Microbial Considerations for the Selection of Aseptic Connectors

The application of single-use, sterile and disposable technology is encouraged by regulators, as the draft to EU GMP Annex 1 indicates. Such technology can increase the level of sterility assurance and reduce the environmental impact of processing at the pharmaceutical facility. Aseptic connectors (and disconnectors) are an important example of such technology, permitting the continuation of the fluid path by using devices designed to ensure no microbial contamination ingress. With aseptic processing, any connection undertaken post- sterilizing grade filter is of particular importance.

It is necessary for pharmaceutical manufacturers implementing aseptic connectors (or changing from one supplier to another) to evaluate the robustness of the connector and its ability to connect, plus aspects of the weld, and the sterilization method, as part of the Contamination Control Strategy. This article considers the microbial risk factors and the areas to examine, which can be used as the basis of questions to be directed at an aseptic connector supplier as part of the quality audit process. There are other considerations for aseptic connectors, which need to be considered as part of Quality by Design, such as product interactions through leachables and extractable testing, disinfectant compatibility, particulate release, and the design ergonomics. While important, these are not addressed in this article; the focus here is with microbial control.

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Aseptic Connectors

Sterile connectors for aseptic processing (commonly referred to as ‘aseptic connectors’) enable two lines of tubing to be joined while maintaining a sterile fluid pathway. Each line of tubing is pre- connected to one connector end. At the end of the connector is a removable membrane which facilitates the two pieces of tubing to connect (mechanical coupling). Once the two connectors ‘connect’ the membrane is removed. Through the act of connection, the process line is maintained as closed and sterility ideally assured. Primary uses of connectors include connecting product vessels to filling lines; connecting filtration assemblies; or connecting biocontainer bags for taking bioburden samples.¹ The design of many connectors also facilitates a fast and safe disconnection by the pushing of an internal plunger that allows the two parts to become separated. The rapid disconnection means that any backflow of fluid is avoided.²

The advantages of aseptic connectors are not only that they provide more robust sterility assurance, the connectors also permit the operation to be conducted within a lower classification for the surrounding cleanroom (for example, where a traditional connector would require ISO class 5 / EU GMP Grade A, and ISO class 7 / EU GMP Grade B area could be used). Furthermore, whereas the risk with traditional connectors includes the operator and hence finger plates are typically taken, the aseptic connector does not require finger plates to be taken as the impact of the operator and the level of bioburden carried on gloved hands will not affect the assurance of sterility as the connection is made. These advantages can only be realized if the device qualification has addressed the primary microbiological risks.

Primary Microbiological Risk Factors

The main microbiological risks when using an aseptic connector relate to:

1. The act of connecting the two components together, and contamination transferring from the surrounding environment.
2. Direct transfer of contamination from the hands of the operator.
3. A weakness with the design of the connector, that leads to contamination ingress during the connection.
4. A weakness with the design of the connector that leads to a weakness occurring at a later time point, perhaps influenced by the speed or pressure associated with the fluid flow or with temperature.
5. A weakness with the design that leads to a loss of integrity across the shelf-life of the connector.
6. Failure to adequately sterilize the device.
7. A weakness with the seal of the outer packaging of the device, leading to loss of sterility.
8. The presence of bacterial endotoxins.

Each of these factors, that could lead to microbial ingress, need to be addressed through qualification testing. When selecting a connector between suppliers, evidence should be sought in relation to the above points.

Microbial Qualification Testing

The microbial assessment involves a challenge with microorganisms, through variants of immersion designed to assess microbial ingress. As with sterilizing grade filter testing,³ the‘default’organism has traditionally been Brevundimonas diminuta (strain reference ATCC 19146), after it has been nutritionally starved as part of the process of achieving smaller cell sizes, an effect that also seems to occur as the culture ages⁴ (typical cell sizes are between 0.3 – 0.4 µm x 0.6 – 1.0 µm).⁵ The origin of the name, as an approximate translation from Latin, means a ‘small bacterium with short wavelength flagellum’.⁶ However, as with sterile filter testing the organism selected can no longer default to B. diminuta without an assessment (at least not within Europe).⁷ There is a requirement to assess the microbiota of the facility to determine if organisms smaller than B. diminuta pose a risk (as could be the case, under certain circumstances, with the common water contaminant Ralstonia pickettii, for example where the typical cell size is between 0.5-0.6 x 1.5-3.0 µm,⁸ and in the context of different exogenous factors influencing cell size, including various physicochemical factors).

