Evaluation of Microbial Stability of Formulations

Microbial stability testing involves the following five steps:

Sampling : The number of containers to be used and volume to be tested are specified in the Pharmacopoeia.

Samples of aseptically manufactured product are collected in the beginning of, during, and end of batch fill, and also after any kind of interventions and stoppages.

Samples from terminally sterilised product are collected from previously identified cool spots within load methods as specified in the Pharmacopoeia.

If the product can be filtered, membrane filtration method is preferred, while direct inoculation is used as an alternative method.

Growth Media : Soybean Casein Digest (SCD) medium and Fluid Thioglycollate Medium (FTM) are used for the detection of aerobic and anaerobic organisms.

The efficiency of media to support growth of a range of low numbers of organisms in the presence of product is demonstrated by validation studies.

Bacterial growth should occur after 3 days, and growth of moulds should occur after 5 days.

Media is either procured or made in-house by validated sterilisation techniques.

Before using the media, its growth supporting qualities (for low number of organisms) should be tested.

Batch number and shelf-life of the media should be allotted.

Incubation Period : The SCD media is incubated at $20-25°C$ and the FTM at $30-35°C$ , both for 14 days in test containers, which should be periodically examined.

Negative Test Controls

Before using the media, either a portion of it or the complete batch should be incubated for 14 days. Incubation may be done simultaneously with the test.

The negative product control items identical in type and packaging to actual product under test should be included in each test session. This makes interpretation of test results easy.

Positive Test Controls

Bactiostasis/fungistasis test should prove that media can support the growth of a range of low numbers of organisms in the presence of product.

Bacterial growth should occur after 3 days, and that of moulds should occur after 5 days.

Results : Any type of growth identified (Genotypic) by automated/semi-automated systems, should be verified from time-to-time using reference strains.

Interpretation and Repeat Tests

No contaminated units should be found.
A test is repeated when it proves out to be invalid due to reasons dissimilar to the product under test.

Methods of Evaluation

1. Method A (Membrane Filtration)
  1. The sample is collected and diluted as per the requirement.
  2. A suitable nutrient or culture medium is selected. The broth is dispensed in a sterile petri dish. As a result, the absorbent pad gets uniformly saturated.
  3. The forceps are passed through flame and then used for removing the membrane from the sterile package.
  4. The membrane filter is placed in the funnel assembly.
  5. The pouring lip of the sample container is flamed, after which the sample is poured in the funnel.
  6. The vacuum is turned on and the sample is completely drawn through the filter.
  7. The funnel is washed with sterile buffered water.
  8. The vacuum is turned on and the liquid is completely drawn through the filter.
  9. The forceps are again passed through flame and the membrane filter is removed from the funnel.
  10. The membrane filter is then placed in the prepared petri dish.
  11. The dish is incubated at suitable temperature for suitable time period.
  12. After incubation the number of colonies formed on the plate is counted under a microscope with 10-15X magnification.
  13. The presence of colonies is confirmed and the results are reported.

A care should be taken to maintain aseptic conditions during the test.

Method B (Direct Inoculation)

The contents of the container(s) to be tested are transferred to the culture medium.

A small number of viable microorganisms (not more than $100CFU$ ) are inoculated to the medium.

A growth promotion test is carried out as a positive control.

All the medium containing containers are incubated for not more than 5 days.

If after incubation the microbial growth is clearly visible, this growth is visually compared to that in the control vessel (without the product). Either the product exhibits no antimicrobial activity under the conditions of the test or the activity has been eliminated.

The sterility test is then performed without any modification.

If microbial growth is not clearly visible in the presence of the product to be tested, this growth is visually compared to that in the control vessel (without the product). The product possesses antimicrobial activity that has not been eliminated under the conditions of the test.

The conditions are further modified to eliminate the antimicrobial activity and the method suitability test is repeated.

The method suitability test is performed due to any one of the following reasons:

When the sterility test has to be carried out on a new product, or
When the experimental conditions of the test are changed.

The method suitability test is simultaneously performed with the sterility test of the product to be examined.

Sterility Test of the Product to be Examined

Sterility test is performed using either the membrane filtration method or by direct inoculation of the culture media with the product to be examined.

Appropriate negative controls are used.

Membrane filtration method is used when the product nature permits for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with or soluble in aqueous or oily solvents. It is important to notice that these solvents should not show their antimicrobial effect on the test conditions.

Endotoxin Test: Endotoxin is a lipopolysaccharide found in the cell wall of gramnegative bacteria. The raw materials, water for injection used in manufacture, and the finished product should undergo endotoxin test, which is of two types:

Gel clot (limit test), and
Gel clot (semi-quantitative test).

The equipment used for endotoxin test should be free from any endotoxins.

Validation of accuracy and dependability of the test method for each product is:

Gel clot method,
Original method,
The official “referee test”,
The sample is incubated with LAL of known sensitivity. A gel clot formation shows positive endotoxin test, and
Gel clot is a quantitative method as it quantifies bacterial endotoxins in the test solution by titration to an end point.

Microbial Limit Test (Test for Specified Microorganism)

This test is designed for determining whether or not a preparation fulfils the established specifications of microbiological quality.
Total Viable Aerobic Count (TVAC).
Total Aerobic Microbial or Bacterial Count (TAMC or TABC).
Trypticase Soy Agar (TSA).
Total Yeast and Mound Count (TYMC or Total Fungal Count).
Sabouraud Dextrose Agar (SDA).
$TVAC=TAMC+TYMC$ .
Report – $ml$ or gm.