Abstract
Frozen-state storage and cold-chain transport are key operations in the development and commercialization of biopharmaceuticals. Today, several marketed drug products are stored (and/or shipped) under frozen conditions to ensure sufficient stability, particularly for live viral vaccines. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial at the target's lowest temperature to ensure container closure integrity and thus both sterility and safety of the drug product. The container closure integrity assessment in the frozen state (e.g., −20°C, −80°C) should include container closure integrity (CCI) of the container closure system (CCS) itself, impact of processing (e.g., capping process on CCI), and impact of shipment and movement on CCI in the frozen state. The objective of this work was to evaluate the impact of processing and shipment on CCI of a CCS in the frozen state. The impact on other quality attributes was not investigated. In this light, the ThermCCI method was applied to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials and to evaluate the temperature limits of rubber stoppers. In conclusion, retaining CCI during cold storage is mostly a function of vial–stopper combination, and temperatures below −40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Regarding the impact of shipment on the CCI of glass vials, no indication was given at room temperature, −20°C, or −75°C when compared with static storage at such temperatures.
LAY ABSTRACT: Today, several marketed products are stored (and/or shipped) under frozen conditions to ensure sufficient stability. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial and ensure container closure integrity and thus both sterility and safety of the drug product. The impact of processing and shipment on the container closure integrity (CCI) of a container closure system (vial, stopper, and flip-off cap) in the frozen state is assessed. A helium-leakage test at low temperature (ThermCCI) was used to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials as well as the temperature limits of rubber stoppers. In conclusion, it was found that retaining CCI during cold storage is mostly a function of vial–stopper combination and that temperatures below −40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Additionally, it was observed that the shipment of the frozen glass vials did not affect the CCI.
- Container closure integrity
- Frozen drug product
- Vial
- Helium-leakage test
- Headspace analysis
- Transportation
- Capping
- Residual seal force
Introduction
Frozen-state storage and cold-chain transport are key operations in the development and commercialization of biopharmaceuticals. The science and technology of these operations have advanced significantly over the past few years, moving from empiricism to fundamental understanding. Today, several marketed products are stored (and/or shipped) under frozen conditions to ensure sufficient stability, particularly for live viral vaccines (1). In addition, drug product storage at −20°C could be a potential mitigation in case of instabilities at 5°C, especially during early stages of development.
If these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial at the target's lowest temperature to ensure container closure integrity and thus both the sterility and safety of the drug product (2⇓–4). Given that the glass transition temperature (Tg) of standard stoppers is in the range of −55°C to −65°C (5, 6), the low temperature may lead to shrinkage and reduced elastomeric properties of the rubber with subsequent ingress of (unsterile) atmospheric air and loss of sterility, even though the elasticity is regained by bringing the stopper back to room temperature. Hence, close consideration should be given to the assessment of container closure integrity (CCI) of a container closure system (CCS) during frozen-state storage and transportation.
The CCI assessment in the frozen state should include CCI of the CCS itself, impact of processing (e.g., capping process on CCI), and impact of shipment and movement on CCI in the frozen state.
The objective of this work was to evaluate the impact of processing and shipment on CCI of a CCS in the frozen state. The impact on other quality attributes was not investigated. The ThermCCI method is described elsewhere (1) and was used to assess pCCI for a variety of container closure systems under variable capping forces as well as after shipment.
Duncan et al. correlated vial seal tightness to CCI at various storage temperatures (7). They observed that sufficient capping compression as well as an appropriate vial and stopper combination are key parameters when considering the capping process of a CCS. For a sealing rubber component, both the elastic property and the viscous property of rubber are important. On the one hand, an applied stress (sealing force) induces a corresponding strain, creating a contact stress. This stored internal energy is the residual seal force (RSF). On the other hand, the viscous property of rubber allows a considerable segmental flow of material. This movement can fill gaps and voids in the sealing surface (8). This study builds on these findings by showing how a variable capping force can influence the CCI at low temperatures, as measured by the ThermCCI method and its comparison to headspace analysis.
This study is based on the hypothesis that storage times should not impact CCI. However, shipment during storage at those temperatures could have a significant effect owing to the impact of vibration and drops on hardened rubber and glass. In this light, we have used the ThermCCI method to evaluate the impact of shipping stress on CCI of frozen vials.