Where alternative organisms are required, bacterial size can be verified from checking exponential phase cultures by phase microscopy to measuring the diameter of the halo of cells developing in media containing 0.3% to 0.7% agar from a very small (say 0.2µl) spot inoculum or a “stab”.⁹ Furthermore, as with microbial assessment of container-closures, often two different organisms are selected, such as B. diminuta plus Escherichia coli, with the latter selected because of its relatively powerful motility and multi-flagella distributed over the cell surface which enables both swimming and tumbling within fluids.¹⁰ A case could also be made for the inclusion of a Gram-positive coccus corresponding to the human skin microbiome, such as a Staphylococcus or a Micrococcus. In terms of the challenge population, this should be sufficiently high to enable any ingress to be assessed. For ingress testing, this involves the use of a microbial suspension in culture media with a target population of between 10⁷ and 10⁸ bacterial cells per milliliter.

Non-Microbial Tests for Integrity

There are three forms of testing typically undertaken to assess the integrity of aseptic connectors in relation to microbiological testing. These are not microbiological tests but the implications for failure pose microbiological risk in terms of the potential for contamination ingress. Each of these is a form of challenge testing, and they can be conducted as part of design qualification, lot release testing and to set the expiry time:¹¹

1. Dip test: With this test, the two pre-assembled halves can be dipped into a preparation of the challenge organism. By ‘dipping’, this is a relatively brief activity designed to be sufficiently long in order to wet the components (such as 30 seconds). Following this, the components are allowed to dry and then culture media is passed through the flow path after assembly. The culture media is then incubated with the acceptance criterion of no visible turbidity.
2. Spray test: With this aspect of testing, each pre-assembled half is sprayed with the challenge organism at the ends to be connected. The components are then allowed to dry, before being sprayed for a second time during paper removal. Growth media is then passed through the flow path after assembly. This test is seemingly more variable than the immersion test (below) as it relies on the rate of sedimentation onto the connector joint to be sufficiently high in bacterial cells (which is difficult to quantify). Furthermore, a bioaerosol test chamber is needed with which the study can be conducted. This test provides a useful adjunct to the immersion test but is not a reliable alternative to it.
3. Immersion test: With the immersion test, the entire and completely assembled connector is immersed in a solution of the challenge organism with growth media in the flow path. The time of immersion needs to be sufficient for the bacteria to move towards the assembly (which can be promoted through gentle agitation). A time of around 5 minutes is recommended. A suitable time for incubation will be five to seven days, alongside a positive control designed to show that turbidity will occur, and diluted plate counts designed to confirm the challenge inoculation. The incubation temperature will be appropriate to the test organism, e.g., 30-35°C for B. diminuta.

During product development, it is best practice to subject the connector to the stresses that can be encountered during manufacturing. This includes conducting testing at the end of the maximum recommended connection, at the end of the expiry date of the connector, and subject to different fluid flows where pressure, temperature and shear forces are set and monitored.

While microbial tests are important, the issues of culturability mean that not every potential contaminant can be accounted for and while the test organism provides assurance that a small sized organism cannot penetrate, the robustness of the integrity should be supported by physical testing. These center on the welding as well as the connection tightness of the complete unit. Variations can also be built in terms of the weld of the connector, to ensure that any inconsistencies of the weld are addressed. While the welding of thermoplastics is well-established and subject to an international standard, (ISO 472) there are different methods of welding.¹² Plastic welding is a process of uniting softened surfaces of materials, with the aid of heat across three sequential stages: surface preparation, application of heat and pressure, and cooling. An important consideration is with weldability of base materials, and it is important that the weld is assessed as part of device qualification and then periodically during the manufacturing process using an AQL approach. As the rheological properties of materials will differ (based on the stress strain relationship for that material at varying temperatures) it is important that leakage testing is sufficiently robust as to account for viscosity, elasticity, plasticity, viscoelasticity, and the materials activation energy as fluid flows through the connector.¹³

The primary tests are:^14,15

• Tensile test: Here a test piece is pulled until it breaks. This test is quantitative and enables an assessment of ultimate tensile strength, strain, as well as the level of energy required to produce failure.
• Tensile impact test: This assessment has the device clamped into a pendulum, and the pendulum is made to swing down and strikes the specimen against an anvil breaking the specimen. This allows the impact energy to be assessed for both the weld seam and base material.
• Three-point flexural test: This test provides values for the modulus of elasticity in bending, flexural stress, flexural strain, and the flexural stress–strain response of the material.
• Creep tests: These tests are designed to consider the long-term weld performance of the connector. Testing is performed at a constant temperature and constant stress, with the objective of providing quantitative data on the long-term weld performance.
• Integrity testing such as a helium leak test: To assess seal integrity through an attempt to force the inert gas through, under conditions designed to mimic processing pressures and temperatures.
• Burst Test: To assess if the connector exceeds established pressure rating when connected. This test confirms a maximum pressure that a connection can withstand.

