Container Closure Integrity Tests (CCIT) for Single-Use Systems

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1 By Lionel Karcher, Head of Microbiology Laboratory, BU Confarma Container Closure Integrity Tests (CCIT) for Single-Use Systems Container Closure Integrity Tests have traditionally been performed using dye testing or microbial challenge testing. The article entitled Development of a Dye Ingress Method to Assess Container-Closure Integrity: Correlation to Microbial Ingress, published in PDA Journal 1 is one of the key reference texts on the subject. There are currently many more sensitive physical methods that in particular allow high-throughput in-process integrity testing; that said, dye tests using methylene blue and microbial challenges remain relevant and offer various advantages.

2 In 2016, the Package integrity evaluation sterile products Chapter 2 of the USP was updated, significantly calling into question the use of traditional methods like dye testing or microbial challenge testing. In this Chapter, the microbial challenge is referred to as Microbial Challenge, Immersion Exposure and the dye test is included among Tracer Liquid methods. These two methods are classified as probabilistic methods. Probabilistic methods are characterized by the random number of events allowing the transfer of liquid into the samples. By contrast, the USP defines deterministic methods as physicochemical techniques that yield quantifiable and reproducible results with clearly defined and predictable detection limits. Principle and key points The microbial challenge test and the dye test both share a similar basic principle: they consist of detecting the transfer of liquid into the container. The transfer of liquid is revealed by coloration or microbial growth. MICROBIAL CHALLENGE The samples are first filled with a medium that allows microbial growth. A standard Trypticase soya (TS) growth medium is generally used. A product containing a sufficient amount of nutrients can be directly used as a growth medium. In this case, the product s fertility compared to the test germ must be assessed before performing the assays. Using the product as the growth medium has the advantage of not producing a specific batch and/or allows the test to be performed in the context of an investigation. Using the product as the growth medium has the advantage of not producing a specific batch and/or allows the test to be performed in the context of an investigation. The samples are then immersed in a microorganism solution. The most commonly used microorganisms are Brevundimonas diminuta, Serratia marcesans and Escherichia coli. In order to challenge a sample with a specific strain, other microorganisms, such as in-house strains may be used. It should be noted that certain microorganisms require special growth parameters that must be taken into account when choosing the test strain. This, for instance, is the case with the strictly aerobic Brevundimonas diminuta, for which the growth medium must contain oxygen. At the end of the immersion cycle, the samples are cleaned and then incubated for a sufficient amount of time to allow the growth of microorganisms. There is no standard in terms of incubation time; seven or 14 days are generally used by default, but if microbial growth can be detected in a shorter time, then this shorter interval may be chosen. After this incubation period, the samples are directly observed to detect any cloudiness. Generally speaking, if the test conditions were correctly chosen, there will be no doubt if there is any growth. That said, an analysis of sample contents may also be performed to detect the presence of any microorganisms. This analysis must be performed if the container or the growth medium does not allow cloudiness to be assessed or if there is any doubt concerning microbial growth.

3 DYE TEST The samples are first filled with product. An empty sample can also be tested, but generally speaking, transfer between two liquids is preferred. The samples are then immersed in a dye solution. Various dyes can be used, but methylene blue has been used traditionally. At the end of the immersion cycle, the samples are cleaned and read. The samples can be read visually or using a spectrophotometer. Positive samples Alongside the tests, positive controls must be performed using samples in which holes have been made. By using samples rather than standard controls, the test can be actually simulated. Compared to certain deterministic methods, using positive controls allows the detection limit on the actual sample to be re-verified during every test session. It should be noted that detection limits vary by container type. The larger the container (several liters, for instance), the higher the detection limit. The detection limit is correlated to the liquid s capacity to penetrate the container. For example, for a 20 liter flexible pouch, the detection limit will generally be greater than 100µm whereas for a syringe or a few milliliter bottle, the detection limit will decrease to 2-5µm given sufficient pressure variations. By comparison, according to the USP, a deterministic method used for rapid in-process testing detects defects of µm. Meanwhile, these methods can be used to detect defects of under 1µm when used out-of-process. Glue Transfer Figure 1: Transfer along the microcapillary In order to create positive controls, microtubes or microcapillaries can be used to simulate microholes in the samples. Creating positive controls is a delicate task. During the preparation and during the insertion of the capillaries in the samples, it is essential not to plug or break the capillaries. Another key point is the attachment of the capillaries. If a capillary is not sealed or correctly sealed to the sample, the liquid will transfer next to it rather than inside of it (see figure 1) even if the capillary is introduced through a rubber stopper. The stopper does not retract perfectly around the capillary and leaves a path for the liquid. There is also a correlation between capillary length and the probability of transfer through it, which makes it necessary to perform a validation before starting routine tests. If a capillary is not sealed or correctly sealed to the sample, the liquid will transfer next to it rather than inside Drawbacks The two methods have certain known drawbacks: They are destructive methods, meaning that the samples cannot be reused in either case, As a direct result of the aforementioned aspect, the generated waste needs to be managed, While the detection limits are relatively high, they can approach those of deterministic methods if significant pressure variations are applied, The analysis time is long, particularly for microbial tests, ranging between one and 14 days, For microbial tests, a medium enabling the growth of the chosen germ must be used.