Finally, the ThermCCI method allows for real-time monitoring of the temperature limits of rubber stoppers. In other words, the break-loose temperature (Tb) can be obtained for a specific primary packaging configuration with/by application of the ThermCCI method. This is the temperature below which the CCS starts to exhibit leak rates higher than the acceptance criterion as the temperature decreases. Thus, it is not considered tight anymore. As reported elsewhere, the impact of process parameters was also studied by finite element simulations, including the Tb (9).
Materials and Methods
Materials
Type I glass vials (2 mL) (Schott AG, Mainz, Germany), with a 13 mm vial neck diameter, and Type I glass vials (20 mL), with a 20 mm vial neck diameter, were sealed using steam-sterilized serum or lyophilization (lyo) rubber stoppers with a Tg of −66.5°C. All vials (2 mL, 20 mL) were sealed using a 13 mm crimp cap or a 20 mm crimp cap made of aluminum and polypropylene, respectively.
Equipment
The Integra Westcapper (Genesis Packaging Technologies, Exton, PA, USA) capping machine was used to seal the vials used in the capping study. Vials for the shipping study were sealed with a semiautomatic capping machine (model Milano, Capsulit S.p.A, Roncello, Italy).
A helium mass spectrometer (model HLT-560, Pfeiffer Vacuum Technology AG, Zürich, Switzerland) was used for the CCI tests. The thermo-chamber was custom-made (A-Z Labortechnik, Allschwil, Switzerland).
A dynamic temperature control system (model Unistat 705w, Peter Huber Kältemaschinenbau GmbH, Offenburg, Germany) was used to control the temperature of the thermo-chamber (Figure 1). A thermocouple connected to a temperature data logger (model Libero Te1P, ELPRO-BUCHS AG, Buchs, Switzerland) monitored the temperature of the vial during CCI testing. The thermocouple is inserted in the thermo-chamber unit, close to the vial neck. The sample preparation for the helium-leakage experiment was performed as described elsewhere (1).
Picture of helium-leakage test setup at low temperatures (right) and its comparison with the test setup at room temperature.
The oxygen headspace analysis was performed in a headspace analyzer (model FMS-760, Lighthouse Instruments, Charlottesville, VA, USA). The carbon dioxide headspace analysis was performed in a headspace analyzer (model FMS-CO2, Lighthouse Instruments). The headspace analyzers were calibrated using NIST-traceable standards containing a mixture of O2/CO2/N2 of the following standard CO2 pressure in torr (0; 7.2; 14.5; 29.0; 57.9; 145.0), as recommended by the manufacturer. The accuracy was 0.5% of the measured value.
The Genesis Packaging Technologies RSF tester was used to measure the sealed vials. The RSF tester's measuring principle is described elsewhere (10). Different cap anvils were used to measure 2 mL vial samples and 20 mL vial samples. The flip-off button was removed prior to the RSF measurement (11, 12). All samples were measured at comparable time points after the capping process.
A magnifying glass with illumination (model RLLQ 48R, Waldmann, Villingen-Schwenningen, Germany) was used to visually inspect samples for defects.
In the transport simulation study, the following elements were used: an IMV Europe Ltd. (Letchworth Garden City, UK) shaker (model V826), a power amplifier (model SA6M-J50EM), a vibration controller (model K2-CE), and the K2 software; a drop table (model AD-160, L.A.B, Itasca, IL, USA); a sensor signal conditioner (model 482C, PCB Piezotronics, Depew, NY, USA); accelerometers (model 4513, Brüel & Kjaer, Naerum, Denmark); and a vacuum chamber (model DN 750, Pfeiffer, Zurich, Switzerland). The test standard ASTM 7386/TS-4 (12) at assurance level I was used in the transport simulation testing without the concentrated impacts and the top-load aspect of vibration, as these test inputs do not add any value to this study.
Methods
Impact of Variable Capping Force:
To study the influence of a variable capping force on CCI at variable temperatures, 2 mL as well as 20 mL vials were stoppered and capped with serum and lyo stoppers. After capping, the RSF was measured, and the CCI was confirmed at room temperature, −20°C and −60°C.
