Trehalose Crystallization During Freeze-Drying - ACS Publications

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Trehalose Crystallization During Freeze-Drying: Implications On Lyoprotection Prakash Sundaramurthi and Raj Suryanarayanan* Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT Lyoprotectants are stabilizers used to prevent denaturation of proteins during freeze-drying and subsequent storage. In order to be effective, lyoprotectants must be retained amorphous. The physical state of the lyoprotectant is usually characterized by powder X-ray diffractometry of the dried cake. While trehalose is widely used as a lyoprotectant, we report its crystallization during freeze-drying and point out why it may not become evident from characterizing the final lyophile. When an aqueous trehalose solution was cooled to -40 °C, ice was the only crystalline phase observed. However, upon annealing at -18 °C, crystallization of trehalose dihydrate was evident. During drying, the dihydrate dehydrated to substantially amorphous anhydrate. Therefore, analyzing the final dried product will not reveal crystallization of the lyoprotectant during freeze-drying. In light of the observed phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigation. SECTION Biophysical Chemistry

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hermolabile pharmaceuticals, specifically macromolecules, are often formulated as freeze-dried products.1 The macromolecule stability can be enhanced, both during freeze-drying and subsequent storage, by the addition of a lyoprotectant to the formulation.2,3 In order to be effective, the lyoprotectant must be retained in the amorphous state (a necessary but not sufficient condition). Crystallization of lyoprotectant during the process or subsequent storage might potentially impact the stability of proteins and hence the formulation performance.4,5 Nonreducing sugars, specifically sucrose and trehalose, are widely used as lyoprotectants in light of their ability to resist crystallization.6-9 While there are no reports on sucrose crystallization,10,11 we report trehalose crystallization during freeze-drying. The genesis of this study was our earlier investigation on the crystallization behavior of succinate buffer components in frozen solutions.12,13 In an effort to inhibit buffer component crystallization, succinate buffer (10 mM) was colyophilized with trehalose since the latter is widely reported to be a noncrystallizing solute. Interestingly, the crystallization of monosodium succinate, a buffer component, was substantially enhanced in the presence of trehalose. We observed that the crystallization of succinic acid and monosodium succinate was followed by that of trehalose dihydrate. We hypothesized that the buffer salts facilitated the crystallization of trehalose dihydrate and vice versa. The observed crystallization of trehalose was surprising in light of the numerous reports documenting amorphous trehalose in the dried cake.3,10,14,15 In an effort to evaluate the crystallization propensity of trehalose, a prelyophilization solution containing trehalose alone was investigated. Trehalose solution was freeze-dried in the sample chamber of an

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X-ray diffractometer. This enabled us to monitor the system during all of the stages of the freeze-drying cycle. Selected experiments were performed in a synchrotron beamline. The “as is” sample trehalose dihydrate (C12H22O11 3 2H2O) was characterized by differential scanning calorimetry, X-ray diffractometry (XRD), and thermogravimetric analysis. Trehalose dihydrate was identified by diffraction peaks at 8.8 (10.10 Å), 16.5 (5.36 Å), and 17.6° (5.04 Å) 2θ, while the peak at 17.8° (4.98 Å) 2θ is attributed to the R-polymorphic form of anhydrous trehalose (C12H22O11). The peaks at 22.7 (3.93 Å), 24.4 (3.65 Å), 25.8 (3.46 Å), and 33.5° (2.68 Å) 2θ were assigned to hexagonal ice.16 Figure 1 is an overlay of the one-dimensional XRD patterns of frozen trehalose solutions recorded during annealing. Pattern a is that of the unseeded solution, while patterns b and c are those of samples seeded, respectively, with succinic acid and trehalose dihydrate. When the solutions were cooled from room temperature (RT) to -40 °C (0.5 °C/min) and held for 15 min, only the peaks of hexagonal ice were observed. There was no evidence of solute crystallization (data not shown). Upon annealing at -18 °C, crystallization of trehalose dihydrate was observed (Figure 1). The annealing temperature was selected based on the thermal events observed in a differential scanning calorimeter (data not shown). It was approximately the midpoint between the eutectic melting (-2.5 °C) and glass transition (∼-35 °C) temperatures of the trehalose-water binary system.6-8 When unseeded, trehalose dihydrate crystallization Received Date: December 1, 2009 Accepted Date: December 16, 2009 Published on Web Date: December 28, 2009

