Sterility Testing Made Simple Key strategies for ensuring reliable and compliant sterility testing in pharmaceutical labs Table of Contents 2
your Sterility Testing Canisters 27 Overcoming Challenges in Sterility Testing Enhance validation processes and achieve precision in sterility testing By focusing on these critical areas, this eBook pro- vides lab managers with detailed, practical guidance to enhance their sterility testing processes, ultimately helping them meet regulatory standards and main- tain high levels of quality and safety in pharmaceuti- cal manufacturing. Sterility testing is a fundamental aspect of pharmaceutical quality control, but it is fraught with challenges that require both technical expertise and careful management. If not properly addressed, the consequences of inadequate testing can be severe-from compromised patient safety to costly product recalls. One such issue arises when working with products, like antibiotics, that possess growth-inhibiting antibacterial and antifungal properties. These substances are designed to inhibit microbial growth, which is beneficial for therapeu- tic purposes but problematic during sterility testing. This can interfere with test results, leading to false negatives, and allowing potentially unsafe products to pass through quality control undetected. To mitigate this risk, specialized techniques and protocols must be employed. For instance, it may be necessary to neutralize the antimicrobial activ- ity using specific additives and/or adopting alternative testing methods, such as membrane filtration. The choice of method and the preparation of samples must be care- fully validated to ensure that the testing process accurately reflects the sterility of the product. The validation process for sterility testing canisters is another area that demands attention. It's recommended that this process be repeated bi-annually and when a change is implemented-new product formulations, changes to product formulations, or changes in experi- mental conditions. Validation involves rigorous testing to demonstrate that the canisters perform as expected under all relevant conditions, including exposure to various types of products and potential contaminants. It also requires thorough documentation to ensure the validation process is traceable and meets regulatory standards.
Sterility Testing A Best Practice Guide Sterility Testing is designed to confirm that sterile products, such as sterile pharmaceuticals and medical devices, do not contain contaminating viable microorganisms. This essential test determines if a manufactured batch of a sterile product is suitable for release. In this guide, we walk you through how best to perform your membrane filtration based sterility test Preliminary Set-up Begin insertion at the tubing guide. Ensure that there is sufficient tubing on either side. Membrane Pre-Wetting Briefly pre-wet the membrane (a couple of ml will suffice) with the rinsing fluid. The Sartochem® membrane has a wetting time of less than 1 second. A low pump speed is recommended for this step (speed setting 50). The rinsing fluid can be filtered directly through the membrane (by closing the venting filter with the tethered plug) OR the canister filled with a small volume of rinsing fluid (by leaving the venting filter open) and subsequently filtered (by closing the venting filter with the tethered plug). Note: more than one sample can be pooled through one sterility testing unit provided that the product does not clog/block the membrane. For such difficult to filter products it is advisable to use one sterility testing unit per product container. Ointments and emulsions can be diluted to 1 % in sterile isopropyl myristate by heating. Heating should not exceed 40°C. Only in exceptional circumstances, pending approval, can a product be heated to not more than 44°C. For liquids that are difficult to filter use Sterisart® Cellulose Acetate canisters (white base). Membrane Rinsing Fluid A (standard) Compatible with most samples Fluid D (standard) For antibiotics and the rinsing of medical devices Fluid K For oily solutions and products that (occasional) are difficult to filter and dissolve The membrane filter is typically rinsed not less than 3 times, with a volume of 50-100 ml. For products with known antimicrobial properties wash the membrane thoroughly. The maximum limit of each rinse cycle being 5 times 100 ml per filter/canister. A low pump speed <70 is recommended to a) prevent potential foaming and b) to maximise contact of the rinsing fluid with the membrane. Note: in select cases there can be a product interaction with the rinsing fluid, leading to the formation of a white precipitate. Such precipitates/ aggregates can clog the membrane. In such an event, try different rinsing fluids and ideally rinsing fluids sourced from different vendors. Differences do exist despite having the same composition. Media Fill Media sourced from some vendors can be darker than others. Ideally opt for a media where visual changes in the media (i.e. turbidity) are easily recognized. The canisters are periodically checked every 3-5 days. If there is no visual change in the growth medium after 14 days of growth, the product is sterile and the sterile product is suitable for release. If a visual change (e.g. turbidity) is observed, implying microbial growth, the test is positive and a detailed root cause analysis should be undertaken.