Several capping parameters were tested and the RSF measured. These included capping parameters to obtain RSF values <20 N on the lower range, as well as capping parameters to obtain RSF values >100 N on the upper range. Capping parameters to obtain RSF middle range values 40–60 N were also tested. This range was chosen on the basis of the ability of the CCS to maintain the pCCI (10).
Impact of Shipping Stress:
First, 2 mL vials were filled with 2.5 mL distilled water and then with helium (g; 99.999%, Carbagas AG, Basel, Switzerland) in an upright position. The vials were closed with serum stoppers and then crimped. The headspace oxygen levels were checked immediately after filling using an oxygen headspace analyzer (results not shown). Accepted residual headspace oxygen levels after helium filling were <5% atm O2, which guaranteed that the oxygen, which had been present initially in the vials, was satisfactorily displaced by the helium.
Artificial leaks were introduced in the samples as positive controls by inserting uncoated copper (Cu) wires (Elektrisola Feindraht AG, Escholzmatt, Switzerland) of a defined diameter (60 μm) between the rubber stopper and the vial. This Cu wire size has been repeatedly shown to generate leaks when inserted between the rubber stopper and the vial; hence, it is used for the positive control vials (1). Vials with artificial leaks were also filled with helium and stoppered and crimped after Cu wire insertion.
The carbon dioxide headspace was measured at the initial time-point, and the vials were visually inspected for defects.
Three transport test specimens were subjected to a transport simulation study. A cold shipper (model CCS-12, va-Q-tec, Würzburg, Germany) was used for the transport simulation at room temperature, with cooling elements at room temperature. A va-Q-tec (CCS-12) cold shipper was also used for the transport simulation at −20°C, with cooling elements preconditioned at −20°C. Finally, a Styrofoam ice chest was used for the transport simulation at −75°C, filled with dry ice. The three transport test specimens were preconditioned at their respective target temperatures for 12 h prior to the tests. The temperature inside the shippers was tracked with ELPRO temperature data loggers (ELPRO-BUCHS AG, Buchs, Switzerland).
Additionally, three groups of control vials were stored in static conditions at the respective temperatures: at room temperature, at −20°C in a freezer, and at −75°C in dry ice.
After the transport simulation study, the vials were visually inspected for defects. The carbon dioxide concentration in the headspace was measured at room temperature. Finally, the CCI of all vials was analyzed by the helium-leakage test at variable temperatures, namely, at room temperature, at −20°C, as well as at −60°C. The results were compared to the CCI of the control group stored under static conditions at the corresponding temperatures.
Holding times at a specific temperature were matched for both controls under static conditions and samples subjected to shipment. The sample size consisted of 10 vials per group. The duration of the transport simulation experiment was approximately 12 h. The time lapse between the transport simulation experiment and sample measurement was approximately one week.
The acceptance criteria for headspace analysis were defined as follows: all vials showing a concentration of ≤0.07% atm CO2 were considered tight (1). Regarding the helium-leakage test, a helium-leakage rate of <1 × 10−7 mbar × L/s implied that the sample was considered tight against microorganisms. The leak rate cutoff for CCI failure was set to 1 × 10−7 mbar × L/s, based on prior data that correlated to microbial ingress (mCCI) and helium leakage rates (pCCI) (1, 9).
Evaluation of Temperature Limits of Rubber Stoppers:
The ThermCCI setup allows monitoring temperature limits of rubber stoppers in real time, i.e., the temperature below which the CCS starts to exhibit leak rates higher than the acceptance criterion and is not tight anymore. The helium-leak rate can be monitored as the system cools the vial to the target temperature. The equilibration time is chosen for the temperature of the system and the temperature of the vial to match ±5°C. Usual equilibration times would be in the range of 15 min to 1 h, depending on the target temperature to reach and the instrument history.
In this study, the CCI was measured at room temperature, −20°C, and −60°C by the ThermCCI method. The ThermCCI setup was started at room temperature, and the vials were inserted in the vial adapter at room temperature. The cooling unit in the setup cooled step by step from room temperature to −20°C and to −60°C (minimum temperature technically feasible with reproducible results), and CCI was measured by helium-leakage test at each of the target temperatures.
The headspace analysis method only allows CCI measurements at room temperature.
