High-Voltage Leak Detection of a Parenteral Proteinaceous Solution Product Packaged in Form-Fill-Seal Plastic Laminate Bags. Part 2. Method Performance as a Function of Heat Seal Defects, Product–Package Refrigeration, and Package Plastic Laminate Lot

Study 1. Detection of Heat Seal Defects

Bag Ear Defects

Visual abnormalities in bag ear heat seals were identified at WAL by microscopic examination of prefilled bags. Examples of bag ear anomalies are shown in Figures 2 and 3. Ear-defect bag samples were filled with product and manually heat-sealed along the opposite bottom end (refer to Part 1 for filling/assembly process). Metal clips were applied to packages as specified by the HVLD method. Bags with ear anomalies were evaluated by HVLD, along with a negative control bag used intermittently to verify HVLD baseline performance.

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Figure 2

Bag 29-1 ear seal defects, magnified images. a) Ear score, b) Ear seal anomaly.

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Figure 3

Bag 29-20 ear seal defects, magnified images. a) Score defect, b) Leak path allowing liquid flow upon bag squeezing, c) Leak path heat sealed to block liquid flow.

HVLD test results of bags with ear defects are presented in Table II. Data sets 1 and 2 show that units with bag ear scores and other anomalies that did not exude visible liquid leakage upon bag squeezing passed HVLD tests, suggesting such defects are cosmetic and pose no risk to package integrity. HVLD failure only occurred when seal gaps permitting visible liquid leakage were present (sets 3 to 5). In these cases, heat sealing the leaking bag ear resulted in passing HVLD tests (sets 4 and 5).

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TABLE II

HVLD of Bag Ear Heat Seal Defects

Both bags in set 5 had visible leakage that the HVLD test failed to find when performed by Method 2 (no clip, bag first); when conveyed through the test zone in the opposite direction, both leaks were found (Method 1: no clip, septum-first). Applying metal clips to the bags improved leak detection regardless of test direction and clip orientation (Methods 3 through 6). These limited results imply better HVLD detection of bag ear leaks if tests are performed septum first; metal clip presence may also improve bag ear leak detection.

Bag Bottom Seal Defects

Bags without any apparent defect were filled and sealed along the bottom edge at WAL using heat seal dwell time set points bracketing the optimal set point of 6.5 (seal examples are shown in Figure 4). Finished units were tested by HVLD by Methods 1 and 2, along with a negative control optimally sealed to verify HVLD baseline performance.

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Figure 4

Bag bottom seals as a function of heat seal dwell time, magnified images. a) Bag 35-2 (setting 3.5), b) Bag 35-3 (setting 4.0), c) Bag 35-5 (setting 4.5), d) Bag 35-7 (setting 5.0), e) Bag 35-10 (setting 8.0).

Table III results indicate HVLD was able to detect leak paths that allowed visible liquid passage, sealed at the worst-case set point 3.5. Both bags sealed at set point 4.0 were identified as leaking, despite the absence of visible leakage. One of the two bags sealed at setting 4.5 visibly leaked; both bags were identified as leaking 1 of 2 times by each Method 1 and 2. Bags prepared at settings 5.0 and above passed all HVLD tests. In conclusion, HVLD is able to detect leaks located in the bag seals. The likelihood of seal leak detection increases as a function of heat seal deterioration and the presence of liquid in the leak path.

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TABLE III

HVLD of Poorly Sealed Bags

Study 2. Detection of Hole Defects as a Function of Product–Package Storage

Physico-chemical stability of the proteinaceous active drug component of the bagged solution formulation is best assured by long-term storage of the product–package at refrigeration with limited exposure to higher, ambient temperature conditions. Therefore, Study 2 explored the ability of HVLD to identify leaks in product-filled packages after extended ambient plus cold temperature storage. This question is especially relevant because protein-based compounds tend to clog leak paths. Clogged leaks may be missed by other test methods that rely on the flow of liquid or gas through the leak path for detection (e.g., dye ingress, mass extraction, and vacuum decay).

The same positive controls used for Part 1 Method Validation Day 1 tests were used for Study 2. The laser-drilled hole sizes ranged from 2.7 to 10.5 μm in nominal diameter and represented six bag locations (left and right seal edges, left and right corners, bottom seal, and center). Following Validation Day 1 tests, these positive controls were kept in an open laboratory environment at ambient conditions for 4 days. Afterward, these bags were stored at 5 ± 3 °C for an additional 17 days for a total of 3 weeks. On the final day, bags were allowed to come to ambient temperature, and were tested by HVLD within a few hours of cold storage removal, along with negative control bags. Testing order duplicated that of Method Validation Day 1.

Test results are found in Table IV, and are highlighted as follows.

• Methods (1, 2, 5, and 6) accurately identified all negative and positive control test samples. (Method 2 negative control bag 33-23 “failed,” but a leak discovered at the bag's seal edge confirmed the accuracy of this result.)

• Method 3 gave one false-positive result out of 12 tests performed (Bag 34-6). An occasional false positive is not surprising given that the Ref Max acceptance criterion was set 3 standard deviation units above the negative control mean (refer to Part 1 for details). This limit was intentionally chosen, as the impact of falsely rejecting a good package was considered inconsequential compared to that of missing a leaking one.