Click to access webinar. The Versatile Role of the Sterisart® Septum The test may be considered invalid and repeated only if it can be clearly demonstrated that the observed growth/contamination is unrelated to the product under examination. Typically, corrective action must be taken before the test can be repeated. A sterility test may be repeated only one time. If contamination is detected in the repeat test the product is not in compliance and the entire batch should be rejected. Note: in select cases there can be a product interaction with the growth media, leading to the formation of a precipitate. If the product renders the media turbid upon completion of the test, incubate the canisters for the 14 days, aseptically transfer a portion not less than 1 ml to fresh vessels of the same medium and incubate the original and transfer vessels for not less than 4 days. In this case we recommend that you use sterility testing canisters equipped with a sampling septum. The Sterisart® septum canisters facilitate aseptic sampling from a canister in a non-destructive manner. Method Validation/Revalidation Fluid K with beef extract and Tween can be tested if Fluid A delivers unsatisfactory results during method suitability tests. Surprisingly, some products selectively precipitate in rinsing fluid sourced from one but not the other vendor. This type of product interaction can prove troublesome. multiple filtration units. Cost cutting measures can at times lengthen the period of validation. As the saying goes - penny wise pound foolish. The 14 day incubation was established to ensure that such dormant forms recover effectively. There was growing frustration that the release of vaccines was stalled pending the completion of the safety tests. Understandably the tests cannot be rushed. There are other analytical tests in the QC release process that take longer that the sterility test. Batch release can take between 21-28 days. So for the moment, no. Sterility Testing is not rate limiting.
E: I will play it safe and say, perhaps! I don't want my comment to age poorly. As you know, the principle of the test remains largely unchanged since the mid-1930's. Our reusable sterility testing systems were launched in the late 1960's and the test has remained largely unmodified since, save the fact we have moved from reusable to single-use systems in the interest of quality and safety. So, like I said, I will answer that with a 'perhaps'. E: I am aware of rapid methods being adopted for the testing of short-shelf life products, such as ATMPs, or cell and gene therapies, and radiotracers used in PETs. Some of these products are meant for immediate use. In the past, some of these therapies have been administered without the completion of a sterility test, given that these products were administered to terminally ill patients who would not survive without treatment. So yes, rapid methods based on ATP-bioluminescence, CO2 detection and nucleic acid amplification are now being adopted to test such short shelf life products. - traditional sterile pharmaceuticals for want of a better word?
E: Again, I will say perhaps! We have partnered with Charles River and have paired the traditional test with a rapid method. Products are filtered, the canisters filled with growth media and incubated for a period of 5-6 days. A sample is then drawn aseptically via the septum port, featured on our canisters and transferred to the Celsis platform for testing. This has shortened the period of incubation and release. However, the method still relies on membrane filtration and microbial culture. There is perhaps no side stepping this requirement and I would like to highlight this. Membrane filtration facilitates the testing of large volumes, eliminates, or at the very least reduces the presence of compounds with antimicrobial properties, and the growth step ensures recovery of slow growing microorganisms. By staying growth-based, the method also ensures that only viable microorganisms are reliably detected. This avoids any undue concern on background or noise, leading to false positives. Perhaps… I missed picking up on this at that point. Could you elaborate?
