Abstract
Due to low optical contrast, the morphology of lyophilized product cakes is difficult to observe and photograph. Furthermore, internal structures are normally not visible unless the cake is fractured. Because most lyophilized substances are hygroscopic and quite fragile, the product cake, once removed from the vial, will rapidly degrade. We propose herein a technique that allows a lyophilized product cake to be preserved, manipulated, and easily observed outside the vial. This technique yields high-quality, cross sectional images that reveal intricate fine structures without the use of expensive specialized equipment.
Introduction
During lyophilization cycle development or investigations of production lyophilization process deviations, a visual inspection of the lyophilizate cake is generally performed as part of the assessment. Particular cake features can provide some clues to events that transpired during the freeze drying operation. For example, a cake that exhibits regions of partial collapse can be indicative of excessive shelf temperature during the primary drying step. Frequently, these regions of partial collapse cannot be seen by external inspection while the the cake remains in the vial.
It is desirable to maintain a photographic record of the various lyophilizate cakes for documentation purpose. However, properly imaging a low-contrast sample through the highly curved surface of a glass vial presents significant challenges. Extracting the cake from the vial can improve the quality of the final images and allow for the observation of internal structures by sectioning with a sharp blade. However, due to their fragile nature, the manipulation of naked cakes is difficult and, for hygroscopic products, the cakes may also “melt” rapidly once exposed to ambient conditions.
Previously, Patapoff and Overcashier (1) introduced a technique that involved embedding a lyophilizate cake in a low-melting-temperature paraffin. Once solidified, the cake-containing paraffin plug could be removed by breaking the vial. The authors have shown that even microscopic, intricate structures can be easily photographed and preserved for a substantial length of time in this fashion. There are, however, several requirements, such as the need to incorporate a fluorescent dye to the liquid prior to freeze-drying and the exposure of the final cake to a temperature of ∼60 °C, which limit the utility of the technique to lyophilization runs carried in the laboratory and to cakes that can withstand higher temperatures without significant structural change.
In this paper we present an improved technique that is not subject to the limitations mentioned above and that produces comparable image quality. The new procedure still involves encapsulation of the lyophilizate cake and a fluorescent dye for imaging, but in this instance a silicone elastomer is used as the matrix and the dye is added just before visual observation. Product vials from both laboratory and large-scale production facilities have been successfully processed and imaged in this manner.
For the purpose of illustration, we have encapsulated and imaged lyophilized samples that had been frozen in a controlled manner and show that the resulting cake morphologies are as expected (1, 2).
Material and Methods
Preparation of Lyophilized Samples
Two solutions were used to demonstrate the usefulness of the embedding and imaging technique. One solution (with no protein) contained 4% trehalose, 50 mM sodium phosphate at pH 6.2, and 0.02% polysorbate 20. A second solution contained 25 mg/mL protein (a monoclonal antibody) in the same base formulation as described above. Vials (10 cc) were filled with 4 mL of the above solutions and frozen by methods as described below, and reported previously (1).
The following procedure was used to prepare vials under the “normal” freezing conditions:
Samples were equilibrated on the lyophilizer shelf at 5 °C for several hours
The shelf temperature was ramped linearly from 5 to −50 °C at 0.3 °C/min
The shelf temperature was held at −50 °C for at least 5 h
This cooling strategy resulted in vials that were supercooled to between −15 to −25 °C prior to spontaneous ice nucleation. Our estimates were based on the shelf temperature and visual observation of the samples during the freezing ramp.
To produce directional ice growth, samples were prepared as follows:
Samples were equilibrated under wet ice
The lyophilizer shelf temperature was ramped from 5 to −50 °C at 0.3 °C/min
Once the shelf temperature reached less than 0 °C, each sample from step 1 was briefly contacted on the bottom of the vial with a piece of dry ice to induce ice nucleation then immediately placed on the lyophilizer shelf
Upon completion of the ramp, the shelf temperature was held at −50 °C for at least 5 h
To produce rapidly frozen ice growth, samples were prepared as follows
Samples were equilibrated under wet ice
The samples were immersed in a slurry of dry ice–ethanol until completely frozen
Each sample was then placed on the lyophilizer shelf that was precooled to −50 °C
The shelf temperature was then held at −50 °C for at least 5 h
These cooling strategies, although impractical for manufacturing, demonstrate the ability of the embedding and imaging method to resolve finer structures found in some cakes.
