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This report describes the first known attempt at quantifying the success of such processes in inducing nucleation on a 56–m2 freeze dryer operating at a load of 195,960 vials.
The freeze-drying community has dedicated resources for artificially inducing nucleation since it was introduced as a means for reliable prediction of product stability, scale-up, and primary drying time reduction. Broadly, there are at least five known techniques for artificially inducing nucleation with the same basic characteristics, but only few studies have tested these processes at large scales of operation (>35-m2 shelf area, >150,000 vials) uniformly. This report herein describes the first known attempt at quantifying the success of such processes in inducing nucleation on a 56-m2 freeze dryer operating at a load of 195,960 vials. It was found that thermal gradients within freeze-dryer chambers typical of the production environment should be considered when designing the scale up of the freezing process in lyophilization.
Freezing, primary drying, and secondary drying are three mains steps of the freeze-drying process. While the primary and secondary drying steps of freeze-drying for the most part are well-controlled, the freezing step remained largely uncontrolled until recently (1, 2). Despite the current variety of nucleation technologies available, there is little or no data to prove scalability of these technologies in large freeze dryers, especially those on production scale (3). The overall efficiency and consistency of the lyophilization process, as well as the reliable prediction of product quality, largely depends on the nucleation temperature (4). Furthermore, having a reliable method to induce nucleation in production freeze dryers simplifies the scale up of processes developed at laboratory scale (5).
The temperature difference between the equilibrium freezing point and the ice nucleation point is known as super-cooling. Lower nucleation temperatures result in more ice-nuclei across the ice frozen matrix with smaller crystals (6). These smaller ice crystals leave behind smaller pores as the product dries, creating a high specific surface area within the product, which presents a greater resistance to sublimation and results in a longer primary drying period. This is undesirable as the primary drying step of the freeze-drying process is quite often the rate-limiting step in the drying process (6, 7). The product may also visually appear to be inconsistent across the batch and thus, reconstitute differently. For this reason, it is beneficial to narrow the range of product temperature at which nucleation can be induced across the product batch.
By contrast, a higher nucleation temperature, or a lower degree of super-cooling, results in fewer ice-nuclei and larger ice crystals forming pores. Larger pores enable higher sublimation rates, resulting in shorter primary drying cycles, as well as reduced reconstitution times and potentially improved finished product attributes. It is also important that all vials nucleate at or near the same temperature to ensure consistency of the product morphology, the resultant cake structure and appearance, and better overall product uniformity.
Induced nucleation methods offer many benefits to freeze-dried products including: increased vial-to-vial uniformity, reduced cycle times, reduced protein aggregation, and better conformance to FDA’s Guidance for Industry, Q10 Pharmaceutical Quality System Section 3.2.1 (8), which states that pharmaceutical manufacturers are expected to “identify sources of variation affecting process performance and product quality for potential continual improvement activities to reduce or control variation.” Therefore, it is of great importance that nucleation methods perform well not only at lab scale, but also in large manufacturing setups. The current work presents some of the latest developments in inducing nucleation in large manufacturing freeze dryers (>35-m2 shelf area).
Ice fog nucleation is the process by which ice crystals are introduced into the chamber during the freezing step to provide nucleation sites for all of the vials in a batch (9). This process results in all of the vials nucleating at the same temperature, thereby improving homogeneity of the ice crystal structure and dried product characteristics across the batch. It has been shown that artificially inducing nucleation can decrease primary drying times, lower residual moisture contents, and improve homogeneity (10).
The nucleation system (Veriseq nucleation, jointly developed by IMA and Linde) induces product nucleation through a process that is retrofittable to most freeze dryers. Before the ice fog is introduced, the product is first stabilized at a predetermined temperature for a given period of time until all of the vials have reached the desired nucleation temperature. This is achieved by using an appropriate shelf temperature, usually 1-2 °C colder than the desired product temperature and adequate time (e.g., 90 minutes at a shelf temperature of -5 °C to have a product temperature of -3 °C). The product temperature required for inducing nucleation varies based on product concentration and composition, fill height, and freeze dryer size. The nucleation system generates an ice fog by mixing sterile liquid nitrogen and steam, and introduces it into the chamber under defined conditions. Ice fog uniformly fills the chamber and enters the vials containing supercooled liquid solution. All of the vials nucleate at the same or similar temperature (within 0.5 °C in most cases), and the freezing process continues.