E: Correct! The volume to be tested and the number of containers to be tested per batch have been specified in the pharmacopeial chapters on sterility testing. For instance, a fixed volume, depending on the final volume, from 2% or 20 containers, whichever is less, is tested for sterility from a batch of 500 containers. So, 20 containers from a batch of 1000 containers or a similar 20 containers from a batch of 100,000 containers. There is therefore discussion on whether the results of a sterility test, either by the traditional method or by rapid methods, are statistically reliable. However, let's not get caught up in this. The industry has developed other means of ensuring sterility through improved manufacturing standards with the help of guidelines such as the EU-GMP Annex 1. The sterility test remains an important check for the foreseeable future and serves to monitor possible gross contaminations. E: There are several guidance documents, besides the pharmacopeia, on sterility testing. The PIC/S, TGA, PDA and the FDA have issued guidance on sterility testing. Most of the guidelines are harmonised, similar to the pharmacopeial chapters. E: I don't quite think so. We've covered a wide range of topics today. Maybe the only thing I would like to mention is that most guidelines on sterility testing recommend that validation be repeated annually and not necessarily only where there is a change implemented. This may not be a pharmacopeial requirement, but it is considered good practice. Application Note December, 2023 Keywords or phrases: Sterisart®, Sterility Testing, Antibiotic Testing, Microbiological quality control
The Sterisart® System Simplifies the Sterility Testing of Therapeutics with Antimicrobial Properties
1 Lab Essentials Applications Development, Sartorius Lab Instruments, Göttingen, Germany 2 Product Management, Lab Essentials Microbiology, Sartorius Lab Instruments, Göttingen, Germany Correspondence
E-Mail: nils.jaeger@sartorius.com and eric.arakel@sartorius.com Abstract Membrane filtration-based sterility testing is particularly suited for products with microbial growth-inhibiting properties, such as antibiotics. These compounds need to be purged from the system to mitigate the incidence of false negatives. This is achieved by constructing the sterility testing canisters with materials that exhibit minimal to no non-specific binding. In this study, the recovery of microorganisms following the filtration of ciprofloxacin for intravenous administration and gentamicin for intramuscular injection using the Sterisart® closed system sterility testing device was evaluated. The results demonstrate that the Sterisart® canisters containing regenerated cellulose membranes are optimal for the sterility testing of antibiotics. Introduction Pharmaceutical products are routinely manufactured under strict GMP guidelines. Despite these strict codes, as a fail- safe prior to batch release, not all pharmaceutical products undergo stringent sterility testing to identify the potential presence of viable microorganisms. It is crucial that pathogenic microbes, such as bacteria and fungi, are detected in contaminated products before patients come in contact with. There have been rare instances where compromised drugs have been released to the market with devastating consequences for patients, and pharmaceutical companies. Sterility tests are performed in accordance with the regulatory requirements defined by the International Pharmacopeia (USP <71>, Ph. Eur. 2.6.1., and JP 4.06) and harmonized in ICH Q4B Annex 8. According to these requirements, sterility testing can be performed either by direct inoculation, or by membrane filtration, which is the method of choice. Products are tested for sterility by direct inoculation only when the properties of the product do not permit membrane filtration. The membrane filtration approach typically relies on a closed filtration unit containing a membrane with a pore size not greater than 0.45 μm and that has reliably demonstrated the retention of microorganisms. Other components of the system include a suitable pressure supply (such as a peristaltic pump) that drives the sample across the membrane filter, an appropriate membrane rinsing solution, and growth media. This closed setup is conventionally cleanroom compliant to eliminate any contamination risks and consequent false positives. Once sample filtration is complete, the closed system is incubated, typically for 14 days, and screened for turbidity as an indicator of microbial contamination. Sterisart® canisters are a closed system for sterility testing based on the membrane filtration