Single-Use System Integrity IV: A Holistic Approach Based on Compiled Scientific Study Data.

This concluding article of the publication series provides an overarching summary of all study results presented in the three previous articles (1-3). Their interdependency in achieving a holistic approach to the integrity assurance of single-use systems (SUSs) employed in (bio)pharmaceutical manufacturing is finally illustrated. Two of those three studies were conducted to understand microbial ingress and liquid leak mechanisms in polymeric film material as determinants of the maximum allowable leakage limit (MALL) for SUSs using artificially created defects. The third study characterized gas flow through these defects-an essential variable for robust validation of physical integrity test methodologies based on gas flow. In all studies, the test samples used were 50 mm round patches of two ethylene vinyl acetate (EVA) multilayer films (300 µm and 360 µm thick) and a polyethylene (PE) multilayer film (400 µm thick). More than 1400 test samples with artificially created leaks were used in sizes ranging from 1 µm to 130 µm. The leaks were laser-drilled into the center of each patch. Microbial ingress and liquid leak testing under various process conditions resulted in a MALL of 2 µm for microbial integrity and the prevention of liquid leakages under most severe use-case conditions. The studies also demonstrated a close relationship between the occurrence of liquid leakage and microbial contamination. Different model solutions were used to evaluate the impact of liquid characteristics, mainly surface tension. The data were applied to build mathematical models for predicting the MALL under any intended use-case condition. By characterizing gas flow behavior over a broad pressure range using various defect sizes, it was possible to create formulas for three different flow regimes. These were suitable for calculating leak size in a defective product directly from the corresponding flow rate or vice versa. Finally, compilation of these different aspects enabled the authors to design and validate non-destructive physical integrity test methods having detection limits correlated to the MALL for SUSs.

Single-Use System Integrity III: Gas Flow Rate through Laser-Drilled Microchannels in Polymeric Film Material.

This study investigated the gas flow mechanism through microchannels in a flexible single-use packaging system composed of multilayer plastic film. The relationship was studied between the gas flow rate and several parameters, which included the differential pressure as an external parameter and channel geometries as internal parameters. Based on the results of this study, empirical formulas were derived that show the different dependency of the parameters for each gas flow regime. It was found that these formulas are suitable for calculating the size of a leak in a defective product directly from the corresponding flow rate. The test samples used were 50 mm patches of an ethylene vinyl acetate multilayer film (300 µm and 360 µm thick) and a polyethylene multilayer film (400 µm thick). Artificial leaks in a range of sizes from 2 µm to 100 µm were laser drilled into the center of each patch. The patches were assembled in a filter holder to form a leak-tight seal and were mounted on the test setup. The test setup included the flow measurement device and the pressure controller that used compressed air to produce a certain differential pressure. Various differential pressures were applied to each test unit to cover the whole range of desired use-case conditions. To understand and interpret the effect of the physics and geometry of the microchannels on flow rate measurement, the microscopic investigations performed in our previous study were used. All measurements were carried out under laboratory temperature conditions of 20°C.

Single-Use System Integrity II: Characterization of Liquid Leakage Mechanisms.

This study investigated the liquid leakage mechanism through microchannels in a flexible single-use packaging system composed of multilayer plastic film. Based on this study, a relationship between the maximum allowable leakage limit (MALL) and the loss of package integrity can be established under different use-case conditions. The MALL is defined as the greatest leak size that does not pose any risk to the product. A specifically designed liquid leak test was used to determine the leakage time, i.e., the time it takes for a package to show leakage. As a result, this method was able to determine the leak size for which no liquid leakage is observed after 30 days. This leak size varied between 2 µm and 10 µm and can be considered the MALL for liquid egress under different use-case conditions. This article also compared the MALL results of this liquid leak test with those of the microbial ingress test, showing a direct correlation between both tests. As test samples, an ethylene vinyl acetate multilayer film (300 µm thick) and a polyethylene multilayer film (400 µm thick) were cut into 50 mm patches. Before the patches were assembled in a filter holder to form a leak-tight seal, artificial leaks in sizes of 2 -25 µm were laser drilled into the center of each patch. The test units were filled aseptically with culture media and mounted vertically on the test setup. Various pressures were applied to each test unit to simulate the constraints that single-use systems may be subject to under real-world conditions. To detect the exact leakage time, electric circuits with timers were attached below each film patch. Microscopic investigations, including light microscopy and computed tomography, were used to interpret and understand the physics and geometries of the microchannels to explain any deviation from the expected results.

Single-Use System Integrity I: Using a Microbial Ingress Test Method to Determine the Maximum Allowable Leakage Limit (MALL).

An aerosol microbial ingress test was specifically designed and used to create a predictive model in order to determine the maximum allowable leakage limit (MALL) of single-use systems (SUSs). The MALL is defined as the greatest leak size that does not pose any risk to the product. The procedure involved taking test samples of film material from single-use bags. As test samples, an ethylene vinyl acetate multilayer film (300 µm thick) and a polyethylene multilayer film (400 µm thick) were cut into 50 mm patches. Artificial defects of 1-100 µm were laser-drilled in the middle of each film patch. The patch was assembled on a holder and properly sealed. The test units were filled aseptically with culture media and placed inside an aerosol chamber. Various pressures were applied to the test unit to simulate the constraints that single-use systems may be subject to under real-world conditions. After an aerosolization cycle with spores of Bacillus atrophaeus, a minimum concentration of 106 CFU/cm2 was reached on the film surface. The test units were incubated for 14 days at 30°C-35°C and visually inspected for bacterial ingress. Thirty samples per defect size were tested. Logistic regression was used to indicate the MALL for a single-use system according to the required risk level. With this method, the probability of the occurrence or absence of ingress for a specific defect size was reported according to the experimental data. In addition to physical parameters, such as the pressure applied and the film material, the effect of the probabilistic nature of the microorganisms in determining the MALL is considered. Although finding an experimental model to predict the MALL for real-life process conditions was the ultimate objective, this paper also presents the microbial ingress test data obtained so far for two extreme conditions. Potential constraints, such as vibration, shock, acceleration, liquid movement, and pressure differentials, observed during normal usage were simulated using two extreme differential pressures, 0 mbar and 300 mbar. The estimated MALL for typical use-case conditions are 10-20 µm for storage applications and 2-10 µm for shipping conditions. The microbial integrity test method used in this article was able to detect bacterial ingress down to 3 µm defect size.LAY ABSTRACT: As use of single-use systems (SUSs) is increasingly expanding into all process steps of commercial manufacturing, integrity failure can significantly impact drug safety, availability, and costs. Consequently, growing industry scrutiny on single-use system integrity (SUSI) is raising the need to develop good science behind reliable determinations of liquid leakage and microbial ingress as well as the appropriate physical integrity testing technologies. In the current study, microbial ingress testing by the aerosol method is used to determine the maximum allowable leakage limit (MALL) for SUSs. To define the MALL, it is generally assumed that a system or product will not show any microbial ingress or leakage at a certain defect size. Statistical analysis of the experimental data in this study indicated the MALL with probability at a certain defect size for each system. As a result, the method studied provides a more accurate way of predicting ingress and increasing safety down the line for drug manufacturers and patients alike.