• Method 4 tests yielded one false-negative result in 12 tests performed (Bag 38), although a retest produced a fail result. This same holed sample also avoided detection during Day 1 Validation tests, likely due to the hole's location on the partially collapsed bag surface. As confirmed in Part 1, a leak in a bag partially or fully collapsed due to low fill or product leakage might evade detection if HVLD electrode brushes fail to contact the defective area during test. Screening packages for improper fill can eliminate this risk of incorrectly passing largely leaking packages.

To conclude, storage of product-filled bags for up to 21 days, including 4 days at ambient temperatures and 17 days at refrigeration, had no measurable impact on HVLD reliability. With the exception of a single false-positive test result, HVLD tests performed on product-filled bags the day of fill-and-seal mirrored those generated after a 21-day bag storage period. This finding is significant given the proteinaceous nature of the active component, known from past experience by Novo Nordisk A/S that this product formulation tends to clogs leak paths, making leak detection by other test methods such as vacuum decay unreliable.

Study 3. HVLD of Cold Product–Packages

All prior study HVLD tests were performed on ambient temperature product-filled bags. Study 3 explored the consistency of HVLD readings for no-defect product-filled bags if tested when still cold from refrigerated storage.

Test units were all product-filled negative control bags prepared from three laminate film lots (designated A, B, and C). During 5 ± 3 °C storage, bags were spaced apart laying face-down on metal trays. The applicable metal tray with bags was removed from refrigerated storage immediately prior to HVLD test start. Each bag was manually transferred from the metal tray to the HVLD test carriage, touching only the outer bag edges. Bags were either tested in “whole sets” (meaning all test bags to be tested by a given method were removed from cold storage simultaneously, then sequentially tested), or they were tested in multiple “subsets” (meaning a smaller group of bags was removed from refrigeration at the same time, then each bag was sequentially tested). The same bags used for whole set tests were also used for subset tests. HVLD tests started within 1 min after bag set storage removal. All data are time-stamped with the first bag test as time zero. Two days of replicate tests were performed. All six HVLD methods were included, although only one bag lot was used per method. Bag lots were matched with methods to ensure the two no-clip methods were evaluated by two different bag lots, and the three with-clip methods were evaluated by at least two different bag lots.

All six HVLD test methods yielded similar data. For brevity, only Bag lot C Method 6 test results are graphically illustrated (Figures 5 to 7). Sequential HVLD voltage readings of the bags tested as one set started low, slowly climbed, and then declined (Figures 5 and 6). This peaking effect was confirmed to correlate to time post-refrigerated storage. When all bags were removed from the refrigerator simultaneously (i.e., as a whole set), one peak was observed (Figures 5 and 6). When these same bags were placed back in the refrigerator and were removed in three smaller subsets, three peaks were observed (Figure 7). In all cases, maximum voltage was observed for bag tests time-stamped within about 3 min post-refrigerated storage removal. Often, highest readings exceeded the Ref Max limit. Interestingly, the rise in voltage readings corresponded to condensation observed on cold bag surfaces upon exposure to room temperature and humidity conditions. Bag surface condensation disappeared about 5 to 6 min post-refrigeration, as voltage readings returned to baseline values, even though bags still felt cool. Peak voltage readings varied from day to day as Figures 5 and 6 illustrate. This variation in maximum peak voltage reading is likely a function of test area relative humidity; however, no exact correlation was attempted. In summary, this work found that bag surface condensation will elevate HVLD voltage readings temporarily, risking false-positive test results. To avoid this, refrigerated bags should be allowed to warm at temperature and humidity conditions that preclude bag surface moisture prior to HVLD tests.

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Figure 5

Bag lot C (whole set) tested post refrigeration by HVLD Method 6, Day 1.

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Figure 6

Bag lot C (whole set) tested post refrigeration by HVLD Method 6, Day 2.

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Figure 7

Bag lot C (3 subsets) tested post refrigeration by HVLD Method 6, Day 2.

Study 4. HVLD as a Function of Package Plastic Laminate Lot

Study 4 explored the effect of laminate film manufacturing lot on HVLD test results. About 33 WAL-made negative controls were prepared for each of three bag film lots, designated lots A, B, and C. All three lots were sourced from a single converter, but lot B ingredients came from a different raw material supplier. All negative controls were HVLD-tested by all six methods on 3 replicate test days. Bags were stored between tests at ambient conditions.

Results are summarized in Table V. Lots A and C performed similarly with no more than 2 units exceeding the Ref Max limit by any given method over 3 test days. Each lot A and C also had one unit to fall below the Ref Min warning limit when tested by Method 1. In contrast, lot B yielded markedly more failing results. This was especially apparent for test Methods 1, 3, 4, and 6, in which false-positive rates ranged from 5% (Method 3) to 15% (Method 1). Voltage reading coefficients of variation were similar for the three lots; therefore, the rise in lot B false positives was not due to greater data variability, but to an upward shift in the HVLD baseline for lot B. The unique characteristic(s) of lot B affecting HVLD readings could not be pinpointed. However, to ensure consistency in HVLD results, steps would be required to confirm bag HVLD performance prior to approving a laminate film supplier.