method. This closed system excludes the need for physically manipulating membrane filters and thereby mitigates the risk of secondary contamination and false positives. Being a method based on the evaluation of microbial growth, it is crucial to distinguish between true product sterility and a false negative. Different ingredients of a pharmaceutical formulation can possess innate bacteriostatic or fungistatic properties that can negatively influence the results of a sterility test. Antibiotics possess such growth-inhibiting antibacterial and antifungal properties. It is therefore recommended to use the membrane-filtration method for the sterility testing of antibiotics or use a suitable sterile inactivating agent (such as penicillinase or cephalosporinase) to supplement the growth media. However, some antibiotics cannot be effectively neutralized. In such cases, non-specific adsorption of inhibitory compounds to the components of the sterility testing system is a major cause for concern. It is therefore critical that the physicochemical properties of the materials used in the construction of the sterility testing canisters must exhibit negligible non-specific adsorption and facilitate thorough rinsing to purge all traces of the antibiotic, and yet deliver on microbial retention. In this report, a method suitability test was performed using the two antibiotics ciprofloxacin, for intravenous administration, and gentamicin, for intramuscular injection. Ciprofloxacin is a fluoroquinolone antibiotic effective against most Gram-negative bacteria such as
Pseudomonas aeruginosa. Gentamicin is a type of aminoglycoside used in the treatment of infections mainly caused by Gram-negative bacteria as well as some Gram- positive bacteria such as
Staphylococcus aureus. The susceptibility of
P. aeruginosa and
S. aureus to ciprofloxacin and gentamicin respectively was the main rationale for choosing these antibiotics. Following dilution and membrane filtration of the antibiotics, the canisters were rinsed, inoculated with microorganisms listed in USP <71>, filled with growth medium and incubated at the prescribed temperature. Uninhibited microbial growth was observed in all the samples well before the prescribed 3-day maximum for bacteria and 5-day maximum for fungi recommended for growth promotion testing. Our results demonstrate that the Sterisart® canisters are optimal for the sterility testing of antibiotics and comply with all pharmacopeial requirements. Materials and Methods Sterisart® Universal Pump 16420 Ampoule breaker for Sterisart® Universal Pump 1ZW---0002 Sterisart® Transfer-Kit for liquids 16472 GBD Sterisart® system for liquids in open containers 16467 GBD Sterisart® system for closed large volume containers, with septum 16466 GSD 6 Incubate at 32.5 °C (±2.5 °C) for FTM and 22.5 °C (±2.5 °C) for TSB for 3 to 5 days / Ciprofloxacin (200 mg / 100 mL) / Gentamicin (80 mg / 2 mL) / Tryptic Soy Broth (TSB) 100 mL BD-257247 Fluid Thioglycollate Medium (FTM) 100 mL BD-257246 Fluid A (peptone water) 300 mL BD-254979 Tryptic Soy Agar (TSA) BD-254086 Pseudomonas aeruginosa ATCC® 9027™ Bacillus subtilis ATCC® 6633™ Staphylococcus aureus ATCC® 6538™ Clostridium sporogenes ATCC® 19404™ Aspergillus brasiliensis ATCC® 16404™ Candida albicans ATCC® 10231™ International pharmacopeias, including USP <71>, Ph. Eur. 2.6.1., and JP 4.06, recommend that for liquid antibiotics, a minimum sample volume of 1 mL from 20 distinct containers should be tested when the batch size exceeds 500 units. Accordingly, 20 mL of the respective antibiotic was tested per Sterisart® canister. Ampoules containing gentamicin were opened with the Sterisart® ampoule breaker. Using the Sterisart® Transfer-Kit-for liquids, 40 mL of either gentamicin or ciprofloxacin were aseptically pre- diluted in 200 mL of Fluid A. Two Sterisart® canisters were positioned in the canister holder and the Sterisart® tubing system was threaded through the pump head. The sterile venting filters were left open, the needle was inserted into the septum of the container containing Fluid A and the pump was switched on (see Table 4). Once 50 mL of Fluid A were transferred into the canisters, the sterile venting filters were sealed using the tethered filter plugs. Pre-wetting the membrane with a rinsing fluid limits non-specific adsorption and is strongly recommended. Next, the needle was inserted into the container containing the pre-diluted antibiotics. To reduce the contact time of the antibiotic-containing solution with the membrane, the tethered filter plugs were left on the sterile venting filters. The pump was switched on and the entire content of