11)

(Revised 1978)

Part A Section 5.5

THE TEST

The number of culturable particles of each final lot shall be determined by an appropriate method approved by the national control authority (See Part A section 3.4.5) The viable count shall have a value within a range approved by the national control authority. By comparison with the results of the test for number of culturable particles carried out on the final bulk, as described in Part A section 3.4.5, the percentage survival on freeze-drying may be calculated and this value should be not less than one approved by the national control authority.


The survival rate usually varies between 75% and 20% although a survival rate as low as 10% is permitted by some national control authorities.

The number of culturable particles of each final bulk shall be determined by an appropriate method approved

by the national control authority.


The medium used in this test should be such that the number of culturable particles may be determined not more than 4 weeks after the medium has been inoculated with dilutions of vaccine.

There are various methods of determining the number of culturable particles in BCG vaccine, and it is desirable that one method should be adhered to for all the vaccine lots produced by a manufacturer (7). It is also desirable that the test should be carried out in parallel with a reference preparation.

In the case of retesting a vaccine, which has been tested before issue by the producer, the test that will give the most essential information, is the Test for Culturable Particles. The total bacterial content, however, is not important, as this will not change. As the test for contamination has been done by the producer, the ampoules should not be contaminated. Such tests will be done, therefore, only if there are special indications for retesting the vaccine.

The importance of checking the number of culturable particles is that it may have changed during transport from the producer, or during the vaccination

e.g. by a failure of the cold chain. Moreover, such information may be useful where vaccines have been stored under correct conditions, but for a long time during which the organisms may have died. This is a good check on the stability of the vaccine.


ADDITIONAL INFO~~TION FOR CARRYING OUT THE TEST

A. In the document TB/77.8 (available from WHO), it is stated that:

The estimation of the number of culturable particles is the most informative viability test, and the method developed is widely used, not only in quality control laboratories but also in most production centres. Traditionally this test was carried out by inoculating on solid medium tenfold different dilutions (of the already highly diluted vaccine), but for the last decade a series of three twofold different dilutions (1:2:4) are chosen (sometimes after a preliminary test) in such a way that estimates are always based on an optimal number of colonies. The use of several containers (tubes, dishes) for each dilution level makes it possible to compute routinely the chi-squares(thePoisson-distribution giving the expected variance). The use of three dilution levels makes it possible to compute "t" values as an indication of the dilution errors, whether systematic or stochastic.

The calculation of these statistics as well as the calculation of the final estimate of the number of culturable particles must be carried out for each sample examined. The computations for each examination, as well as the copy-typing of the report, is therefore a major task that requires utmost accuracy. Since standard procedures are used, however, the data are nowadays easily processed electronically.

Solid media sometimes vary in quality, and this could cause serious errors in comparisons of vaccines. Also, incubation might affect the different containers in an investigation in different ways. The containers of solid media used in ay investigation are therefore randomized and coded. Also in this respect the use of the computer is profitable: the sheets of random permutations according to which the prenumbered containers are selected are easily prepared by computer. Moreover no decoding is needed (which excludes bias): thF


counts for each container number are punched and the computer decodes before it compute.s. and prints the results.

Thus all the painstaking arithmetic, which for many laboratory workers made the determination of the number of culturable particles a relatively "complicated" test, is now done by the computer, which moreover presents the final result directly in printed form.

B. Document TB/77.9 (available from WHO) gives the following data about the materials and design of the test:

Materials

No standard diluent, container, or solid medium is specified here. Tools or recipes that have come to work well in one laboratory (after much trial and error) do not necessarily work if copied elsewhere; it would scarcely be possible, for example, to prescribe a particular medium for viability counts as the standard to be used in all laboratories.

