Water Sorption and Glass Transition Behaviors of Freeze‐Dried Sucrose–Dextran Mixtures

Water Sorption and Glass Transition Behaviors of Freeze‐Dried Sucrose–Dextran Mixtures

Water Sorption and Glass Transition Behaviors of Freeze-Dried Sucrose–Dextran Mixtures KOREYOSHI IMAMURA, ATSUSHI FUKUSHIMA, KEISUKE SAKAURA, TAKUO SU...

116KB Sizes 3 Downloads 85 Views

Water Sorption and Glass Transition Behaviors of Freeze-Dried Sucrose–Dextran Mixtures KOREYOSHI IMAMURA, ATSUSHI FUKUSHIMA, KEISUKE SAKAURA, TAKUO SUGITA, TAKAHARU SAKIYAMA, KAZUHIRO NAKANISHI Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan

Received 20 November 2001; revised 10 April 2002; accepted 10 May 2002

ABSTRACT: The water sorption and glass transition behaviors of freeze-dried disaccharide–polysaccharide mixtures at various contents were investigated at relative humidities (RHs) of 0, 11, 23, and 33%. Sucrose and three types of dextrans, which differ in molecular weight, were used as model di- and polysaccharides, respectively. The relationship between the dextran and water contents of the sucrose–dextran mixture at different constant RHs indicated that a mixture of sucrose and dextran was lower than that calculated by the Lang and Steinberg mass balance equation. In the RH range of 0– 23%, the glass transition temperature, Tg, increased to a considerable extent when the dextran content was equal to or higher than 50%, while the increase in Tg at dextran contents lower than 50% was small. A marked increase in Tg was observed at RH 33% for dextran contents of 0–25% as well as in the range above 50%. This suggests that the physical stability of the highly hydrated amorphous disaccharide is effectively strengthened by the addition of a small amount of polysaccharide. These tendencies were similar for the three dextrans of different molecular weights. Furthermore, it was demonstrated that the addition of a small amount of dextran is quite effective in preventing the collapse of amorphous sugar during freeze drying. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2175–2181, 2002

Keywords: freeze drying; water sorption; glass transition; disaccharide–polysaccharide mixtures

INTRODUCTION When labile proteins are used as components of commercial products, they are often freeze dried in the presence of sugar and embedded in amorphous matrices of sugars.1–3 By embedding in an amorphous sugar matrix, the protein is stabilized against physical and/or chemical denaturation during dehydration and storage.4–8 The stability of proteins in an amorphous sugar matrix is considered to depend mainly on the following two factors. One is the extent of the stabilizing

Correspondence to: Kazuhiro Nakanishi (Telephone: 81 86 251 8200; Fax: 81 86 251 8264; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 2175–2181 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

effect of the sugar, which varies with the specific sugar used.9,10 The other is the physical stability of the amorphous sugar matrix: when an amorphous sugar is exposed to high temperature or high humidity above certain levels, the glass transition, and subsequent loss of the stabilizing effect of the amorphous sugar typically result.11,12 However, some studies have indicated that an amorphous sugar that has a higher stabilizing effect for protein frequently shows a poor physical stability.13,14 Namely, the enhancement of both the stabilizing effects and the physical stability of the amorphous sugar seems to be difficult. Recently, to overcome this dilemma, Allison et al.14 proposed the use of two types of saccharides, a disaccharide and a polysaccharide, as a stabilizing agent for proteins during freeze drying and storage. In the mixed sugar matrix, the





disaccharide would stabilize the protein, while the polysaccharide would improve the physical stability of the amorphous matrix. They demonstrated that, by adding both the di- and polysaccharides to a protein solution followed by freeze drying, the protein stability during freeze drying and storage was improved, compared to the case where a di- or polysaccharide alone was used. As the result of extensive development in bioscience and biotechnology, a number of proteins that have potential for use in pharmaceutical formulations have been reported.15 Thus, the importance of mixed sugar matrices becomes a more pressing issue. However, a serious lack of the information on the physical properties of mixed sugar matrices exists. In this study, we report on an investigation of the physical properties of amorphous matrices composed of a disaccharide and a polysaccharide. As the disaccharide and polysaccharide, sucrose and three types of dextran with different mean molecular weights were used. Various mixtures of sucrose–dextran mixtures were prepared by freeze drying. The equilibrium water content and glass transition temperature, Tg, for these mixtures, at different relative humidities (RH), were then measured. The influences of the content and molecular weight of the dextran on water sorption and glass transition behaviors of the mixtures were investigated. Furthermore, the effect of dextran on preventing the collapse of amorphous sucrose during freeze drying was also addressed.