Once frozen all the samples were dried under the following conditions:
The chamber pressure was set to 150 μm Hg pressure absolute
The shelf temperature was ramped linearly from −50 to 20 °C at 0.25 °C/min
The self temperature was held at 20 °C for 47 h (this is the combined primary and the secondary drying steps)
Upon completion of the drying, the vials were stoppered under vacuum and stored at ambient temperature.
Encapsulation
We used two different methods for encapsulating lyophilized cakes in Dow Corning Sylgard 184 polydimethylsiloxane (PDMS) polymer, as discussed below.
The polymer, received as a two-component kit comprised of a base elastomer and a curing agent, was prepared according to the manufacturer's instructions. After mixing, repeated cycling between 10–20 kPa absolute pressure and ambient pressure greatly facilitated the removal of entrained and dissolved air from the PDMS. The mixture has a working time of several hours (>2 h according to manufacturer documentation), providing ample time to perform all subsequent operations.
During the encapsulation procedure, each sample vial must be uncapped and briefly exposed to ambient conditions. To minimize the possibility of excessive moisture pickup by the lyophilized cake, a gentle dry gas purge over the opened vial can be used.
Method 1
Each vial containing a lyophilized cake for encapsulation was set up as shown in Figure 1a. A modified 50 mL Eppendorf Combitip Plus pipette tip was used as the reservoir for the PDMS. The tip of the Combitip was trimmed down to the base leaving ∼5 mm, and a small Parafilm M plug was inserted in the opening. Some notches were made in the plastic plunger of the pipette tip to allow for venting. A retaining bracket fashioned from wire (here copper was used) served to prevent the lyophilized cake from floating atop the PDMS once the polymer is introduced in the vial. The wire bracket and the Combitip funnel were secured to the vial using adhesive tape. Sufficient amount of PDMS was poured into the funnel, and the plunger was set in place. The assembly was placed inside a lyophilizer and the pressure was lowered to ∼5 kPa absolute; the shelf temperature remained at ambient. At this pressure additional degassing of the PDMS occurred but most of the bubbles dissipated reasonably rapidly. The lyophilizer shelves were then partially collapsed, just enough as to sufficiently push the plunger down and dislodge the Parafilm plug. The PDMS was allowed to flow from the funnel and flood the vial for up to 1 h. Subsequently, the lyophilizer chamber was brought back to ambient pressure, forcing the polymer into the lyophilized cake interstitial space. The vial assembly was unloaded from the lyophilizer, and the funnel and wire bracket were removed. The vial was then placed into an oven set at 35–40 °C for 3–4 days to allow the PDMS to fully cure. Note that the PDMS can be cured at still lower temperatures but the curing time will be lengthened.
Encapsulation setups. a) Method 1, b) Method 2. (1) modified 50 mL Eppendorf Combitip, (2) parafilm plug, (3) wire retaining bracket, (4) 50 mL centrifuge tube funnel.
Method 2
The setup for this method is simpler than for Method 1 in that the PDMS is introduced into the vial prior to the application of vacuum. However, upon reducing the pressure, substantial amount of foaming occurs. Therefore, a funnel attached to the vial opening is needed to prevent the PDMS from spilling out. Figure 1b shows a vial assembly ready to be filled with the polymer. In this case, the funnel comprises a section of a 50 mL centrifuge tube carefully attached to the vial mouth using adhesive tape so as to prevent the PDMS from leaking out. A retaining bracket for the lyophilized cake must also be in place before introduction of the polymer.
