Introduction
There are many reasons why the traditional sterility test, with its lengthy 14 days incubation time, should be replaced with a Rapid Sterility Test. As a consequence to the 14 days incubation time, possible product-contaminations and the respective corrective actions are delayed resulting potentially in an enlarged amount of affected product batches. Another important advantage of shorter incubation time is for example reduction in the release time for sterile products, therefore reduction of stock keeping costs and earlier market delivery. Furthermore, the use of RMMs (rapid microbiological methods) is highly supported by regulatory guidance documents, which request pharmaceutical microbiologists to use these methods.
After the decision had been made at Novartis that a rapid microbiological method for the replacement of the traditional pharmacopoeial sterility test should be validated, the appropriate method for replacement had to be chosen. From many available systems and methods on the market, an appropriate choice for a Rapid Sterility Test based on ATP Bioluminescence of micro-colonies was made.
Before starting the validation work, several pre-studies had to be conducted to determine a suitable range of stressed challenge microorganisms, to evaluate suitable nutrient media and to define the “rapid” incubation time. The main work had been the method validation for the Rapid Sterility Test following the guidance documents Ph.Eur. 5.1.6, USP <1223> and PDA Technical report 33 on how to validate alternative microbiological methods. Product specific validations in the Rapid Sterility Test followed after finalization of the method validation and were conducted successfully.
Important end of the story is the road map to regulatory acceptance of this Rapid Sterility Test, now approved by FDA CDER, FDA CBER, MHRA and by the European Medicines Agency for a number of Novartis sterile products.
Advantages of a Rapid Sterility Test
A sterility test is sometimes performed as in process sterility test or it is performed on the finished drug product. The sterility test on the finished drug product is the release test for aseptically filled or terminally sterilized drug products. In the pharmaceutical production of Novartis Pharma, only the release sterility test has to be performed. In contrast to that, at Novartis Vaccines and Diagnostics the vaccines production has additional in process sterility tests.
With routine Rapid Sterility Tests performed at one of the Novartis Vaccines and Diagnostics manufacturing sites, the timeframe from start of production until release could be reduced from 23 days to 13 days for one drug product. In Pharma, a reduction from 14 days to 7 days until release could be achieved. These 7 days consist of the 5 days incubation time of the Rapid Sterility Test and additional days to wait for the results of environmental monitoring, bioburden testing and water analysis (WFI, water for injection). With this a reduction of the storage costs and a more flexible planning could be achieved.
The Rapid Sterility Test incubation time of five days allows faster knowledge about product contaminations; therefore faster corrective actions can be made to reduce the amount of potentially affected product batches. Discarding one batch of sterile drug product can result in high costs. Additionally to this, even more than one batch could potentially be at risk.
One of the differences between the traditional and the Rapid Sterility Test is the early detection of micro-colonies instead of macro-colonies. This is considered to be an advantage due to earlier detection. In the Rapid Sterility Test, the detection threshold starts at around 10-100 yeast/mold cells and 1000 bacterial cells per micro-colony after a growth phase in the incubation time arising from a single contaminating cell [8]. In contrast to that, the detection threshold of the human eye starts at around 106 -109 cells per colony [5].
Another advantage is the nutrient medium used for Rapid Sterility Testing [4]. Since more than 30 years, the growth promoting properties of Fluid Thioglycollate Medium of the traditional sterility test were questioned [1], an appropriate nutrient medium could now be identified for the Rapid Sterility Test. In the Rapid Sterility Test, a solid nutrient medium is used, being in line with reports that solid media have advantages over fluid media [14].
Stressed micro-organisms have been used for the validation of the Rapid Sterility Test due to the fact that most contaminants in sterile products are expected to be stressed, for example following a treatment with disinfectants, exposition to heat or dehydration. The nutrient medium used for the Rapid Sterility Test has been evaluated with stressed micro-organisms and was found to be superior to the nutrient media used in the traditional sterility test.
Regulatory Guidance on RMMs
In regulatory guidance documents and in the pharmacopoeia, more and more views on rapid methods are shared. For example the 2-year FDA initiative “Pharmaceutical cGMPs for the 21st Century: A risk based approach”, which was started in 2002 integrated RMMs into the PAT initiative.
Additionally to that in 2004 the FDA Guidance for Industry “Sterile Drug Products produced by Aseptic Processing- current Good Manufacturing Practice” stated the following sentence: “Other suitable microbiological test methods (e.g. rapid test methods) can be considered for environmental monitoring, in-process control testing, and finished product release testing after it is demonstrated that the methods are equivalent or better than traditional methods (e.g. USP).” The validation for alternative microbiological methods should follow the guidance in [11, 13] and in [9].