MATERIALS AND METHODS Materials Sucrose was purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan). The three dextrans, DX1500 (MW 1500, by enzymatic synthesis), DX6000 (MW 6000 from Leuconostoc sp.), and DX20000 (MW 15,000–20,000 from Leuconostoc sp.) were products of Fluka Chemie GmbH (Buchs, CH, Switzerland). P2O5, LiCl, CH3COOK, and MgCl2 were obtained from Wako Pure Chemical Industries. All other chemicals were of reagent grade. Preparation of Samples Typically, sucrose and dextran were dissolved in distilled water at a total concentration of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 10, OCTOBER 2002

50 mg/mL. The dextran content in the total concentration of the solutes was varied from 0–100%. Three milliliters of the sucrose–dextran mixture were transferred to a 20-mL vial and instantaneously frozen with liquid nitrogen. The vial containing the frozen sample was then placed in a glass chamber, which was evacuated using a vacuum pump (GLD-100, ULVAC Japan, Ltd., Tokyo, Japan) connected to a vapor condenser (UT-80, Eyela Tokyo Rikakikai Co., Tokyo, Japan). During the freeze drying, the glass chamber was immersed in an ethanol bath (NCB-2400, Eyela Tokyo Rikakikai Co.), the temperature of which was usually kept at 308C for 48 h in the first step, 108C for 12 h in the second step, and 08C for 12 h in the third step. The sample temperature during freeze drying was measured using a K-type thermocouple. The thermocouple was introduced into the glass chamber of the freeze dryer by passing through a orifice of the needle inserted into a rubber tube connected to the glass chamber, and the thermocouple junction was placed at the bottom of the frozen sample. The sample temperature was found to be below 36  18C during sublimation of freezable water (the early period of the first step). The remaining water contents at each end of the three steps of freeze drying were 0.074, 0.057, and below 0.002 g/g-dry matter, respectively. Judging from these results and the water content dependency of the glass transition temperature reported previously, 16,17 we concluded that the sample temperature was lower than the glass transition temperatures through the freeze drying. Some samples were freeze dried at room temperature (308C) without any temperature control. The sample temperature during the freeze drying at the room temperature was measured in the same manner as that described above. In this case, the freeze drying was complete within 24 h. After further drying at room temperature for 1 h, the samples were thoroughly dehydrated at 258C in a vacuum desiccator over P2O5 for 7 days. The samples were then rehumidified at 258C in a vacuum desiccator in the presence of saturated solutions of LiCl, CH3COOK, and MgCl2 for about 1 week. The relative humidities in the desiccators with saturated LiCl, CH3COOK, and MgCl2 solutions are 11, 23, and 33, respectively.18 Rehumidification of the sample was confirmed as being at equilibrium within 3 days19 in most cases. Because the samples containing 0–12.5% dextran showed significant collapse at RH 33%, the moisture contents and Tgs for these samples



equilibrated at RH 33% for more than 3 days could not be determined. Hence, values for the sample containing 5 and 12.5% dextran after rehumidification for only a 2-day period were measured. Water Content Analysis The water content of the sucrose–dextran mixture was measured from the difference between the weight of the sample that had been dehydrated thoroughly in the desiccator over P2O5 and that after the rehumidification in the presence of saturated salt solutions.