For CCI method comparison purposes, prior to CCI measurement, either by ThermCCI method or by headspace analysis, the samples were stored at room temperature, −20°C, and −75°C for a defined time. These specific temperatures were chosen to mimic the effect of storage and transportation of vials at such temperatures and to further allow indirect comparison of the ThermCCI method with the headspace analysis method. Room temperature was chosen as the reference temperature (control). The temperature −20°C was chosen as a temperature below zero but above Tg. The temperature −75°C was chosen as the temperature of the samples when stored in dry ice, where the temperature is below Tg and a high concentration of CO2 is present. In the case that the sealing of the vial and the stopper would allow for a leak at such temperature, the CO2 could ingress in the vials and remain trapped upon vial warmup. The trapped CO2 could subsequently be detected by headspace analysis at room temperature. The authors reported this approach previously (1).
Finally, the CCI results obtained by CO2 headspace analysis (measurement at room temperature after storage at −75°C and thawing to room temperature) were compared with the results obtained by the helium-leakage test (ThermCCI) at −60°C (measurement directly at −60°C after storage at −75°C and thawing to room temperature).
The thawing step is not expected to have an impact on the properties of the rubber or the CCI results, as the rubber properties allow for the recovery of elasticity after temperature cycling. If there is a leak present at low temperature, the ThermCCI method can detect it directly at the target low temperatures. However, the headspace analysis method needs a surrogate (CO2 ingress) to detect any transient leak during cold storage. Therefore, the defined transportation and storage temperatures are merely used to have a common baseline to compare both methods and are not expected to match the actual measurement temperature by the ThermCCI method, which is the only method here presented that can measure CCI directly at temperatures below zero.
Results and Discussion
Impact of Variable Capping Force
Table I shows the correlation between RSF at lower, middle, and upper ranges and CCI values at variable temperature.
Comparison of the Influence of Capping Force on the CCI of 2 mL and 20 mL Vial Neck Size Using Serum (Liquid) and Lyo (Lyophilization) Stoppers
The results show that crimped 2 mL vials sealed with serum and lyo stoppers are all tight in the range of tested RSF, both at room temperature and at −20°C.
The results show that crimped 20 mL vials sealed with serum and lyo stoppers are all tight in the middle and upper ranges of capping forces, both at room temperature and at −20°C.
CCI testing at −60°C shows most vials leaking, regardless of capping force and CCS configuration. At such temperatures, the loss of integrity varies unpredictably.
In the lower range of capping forces, un-tight vials were only observed for the 20 mL serum configuration at room temperature and at −20°C. The lower RSF range chosen for these vials (18 ± 3 N) seems to be in the lower limit, hence not ensuring CCI. On the other hand, the 2 mL vials were tight at all instances.
These observations (2 mL vs 20 mL) could be explained by the different fitting of stopper and vial, as the material of both stopper sizes (13 mm and 20 mm) is the same; therefore, its elastomeric properties are alike. As Lam et al. described, the performance of vial–stopper combinations depends on many design aspects (14).
All vials that are un-tight at room temperature are also un-tight at −20°C. In contrast, all vials that are tight at room temperature are also tight at −20°C.
It was not observed that lower capping force would systematically correlate to high CCI failure during deep cold storage as compared to room temperature.
Impact of Shipping Stress
Table II presents a summary of the CCI testing results before and after subjecting the vials to shipping stress. As mentioned, the acceptance criteria for the helium-leakage method are defined as follows: vials are not considered tight if the helium-leak rate is ≥1 × 10−7 mbar × L/s.
Container Closure Integrity Testing Results of 2 mL Vials with Serum Stoppers Under Static and Under Dynamic Conditions, as Measured by the ThermCCI Method at Variable Temperatures
As expected, all positive controls (60 μm Cu wire; results not shown) were leaking.
As shown in Table II, no evident impact on CCI by transportation was observed. Hence, the results under static conditions and under dynamic conditions were corresponding well. Both under static conditions and under dynamic conditions, all vials were tight at room temperature as well as at −20°C, and their behavior was unpredictable at −60°C. The measurement procedure by the ThermCCI method has been described elsewhere (1). For the selected CCS, it can be stated that transportation does not create permanent damage to the CCS.
However, we hypothesize that shipment during storage at low temperatures could affect CCI owing to the impact of both vibration and drops on hardened rubber and glass. The headspace analysis results thus help to understand the potential loss of CCI during transportation in frozen state (Table III).