Choosing the Appropriate Method to Replace the Traditional Sterility Test
Commercially available rapid microbiological methods were evaluated on their ability to serve as basis for the development of a Rapid Sterility Test. Close proximity to the traditional test led to the decision to use a growth based method with ATP-(Adenosine triphosphate) Bioluminescence as principle of detection. Firefly ATP-Bioluminescence is known since McElroy realized the importance of ATP in the ATP Bioluminescence reaction in 1947 [6].
Therefore, a rapid microbiological method was chosen for Rapid Sterility Testing, in which micro-colonies are detected on a membrane filter incubated on a solid nutrient medium. The system is able to get information about the extent of contamination. Quantification is generally not required for sterility testing, but might become useful in certain cases. For the use of the chosen system, a solid nutrient medium became necessary, which should also enable the growth of stressed micro-organisms.
The chosen system has several advantages regarding an application for the qualitative Rapid Sterility Test: It is growth based and therefore similar to the traditional sterility test. Most microbiological tests in pharmaceutical industry are growth based methods as this is still state-of-the-art in pharmaceutical microbiology. It was proven that the prerequisite of sterility testing, the detection of 1 CFU following incubation, can be fulfilled. Furthermore, the principle of membrane filtration is feasible and the system was adaptable to sterility testing.
Workflow of the Rapid Sterility Test
The test takes place inside a sterility test isolator (cleanliness zone A). After successful decontamination of the isolator and the material inside (30 vials of certain drug product, rinsing fluids, the solid nutrient medium RSTM = Rapid Sterility Test Medium, filtration funnels, transfer unit, sterile bottle, sterile forceps, all necessary material for environmental monitoring), the following actions are performed:
30 product vials are pooled in a sterile bottle via a transfer unit using a peristaltic pump.
The amount of vials needed for the test is described in the Pharmacopoeia - for the mentioned product 10 vials are needed per membrane.
The pooled sterility test samples are filtered over three filters; a possible contamination would be trapped on the filter membrane (0.45μm pore size).
Afterwards, the rinsing of the membrane (which contains product residue) using a validated kind and amount of rinsing fluids (validated during product-specific Bacteriostasis and Fungistasis test) is performed e.g. using 2 times 50ml Fluid A per membrane. This is done using the transfer unit used beforehand to pool the sterility test sample to obtain possibly present micro-organisms sticking to the side of the tubing of the transfer unit.
The three filters are applied to the validated solid nutrient media (RSTM).
The lid of one of the three membranes with cassette is punched with a hot tool attached to the side wall of the sterility test isolator to prepare the membrane for anaerobic incubation. Due to the hole in the lid, appropriate gas exchange is ensured. The punched membrane with attached RSTM-cassette is put into an anaerobic jar. The anaerobic jar is equipped with an anaerobic indicator strip and closed.
The environmental monitoring of the sterility test isolator is performed at the end of the described sterility test session. The sterility test isolator is opened to unload the sterility tests (the sterility test cassettes and anaerobic jar) as all following work steps are conducted outside of the sterility test isolator - in the incubator, in the laminar flow and on the laboratory bench.
Incubation of the filters on the RSTM-cassettes takes place aerobically at 20-25°C, aerobically at 30-35°C and anaerobically at 30-35°C (anaerobic jar is first filled with anaerobic gas). Growth of potential contaminant(s) to “micro-colonies” can take place. The necessary incubation time has been determined during validation and is 5 days; the incubation time describes the time needed to detect a “worst-case micro-organism” regarding slow growth (stressed Propionibacterium acnes), including additional time to account for variability and testing in the laboratory.
The laminar flow, the autospray station and detection tower (located within the laminar flow; zone microbiologically tested against class 100) are prepared for adequate environmental conditions prior to each reading of the Rapid Sterility Test. This is done by wiping with a disinfectant. The autospray station is prepared by performing the cleaning/rinsing cycle (decontamination is performed once per week).
At the end of the incubation time, filters are separated from the RSTMcassettes. A visual check for microbial growth is performed. If no growth is visible, the following steps are performed. The membrane is dried under Laminar Flow for 5 minutes and subsequently sprayed with ATP releasing and bioluminescence reagent.
The entire steps after incubation are not critical steps regarding lab contamination, since micro-colonies need a certain size which is necessary for detection in the Rapid Sterility Test. If an environmental or operator-derived micro-organism is added to the membrane during the steps after incubation, it would not be detected due to the lack of incubation and therefore the lack of sufficient amounts of ATP.