Figure 1. Water contents (a) and Tg (b) of amorphous (*) sucrose, (~) DX1500, (}) DX6000, and (!) DX20000 at different RHs. The water content and Tg for amorphous sucrose at RH 33% represent values reported in the literature.17,19

Differential Scanning Calorimetry Initially, 2–5 mg samples were transferred into 20 mL aluminum pans in a nitrogen atmosphere and hermetically sealed. The samples were prewarmed to a temperature at least 108C higher than the Tg to eliminate structural enthalpy relaxation.20,21 After prewarming, the samples were scanned at a rate of 108C/min from a prescribed temperature (at least 508C lower than the Tg) to 1808C, using an empty aluminum pan as a reference. A Perkin-Elmer DSC Pyris was used for the measurement. Calibrations for temperature and heat flow were made with distilled water (Tm ¼ 0.08C, DHm ¼ 333 J/g) and indium (Tm ¼ 156.68C, DHm ¼ 28.45 J/g). From the obtained thermograms, Tg values were determined as the onset temperatures of shifts in apparent specific heat due to transitions.

increasing molecular weight of dextran. Such a tendency has been generally observed,22 although the mechanism is not well known. The Tg values for amorphous samples composed of a single component as a function of RH are shown in Figure 1b. The Tg value for amorphous sucrose at RH 33% represents value reported in the literature.17 The Tg value decreases with increasing water content by the so-called plasticizing effect of the sorbed water.17,23 In Figure 1b, the Tg value for dextran increases with increasing molecular weight probably because covalent (ether) bonds between glucopyranose units would contribute more largely to the stabilization of the amorphous matrix than intermolecular hydrogen bonds.


Water Sorption Behavior of Sucrose–Dextran Mixtures

Moisture Contents and Glass Transition Temperatures for Amorphous Sucrose and Dextran In Figure 1a, the water contents for sucrose alone and dextran alone are shown as a function of RH. The water content of sucrose alone at RH 33% could not be determined because of the significant collapse of the sample and subsequent crystallization that occurred during humidification. Hence, as the corresponding water content, the value previously determined from the sucrose content dependency of the water content for sucrose–protein mixtures at RH 33%19 was used. Figure 1a shows that the water content increases with increasing molecular weight of dextran, indicating that the affinity of the amorphous matrix to the sorbed water becomes stronger with

In Figure 2, the equilibrium water contents for sucrose–dextran mixtures at different RHs are plotted against the dextran content. The lines in Figure 2 indicate the theoretical dependency of the water content on the dextran content calculated from the values for sucrose alone and dextran alone using the Lang and Steinberg mass balance equation24 given as Wcal ¼ wsucrose ð1  Mdextran =100Þ þ wdextran Mdextran =100


where Wcal and Mdextran are, respectively, the calculated water content and the dextran content. wsucrose and wdextran represent the water contents for sucrose alone and dextran alone, respectively, which are shown in Figure 1a. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 10, OCTOBER 2002



experimental water content of the sucrose– dextran mixture would be lower than the calculated value as shown in Figure 2. Glass Transition Behavior of Sucrose–Dextran Mixtures

Figure 2. Water contents of freeze-dried sucrose containing (a) DX1500, (b) DX6000, and (c) DX20000 at RH (}) 11%, (*) 23%, and (~) 33%. Water content for the sucrose-alone sample is the reported value.19 Dashed lines are theoretical values calculated from individual water contents for sucrose- and dextranalone samples using the Lang and Steinberg equation24 given as eq. (1).

As shown in Figure 2, the moisture contents of the sucrose–dextran mixtures are lower than those calculated using eq. 1 for all dextrans and RHs. This indicates that the amount of the water sorbed on sucrose and/or dextran in the mixture were lower than for sucrose alone and dextran alone. Due to the steric hindrance in the dextran molecule, the amorphous matrix of dextran is expected to contain some dead spaces, which are free of inter- and intramolecular interactions (hydrogen bonds). When an amorphous matrix is constructed solely of dextran, the hydroxyl groups in the dead spaces represent the hydration sites for interacting with water molecules. Whereas, in the sucrose–dextran mixture, the added sucrose molecules would be, more or less, integrated into the dead spaces because of the smaller size of a sucrose molecule. Because some of the hydration sites in the dextran molecules are occupied by the integrated sucrose molecules, the amount of the sorbed water for the sucrose– dextran mixture may become less than that for the case of the dextran alone sample. Thus, the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 10, OCTOBER 2002