4 Advantages Despite the relatively significant drawbacks of these methods, they still offer some advantages. 1 PRESSURE VARIATIONS One of the main advantages is the possibility to expose the samples to different pressure variations. At present, various companies challenge their products using specific conditions that most closely mimic the transportation conditions of their products. Some samples are thus immersed in a solution and undergo a series of dips in pressure to simulate multiple flights. That said, the pressure variation must be large enough to set air bubbles free that prevent the transfer of the liquid through the microtubes. The difference in pressure is therefore directly correlated to the test s detection limit. As mentioned above, using significant pressure variations allows us to approach the detection limits offered by deterministic technologies, but the structure of the samples must be taken into account. For samples that have a mobile component, such as the rubber stoppers of syringes, an excessive variation in pressure significantly shifts the stopper and presents the risk of a false positive result. By using pressure that is equal to the pressure variations that the product is subject to during air transport, the shifting of mobile components can be mimicked without generating false positive results. This latter point allows the verification of the risk of microbial contamination when the product s mobile components shift as a result of pressure variations. We should keep in mind that pressure variations mimicking air transport are relatively minor, therefore the detection limits of these tests are relatively high. 2 LARGE VOLUMES For many sample integrity testing methods, sample size can quickly become a limiting factor. For dye testing and microbial challenge testing, the size limit for conducting a test is relatively high. To conduct the tests, special large size containers are manufactured (see photo 1). Then a large volume of immersion solution must be prepared to make sure that all sample surfaces are challenged. Photo 1: Containers used for CCIT The containers are then introduced into a chamber large enough to perform cycles of pressure variations (see photo 2). When studying large or very large volume containers, the studies are relatively long because every sample needs to be tested individually. In addition, for microbial tests, the incubation chamber must be sufficiently large. Lastly, the destruction of samples is also more difficult. Photo 2: Pressure variation chamber

5 3 EASE OF SET-UP Dye tests and microbial challenges also have the advantage of being relatively simple to set up. For microbial challenges, if the reading is performed visually, only a device for implementing pressure variations is needed in addition to standard microbial laboratory equipment. What is needed: A sterilizable and/or sterile container to immerse and challenge all of the sample surfaces, Samples containing a medium enabling the growth of the germ, A device for implementing changes in pressure, Incubators or incubation chambers, Growth media and microorganisms to prepare the immersion solutions, Capillaries or microtubes if these are chosen to make the positive controls. To perform dye tests, standard chemical laboratory equipment is enough in addition to the equipment for implementing pressure variations. A container for immersing the samples, Samples, A device for implementing changes in pressure, A reading booth or a spectrophotometer depending on the type of reading, Capillaries or microtubes if these are chosen to make the positive controls. In these two cases, a single device may be used to conduct the tests. In function of the size of the device, containers of various volumes can be assessed. Other applications There are other applications for microbial tests and for the dye test that is not described in the USP. These include two often used examples: In the case of the dye test, there is a test method that detects seal leaks in porous packaging (ASTM F ). When performing this test, there are two possibilities: either the packaging is filled with dye or conversely, the packaging is dipped into solution. For microbial challenges, another option is to use bacteria in aerosol form to challenge the integrity of the sample by simulating an atmosphere saturated with microorganisms.

6 Conclusion In recent years, traditional dye tests using methylene blue and microbial challenge tests have been seeing competition from highly effective new technologies. These technologies, referred to as deterministic methods, guarantee known detection limits. But despite the known drawbacks of traditional methods, they nevertheless remain useful thanks to their relative simplicity, rapid set-up and low cost. Moreover, these methods offer a solution for the integrity testing of large-volume products. Even more importantly, they can be used to test the impact of any shifting of a product s mobile components (such as the stopper) on the risk of microbial contamination. References 1. USP 39 (1207.1) Package integrity testing in the product life cycle - test method selection and validation. 2. PDA, Development of a Dye Ingress Method to Assess Container-Closure Integrity: Correlation to Microbial Ingress 3. ASTM, F 1929 Standard Test Method for Detecting Seal Leaks in Porous Medical Packaging by Dye Penetration Originally published in French in La Vague in October 2017 Confarma, a Solvias Group company, provides biological analysis Confarma France SAS ZI rue du Canal d Alsace, Hombourg, France info@confarma.fr