The following reaction chemistry [7] forms the basis of the detection:
Luciferase, Mg2+
Luciferin + ATP ─────▶ Oxyluciferin + AMP + hν (λ=562nm)
(AMP Adenosine Monophosphate, hν emitted photon, λ wavelength)
The next step is the transfer of the sprayed membrane within a validated time period from the autospray station to the detection tower. This has to be done due to the fact that the bioluminescence reaction starts immediately once the reagent is applied, this time period has to be below 90 seconds in order to catch the maximum of emitted photons. The possibly present light signals are detected with a CCD-camera (charge coupled device), the software algorithm calculates the amount of micro-colonies and removes background signal. Micro-colonies, which can be detected in the Rapid Sterility Test follow the detection threshold of the system and have an approximate size of: 10-100 yeast cells, 1000 bacterial cells.
Validation of Method
Prestudies to the Validation
At first an appropriate nutrient medium capable of replacing the liquid media of the traditional sterility test had to be found. During the growth promotion studies, three incubation parameters were selected: 20-25°C aerobic incubation, 30-35°C aerobic incubation and 30-35°C anaerobic incubation. The selected nutrient medium furthermore had to show that no bioluminescent background is interfering with the ATP-Bioluminescence detection of the Rapid Sterility Test. Another important part was for example the determination of the incubation time for the Rapid Sterility Test. This time is dependent on the worst-case micro-organism regarding slow growth. The worst-case micro-organism in our case, stressed Propionibacterium acnes, can reproducibly be found with the Rapid Sterility Test after 91 hours of anaerobic incubation at 30-35°C. These 91 hours were rounded up to full four days and an additional day was added to account for variability and testing in the laboratory [4]. Therefore, the incubation time for the Rapid Sterility Test is 5 days. A nutrient media evaluation with stressed micro-organisms (ATCC strains and environmental isolates from the Novartis Pharma manufacturing site) was conducted and the appropriate nutrient medium for the Rapid Sterility Test was identified.
This nutrient medium is called RSTM for Rapid Sterility Test Medium and is based on Schaedler Blood Agar, which has been modified for gamma-irradiation and to make it suitable for the spectrum of rapid sterility challenge micro-organisms. Last but not least in the row of prestudies an integrity study (towards ingress of hydrogen peroxide into the expendables used in the Rapid Sterility Test decontaminated in the sterility test isolator) and a D-value study using Geobacillus stearothermophilus spores for all expendables for the Rapid Sterility Test placed inside the isolator were performed [2].
Method Validation: “The Rapid Sterility Test”
All parameters which one is obliged to show for a successful validation mentioned in the pharmacopoeial guidelines were tested (Robustness, Ruggedness, Repeatability, Limit of Detection, Specificity, Accuracy and Precision). The choice and use of environmental isolates in addition to ATCC strains for the validation of the Rapid Sterility Test was done following the idea to challenge the rapid method. The chosen strains were environmental isolates from the production facility collected in zones A and B, from positive bioburden tests and sterility failures. The isolates used during the entire validation works were used in a stressed state. This stressed state has been experimentally shown to reduce the growth rate of the micro-organisms making their detection more challenging compared to unstressed ATCC strains.
A special study of the method validation was Limit of Detection (LOD), in which the Rapid Sterility Test was compared to the traditional sterility test. The results show that the Rapid Sterility Test method is numerically superior and statistically non- inferior to the traditional sterility test method with respect to the limit of detection for the micro-organisms evaluated.
The limit of detection was shown in two different approaches: results for the first approach for 22 stressed microbial strains using a low level inoculum in the range of 1-5 CFU were evaluated using a χ2 –Test. The p-value of this χ2 –Test was higher than 0.05, meaning that there is statistically no significant difference between the Rapid Sterility Test and the traditional sterility test on the 95% confidence level. In the study, 7 ATCC strains and 15 isolates from one of the Novartis Pharma manufacturing sites were used. The chosen range of microorganisms included yeasts/molds, grampositive sporulating bacteria, gramnegative rods, grampositive cocci and grampositive rods (both aerobic and anaerobic micro-organisms). For the study, all microorganisms were used in a stressed state.
