Testing for sterility: background, performance and verification (method suitability test)
In today's blog article, we’d like to take a look at sterility testing and its verification. As this is a compendial method, we should talk about verification instead of validation in this context, even though unfortunately we often still read about validation of this method. To stick with the wording, in the pharmacopoeias this is referred to as method suitability test (MST), which in my opinion fits very well 😉
What is sterility?
Simply put: the "absence of viable microorganisms".
Where are sterility tests performed?
Sterility testing is used for all drug products for which it is prescribed in the pharmacopoeia, e.g. for parenterals, eye drops, immune sera, vaccines or suture materials. Accordingly, the microbiological laboratories of the pharmaceutical manufacturer's quality control department or commissioned contract laboratories carry out this test as part of the release analysis of the batches produced.
However, medicines are not only produced in industry. For example, so-called pain pumps can be produced by hospital pharmacies by the pharmacy filling sterilized pumps with the corresponding analgesic infusion under aseptic conditions. A batch of such pain pumps manufactured in the pharmacy must also be tested for sterility prior to use.
Why is such a test important?
Without sterility testing, it can’t be proven that the manufactured product (be it an industrially produced infusion solution or a pain pump manufactured in a hospital pharmacy) is free of potentially harmful microorganisms, thus posing a potential health risk to the patient to whom it is administered. Depending on the type and quantity of microorganism, a medication contaminated with microorganisms can cause severe health problems such as blood poisoning.
How is the sterility test performed?
The exact procedure is described in the respective chapters of the relevant pharmacopoeias (e.g. Ph. Eur. 2.6.1., JP 4.06 or USP <71>, [1-3]). A distinction is made between the direct inoculation method (direct feeding) and the membrane filtration method. If possible, membrane filtration is preferable. Working is strictly performed under aseptic conditions, e.g. in a sterile test isolator.
For membrane filtration, the product to be tested must be filterable and aqueous, ethanolic or oily. Appropriate filtration units with a nominal pore size of maximum 0.45 µm with proven bacterial retention capacity, a diameter of approx. 50 mm and a filter material suitable for the product to be tested (e.g. Steritest from Merck) are used. After wetting the membranes with a buffer solution (depending on the product: Fluid A, Fluid D or Fluid K), the product to be tested is filtered, whereby microorganisms present remain on the filter membrane in the event of contamination. This is followed by a rinsing step with buffer solution (usually 3x 100 mL), which serves to remove potentially existing antimicrobial properties of the product to be tested. Subsequently, 2 different liquid culture media (TSB and Fluid Thioglycollate Medium = FTM) are added to the filtration units and incubated for 14 days (unless growth has been observed earlier) at the appropriate temperature. The units are visually checked for turbidity (i.e. growth) at various times.
If the product is not filterable, e.g. surgical suture material, the direct inoculation method is used, in which the product is introduced directly into the culture media (whereby it’s not allowed to make up more than 10% of the culture medium’s volume), and also checked for turbidity after incubation.
How does the method suitability test for the sterility test is performed?
The method suitability test (MST, verification) is intended to prove that the compendial method also meets the requirements of the pharmacopoeia for its intended use under the current conditions in the laboratory in which it is to be used. The method suitability test provides evidence that the product to be tested has no negative influence on the growth of potential microorganisms, i.e. that it has no antimicrobial properties or that these have been sufficiently eliminated. Put simply, we want to ensure that no false-negative results are obtained in case of a contaminated product.
Therefore, as part of the method suitability test, a growth control is carried out for each of the two culture media used with various microorganisms capable of multiplication, which are added to the culture media in defined quantities. This is done once in the absence and once in the presence of the product to be tested. The test in the absence of the product acts as a positive control, as we expect the added microorganisms to grow. If, after a certain incubation time, the two preparations (1. with product and 2. positive control) are compared with each other and both show comparable growth, the method suitability test has been successful. If the culture medium with product shows a lesser growth, the product has growth-inhibiting properties that haven’t been sufficiently eliminated yet under the selected conditions. The test conditions must be adapted accordingly, e.g. by creating dilutions or using neutralizing or – in case of antibiotics as product - inhibiting agents, such as e.g. polysorbates or β-lactamases.
