All batches of starting materials like substances of animal origin (e.g. serum, skimmed milk and heart infusion), mycoplasma seed and media as well as other ingredients used during the manufacturing process shall be tested for sterility.
The test for sterility should also be carried out following the pooling of individual mycoplasma vaccine harvests, following the addition of stabilizer and on the final lot of vaccine as well the reconstitution fluid.
The following culture media have been found suitable for the test for sterility:
Fluid Thioglycollate Medium is intended primarily for the culture of anaerobic bacteria but will also sustain the growth of aerobic bacteria.
Soyabean Casein Digest Medium (Tryptic Soya Broth) is primarily intended for the culture of aerobic bacteria but will also sustain the growth of fungi.
Media for the test may be prepared as described below, or dehydrated similar formulation reconstituted as directed by the manufacturer may be used.
A. Fluid Thioglycollate Medium/Fluid Mercaptoacetate Medium
|Sodium Chloride||2.5 g|
|D-Glucose monohydrate (C6H12O6H2O)||5.5 g|
|Agar, granulated (< 15% moisture)||0.75 g|
|Yeast extract (water-soluble)||5.0 g|
|Pancreatic digest of casein||15.0 g|
|Sodium mercaptoacetate||0.5 g|
|( or Mercaptoacetic acid||0.3 ml)|
|Resazurin sodium solution||1.0 ml|
|(1 in 1000, freshly prepared)|
|Distilled water to||1000 ml|
Mix the L-cystine, sodium chloride, D-glucose, agar, yeast extract and pancreatic digest of casein with the water and heat until complete dissolution. Add the sodium mercaptoacetate or mercaptoacetic acid and, if necessary, adjust the solution with 1M sodium hydroxide to give a final pH of 7.1 +/- 0.2 after sterilization. Pass through a course filter while the solution is still hot. Add the resazurin solution and distribute in suitable vessels e.g. 10 ml into 16 ml test tubes, which provide a ratio of surface to depth of medium such that during subsequent incubation, not more than the upper third of the medium has undergone a colour change indicative of oxygen uptake. Sterilize in an autoclave at 121°C for 20 minutes.
If more than the upper one-third has acquired a pink colour, the medium may be regenerated just before use by heating in a steam water bath until the pink colour disappears and then cooling rapidly.
Use the medium by inoculating it under aerobic conditions.
B. Soyabean Casein Digest Medium/Tryptic Soy Broth (SBCDM)
|Pancreatic digest of casein||17.0 g|
|Papain digest of soyabean||3.0 g|
|Sodium chloride||5.0 g|
|Di-potassium hydrogen phosphate||2.5 g|
|D-Glucose monohydrate (C6H12O6H2O)||2.5 g|
|Distilled water||1000 ml|
Dissolve the solids in the water, warming slightly to effect solution. Cool the solution to room temperature, and, if necessary, adjust with 1M sodium hydroxide in order to obtain pH 7.3 +/- 0.2 after sterilization. Filter, if necessary, to clarify and dispense into suitable vessels such as 16 ml test tubes at 10 ml medium per tube. Sterilize by heating in an autoclave 121°C for 15 to 20 minutes.
Use the medium by inoculating it under aerobic conditions.
The test for sterility shall be carried out under aseptic conditions in a laminar air flow cabinet in order to avoid accidental introduction of contaminants during the test thereby resulting in false positive readings. The precautions taken to avoid contamination must be such that they do not adversely affect any microorganisms that should be revealed in the test. The working conditions should be monitored regularly by sampling the air and surfaces of the working area. The media used should comply with sterility carried out before and in parallel with the test on the preparation being examined. For this purpose, incubate portions of the media intended for the detection of bacteria at 30°C to 35°C and portions of those intended for the detection of fungi at 20°C to 25°C for not less than seven days. No growth of microorganisms should occur. The media used should also be tested for nutritive properties and for effectiveness in the presence and absence of the preparation being examined. These tests shall be carried out in accordance with the methods contained in the British Pharmacopoeia.
In determining the number of containers to be tested, the manufacturer should have regard to the environmental conditions of manufacture and the volume of preparation per container. The following table gives guidance concerning the minimum number of items recommended to be tested in relation to the number of items in the batch.
|Number of items in the batch||Minimum number of items recommended to be tested|
|a.||Starting materials Less than 50 containers||20 per cent or 4 containers whichever is the greater.|
|More than 50 containers||2 per cent or 10 containers whichever is the greater.|
|b.||Individual or pooled harvest of vaccine||Irrespective of the number each container should be tested.|
|c.||Batch/final lot of vaccine||1 percent of the containers in each batch, with a minimum of three and a maximum of ten.|
In performing sterility tests it is important to observe the following considerations:
1. Clean thoroughly the exterior of vaccine container (ampoules, vials or bottles and their closures) using a suitable decontaminating agent e.g. 70% ethanol and gain access to the contents in an aseptic manner. Use not less than the number of containers specified in the table above.
2a. Freeze dried vaccine
Reconstitute the contents of a vial of freeze dried vaccine using 5 ml of cold, sterile distilled water.
Inoculate 0.55 ml of reconstituted vaccine into each tube of a series of six tubes of SBCDM and three tubes of thioglycollate medium each containing 9 ml of medium (i.e the whole content of reconstituted vaccine vial must be tested so as to give a ratio of vaccine suspension to test medium of between 1:10 and 1:20).
Inoculate each of 2 tubes of SBCDM and 1 tube of thioglycollate medium with 1 ml of the fluid used for reconstitution (these are the diluent controls).
For each test two tubes of SBCDM and 1 tube Thioglycollate medium are included as uninoculated medium controls.
Incubate all the thioglycollate and one half of the SBCDM test media tubes for 7 days at 35°C to 37°C in the test intended to detect bacteria and the other half of SBCDM media tubes at 20 to 25°C (room temperature) in the test intended to detect fungi.
After 7 days following incubation subinoculate 1 ml from each tube of the previously inoculated media into a corresponding series of fresh media. These are then incubated as earlier described for a further minimum period of 7 days.
2b. Liquid Vaccine
Using a minimum of 15 ml of vaccine from each bottle, inoculate 10 tubes of SBCDM and 5 tubes of Thioglycollate medium using 1 ml of inoculum per tube.
Incubate as described in 2 (a) above for a minimum of 14 days. Uninoculated media controls for each incubation temperature should also be included as previously described.
3. Examine the media visually for growth several times during the incubation period, and on the last day of the test period. In the case of Thioglycollate medium used for the detection of anaerobic microorganisms, shaking or mixing should be kept to a minimum in order to maintain anaerobic conditions.
4. Record the observations.
III. Interpretation of Results
If no growth of microorganisms is observed in any of the broths/media during the incubation period and at its conclusion, the preparation being examined passes the test for sterility. Growth is indicated by turbidity of the medium/broth.
The test is valid if no growth is observed in any of the controls, i.e.
If growth occurs in either control, the test should be repeated.
If the test is valid and growth is observed in the media inoculated with the test material, the preparation fails.