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DOI: 10.1021/jz900338m |J. Phys. Chem. Lett. 2010, 1, 510–514

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sample, when ice was “seeded” either with succinic acid or trehalose dihydrate, as expected, only the peaks of ice were observed. When the seed crystals were sprinkled on the surface of the frozen sample and annealed, spherulites emanated from the seeds and covered the entire sample surface (data not shown). Since the laboratory XRD experiments were conducted in the reflection mode, the possibility of an experimental artifact cannot be ruled out. In other words, we were not confident that the crystallization had occurred uniformly throughout the frozen mass. However, the results obtained using the synchrotron beamline, where the experiments were conducted in the transmission mode, confirmed that crystallization occurred in the entire frozen mass (Figure 2). In this case, the frozen mass dimensions were 5 5 x 2 mm, and as before, the seeds were sprinkled on the surface of the frozen mass. The instrumental setup ensured that the synchrotron beam [100 (vertical) 200 (horizontal) μm] sampled the lower half of the frozen mass. The sample cell dimensions and the beamline experimental setup were explained, in detail, in our earlier publication.17 While there was unambiguous evidence of trehalose crystallization (Figures 1 and 2), the literature reports strongly suggest that trehalose is amorphous in the final dried cake.18,19 This conclusion is based on characterization studies, carried out by numerous groups, on the final dried cake.20 This raises an interesting question: Can trehalose crystallize in the frozen solution and then undergo a crystalline to amorphous transition during drying? There are numerous examples of both inorganic and organic compounds which undergo a crystalline hydrate to amorphous anhydrate transition during drying.4,21 The kinetics of water removal can often be a determinant of the physical form of the product phase. During freeze-drying,

was evident but only after 3 days of annealing (pattern a). Upon seeding, with either succinic acid or trehalose dihydrate, crystallization was facilitated, and the trehalose dihydrate peaks were observed after 12 h of annealing (patterns b and c). Our earlier observations had suggested that succinic acid facilitated trehalose crystallization in frozen solutions.12 Interestingly, much higher peak intensities were observed when trehalose dihydrate was used as the seed. In a control

Figure 1. Overlaid XRD patterns of trehalose solutions recorded during annealing at -18 °C. The prelyophilization solutions containing trehalose (4% w/v) were cooled from room temperature (RT) to -40 at 0.5 °C/min, held for 15 min, warmed to -18 °C, and annealed. The XRD pattern a was recorded after 3 days of annealing, while b and c were recorded after 12 h of annealing. Prior to annealing, samples b and c were, respectively, seeded with crystals of succinic acid and trehalose dihydrate.

Figure 2. Two-dimensional synchrotron XRD image of frozen trehalose solution recorded after annealing for 24 h. The sample was prepared by cooling the solution (4% w/v trehalose) from RT to -40 °C, holding for 15 min, and then warming to -18 °C. The solution was seeded with trehalose dihydrate and annealed. The cooling and heating rate was 0.5 °C/min. Some characteristic Debye rings of hexagonal ice and trehalose dihydrate are pointed out.


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Figure 3. Overlaid XRD patterns of trehalose solution recorded during the drying stage of lyophilization. Prior to the initiation of primary drying, this sample was cooled from RT to -40 at 0.5 °C/min, seeded with trehalose dihydrate, and annealed at -18 °C for 24 h. The characteristic peaks of trehalose dihydrate and anhydrous (R-polymorph) trehalose are pointed out. The experiments were conducted in triplicate, and a representative example is shown here.

dehydration of disodium phosphate dodecahydrate and raffinose pentahydrate yielded the respective amorphous anhydrous phases.4,21 Recently, we reported the formation of amorphous anhydrate following the dehydration of disodium succinate hexahydrate.22 Hence, in order to monitor such phase transformation during freeze-drying of trehalose solution, the entire freeze-drying cycle was simulated in the X-ray diffractometer. Figure 3 shows the XRD patterns of frozen trehalose solution, recorded during primary and secondary drying. Hexagonal ice crystallized first during cooling, followed by trehalose dihydrate but only after annealing the frozen solution for 12 h. Upon further annealing, the intensity of the characteristic peaks (8.8 and 17.6° 2θ) of trehalose dihydrate increased, indicating further crystallization (data not shown). When primary drying was performed at -25 °C, ice sublimation was evident from the gradual decrease in ice peak intensities, which disappeared after 15 min of primary drying. This was followed by the gradual decrease in the intensities of trehalose dihydrate peaks, indicating dehydration. Since this was not accompanied by the appearance of any new peaks, dehydration yielded an amorphous anhydrate. Even after 3 h of primary drying, dehydration was not complete. During secondary drying at -10 °C, there was a pronounced


