The sterility testing method in pharmaceuticals is a critical quality control procedure designed to confirm that a product is free of viable contaminating microorganisms. This test is essential for sterile pharmaceutical products, such as injectables, ophthalmic solutions, and certain medical devices, to ensure patient safety. The process adheres to pharmacopeial guidelines (e.g., USP, EP) and is mandatory for sterile drugs, medical devices, and parenteral preparations to ensure safety and compliance.
What is Sterility?
Sterility refers to the complete absence of viable microorganisms, including bacteria, fungi, and spores, in a product or environment. In pharmaceuticals, sterility ensures that products like injectables, surgical instruments, and other sterile dosage forms are free from any microbial contamination, making them safe for patient use.
Types of Sterility Testing Method
A. Direct Inoculation Sterility Testing Method
Product is directly inoculated into media capable of supporting microbial growth.
Incubate at two temperatures:
20–25°C for fungi and aerobic microorganisms.
30–35°C for bacteria.
Observe for turbidity or microbial growth for 14 days.
B. Membrane Filtration Sterility Testing Method
The product is filtered through a 0.45 µm or smaller membrane filter.
The filter is transferred to sterile growth media.
Incubate at the same temperatures as above for 14 days.
Advantages:
Preferred for products with antimicrobial properties or large volumes.
Reduces the chance of inhibitory substances in the product affecting the test.
Limitations of Sterility Testing Method
The test detects only viable organisms; it does not identify non-viable contaminants.
It is a destructive test, meaning the tested samples cannot be returned to the batch.
False positives or negatives may occur due to contamination or inhibitory substances.
Acceptance Criteria
The product must exhibit no evidence of microbial contamination in all test samples.
A single failure requires batch investigation and potentially invalidates the lot.
If retesting is justified:
Retests must include double the initial number of samples.
Retest results must also show no microbial growth.
Key Points For Sterility Testing
1. Sample Size
The sample size is typically determined based on the volume or number of units in the batch.
For liquids: A typical sample size is 100 mL per container, or 1 unit (e.g., vial, ampoule) if the unit is small enough.
For solid products (e.g., tablets, capsules): A recommended sample size is 10 units.
For larger batch sizes, the total volume to be tested should correspond to the specific volume or number of units per batch as prescribed by compendial standards (e.g., USP or EP).
2. Growth Media
The growth media are essential to support the growth of microorganisms if present in the sample.
Fluid Thioglycollate Medium (FTM):
Primarily designed to support the growth of anaerobic and aerobic bacteria.
Used for sterility testing of clear, water-soluble products, oily solutions, and materials containing viscous substances.
Ideal for detecting anaerobic contaminants.
Soybean Casein Digest Medium (SCDM) or Tryptic Soy Broth (TSB):
Supports the growth of aerobic and facultative anaerobic bacteria as well as fungi (yeasts and molds).
Commonly used for sterility testing of a wide range of pharmaceutical products, including injectables and ophthalmic solutions.
Ideal for detecting aerobic bacteria and fungi.
3. Controls
Controls are essential to verify the accuracy and reliability of the sterility test.
Negative Control:
The negative control ensures that there is no contamination in the test setup or the media itself.
Negative control is typically represented by un-inoculated growth media that are incubated under the same conditions as the test sample.
If no growth is observed in the negative control, it confirms that the test media is sterile.
Positive Control:
The positive control ensures that the test system is capable of detecting microbial contamination if present.
Positive control involves inoculating the growth media with a known quantity of microorganisms (usually specific strains like Bacillus subtilis for aerobic or Clostridium sporogenes for anaerobic testing).
The growth of microorganisms in the positive control confirms the effectiveness of the test conditions and media.
4. Incubation Conditions for Sterility Testing
The test sample is incubated at specific temperatures, generally 20-25°C and 30-35°C, for a defined period (usually 14 days).
Test Duration: The test typically lasts for 14 days to allow time for the growth of slow-growing organisms.
Test Methodology: The test can be conducted via direct inoculation or membrane filtration methods, depending on the product’s nature and regulatory guidelines.
The sterility test provides assurance that the pharmaceutical product is safe for use, free from harmful microorganisms, and complies with the required quality standards.
Regulatory Guidelines
USP <71> Sterility Tests
EP Chapter 2.6.1
JP Chapter 4.06
Abdus Sobhan Salim is professional experienced pharmacist in pharmaceuticals, author and founder of pharmabossbd.com, the first Bangladeshi pharmaceutical blogger since 2019.