While there are various methods to induce nucleation in smaller freeze dryers, few have been tested on production-scale freeze dryers. This work describes the challenges associated with inducing nucleation on production scale freeze dryers, and the authors’ findings in using this nucleation method to successfully nucleate 195,960 vials on a 56-m2 freeze dryer, believed to be among the largest freeze dryers in operation in the pharmaceutical industry.
Traditionally, it was believed that the greatest challenge of the ice fog technique is the ability to successfully scale up to production-scale freeze dryers due to the thermal gradient within a freeze dryer (10). Coupled fluid thermal models were used to compute the thermal gradients for a 56-m2 shelf area freeze dryer. It was found that in the region covered by the vial pack, the largest gradient could be as high as 2 °C as shown in Figure 1. This thermal gradient provides the first challenge associated with the ice fog method on production scale freeze dryers.
Figure 1. Computational modeling of the thermal gradient existing in production-scale freeze dryers.
To better understand the effect of the thermal gradient within a freeze dryer, a study was conducted to determine the variables that could be adjusted when scaling to production scale freeze dryers. In this study, a nucleation system (Linde’s Veriseq nucleation) was connected to a freeze dryer (IMA Life LyoMax 15) with one two-inch port on top of the freeze dryer used as the inlet for ice fog, and a single two-inch port located on the mechanical space door for the outlet. The locations of the inlet and outlet ports were chosen due to the expected buoyant forces during typical freezing cycles in a freeze dryer.
Clusters of 10-cc tubing vials (Schott, Lebanon, PA) with 3 mL of a 3% w/w mannitol solution with one internal and one external thermocouple taped to the side of the vial were placed throughout the top, middle, and bottom shelves within the freeze dryer. Internal thermocouples were used to monitor the product temperature, while external thermocouples were used to monitor product nucleation without providing a nucleation site. The wall temperature was also monitored with a thermocouple to monitor the effects of radiation from the walls.
First, the product vials were allowed to equilibrate at -6 °C for 90 minutes before proceeding with the introduction of the ice fog. After stabilization, ice fog was injected into the product chamber to observe the effect of the thermal gradient (the thermal gradient is from top to bottom). As anticipated from the computational models, vials on the top shelf directly facing the door did not nucleate due to the increased effects of radiation from the walls.
Figure 2. Comparison of the Veriseq nucleation method with and without adjusting chamber wall temperature. Reduced wall temperature and radiative effects result in successful nucleation throughout the batch.
In the following test, product vials were allowed to equilibrate at -6 °C for 90 minutes with the walls cooled to 10 °C (compared to 16 °C in the previous test). After the 90-minute stabilization period, ice fog was injected into the product chamber with all vials successfully nucleating during the ice fog injection phase. Figure 2 provides a summary of the optimization study. The study proved that successfully inducing nucleation at production scales may require a reduction of the existing thermal gradients. The following section discusses results from a 39-m2 shelf area freeze dryer.
45,540 vials in a 39-m2 machine. Given that the thermal gradient of the wall temperature (and thus the radiation from the walls) plays a crucial role in the success of artificially inducing nucleation, the next test focused on finding a means to mitigate the thermal gradients on a 39-m2 freeze dryer. In this study, a nucleation system was connected to the freeze dryer with one two-inch port on top of the freeze dryer that was used as the inlet for ice fog, with a single three-inch port located on the mechanical space door that was used as the outlet. In an ideal scenario, three-inch ports are recommended to promote recirculation through the ice fog ejector.
A total of 45,540 vials were placed on the top, middle, and bottom shelves of the freeze dryer; 29,700 vials contained the standard concentration of product used by the client, while 15,840 vials contained 20x the concentration in a larger vial, 3 mL and 60 mL vials, respectively. Both, internal and external thermocouples were used throughout the batch. Vials were manually loaded, with the shelf temperature set at 2 °C during the loading phase. The shelf temperature was then set to -7 °C, and after an initial equilibration period of approximately 90 minutes, ice fog was introduced under defined conditions.