Comparison of CCI Results Obtained by Headspace Analysis (Performed at Room Temperature, After 1 Day of Storage in Dry Ice) and by the Helium-Leakage Test (ThermCCI) Measured Directly at −60°C (After 1 Day of Storage in Dry Ice) in 2 mL Vials with Serum Stoppers
In Table III, the headspace analysis indicates that all vials are tight under static and under dynamic conditions.
The comparison of the CCI results obtained byheadspace analysis with the results obtained by the helium-leakage test (ThermCCI) at −60°C indicates that headspace analysis is much less sensitive than the helium-leakage test. In all cases, a higher number of leaking vials was detected by the ThermCCI method other than by CO2 headspace analysis.
The CO2 headspace analysis after storage under static conditions showed that none of the vials (no wire) was leaking after having been exposed to dry ice for 1 day, as described elsewhere (1). However, positive control vials (60 μm Cu wire) showed significant leakage (elevated CO2 ingress, approximately 15% atm) as expected (results not shown) when stored in dry ice.
The helium-leakage method at −60°C after storage under static conditions shows leaking vials at all conditions. Hence, the ThermCCI method resulted in higher sensitivity than headspace analysis, corresponding well to prior experiments (also under static conditions) (1).
CO2 headspace analysis after dynamic storage conditions (simulated transport) showed that none of the vials (no wire) was leaking, regardless of the storage and transportation temperature. After shipment, the helium-leakage method at −60°C showed leaking CCS. Hence, greater sensitivity of the ThermCCI method over the CO2 headspace analysis method is presented.
Additionally, no significant differences were observed when comparing the number of leaking vials, measured by ThermCCI after dynamic conditions, to the number of leaking vials, measured after static storage conditions, indicating that the shipping stresses do not introduce permanent damage to the CCS.
No evident impact of shipment at room temperature, −20°C, or −75°C on the CCI of glass vials was observed in this study.
Temperature Limits of Rubber Stoppers: Tb Temperature
Shrinkage of the stopper owing to low temperatures is assumed to be the main factor for leakage. Tb is the temperature at which the vial starts to exhibit leak rates higher than the acceptance criterion as the temperature decreases.
Applying this method, the low temperature limits for rubber stoppers were determined. Table IV summarizes the evaluation of temperature limits (Tb) for the lyo stopper in 20 mL vials. Overall, it was observed that the temperature at which the vials exhibit leak rates above the threshold value of 1 × 10−7 mbar × L/s is below −40°C. Tb varies slightly depending on the configuration.
Evaluation of Rubber Stopper Temperature Limits in Lyo Stoppers with 20 mm Vial Neck Size
Conclusions
Conclusions regarding the impact of variable capping force are as follows:
At −20°C and at room temperature, 2 mL vials exhibit no impact of capping parameters on the helium-leakage rate in the range of measured RSF from 10 to 110 N for both serum and lyo stoppers.
At −20°C, 20 mL vials with a serum stopper show leakages at measured RSF values of approximately 15 N. No impact on the helium leak rates was observed for the CCS consisting of a 20 mL vial and a lyo stopper.
Conclusions regarding the impact of shipping stress are as follows:
The helium-leakage test (ThermCCI) method is more sensitive than the CO2 headspace method.
The CCS were tight when stored and shipped at both room temperature and −20°C under identified standard middle- and upper-range capping parameters.
The CCS stored and shipped in dry ice were not tight when measured by the ThermCCI helium method at −60°C. At such temperatures, the loss of integrity varies unpredictably.
According to the standard ASTM 7386/TS-4, shipping stress should not create permanent impact on/damage to the CCS.
No significant differences were observed by ThermCCI when comparing the number of leaking vials after dynamic conditions to the number of leaking vials observed after static conditions. Hence, there is no evident impact of shipment on the CCI of glass vials at room temperature, −20°C, or −75°C.
The temperature at which the tested configurations exhibited leak rates above the threshold value of 1 × 10−7 mbar × L /s was below −40°C.
Retaining CCI during cold storage is mostly a function of vial–stopper combination.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
Acknowledgement
The authors would like to acknowledge Professor Dr. Hanns-Christian Mahler (Lonza AG, Basel, Switzerland) for his valuable input and Daikyo Seiko Ltd. for the low Tg parts.
- © PDA, Inc. 2018
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