the bottle was pumped in equal volumes between the two Sterisart® canisters. The pump was switched off and the sterile vent filters were uncapped. To eliminate potential droplets of antibiotics at the canister walls, the needle was inserted into the container with Fluid A and the two canisters were filled with a pre-defined volume of 100 mL, by switching the pump on. The sterile vent filters were capped, and the membrane was rinsed with the contents of the canister. As recommended by the international pharmacopeia, the membranes were washed not more than 5 times with 100 mL per canister and filter. The fifth and final rinsing volume was spiked with <100 colony forming units (CFU) of certified test microorganisms and filtered through the Sterisart® canisters. For cell number quantification, the same amount of the spiked test microorganisms was transferred onto Tryptic Soy agar plates (TSA) using spread plate method and incubated under the same conditions as the respective spiked Sterisart® canisters. After the last rinsing step, the outlet of each Sterisart® canister was sealed using the enclosed wing nut plugs. The two sterile vent filters were uncapped. The yellow tube A clamp at the outlet of the Y-distributor was opened and the 1 TSB / / adjacent white tube clamp closed. The needle was inserted into a bottle containing either 100 mL Fluid Thioglycollate Medium (FTM) or Tryptic Soy Broth (TSB) and the Sterisart® 2 Universal pump was switched on. The Sterisart® canisters B FTM either TSB or FTM (see Table 5). The tubing was sealed using the two clamps above the inlets of the canisters. The tubing was cut at an appropriate distance away from the Ciprofloxacin / 7 8 1 2 3 4 5 6 Results Although both gentamicin and ciprofloxacin are highly potent against Gram-negative microorganisms, the canisters containing FTM spiked with
P. aeruginosa ATCC® 9027™ exhibited clearly visible growth that was no different from the positive control (see Figure 1). Canisters containing FTM medium spiked with
C. sporogenes ATCC® 19404™ turned fully turbid after 3 days of growth. FTM medium containing
S. aureus ATCC® 6538™ exhibited clearly visible growth throughout the culture media column within the canister, with colonies growing directly on the membrane. Ctrl. Gent. Cipr. Ctrl. Gent. Cipr. Ctrl. Gent. Cipr. Without prior agitation, all canisters filled with TSB showed clearly visible growth throughout the culture media within the canister or directly above the membranes for
A. brasiliensis ATCC® 16404™,
C. albicans ATCC® 10231™, and
B. subtilis ATCC® 6633™ (see Figure 2). In summary, no growth inhibition was observed in any of the canisters used for filtration of either gentamicin or ciprofloxacin, compared to the positive controls, where no antibiotics were filtered. Growth was not observed in any of the negative controls (see Figure 3). Conclusion This study demonstrates that Sterisart® canisters are optimal for testing sterile pharmaceuticals with antimicrobial properties, including those products that cannot be effectively neutralized. Rinsing each membrane with 5x100 mL of Fluid A solution guarantees adequate removal of gentamicin and ciprofloxacin. The Sterisart® Sartochem® Regenerated Cellulose Membrane stands as an universal membrane, characterized by minimal to no non-specific binding properties. As a result, there is no imperative need for segregation into products with or without antibiotic properties before conducting sterility tests. In addition to the tests performed in this study, we have conducted detailed adsorption and desorption tests of compounds with antimicrobial properties using Reverse Phase HPLC. Please see sections 5.1-5.2 of our validation guide for further details. Our extensive Sterisart® sterility testing portfolio has been designed for simplicity and is fully compliant with every pharmacopeial need. The unique Sterisart® septum port eliminates the risk of false positives and eases aseptic supplementation for antibiotic inactivation or aseptic sampling for identification, sub-culturing or rapid microbial release. References: Correspondence:
E-Mail: eric.arakel@sartorius.com Abstract In this study, we evaluated the Sterisart® closed system sterility testing device, with a septum, for the recurrent sterile extraction of samples. The results demonstrate that even after more than 100 repeated septum sampling events, which far exceeds any foreseeable sampling requirements, the septum remains intact and the growth media contained in these canisters remains sterile. The Sterisart® septum allows easy inoculation and sampling, and enables the coupling of the conventional closed system sterility testing with rapid detection methods. Introduction Pharmaceutical products are routinely manufactured under strict GMP guidelines. Despite these strict codes, as a fail-safe prior to batch release, all pharmaceutical products undergo stringent sterility testing to identify the potential presence of viable microorganisms. It is crucial that patho- genic microbes, such as bacteria, viruses and fungi, are detected in contaminated products before patients come in contact with them. There have been rare instances where compromised drugs have been released to the market with devastating consequences, for the patients and also the pharmaceutical companies. Sterility tests are performed in accordance with the regulatory requirements defined by the International Pharmacopeia (USP <71>, EP 2.6.1, JP 4:06). Sterility testing can be performed either by direct inoculation | transfer, or membrane filtration, which is the method of choice. Products are tested for sterility by direct inoculation only when the properties of the product do not permit mem- brane filtration. The membrane filtration approach typically relies on a closed filtration unit containing a membrane with a pore size not greater than 0.45 μm and that has reliably demonstrated the retention of microorganisms. Other components of the system include a suitable pressure supply (such as a peristaltic pump) that drives the sample across the membrane filter, an appropriate membrane rinsing solution, and growth | culture media. This closed setup is conventionally cleanroom compliant to eliminate any contamination risks and consequent false positives. Once sample filtration is complete, the closed system is incubated, typically for 14 days, and screened for turbidity as an indicator of microbial contamination. Sterisart® canisters are a closed system for sterility testing based on the membrane filtration method. This closed system excludes the need for physically manipulating mem- brane filters and thereby mitigates the risk of secondary contamination and false positives. However, sample extraction is a prerequisite, when the growth media is ren- dered turbid by microbial growth, following the prescribed 14 days of incubation. If microbial growth is detected, the identity of the microorganism and the source of the con- tamination is determined, and the sterility test is declared invalid and then repeated. Aseptic sample withdrawal or aseptic enzyme supplementation, for instance to deactivate antibiotics that might result in false negatives, may also be required after filtration or during incubation. Precipitation of the filtered test sample, or an adverse color change due to the inherent properties of the compound, can also render the growth media turbid, even prior to incubation at the prescribed temperatures. This convolutes the interpretation of the sterility test and the certification of the batch for release; the batch may require additional testing by sample extraction from the canister and subse- quent sub-culturing. Sample extraction in conventional sterility test systems involves puncturing or cutting the tubing leading to the inlet of the canister and then attempting to carefully extract a sample, without compromising the integrity of the canister or its contents. Sample extraction by cutting the tubing precludes repeated sampling. Multiple sampling using other approaches can increase the risk of contamination by compromising the closed system. The Sterisart® septum was designed to facilitate repeated sampling during incubation of the growth promotion test. In this report, we show that multiple sampling performed through the Sterisart® canister septum - over 100 times - exceeding any conceivable requirement for aseptic sampling, does not lead to the contamination of the system. Materials and Methods Tryptic soy broth (TSB) (Gila/BD), Fluid thioglycollate medium (FTM) (Gila/BD), TSB (Merck), FTM (Merck), Tryptic soy agar (TSA) (Merck), Glass reaction tubes, 30 ml (Borosil), Needle - 0.90 × 70 mm, 2OG × 2 ¾ (Sterican - B. Braun), Syringe - F Luer (Omnifix - B. Braun). Sterisart® universal pump, Incubator (Sartorius Stedim Biotech GmbH), Combisart® 3-branch filtration manifold (Sartorius Stedim Biotech GmbH), e.jet Pump (Sartorius Stedim Biotech GmbH). 