Marc Hogreve is senior engineer, Integrity Testing Solutions at Sartorius Stedim Biotech.

Marc Hogreve

Single-use technologies have transformed biopharmaceutical manufacturing by providing opportunities to reduce costs, improve flexibility, and shorten cycle times. Manufacturers of biopharmaceuticals want to maximize the benefits they can derive from single-use technologies and are becoming increasingly confident when integrating single-use assemblies into processing steps that have a greater impact on product quality. Today, the expansion of such technologies into more critical applications, such as drug substance and drug product storage, has naturally raised new challenges for the industry to address. Industry surveys show that these challenges include quality assurance, supply chain reliability, supplier change control, raw material transparency, and maintaining the integrity of assemblies (1, 2). “A lack of robustness can lead to contamination of process fluids or drug products and, subsequently, loss of time and materials,” Weibing Ding, PhD, principcal scientist, Process Development at Amgen said in a statement. The cost of bag failures could be between $100,000 to $1 million per bag.

	To avoid these costs, established suppliers of single-use bags provide assurance of container-closure integrity across the entire product lifecycle. They do this by applying quality-by-design principles, performing process validation, and ensuring process control. Quality control policies ensure the integrity of the film, the welds, and the bag chamber.

	As part of their quality risk management strategy and in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q9 biomanufacturers can reduce the risk of losing high-value product and enhance patient and operator safety by performing a non-destructive point-of-use leak test on all single-use bags used in critical process steps (3). This ensures no damage occurred to the single-use bags during shipping, storage, and handling at the user site.

	In this article, a leak test method was validated to detect leaks by means of pressure decay for 2D storage bags (Flexboy 2D, Sartorius Stedim Biotech). This article describes the validation of a pressure decay test method used for the point-of-use leak test at the user site of Flexboy bags from 50 mL to 50 L with the FlexAct BT and the Sartocheck 4 plus Bag tester. A preliminary parameter study was first performed to pre-determine the test pressure, stabilization time, and test time. A complete validation study was then carried out to validate the parameters, the maximum allowable pressure decay, and the leak detection limit.

Bag integrity test hardware and instrument


	Test method development and validation were performed using the FlexAct BT system with Sartocheck 4 Plus Bag tester. The bag tester was equipped with two bag holders, each consisting of two metal plates with porous spacers. By using porous spacers, the film surface of the bag is not in direct contact with the stainless steel holder during the test. Any potential masking effect is eliminated and environmental heat transfer is reduced. The holders allow performance of the leak test with a small and reproducible inflating bag volume and at a higher test pressure. This is critical for achieving the test sensitivity and reliability required. Furthermore, the holders protect the bag from mechanical stress during the test.


	The designs of 50 mL to 50 L Flexboy 2D bags for pre-use have been adapted to meet the specific requirements of critical process applications and pre-use leak testing. This requires the installation of a sterile vent filter line to permit the performance of the test under conditions that maintain the integrity and the sterility of the system (

ASTM F2095-01: Standard Leak Test for Pressure Decay Leak Test for Nonporous Flexible Packages with and without Restraining Plates 

(4). Once the test pressure has been set and allowed to stabilize, the system measures the pressure decay and compares the result to an acceptance criteria determined during the development and validation of the method (

). The pressure decay test method developed detects defects according to the leak-rate specification on the film, welds, and ports of the bags. Because the validated test method is non-destructive, it is compatible with performing pre-use leak tests on 100% of bags used at a biologics production facility.


	The aim of the initial phase of the study was to pre-determine the stabilization time and test time parameters necessary to detect a defect reliably over the volume range of the bag configurations. The range contains bags with 10 different volumes from 50 mL to 50 L. For each of the 10 bag sizes, three non-defective test samples, and three defective test samples were prepared. Defects were introduced into film samples with a laser drill and flow calibrated hole.

	All 60 samples were tested at a fixed 300-mbar test pressure. For each test run, four different stabilization times of 60 seconds, 120 seconds, 180 seconds, and 240 seconds were used. The pressure drops were continuously measured and reported every second across the entire test time from 0 to 240 seconds during the four different stabilization time test runs.

	The minimum, the mean, the maximum, and the standard deviations (σ) of the measured pressure drops were calculated for the four different stabilization times separately, with non-defective and defective test samples for each different bag volume. The optimum stabilization time and test time were chosen to provide a selective test method capable of differentiating defective bags form non-defective bags (i.e., the points where the error bars [± 3 σ] from the defect is distinguished from the error bars [± 3 σ] from the non-defective measurements). Initial results showed that, for tests performed with a 120-second stabilization time and 90-second test time, a difference between the observed pressure drops of defective and non-defective test samples could be detected with a probability of 99.9%. A safety margin was then applied by doubling the stabilization and test times to avoid false positive and false negative results during normal operations. These timings were selected for the subsequent validation study.


	The purpose of the validation study was to verify the ability of the pre-established test method and test parameters to detect a defect reproducibly and accurately over the volume range of the bags. The validation study was performed with a statistically significant number of bags from different routine production lots to provide a robust validation and test method. For each of the 10-bag volumes, 32 non-defective test samples from production with representative raw material and process variability, and 32 test samples with a defect were used. This represented a total of 640 samples tested during the validation study. Every defect film sample was checked for its calibrated hole size before it was used. Tests were performed using the pre-determined test pressure of 300 mbar, stabilization time of 240 seconds, test time of 180 seconds, and a defect size.

	This study allowed the validation of the pre-established test parameters and the setting of a maximum allowable pressure decay specification at 3.1 mbar. The validated pressure decay method was capable of reliably detecting defective bags from non-defective bags with a given leak detection limit in less than 10 minutes including installation and testing.

	The 3.1 mbar maximum pressure decay specification was established with a 6σ interval of confidence for the full range of bags from 50 mL to 50 L to avoid false positive or false negative results under real testing conditions (

). The final test parameters established during these studies are provided in 

	The authors developed and successfully validated a pressure-decay leak test for 2D bags using commercially available equipment and proved that it is a robust and predictive method for the reliable detection of leaks. Using the method, non-defective bags gave results below the maximum pressure drop specification. The bags into which a defect was deliberately introduced gave results above the maximum pressure drop specification and failed the test. The method is, to the authors’ knowledge, the first point-of-use leak test capable of detecting down to 10 µm defects in 2D bags, irrespective of their volume. The sensitivity of the test is independent of 2D bag size.

	1. Aspen Brook Consulting LLC, 6th Annual Survey of the Single Use Bioprocessing Market 2014 (Aspen Brook Consulting LLC, 2014). 
	2. Bioplan Associates, 12th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production (Bioplan Associates, 2015).
	3. ICH, Q9 Quality Risk Management (ICH, 2005). 
	4. ASTM F2095-01: Standard Leak Test for Pressure Decay Leak Test for Nonporous Flexible Packages with and without Restraining Plates (West Conshohocken, 2001).

	Carole Langlois is senior product manager, Fluid Management Technologies; Marc Hogreve is senior engineer Integrity Testing Solutions; and Jean Marc Cappia is group VP Marketing & Product Management Fluid Management Technologies all at Sartorius Stedim Biotech.

	When referring to this article, please cite it as C. Langlois, M. Hogreve and J. Cappia, "Point-of-Use Leak Testing of Single-Use Bag Assemblies,”

Articles

The authors describe the development and validation of a highly sensitive point-of-use pressure decay test.