Figure 3a presents a comparison of thermocouple data with and without nucleation. As expected, the use of induced nucleation provides a reduction of nucleation time and nucleation temperature range from 152 minutes to < 2 minutes and from 9 °C to 0.8 °C. This reduction in nucleation temperature range results in increased homogeneity throughout the product batch. Figure 3b provides a comparison between identical freeze drying cycles, with the only change being the use of the nucleation method. By using nucleation, the time that primary drying begins to slow down as indicated by the nitrogen bleed valve opening occurs 12.4 hours earlier. The end of primary drying occurs 5.4 hours earlier as indicated by the reduction in Pirani data for chamber pressure. It is important to note that the freeze-drying cycle can be further optimized (e.g., by increasing shelf temperature using ice fog with an expected reduction in product resistance) to potentially reduce the primary drying time even further. Table I compares testing in a 39-m2 freeze dryer with and without nucleation.
195,960 vials in a 56-m2 machine. Although processing conditions such as loading temperature can be favorable to promote successful ice fog injection, some products may be too restrictive to cycle changes. The objective of this test was to determine if the freeze dryer jacket could be cooled during the loading phase to reduce the thermal gradient during the freezing phase, as well as prove the scalability of the nucleation system to such large systems.
In this study, the nucleation system was connected to a freeze dryer (IMA Life LyoMax 56) with one three-inch port on top of the freeze dryer that was used as the inlet for ice fog, with a single three-inch port as the outlet.
Table II. Parameter comparison of testing in a 56-m2 freeze dryer with and without Veriseq nucleation. Note that the cycle has been not optimized in this case, hence, the total time to end secondary drying was increased.
The freeze dryer was fully loaded with 195,960 3-cc vials with a 1.2-mL fill. Internal and external thermocouples were placed near the top, middle, and bottom shelves of the freeze dryer. Vials were loaded using the client’s automated loading system. The loading shelf temperature was set at 5 °C. During the loading and freezing ramps, the jacket of the freeze dryer was cooled with the silicone oil used to maintain the temperature of the shelves. After loading, the shelf temperature was held at -7 °C for approximately 90 minutes, at which point ice fog was introduced under defined conditions. Table II shows a comparison of testing in a 56-m2 freeze dryer with and without nucleation.
Figure 4. Cycle plot using Veriseq nucleation.
Figure 4 provides the cycle data during the ice fog injection phase. By using artificially induced nucleation, the primary drying duration (as indicated by the time the last thermocouple reaches the shelf temperature) was reduced by 19%. While the overall cycle time did increase from 1514 minutes to 1854 minutes (since the original cycle cold loaded the product on -40 °C shelves), it should be noted that the cycle can be optimized (e.g., by increasing shelf temperature since the product resistance is expected to have reduced), which may reduce overall cycle time. The overall product uniformity was improved by 40%, further demonstrating the increased batch homogeneity associated with induced nucleation.
While there are several known techniques to artificially induce nucleation with the same basic characteristics, few studies have tested these processes at large scales of operation (> 35 m2 shelf area, >150,000 vials). The data presented here show that to successfully induce nucleation at such large scales of operation, the existing thermal gradients within the freeze dryers need to be minimized. While some dryers/product combinations can withstand cooler loading, others may require the jacket of the freeze dryer to be cooled along with the shelves during the freezing stage. This was demonstrated at full scale on two different freeze dryers--a 39-m2 shelf area freeze dryer with a product load of 45,540 vials and a 56-m2 shelf area freeze dryer with a load of 195,960 vials. While the former was tested successfully with a cooler shelf loading temperature of 2 °C, the latter required jacket wall cooling to successfully induce nucleation across the batch.
Further, inducing nucleation at warmer temperatures reduced primary drying times of the product by up to 19% with an improvement of batch homogeneity up to 40%. The nucleation system used to be capable of generating an ice fog distribution that is robust up to a 56-m2 shelf area freeze dryer with a load of 195,960 vials.
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Vol. 29, No. 12
Pages: 36–41, 55
When referring to this article, please cite as J. Azzarella et al., “Increasing Vial-to-Vial Homogeneity: An Analysis of Using Veriseq Nucleation on Production-Scale Freeze Dryers," BioPharm International 29 (12) 2016.