The basic suspension should normally be vaccine ready for use in the standard strength recommended by the manufacturer. Freeze-dried products should be reconstituted as recommended by the manufacturer. The sample to be tested may be an ampoule of liquid or reconstituted vaccine or a pool of two or more ampoules (to even out possible ampoule-to-ampoule differences), or it may be a sample of the stock suspension not yet in ampoules (either in original concentration or diluted down to normal vaccine strength). Further dilution is in terms of volume, i.e. in terms of millilitre of vaccine and not in terms of milligramme semi-dry weight (though a note is made of the nominal strength, as specified by the manufacturer, in terms of mg per ml for the liquid or reconstituted vaccine).


Commonly used media for diluting the reconstituted vaccine are: liquid Sauton medium1 (as used for BCG cultures) diluted 1:4 (i.e. 1 + 3); isotonic saline with or without buffer. A wetting agent (e.g. Tween 80) or bovine albumin is sometimes added to the diluent.

Reference is made to descriptions in t£e litera2ure of the Lowenstein-Jensen medium, theogawa medium3 and the Oleic-acid-albumin-agar medium with blood, which are the most commonly used solid media. A variety of containers are used for the solid medium, such as cotton stoppered glass tubes sealed with paraffin, Legroux flasks, Petri dishes, screw-capped tubes, or flat screw-capped bottles. Major considerations in selecting such a container are the control of ventilation, the avoidance of contamination, and the size of, the surface area. Only solid or semi-solid media should be used, as these allow the number of colonies to be counted. Liquid media, which yield only "growth" or "no growth" for each tube, require the use of a lar.ge number of tubes for reasonably accurate estimates.+ and are therefore not recommended for routine use.

1

2

3

4

Jensen, K.A. Bulletin of the International Union against Tuberculosis, 24: 78-112 (1954)

Obayashi, Y. Dried BCG Vaccine, World Health Organ. Geneva (1955)

Dubos, R.J. & Middlebrook, G.,American review of tuberculosis and pulmonary disease, 56: 334-345(1947)

For statistical handling of such data, see Fisher, R.A. & Yates, F. (1957) Statistical tables for biological, agricultural and medical research 5th ed., Edinburgh, Table VIII2

Design


If the viability is roughly known in advance, a dilution level can be chosen that is likrly to yield a number of colonies optimal for counting. In anticipation of variations in viability, three dilution levels are seeded at the same time, in dilution steps of 1:2:4. The advantage of twofold dilutions (as compared with fourfold or tenfold) is that the validity of the levels will overlap (with a tenfold series, one level may give confluence and the next one too few colonies). Furthermore, two or even all three dilution levels will contribute significantly to the estimate for as long as they show neither confluent nor zero growth. Examples of possible steps in the dilution process are shown in Appendix 1, Sheet 3.

In selecting the dilution levels, it is necessary first to define an optimal number of colonies per container, i.e., a number not too small yet small enough to assure discrete, easily counted colonies. One aims at obtaining this number at the intermediate dilution level, so that for moderate variations (within the range 1:4) one of the three levels will always yield an adequate number of colonies. In the statistical analysis full weight is given to colonies for dilution levels that yield, on the average, the optimum number or less, while reduced weight is given to a dilution level that yields up to twice the optimum, and little or no weight to levels yielding more than twice the optimum. Colonies are nevertheless counted up to 2.5 times the optimum, beyond which counts are reported as "exceeding limit". As will be seen later. tpe

1 If the viability is not known at all, a preliminary viability test must be carried out. The design might be in tenfold steps (1:10:100) or in more than three twofold steps, e.g. 1:2:4:8:16:32:64:128. In the latter case a single container of solid medium per dilution level may suffice, since only a preliminary estimate is aimed at.


statistical interpretation of the latter class is quite different from that of "missing data", e.g. in case of contaminated containers.

For the most concentrated suspension and for the middle suspension the same number of containers is inoculated, e.g. five for each. For the most diluted suspension twice the number of containers is inoculated, in this case 10. Or the respective number of containers may be three, three, and six. It may be noted that in this design the amount of vaccine inoculated at the most concentrated level is the same as the sum of the amounts inoculated at the more dilute levels (1 = 0.5 + 2 x 0.25). Similarly, the amounts inoculated at the two more dilute levels are equal (0.5 = 2 x 0.25). Thus the number of colonies counted will be at least 10 times (or six times respectively) the optimum count for a single container, on the condition that the count for the most concentrated level is not below the optimum and for the most dilute level not above the optimum. Thus the precision will be fairly uniform over this range.