16467--------GSD, 16475--------GSD, 16466 GSD Ten individual Sterisart® canisters from the three types of septum variants (30 in total) were analyzed in the septum sampling tests. One of the ten canisters (from each Sterisart® canister type) served as a negative control (i.e. samples were not extracted from this canister until day 24 of the test). The Sterisart® canisters were filled with growth media under aseptic sterile conditions in a biosafety cabinet. The two Sterisart® canisters were positioned in the pump holder and the Sterisart® tubing system was thread through the pump head. The outlet of each Sterisart® canister was sealed using the enclosed wing nut plugs. The two sterile vent filters were left uncapped. The yellow tube clamp at the outlet of the Y-distributor was opened and the adjacent white tube clamp closed. The dual-needle metal spike was inserted into a bottle containing FTM and the Sterisart® Universal pump was switched on. The pump was switched off once a predefined volume (75 ml) of medium was transferred into the first canister. The white tube clamp at the outlet of Y-distributor was opened and the adjacent yellow clamp closed. The dual-needle metal spike was inserted into a bottle containing TSB and the Sterisart® Universal pump was switched on. A similar volume (75 ml) of medium was transferred into the second canister. The tubing was sealed off using the two clamps above the inlets of the canisters and the tubing was cut off. Please refer to our
Sterisart®
NF gamma user manual for a pictorial depiction of the described process. Sterisart® canisters containing TSB, the recommended growth media used in the detection of low incidence fungi and aerobic bacteria, were incubated at 22.5° C for 24 days. Sterisart® canisters containing FTM, the recommended growth media for cultivating aerobic, microaerophilic, and anaerobic microorganisms were incubated at 32.5° C for 24 days. Samples were extracted from the Sterisart® canisters under sterile conditions in a biosafety cabinet. Three samples of 100 µl each were extracted twice a day from the top, middle, and bottom of the Sterisart® canister, over a period of 17 days (3 × 2 × 17 = 102 samples). The extracted samples were transferred into the glass reaction tubes containing the sterile liquid media, FTM and TSB. The vials containing TSB were incubated at 22.5° C for 14 days, and the vials containing FTM were incubated at 32.5° C for 14 days. The results were recorded by photographing each Sterisart® unit and the corresponding extracted sample. A final inspection was performed using a black gridded membrane filter placed in a sterile Sartorius Combisart® filtration unit and connected to an e.jet pump. 60 - 70 ml of TSB (following the 24 day incubation period of the Sterisart® canisters) was filled into the funnel and filtered through the black membrane filter. The filter was transferred using sterile forceps onto a TSA plate, and the plate was incubated at 36° C for 3 - 5 days. These plates were then inspected for microbial contamination. Results and Discussion After 102 septum piercings and repeated sample 8 1 withdrawals, it was established that all Sterisart® canisters 2 (3 × 9 containing FTM, and 3 × 9 containing TSB; the 10th canister containing FTM and TSB serving as their re- 3 spective controls) were sterile and showed no detectable 4 microbial contamination after 24 days. (Figure 1) 5 A B Similarly, the extracted samples were likewise sterile and free of microbial growth demonstrating that the Sterisart® septum promotes efficient and highly reliable aseptic sampling. (Figure 2) 7 6 C D Figure 1: No microbial contamination after repeated sample extraction. Representative images of the Sterisart® 16466 GSD version filled with TSB (A and B) FTM (C and D) incubated for 24 days at 22.5° C and 32.5° C respectively. Negative controls are shown in A and C. Canisters in B and D were pierced 102 times for sample extraction. Figure 2: No microbial contamination in samples extracted from Sterisart® 16466 GSD after incubation in glass vials. Samples were inoculated into glass vials containing TSB (upper panel) and FTM (lower panel) were grown at 22.5° C and 32.5° C, respectively, for 14 days. Coring can occur when a septum has been punctured multiple times or if an inappropriate needle type is used. Only after 36 piercings were small particles observed in some Sterisart® canisters. These particles were collected after 24 days on a black membrane filter and monitored for their ability to form colonies on TSA plates. These particles did not demonstrate any growth even after an incubation period of five days, suggesting that these particles are not biological in nature. Based on their morphology, we con- clude that these inert particles are fragments of rubber that are sheared off the septum during repeated piercing with syringe needles. These fragments do not influence the efficacy of the sterility test and are barely visible in the growth medium. We recommend that septum