In general, the analyses for a MST are performed with 3 different batches of the product. The number of samples to be tested per batch depends on the total batch size; the pharmacopoeias provide precise specifications for this.
The MST is performed in accordance with the conditions specified by the pharmacopoeia for the routine test procedure (especially also under aseptic conditions 😉). In the membrane filtration method, the test microorganisms prescribed by the pharmacopoeia (Staplylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans and Aspergillus brasiliensis, maximum 100 CFU each) are added while performing the last rinsing step. These germs are representative of potential contaminants occurring during drug product manufacturing. In addition to these germs, it can be argued that it also makes sense to test representative house germs, which were detected during environmental monitoring of the production, as part of the method suitability test. Afterwards, the culture medium specified by the pharmacopoeia for the respective bacterial species is added (TSB for B. subtilis, C. albicans, A. brasiliensis and FTM for S. aureus, P. aeruginosa, C. sporogenes), followed by incubation at the temperature specified by the pharmacopoeia for the respective bacterial species (20-25 or 30-35°C) for a maximum of 3 (for bacteria) or 5 days (for yeasts and molds). Visual inspection for turbidity is then performed daily.
In the test setup above, we have described the procedure in the presence and absence of the product but have not yet considered any other controls. In addition to procedure above for the MST, the test is also performed in parallel under routine conditions (i.e. without addition of test microorganisms) and a negative control is also run. At the end of the incubation, there shouldn’t be any growth in the routine procedure and in the negative control. The following table illustrates the 4 approaches and their respective purpose:
| In the presence of product | In the absence of product (positive control) | Routine conditions | Negative control | |
| 1 | Wetting the membrane | |||
| 2 | Filter the product | - | Filter the product | - |
| 3 | Rinsing + addition of test microorganisms | Rinse only (NO addition of test microorganisms) | ||
| 4 | Addition of culture medium | |||
| 5 | Incubation | |||
| Purpose | Statement on the method suitability test | Proof that the product batches used in the MST were sterile | Proof that all reagents used were sterile | |
Finally, a few words about the culture media. Before carrying out the MST, the sterility and growth-promoting properties of the culture media batches to be used in the MST should have been verified during the incoming goods inspection. In addition, the growth-promoting properties of the culture media must of course still be present at the end of the incubation period. This can be verified with a so-called stasis test as part of the MST. This should also show that the drug product has not impaired the growth-promoting properties of the culture medium. For this purpose, the corresponding test microorganisms are added after the end of the incubation period of 14 days of the routine preparation and incubated further (stasis preparation). At the same time, a positive control is added in which fresh medium has been inoculated with the test microorganisms. If the growth of the stasis preparation is comparable to that of the accompanying positive control, the drug product has no negative influence on the growth-promoting properties of the culture medium and proof is provided that these are still guaranteed even after more than 14 days of incubation. Of course, a negative control should also be run in parallel 😉 Such a check of the growth-promoting properties after 14 days of incubation is particularly useful for antibiotics as product to be tested combined with closed sterile test filter units, as it has been observed that small amounts of the antibiotic diffused into the sealed membrane filter edge, were not washed out during rinsing, then diffused back into the culture medium during the 14-days incubation period and were sufficient to inhibit the growth of the added test microorganisms [4].
An example of the difficulties encountered during verification of the sterility test for vancomycin eye drops is shown in the poster of Nicolas Cormier's working group at CHU Nantes, which was presented at the 25th European Scientific Days of GERPAC in October 2022 [5].
References and further literature
[1] European Pharmacopoeia (Ph. Eur.), Chapter 2.6.1 "Sterility"
[2] Japanese Pharmacopoeia (JP), Chapter 4.06 "Sterility Test"
[3] United States Pharmacopoeia (USP) <71> "Sterility Tests"
[4] Concept Heidelberg (2015) Analytische Qualitätskontrolle und pharmazeutische Mikrobiologie (Analytical quality control and pharmaceutical microbiology), Editio Cantor Verlag, ISBN: 978-3-87193-424-7
[5] Rodrigo L., Bourget M., Olivier E., Cormier N. (2022) Development and validation of sterility method by membrane filtration of 5% vancomycin eye drops
PIC/S (2007) „Recommendations on Sterility Testing” (PI 0012-3)
TGA guidelines for sterility testing of therapeutic goods (2006)