From one ampoule, or pool of ampoules, may be prepared either one dilution series, to be inoculated in 5 + 5 + 10 containers, or two dilution series each to be inoculated in 3 + 3 + 6 containers. The purpose of the latter design is to checf the experimental error inherent in the dilution process.

1 The same design cannot immediately be used for checking the ampoule-to-ampoule variation (by preparing one dilution series from each of two different ampoules) because the estimate of experimental error in this case would confound ampoule-to-ampoule variation with dilution error. A proper check on ampoule-to-ampoule variation requires four dilution series as a basic design, two for each ampoule.


C. Document TB/77.9 (available from WHO), gives a randomization, the reporting and the statistical analysis. If it is planned to perform this test it is essential to study the complete documentrB /77.9, which is available from the Tuberculosis Unit of the WHO, Geneva.


CLASS OF TEST B

BCG-6

VACCINE: BCG

TEST: Test for Stability


(Revised 1978)

Part A Section 5.6

STABILITY TEST

Each final lot shall be tested for its degree of stability by a method 1approved by the national control authority.

The object of this test is to obtain experimental evidence on which to base the statements concerning storage temperature and expiry dates that appear on the label and the leaflet as required in Part A, Section 8.

The test shall involve the determination of the number of culturable particles before and after the samples have been held at appropriate temperatures and for appropriate periods.

An accelerated degradation test shall be carried out by_taking samples of the vaccine and incubating them at

1 Part A, Section 3.4.5 concerns the determination of the number of culturable particles and is discussed in detail in doc. "BCG-5".


-u 37 C for 28 days. The percentage fall in the number of culturable particles is Shen compared with that of the same vaccine stored at 4 C. The number of culturable particles in the vaccine after heating shall be not less than 20% of that in the refrigerated vaccine. The absolute value shall be approved by the national control laboratory. The test shall also be made in parallel with a reference preparation. One method of determining the number of culturable particles shall be adhered to, as suggested in Part A, Section 3.4.5.

The purpose of including a reference preparation is to have a check on the quality of the medium used for the determination of the number of culturable particles. It is not intended to adjust the count of the vaccine by comparison with the reference preparation.

All manufacturers shall keep their product for the permitted storage period and shall determine the number of culturable particles to demonstrate that this has been maintained at an adequate level.

In some countries, the stability test is carried out only after the vaccine has been stored for 3-4 weeks after drying, since it is considered that the degree of stability during the first 3 weeks may not be related to the longterm stability of the product.

As a guide to stability, some manufacturers of dried BCG vaccine determine the residual water content of the final vaccine, since failure to achieve a certain degree of desiccation results in an


unstable product. However, such a test cannot be regarded as an alternative to tests involving the determination of the number of culturable particles

BCG VACCINE

VACCINE: BCG



CLASS OF TEST B

BCG-1


(Revised 1978)

Part A Section 5.1

THE TEST

Identity Test

An identity test shall be performed on samples of vaccine from each final lot.

The identity of each filling lot of vaccine shall be verified by the morphological apperance of the bacilli in stained smears and by the characteristic appearance of the colonies grown on solid media.

The medium used is generally the Lowenstein-Jensen or a similar medium. The smears are stained by the Ziehl- Neelsen method.

Lowenstein-Jensen Medium

Lowenstein's medium with 0.75% Glycerin (usually called Lowenstein-Jensen medium) (Zentralblatt fur Bakteriologie, Parasitenkunde u. Infektionskrankheiten I. Abt. Orig. 125, 222, 932 and Advances in the Control of Zoonoses, p. 11 and 35, World Health Organization, Geneva 1953).