In situ leak testing, integrity testing and PUPSIT for flow kits using the manual drug product filtration system

Table 1. Equipment used for this study

We used single and redundant flow kits for this study. Single flow kits are used to process through a single sterilizing filter, while redundant flow kits are used to process through multiple filters in series, providing an additional layer of assurance by verifying the integrity of each filter independently.

Table 2. Flow kits used for this study

The manual drug product filtration system includes six movable valves that can be manually removed and repositioned as needed to manage configurations effectively. Additionally, two flush bag trays can be positioned on shelves to hold 5 L, 10 L, 20 L or 2 × 20 L flush bags, with all flow kits gamma sterilized (except for the non-gamma sterilized FAT flow kits).

Flow kit leak testing

We performed flow kit leak testing prior to use. Two types of flow kit leakage tests can be applied using the Palltronic Flowstar IV LGR; the leak test applied to check for potential leaks in flow kit tubing and the single-use system (SUS) leak test performed to check biocontainers for potential leaks. With the manual drug product filtration system, we can check the entire installed flow kit for potential leaks. The following tests can be performed covering different flow path areas:

• Leak test 1: Covers the full flow path from valve position MV001 to the flush bag clamp, keeping product outlet (MV006) closed. This is a flow kit leak test which checks potential leakage of the flow kit tubing.
• Leak test 2: Covers the flow path from valve position MV001 to the flush bag, keeping the flush bag valve (MV005) open and product outlet (MV006) closed. This is a SUS leak test which checks potential leakage of the connected 2D flush bag.

Fig 2. The picture shows leak test 1 (left) and leak test 2 (right) for the redundant filtration configuration of the manual drug product filtration system. The general flow path defined for each leak test is the same irrespective of configuration or size. Filter positions 1 and 2 are highlighted in the left picture. All six valve positions are highlighted in blue.

We leak tested the full range of flow kit combinations. The full flow kit list and parameters input for leak testing can be found in Tables 2 and 3, respectively. The maximum acceptable diffusive gas flow limit for each flow path was based on the volume and type of components used within each flow path studied. These values can be found in Table 7. Leak tests were performed at ambient temperature (15°C to 25°C). During the test we controlled the temperature to ± 1°C of the temperature at the start of the test. After completion of the SUS leak test, we fully evacuated the 2D biocontainers using an air evacuation mat with the required size.

Table 3. Leak testing parameters used in this study to perform leak tests on flow kits

* Maximum acceptable air flows can be found in Table 7.

We started the leak test sequence using the Palltronic Flowstar IV LGR integrity test instrument with a pressurization phase to the entered test pressure (see Fig 3). After this, the stabilization phase began with the stabilization time automatically controlled by the integrity test instrument; it finished as soon as the flow stabilized within defined limits. Once the stabilization phase was complete, the measurement phase commenced, and the diffusive gas flow was measured over the set test time.

During the first 150 s, the integrity test instrument uses values measured during the stabilization phase to calculate the average flow. The flow reported on the graph is the average flow value for the previous 150 s (see Fig 3). If the measured diffusive gas flow is less than or equal to the specified maximum acceptable flow limit (mL/min) the test is a “PASS”. If the measured diffusive gas flow is greater than the specified maximum acceptable flow limit (mL/min), the test is a “FAIL”.

Fig 3. Typical example of a flow kit leak test sequence, (each phase of the leak test is labelled with vertical reference lines showing the transition point between each phase (^#pressurisation phase and ^##vent phase after test completion).

Filter integrity testing

The system accommodates in-line filters sized from KA02 to NP6 (0.2 m² to 5.5 m²) and includes six valve positions. The valves can be manually removed and repositioned as needed to manage configurations effectively. Our study encompassed the full range of filter sizes available, positioned in both the filter 1 and filter 2 positions (Fig 2), and single or redundant filtration configurations. The PUPSIT and the post-use integrity testing of the filters were conducted using a Palltronic Flowstar IV LGR integrity testing device. Filter integrity tests were performed at ambient temperature (15°C to 25°C). At the start of the testing we controlled the temperature at ± 1°C of the temperature at the start of the test, to confirm the filter flow rate was stable before proceeding to measurement.

For PUPSIT, we prepared the filters and integrity tested in situ. We primed all filter capsules at 0.10 L/min (with the exception of KA02 being primed at 0.036 L/min and KA1 being primed at 0.073 L/min). This allowed a slow capsule filling of ~ 1” of filter cartridge media per minute and ensured effective venting. Each filter type was flushed to remove extractables and leachables and achieve successful membrane wetting (see Table 4). Forward flow and bubble point integrity testing were completed for each liquid filter installed. For integrity testing parameters see Table 5. RO water was used as product for the filtration runs.

Table 4. Filter flushing flow rate and time per capsule size

Table 5. Filter integrity test parameters used in this study for forward flow and bubble point tests (minimum bubble point and forward flow acceptable air flow can be found in Table 7)

We started the forward flow integrity test sequence using the Palltronic Flowstar IV integrity test instrument with a pressurization phase to the validated test pressure (see Fig 4). After this, the stabilization phase began with the stabilization time automatically controlled by the integrity test instrument; it finished as soon as the flow stabilized within defined limits. Once the stabilization phase was complete, the measurement phase commenced and the diffusive gas flow through the wetted filter membrane was measured over the set test time.

During the first 150 s the integrity test instrument uses values measured during the stabilization phase to calculate the average flow. The flow reported on the graph is the average flow value for the previous 150 s, (see Fig 4). If the measured diffusive gas flow is less or equal than the specified maximum acceptable flow limit (mL/min) the test is a “PASS”. If the measured diffusive gas flow is greater than the specified maximum acceptable flow limit (mL/min) the test is a “FAIL”.

Fig 4. Typical example of a forward flow integrity test sequence (each phase of the integrity test (pressurization, stabilization and measurement) is labelled with vertical reference lines showing the transition point into each phase. ^#pressurisation phase. ^##vent phase after test completion).

The bubble point integrity test sequence using the Palltronic Flowstar IV LGR integrity test instrument starts with a pressurization phase to 80% of the entered minimum bubble point pressure. After this, and prior to the actual bubble point measurement phase, a leak test phase is performed to confirm that there is no gross leak/defect in the filter system under test. During the leak test phase, the instrument measures the gas flow occurring at this gas pressure and compares this against a limit value which is determined by setting a "module factor”. If the gas flow measured is below the limit value, the bubble point test proceeds and enters the measurement phase. Applied module factors for bubble point testing of filters are shown in Table 6. In the bubble point measurement phase, the bubble point is identified when two sequential pressure decay values are obtained that are significantly greater than the preceding values. If the measured bubble point pressure is equal or greater than the membrane specified minimum bubble point pressure, the test is a “PASS”. If the measured bubble point pressure is less than the membrane specified minimum bubble point pressure, the test is a ‘FAIL’.

Fig 5. Typical example of a bubble point integrity test sequence (each phase of the integrity test (pressurization, leak test and measurement) is labelled with vertical reference lines showing the transition point into each phase. ^#pressurisation phase. ^##vent phase after test completion).