sampling be performed only after unplugging the sterile vent (i.e. uncapped) in a controlled environment. Our results demonstrate that Sterisart® canisters remain a closed and sterile unit, even after successive sampling for a tested total of 102 extractions. Figure 3: Representative image of the Sterisart® septum after 102 sample extractions. Conclusion In summary, we demonstrate that the Sterisart® septum is exceedingly robust and maintains an intact sterile environ- ment even after more than a hundred sample extractions. The presence of preservatives and anti-microbial agents, in products tested for sterility, have to a great extent impeded the adoption of rapid detection methods that rely on direct inoculation. Membrane filtration, and subsequent membrane rinsing, of such products curtails the risk of false negatives during sterility testing. By also facilitating the analysis of large volumes through membrane filtration and by enabling the extraction of samples, we afford our users the ability to integrate closed system sterility testing with rapid sterility testing methods. However, some slow growing anaerobes can be difficult to detect using some rapid sterility methods. Given that septum sampling does not compromise the integrity of the closed system sterility test, we provide our customers with the potential to sample for rapid sterility testing, yet re-incubate the sterility tests for the stipulated 14-day period of incubation.
Points to Consider when Validating your Sterility Testing Canisters Sterility testing is an integral part of all pharmaceutical microbiology laboratories and is designed to detect the presence of viable microbial contaminants in sterile pharmaceuticals. Being a method based on the evaluation of microbial growth, it is crucial to distinguish between true product sterility and a false negative (Aseptic Guideline 2004). Certain ingredients used in the formulation of drugs can possess innate anti-microbial properties and prevent a sterility test from reliably reporting on the presence of viable microorganisms. Validation is therefore performed for all new product formulations, whenever there is any change made in product formulation or if there are changes in experimental conditions. This includes selecting or making a change between a primary | secondary supplier of your sterility testing equipment. Even in the absence of a change, it is recommended to routinely revalidate all processes on a regular basis. Sterility Testing This involves the continual collection, evaluation and documentation of data. As a general rule, it is advisable to seek guidance and feedback from local or international regulatory bodies or advisors on the proposed methodology early in the process and prior to undertaking a validation exercise to ensure these will comply with their requirements. There are several detailed guidelines for sterility testing, besides the pharmacopeial chapters. We have compiled the following points to be considered during the validation | revalidation of your sterility testing canisters. Validation of Sterisart® Sterility Testing Canisters Sterility testing canisters must be compliant with the pharmacopoeia guidelines used in the facility, and a manufacturer's validation guide should be available. Ultimately, methods validation studies should demonstrate that the method does not provide an opportunity for false negatives (Aseptic Guideline 2004). The following points should be considered when selecting or making a change to suppliers of sterility testing canisters or any other critical component of manufacture or testing of a product. Reports should be reviewed by concerned departments and approved by the head of Quality Assurance or their designated authority. Report approval shows that the validation was completed successfully and according to the validation protocol. References U.S. Pharmacopeia. USP <71> Sterility Test European Pharmacopoeia. Ph. Eur. 2.6.1 Sterility Japanese Pharmacopoeia. JP 4.06 Sterility test World Health Organization (WHO); 3.2 Test for sterility TGA guidelines for sterility testing of therapeutic goods, 2006 21 CFR 610.12 - General Provisions PIC/S PI 012-2 Recommendation on Sterility Testing FDA Aseptic Guideline (Sterile Products Produced by Aseptic Processing, 2004) Sartorius Lab Instruments GmbH & Co. KG Otto-Brenner-Strasse 20 37079 Goettingen Phone +49 551 308 0 Sartorius Corporation 565 Johnson Avenue Bohemia, NY 11716 Phone +1 631 254 4249 Toll-free +1 800 635 2906
For further contacts, visit www.sartorius.com Specifications subject to change without notice. Copyright Sartorius Lab Instruments GmbH & Co. KG.