In addition, it is useful to have operators examine the connectors for any visible damage before use. According to the standard DVS2202-1,¹⁶ DIN 91441,¹⁷ and DIN 32502,¹⁸ the ‘inspector’ should examine the seal for:

• Discolorations
• Weld defects,
• Discontinuities,
• Porosity,
• Notches,
• Scratches

A visual inspection scale is provided in the standard to assist inspectors.

Sterility and Bacterial Endotoxin

The connectors should be subject to bioburden controls during manufacturing, which will ensure that the devices are unlikely to be heavily contaminated, and then subjected to an appropriate sterilization process, such as moist heat or irradiation. The sterilization cycle should deliver a Sterility Assurance Level of 106 or greater. In addition, the devices should be assessed for bacterial endotoxin as part of release testing. The acceptable level of endotoxin is typically <0.25 EU/mL.¹¹

Summary

As with any technology that begins as a ‘niche’ and progresses to ‘mainstream’, as with single-use fluid-management solutions applied in critical downstream and final filling applications, it is important to ensure that cGMP appropriate standards are in place. With devices like sterile connectors for aseptic processing, the user will not be equipped to undertake much in the way of confirmatory testing. Therefore, the selected supplier will need to provide evidence that appropriate qualifications have been undertaken (ideally through a third-party independent laboratory). The issues raised in this article can provide some of the questions to ask during supplier assessment, quality audit, and data evaluation.

References

1. Andrews, T. (2018) A deep dive into the process of designing and developing a single- use aseptic connector. In “Single-Use Technologies III: Scientific and Technological Advancements, Ding, W., Martina A., Micheletti, R.R. (Eds.) ECI Symposium Series, https:// dc.engconfintl.org/sut_iii/57
2. Peyrache, S. and Masy, C. (2018) Technologies de connexion à usage unique : situation actuelle et tendances, La Vague, 58: 35-40
3. Lee, S., Lee, S., and Kim, C. (2002) Changes in the Cell Size of Brevundimonas diminuta Using Different Growth Agitation Rates. PDA Journal of Pharmaceutical Science and Technology. 56 (2): 99-108
4. Sundaram, S. , Auriemma, M., Howard Jr, G. (1999) Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes, PDA J Pharm Sci Technol. 53(4):186-201
5. Sandle, T. (2013) Sterilisation and Sterility Assurance for Pharmaceuticals, Elsevier, Cambridge, UK, pp143-155
6. Euzéby J (1997) List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. International Journal of Systematic and Evolutionary Microbiology. 47 (2): 590–2
7. EMA. Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container, EMA/CHMP/CVMP/QWP/850374/2015, European Medicines Agency, 2019, at: Sterilisation guideline - adopted by CxMP - 10.12.2018 (europa.eu)
8. Ryan, M.P.; Pembroke, J.T.; Adley, C.C. (2007) Ralstonia pickettii in environmental biotechnology: potential and applications. Journal of Applied Microbiology. 103 (4): 754–764
9. Millikan, D.S. and Ruby, E.G. (2003) FlrA, a 54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization. Journal of Bacteriology; 185(12): 3547–3557
10. Sandle, T. (2012) Container Closures for Pharmaceutical Preparations: A review of Design and Test Considerations, BioPharm International; 25 (12): 32-36
11. Sandle, T., and Saghee, M. (2011) Some considerations for the implementation of disposable technology and single-use systems in biopharmaceuticals. J Commer Biotechnol 17, 319–329
12. ISO 472:2013 Plastics — Vocabulary, International Standards Organization, Switzerland
13. Balkan, O., Demirer, H., Ezdeşir, A., Yıldırım, H. (2008) Effects of welding procedures on mechanical and morphological properties of hot gas butt welded PE, PP, and PVC sheets. Polymer Engineering & Science. 48 (4): 732–746
14. Czichos, Horst (2006). Springer Handbook of Materials Measurement Methods. Berlin: Springer. pp. 303–304
15. Stokes, V.K. (1989) Joining methods for plastics and plastic composites: An overview. Polymer Engineering & Science. 29 (19): 1310
16. DIN - DVS 2202-1: 2008 Imperfections in thermoplastic welded joints Features, description, evaluation, Germany
17. DIN SPEC 91441: 2019 Packaging - Test method for determination of the peel strength of sealable packaging materials, Germany
18. DIN 32502: 1985 Imperfections in plastic welded joints; classification, terminology, Germany