To encapsulate a lyophilized cake, an appropriate amount of PDMS was poured into the funnel and the assembly was transferred to a lyophilizer (a vacuum oven can also be used in lieu of a lyophilizer). The chamber pressure was then reduced to ∼5 kPa absolute. Depending on the size of the cake, more than 1 h may be required for the foam to collapse sufficiently before the pressure can be restored to ambient conditions. Once the PDMS fully penetrated the cake, the vial assembly was unloaded from the lyophilizer and the funnel and wire bracket removed. The vial was then placed into an oven set at 35–40 °C for 3–4 days to allow the PDMS to fully cure.
Extraction of the Embedded Lyophilized Cake
The embedded cake was extracted by placing the vial in a plastic bag and a vise was used to carefully shatter it. Methanol or isopropanol can be used as wetting agent to facilitate the removal of adhered glass fragments from the PDMS plug.
Visualization
The embedded cake that was released from the vial was sectioned by cutting it with a sharp blade. Longitudinal and cross-sectional cuts were made and imaged under white illumination or illumination to enhance fluorescence. For the latter, a fluorescent dye was applied from a felt tip marker (Sharpie Accent Liquid Highlighter, orange color). The water-based dye did not adhere to the hydrophobic polymer, and excess dye was easily wiped off with a clean cloth.
For fluorescence imaging, the sample was lighted with a Prolight High Power 1 watt UV LED (UV-1WS, 400 nm) equipped with a 15° focusing lens and powered by a 350 mA Buckpuck LUXDRIVE (03021-DE-350) LED driver. A Hoya B-390 blue filter was added to the front of the LED focusing lens to minimize higher wavelength light. Photographs were taken through a Leica MZ16 stereo microscope equipped with a PLAN APO 0.63× objective and a Canon 5D SLR camera. A Hoya K2 yellow filter was placed in front of the microscope objective to further enhance contrast.
Results and Discussion
In the majority of cases, either proposed encapsulation method can be used successfully. Method 1 may reduce the potential for entrapping bubbles, particularly with larger cakes because vacuum is applied prior to the introduction of the polymer. If the cake volume is more than half the vial volume, then Method 2 should be used to prevent overfilling the vial before the polymer can penetrate the cake.
While the PDMS has relatively high pre-cure viscosity (3900 cP), it exhibits very low surface tension. In most instances, full penetration and “wetting” of lyophilized cake has not been an issue in our experience. The polymer cures into a hydrophobic, crystal-clear, medium-soft material (Shore A durometer value of 48) with excellent dimensional stability.
Figures 1⇓⇓–4 are photographs of samples that have been encapsulated and sectioned both vertically and horizontally at about mid-section. The images, usually taken a few minutes after sectioning the cakes, demonstrate the resolving power of this technique, and the advantage of using fluorescent staining is dramatically apparent. Note that once a sample has been sectioned and stained, the lyophilizate will melt. However, no loss of optical resolution occurs because the lighting highlights the edges of all cake features that were imprinted in the PDMS. It is also possible to see features below the exposed surface because the polymer is clear and the fluorescent dye tends to penetrate some distance into the lyophilizate.
Encapsulated cake containing 25 mg/mL protein from normal freezing regiment, liquid was subjected to supercooling and ice nucleated between −15 and −25 °C. a–c) vertical section, d–f) horizontal section. Frames a and d are sections photographed under white light before dye staining; all others are from UV lighting post-staining. White outline in frames b and e indicate magnified areas shown in frames c and f.
Encapsulated cake containing 25 mg/mL protein from directional freezing regiment; liquid was equilibrated on wet ice and ice nucleation was induced by briefly touching a piece of dry ice to the vial bottom. The vial was then placed on back the lyophilizer shelf at slightly <0 °C. a–c) vertical section, d–f) horizontal section. Frames a and d are sections photographed under white light before dye staining; all others are from UV lighting post-staining. White outline in frames b and e indicate magnified areas shown in frames c and f.