The Tg values for the sucrose–dextran mixture against dextran content are shown in Figure 3. At RHs 0, 11, and 23%, the increase in the Tg value is insignificant for the dextran content up to 40%, and the Tg values then increase remarkably for dextran contents higher than 40%. Such a drastic increase in Tg for the sugar–polymer mixture at the polymer content higher than 40% has been shown also in other sugar–polymer systems.25–27 On the other hand, the Tg value at RH 33% increases, even at the dextran contents lower than 25%. The increase in the Tg value between dextran contents of 0 and 25% is about 208C. The Tg value then becomes constant in the range of 25–50%, and a further increase in the dextran content above 50% rises the Tg value drastically, which is similar to the results for RH 0, 11, and 23%. The difference between Tg value of the sample containing 5% dextran humidified for 1 day and that for 2 days at RH 33% was around 0.88C. From

Figure 3. Tg values of freeze-dried sucrose containing (a) DX1500, (b) DX6000, and (c) DX20000 at RH (!) 0% (}) 11%, (*) 23%, and (~) 33%. The Tg value for the sucrose-alone sample is cited from the literature.17


this fact, we can deduce that the samples with the low dextran content would be almost equilibriated at RH 33% within 2 days. It has been reported that Tg of an amorphous solid is determined by the strength of the interactions forming the matrix.28 Hence, the relationship between the dextran content and Tg values is discussed below, from the standpoint of interactions forming the amorphous matrix and their strengths. The sucrose–dextran mixture is generally thought to be formed by three intermolecular interactions at RH 0%. The first is the interactions between sucrose molecules (sucrose–sucrose interactions), the second involves interactions between dextran molecules (dextran–dextran interactions), and the third one is interactions between sucrose and dextran molecules (sucrose– dextran interactions). At the very low dextran contents, an amorphous matrix is formed mainly by sucrose–sucrose interactions, and the number of sucrose–dextran interactions would increase with increasing the dextran content. However, the Tg value is nearly constant at the dextran content lower than around 35% (Fig. 3). This fact would suggest that the strength of the sucrose– dextran interaction is similar to or only slightly stronger than that of sucrose–sucrose interaction. When the dextran content increases above a certain level, the number of the dextran–dextran interactions would increase and become predominant. Namely, the majority of the interactions in the matrix changes from sucrose–dextran to dextran–dextran interactions. Thus, the remarkable increase in the Tg value at high dextran contents of above 50% would be due to the presence of dextran–dextran interactions, which would be much stronger than sucrose–sucrose interactions, as indicated by the extremely higher Tg value (Fig. 1b). When the RH values are 11, 23, and 33%, a sucrose–dextran matrix contains the sorbed water as a third component. Due to the wellknown plasticizing effect,17,23 the sorbed water weakens the strength of the three sucrose– sucrose, sucrose–dextran, and dextran–dextran interactions as mentioned above. In fact, as shown in Figure 3, the curves for the Tg values shift to lower temperatures with increasing RH. It should be noticed that the Tg values increase markedly in the low dextran content range of 0–20% at RH 33% is observed. This fact seems to suggest that sucrose–dextran interactions strengthen the amorphous matrix at such a high RH. Such a