decrease in the intensity of trehalose dihydrate peaks, indicating that dehydration was facilitated at the elevated temperature. Interestingly, the most intense peak of the anhydrous (Rpolymorph) trehalose (17.8° 2θ; 4.98 Å) appeared at this stage. However, the substantially amorphous nature of the dried product is evident from the fact that only one broad peak of the anhydrous phase was observed. When the drying temperature was increased to 0 °C, the trehalose dihydrate peak intensities decreased further and disappeared completely when the drying temperature was increased to 10 °C. Thus, there is clear evidence of annealing-induced crystallization of trehalose dihydrate in frozen solutions. Interestingly, dehydration during drying resulted in a substantially amorphous dried cake. This series of phase transformations may not be discernible by characterizing the dried product alone. The crystallization of trehalose in the frozen state may seriously impair its ability to serve as a lyoprotectant. The dry-state stabilization of proteins by sugars is believed to be brought about by the hydrogen bonding between the hydroxyl groups of the sugar and the protein polar residues.23 Thus, stabilization requires both lyoprotectant vitrification (glass formation) and its direct interaction with protein.10,24 Even if the lyoprotectant is amorphous in the dried state, if it phase separated (crystallized) in the frozen state (i.e., during

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ACKNOWLEDGMENT S. Kumar (Kent State University) and D. Robinson (Argonne National Laboratory) are thanked for their help. Parts of this work were carried out in the Institute of Technology Characterization Facility, University of Minnesota, a member of the NSF-funded Materials Research Facilities Network (www.mrfn.org).

freeze-drying), its effectiveness as a lyoprotectant may be seriously compromised. While the possibility of trehalose crystallization is evident from this work, the extensive use of trehalose in protein formulations attests to its effectiveness as a lyoprotectant. We should point out that annealing was necessary to induce trehalose crystallization. In the absence of annealing, the trehalose may be retained in the amorphous state. Moreover, even when there is trehalose crystallization, it is likely that only a fraction of the trehalose crystallizes. The uncrystallized fraction may be sufficient for lyoprotection. Finally, the other formulation components, specifically the noncrystallizing solutes including macromolecules, may effectively inhibit trehalose crystallization. Notwithstanding the above, the potential for trehalose crystallization during freeze-drying warrants serious investigation. The presence of crystallizing solutes may facilitate trehalose crystallization as was observed with succinic acid. Since bulking agents such as mannitol and glycine crystallize readily, it will be interesting to evaluate their ability to induce trehalose crystallization. Finally, it is possible that nucleation but not crystallization may be induced during freeze-drying. The growth of crystals may then occur during product storage and may be particularly facilitated by the residual moisture in the dosage form. Depending on the storage temperature, this process may occur very slowly but still on time scales of pharmaceutical interest. In light of the stochastic nature of crystallization, there is room for batch to batch variation in product performance brought about by trehalose crystallization. Since we freeze-dried an aqueous solution of trehalose which contained no other formulation components, one peak of R-trehalose was readily discernible in the final dried cake (Figure 3). However, in actual formulations, the presence of other formulation components will attenuate the trehalose peak intensity. Moreover, noncrystallizing solutes including proteins may also inhibit the crystallization of anhydrous trehalose. In summary, crystallization of trehalose as trehalose dihydrate during lyophilization was observed in frozen solutions. Dehydration of the crystalline trehalose dihydrate to substantially amorphous anhydrate occurred during drying. Therefore, analyzing the final dried product alone may not reveal crystallization of the lyoprotectant during freeze-drying. The lyoprotectant crystallization can only become evident by continuous monitoring of the system during the entire freeze-drying cycle. In light of the phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigation.

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SUPPORTING INFORMATION AVAILABLE Details of the

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experimental procedure and the solubility of trehalose as a function of temperature are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author:

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*To whom correspondence should be addressed. Phone: 612-6249626. Fax: 612-626-2125. E-mail: [email protected].


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Trehalose Crystallization During Freeze-Drying - ACS Publications

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