Encapsulated cake without protein from directional fast freezing regiment, liquid was equilibrated on wet ice and completely frozen in a dry ice-ethanol bath. The vial was then placed on the lyophilizer shelf at −50 °C. a–c) vertical section, d–f) horizontal section. Frames a and d are sections photographed under white light before dye staining; all others are from UV lighting post-staining. White outline in frames b and e indicate magnified areas shown in frames c and f.
Figure 2 shows photographs of a protein containing sample frozen under normal condition (cooled from 5 to −50 °C at 0.3 °C/min) that resulted in a cake that exhibits sponge-like morphology. This is the expected outcome for liquid vials that have undergone supercooling (ice nucleation occurred between −15 to −25 °C) and are subsequently frozen rapidly (1, 2). In this instance, some cake shrinkage and cracking also occurred during the drying. The spatial relationship between the various parts of the cake has been captured by the PDMS, and the internal fractures are easily observable. Some liquid phase separation may also have occurred in this sample as evidenced by the larger and less dense cell structure near the upper portion of the cake (3). This latter feature would have been very difficult detect without the support of the cake structure by PDMS.
The protein-containing sample shown in Figure 3 underwent directional freezing from bottom up, resulting in a final lyophilized cake that exhibits large lamellar structures. This morphology is consistent with a solution where ice nucleation occurred close to the freezing point and ice growth proceeded relatively slowly such as to form large ice crystals (1, 2). The voids seen in areas near the vial wall may be localized cake fractures due to moderate cake shrinkage.
Figure 4 shows a non-protein sample that underwent rapid directional freezing from the bottom and sides of the vial. This freezing regime resulted in very fine lamellar structures oriented toward the center of the vial. In addition, there is also evidence of cake shrinkage causing large cracks in the periphery as well the center of the cake. This example also demonstrates the ability to apply the fluorescence stain to samples that do not contain protein.
Figure 5 are images of actual protein products prepared in a pilot-scale lyophilizer. Figures 5a and 5b show the vertical and horizontal cross-section, respectively, of a cake that exhibits partial collapse on the bottom half. In addition, strong neighboring vial effects are present as evidenced by the hexagonal shape of the cake bottom. Figure 5c and 5d show the resulting cake structure from a vial that contained a monitoring thermocouple as compared with a vial without. It is known that the presence of thermocouples tends to induce ice nucleation at higher temperatures (lower degree of supercooling). The sample in Figure 5c seems to include structures formed by directional ice crystals, consistent with slower ice crystal growth rate, whereas that in Figure 5d exhibits a mostly sponge-like morphology, consistent with fast ice crystal growth. The sample in Figure 5d was also included to demonstrate the effect of incomplete encapsulation, where the bright areas at the center are regions the PDMS did not fully penetrate. These regions subsequently collapsed due to moisture absorption from the water-based fluorescent dye staining after the cake was sectioned.
Encapsulated cakes of actual protein products prepared in pilot scale lyophilizer. a) vertical cross-section, b) horizontal cross-section take near the bottom (approximately indicated by white line) of a sample containing 20 mg/mL protein in 0.5 M arginine succinate. c & d) vertical cross sections of samples containing 20 mg/mL protein formulated with 60 mM trehalose. Sample c was sectioned as close to the thermocouple as possible.
In addition to the examples presented above, we have successfully employed this technique to image a variety of lyophilized substances, including products formulated with either sugars (i.e., sucrose, trehalose) or organic salts (i.e., arginine phosphate) and of solids contents ranging from less than 5 wt % to over 12 wt %. Sample size ranged from a few milliliters in a 5 mL vial to over 40 mL in a 100 mL vial. With care, even highly hygroscopic material can be processed with good results. However, very fragile lyophilized cakes may present difficulties because they can be crushed by the weight of the polymer when it is introduced into the vial. The technique can easily be refined and adapted with experimentation to suit individual requirements.
Conclusion
We have presented an improved technique that is facile and can be used to image lyophilized products cakes without the need for specialized or expensive equipment. We believe this to be a valuable tool for lyophilization research and development as well as manufacturing investigations.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
- © PDA, Inc. 2011