case could happen when sucrose–sucrose interactions are weakened more significantly by hydration than sucrose–dextran interactions. The reduction of hydration levels of dextran and/or sucrose due to the formation of sucrose– dextran interactions could be considered as another possible reason for the increase in the Tg value at the low dextran content. However, this contribution might be small, because the Tg values at RH 11 and 23% are not significantly increased in the low dextran content (Fig. 3) despite an appreciable reduction of the water content, as shown in Figure 2. Generally, to prepare a freeze-dried cake of sugar that does not collapse, the temperature of the frozen sample must be kept lower than its Tg. Thus, when the Tg value of the frozen sugar sample is low, the drying rate is quite limited, resulting in an extended freeze-drying period. On the other hand, as shown in Figure 3, the addition of a small amount of dextran increases the Tg sufficiently when the Tg value for amorphous sucrose is lower than 258C. Thus, the addition of a small amount of dextran may increase the drying rate by raising the Tg value of the sample containing a high water content. We conducted freeze drying for 24 h at room temperature (308C). The course of the sample temperature during freeze drying of the sucrose alone sample is shown in Figure 4. As shown in Figure 4, the sample temperature is 28  28C at the early period of freeze drying. At about 7 h from the start, the temperature starts to increase, indicating the disappearance of freezable water, and then gradually increases up to 308C at 13 h. Table 1 shows the relative volumes of the freeze-dried samples with different contents of dextran to the volume of the dextran alone showing no shrinkage due to collapse. The presence of 10 or 20% dextran remarkably reduced the shrinkage of the sample during freeze drying while no significant increase in Tg was found at RH 0–23%. This result

Figure 4. Temperature of sucrose alone sample during freeze-drying at room temperature (308C). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 10, OCTOBER 2002



Table 1. Relative Volumes of Sucrose–DX1500 Mixtures Freeze Dried at Room Temperature to That of DX1500-Alone Sample Dextran Content (%)

Relative Volume (%)

0 10 20 50

36 69 92 100

demonstrates that the addition of a small amount of polysaccharides would suppress the collapse. In this study, it was found that more than 50% dextran is required for the improvement of the physical stability of an amorphous sugar matrix at RHs equal to or below 23%. However, the addition of such a high content of polysaccharide would lower the stabilizing effect of the sugar matrix on dried proteins during storage. Thus, to improve both the stabilizing effects and the physical stability of an amorphous sugar mixture, suitable polysaccharides, which strengthens the amorphous matrix effectively at a low content, will be needed.

CONCLUSIONS We investigated the glass transition and water sorption behaviors of freeze-dried sucrose– dextran mixtures at RHs 0, 11, 23, and 33% in this study. The three types of the sucrose– dextran mixtures, differing in the molecular weight of the dextran, showed the identical dependencies of the water content and Tg on the dextran content and RH. The water content of the sucrose–dextran mixture was lower than that calculated by the Lang and Steinberg mass balance equation, indicating that the number of hydration sites for sucrose and dextran was lowered due to the formation of interaction between sucrose and dextran molecules. The dependency of Tg of the sucrose–dextran mixtures on the dextran content changed with RH. Namely, a remarkable increase in Tg was found only at high dextran contents above 50% in the RH range of 0–23%, whereas Tg increased remarkably in the dextran content range of 0–25% as well as higher than 50% at RH 33%. The addition of a small amount of dextran was found to suppress the shrinkage of the freeze-dried sample during freeze drying at room temperature while in the absence of dextran a serious collapse took place. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 10, OCTOBER 2002

ACKNOWLEDGMENTS This work was supported by a grant-in-aid for the Encouragement of Young Scientists, Nos. 10750548 and 12750664, from the Ministry of Education, Science, Sport and Culture of Japan.