The study was performed using a challenge inoculum of approximately 1-5 CFU with ten replicates for each strain with both sterility tests. The statistical analysis was performed using the χ2-Test and showed no significant statistical difference between the two test methods on a 95% confidence level. The results were additionally evaluated using Fisher’s exact test and the Fisher test for one sided equivalence. Taking these two additional statistical tests into account, it could be confirmed, that the rapid and the traditional method are not significantly different with regard to limit of detection. The second approach for limit of detection was performed with inocula of 50 CFU, 5 CFU, 0.5 CFU and 0.05 CFU. These inoculum levels were tested for the stressed environmental isolates Kocuriarhizophila, Acinetobacter lwoffii, Bacillus clausii, Penicillium spec. and Propionibacterium acnes in ten replicates. The analysis of all limit of detection tests (approach 1 and approach 2, all test runs) shows that both sterility tests can be considered equal regarding the challenge of detection of low level inocula.
During this study, the specificity of the Rapid Sterility Test was also shown. The specificity describes the ability of the rapid method to detect a range of micro-organisms (the specificity mainly depends on the quality of the used nutrient medium). Besides Propionibacterium acnes, which was not always detected using the traditional sterility test, all tested strains were detected with both methods.
Product Specific Validation
Any product which should be tested with the herewith presented Rapid Sterility Test has to have some prerequisites. At first, the product has to be filterable. Products, which cannot be filtrated, have to be tested for sterility with Direct Inoculation [12, 10] and cannot be tested with the chosen rapid sterility test. Another important prerequisite is absence of disturbing bioluminescent background in the detection system. For this reason, every drug product has to show that no interfering signals are present.
The validation test for the kind and amount of rinse fluid, the Bacteriostasis and Fungistasis test [12, 10], have to be performed according to the pharmacopoeial guidelines.
The equivalence test for one drug product included a comparison of 90 Rapid Sterility Tests against 90 traditional sterility tests. In this, it was statistically evaluated with a low level inoculum of approximately 1-5 CFU using three different strains of micro-organisms, if equivalence between both test methods could be shown. The statistical analysis was performed with the χ2-Test, Fisher’s exact test and the Fisher test for a one sided equivalence. Many product specific validations were successfully performed and the products were shown to be fully suitable for Rapid Sterility Testing.
The Road to Regulatory Acceptance
Important criterion of starting any rapid microbiological methods project is to bring in advice for regulatory acceptance. Very early in the project of the Rapid Sterility Test it had to be decided which products were in the scope of the test. Suitable products are definitely vaccines for the seasonal influenza and any pandemic vaccine, as the release time for the vaccines is critical in pandemic situations. For Novartis Pharma, it was decided to start the project on a fairly new product with high market volumes. In a first submission to a health authority, FDA CDER received a comparability protocol, which contained the validation master plan. First approval for the comparability protocol on the first drug product was granted in June 2008 four months after submission, further approvals for ten other Pharma drug products followed in April 2009. In February 2010, CHMP of EMA granted approval for the first Rapid Sterility Test submission to Novartis. FDA CBER and MHRA acceptance for the Novartis Vaccines and Diagnostics site in Liverpool followed in May 2010.
Conclusion
Introducing new technology into microbiological testing can be interesting, but takes a lot of time and capacity. On the other hand, it helps you understand more than just traditional microbiology – in the end you are an expert in registration, finances and of course in rapid microbiology. It means building a project team consisting of a regulatory advisor, a controller and microbiologists. Be sure it is worth it to validate a rapid microbiological method!
Acknowledgements
We thank Matthias Schaar, Christian Vogt, Jasmin Kalt, Nathalie Le Goff, Marika Udvardi, Melanie Nast, Oliver Gordon and Celine Inderbitzin from the Rapid Sterility Test team involved in the project for their excellent support.
References
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Author Biographies
Jennifer C. Gray studied Biology (major in Microbiology; Cell Biology and Biochemistry) at the University of Basel, Switzerland and Freiburg in Breisgau, Germany. Since 2006 she’s working at Novartis Pharma AG in Switzerland in the QA/QC-Microbiology department. In her function as Rapid Microbiology Specialist she is playing a leading role in Rapid Microbiological Methods. Her responsibility is the stepwise replacement of Traditional Microbiological Methods (TMMs) against Rapid Microbiological Methods (RMMs) and the method transfer of RMMs to other Novartis sites. In 2010 she will finalize her PhD thesis at the Albert- Ludwigs-University of Freiburg, Germany.
Alexandra Staerk studied Hygiene-Technology at the technical university of Sigmaringen, Germany. Since 1995 she’s working at Novartis Pharma AG (former Sandoz AG) in Switzerland in the QA/QC-Microbiology department. In her function as Head of QA/QC Microbiology she is playing a leading role in microbiological isolator qualification, defining media fill standards and in Rapid Microbiological Methods.