REFERENCES 1. Pikal MJ. 1994. Freeze-drying of proteins. Process, formulation and stability. In: Cleland JL, Langer R, editors. Formulation and delivery of proteins and peptides. ACS Symposium Series, Vol. 567: Washington, DC: American Chemical Society. pp 120–133. 2. Wang YJ, Hanson MA. 1988. Parenteral formulations of proteins and peptides: Stability and Stabilizers. J Parenter Sci Technol 42:S2–S26. 3. Carpenter JF, Prestrelski SJ, Anchodorquy TJ, Arakawa T. 1994. Interactions of proteins with stabilizers during freezing and drying. In: Cleland JL, Langer JL, editors. Formulations and delivery of proteins and peptides. ACS Symposium Series, Vol. 567: Washington, DC: American Chemical Society. pp 134–147. 4. Carpenter JF, Crowe JH. 1988. Modes of stabilization of a protein by organic solutes during desiccation. Cryobiology 25:459–470. 5. Carpenter JF, Crowe JH. 1989. An infrared spectroscopic study of interactions of carbohydrates with dried proteins. Biochemistry 28:3916–3922. 6. Franks F. 1991. Long-term stabilization of biologicals. Biotechnology 12:38–42. 7. Levine H, Slade L. 1992. Another view of trehalose for drying and stabilizing biological materials. BioPharm 5:36–40. 8. Franks F, Hately RHM, Mathias SF. 1994. Material science and the production of shelf-stable biologicals. BioPharm 4:253–256. 9. Izutsu K, Yoshioka S, Takeda Y. 1991. The effect of additives on the stability of freeze-dried b-galactosidase stored at elevated temperature. Int J Pharm 71:137–146. 10. Liu WR, Langer R, Klibanov AM. 1991. Moistureinduced aggregation of lyophilized proteins in the solid state. Biotechnol Bioeng 37:177–184. 11. Chang BS, Beauvais RM, Dong A, Carpenter JF. 1996. Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: Glass transition and protein conformation. Arch Biochem Biophys 331:249–258. 12. Suzuki T, Imamura K, Fujimoto H, Okazaki M. 1998. Relation between thermal stabilizing effect of sucrose on LDH and sucrose-LDH hydrogen bond. J Chem Eng Jpn 31:565–570. 13. Prestrelski SJ, Pikal KA, Arakawa T. 1995. Optimization of lyophilization conditions for recombinant



15. 16.





human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy. Pharm Res 12:1250–1259. Allison SD, Manning MC, Randolph TW, Middleton K, Davis A, Carpenter JF. 2000. Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran. J Pharm Sci 89:199–214. Thayer AM. 1991. Biopharmaceuticals overcoming market hurdles. C&EN Feb 25:27–48. Slade L, Levine H. 1988. Non-equilibrium behavior of small carbohydrate-water system. Pure Appl Chem 60:1841–1864. Roos Y, Karel M. 1990. Differential scanning calorimetry study of phase transitions affecting the quality of dehydrated materials. Biotechnol Prog 6:159–163. Greenspan L. 1977. Humidity fixed points of binary saturated aqueous solutions. J Res NDS A Phys Chem 81:89. Imamura K, Iwai M, Ogawa T, Sakiyama T, Nakanishi K. 2001. Evaluation of hydration states of protein in freeze-dried amorphous sugar matrix. J Pharm Sci 90:1955–1963. Fox TG, Flory PJ. 1950. Second-order transition temperatures and related properties of polystyrene. I. Influence of molecular weight. J Appl Phys 21: 581–591.


21. Tant MR, Wilkes GL. 1981. An overview of the nonequilibrium behavior of polymer glasses. Polym Eng Sci 21:874–895. 22. Roos Y, Karel M. 1991. Phase transitions of mixtures of amorphous polysaccharides and sugars. Biotechnol Prog 7:49–53. 23. Roos Y, Karel M. 1991. Plasticizing effect of water on thermal behavior and crystallization of amorphous food model. J Food Sci 56:38–43. 24. Lang KW, Steinberg MP. 1980. Calculation of moisture content of a formulated food system to any given water activity. J Food Sci 46:1450– 1452. 25. Shamblin SL, Huang EY, Zografi G. 1996. The effects of co-lyophilized polmeric additives on the glass transition temperature and crystallization of amorphous sucrose. J Therm Anal 47:1567–1579. 26. Shamblin SL, Taylor LS, Zografi G. 1998. Mixing behavior of colyophilized binary systems. J Pharm Sci 87:694–701. 27. Oliver AE, Crowe LM, Crowe JH. 1998. Methods for dehydration-tolerance: Depression of the phase transition temperature in dry membranes and carbohydrate vitrification. Seed Sci Res 8:211–221. 28. Lin AA, Kwei TK, Reiser A. 1989. On the physical meaning of the Kwei equation for the glass transition temperature of polymer blends. Macromolecules 22:4112–4119.