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J. Phys. Chem. B 2003, 107, 6614-6620
An Investigation into the Crystallization of r,r-Trehalose from the Amorphous State Orla S. McGarvey, Vicky L. Kett, and Duncan Q. M. Craig* School of Pharmacy, The Queen’s UniVersity of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, U.K. ReceiVed: June 11, 2002; In Final Form: April 17, 2003
The nonisothermal behavior of amorphous R,R-trehalose has been studied in order to identify the factors influencing the recrystallization process. Particular emphasis has been placed on examining the effect of differential scanning calorimetry (DSC) pan type and initial water content on the thermal response. Samples of freeze-dried trehalose with water levels up to 6% w/w were sealed in pinholed and hermetically sealed pans and analyzed using DSC at a scanning rate of 10 °C/min in conjunction with thermogravimetric analysis (TGA), with the dihydrate studied in pinholed pans for comparison. The dihydrate was found to exhibit two endotherms at ca. 100 °C and 122 °C, followed by a further endotherm at 211 °C that was attributed to melting of the anhydrate. Isolation of the material following the first endotherm indicated that the material generated at this temperature was the Tγ form. Amorphous material heated in pinholed pans exhibited an exotherm at ca. 100-150 °C (the value depending on water content) when the initial water levels were 4.1% or below. This response was accompanied by dehydration and was attributed to the formation of the crystalline anhydrate. Higher water content samples, however, showed an exotherm followed immediately by an endotherm, with the water loss taking place in two distinct stages. XRD studies indicated that these samples initially crystallized into the Tγ form, followed by subsequent dehydration to the anhydrate. Samples analyzed in hermetically sealed pans showed a single exotherm between 100 and 150 °C followed by a broad endotherm between 150 and 200 °C, with higher water content samples having lower values for both events. XRD indicated that the exotherm and endotherm corresponded to the formation and fusion of the Tγ form, respectively. The study has therefore shown that the recrystallization behavior of amorphous trehalose is dependent on both the initial water content and the environment in which recrystallization takes place.
Introduction Trehalose (R-D-glucopyranosyl, R-D-glucopyranoside) is a nonreducing disaccharide that has been investigated in recent years for use as a cryoprotectant for proteinaceous and other drugs that may be susceptible to degradation during freezedrying.1 Trehalose occurs naturally as the R,R-isomer in yeasts, certain varieties of insects, algae, and higher plants such as Rhizobium leguminosarum, although other isomeric forms (R,βtrehalose and β,β-trehalose) have been prepared synthetically. The organisms in which trehalose occurs naturally have been shown to induce production of the disaccharide on desiccation, resulting in cell preservation until rehydration takes place; such behavior has been extensively studied as a means of preserving cells and biological membranes via exogenous addition of the cryoprotectant.2,3 In common with the majority of cryoprotectants, trehalose exists in the amorphous form following freeze-drying. An understanding of the properties and stability of this amorphous material is essential in order to optimize processing and storage conditions, both because there may be an association between the physical form of the disaccharide and the corresponding protective activity and also because any products based on this material must be physically stable over the shelf life of the product.4 More specifically, it has been suggested that the tendency of amorphous trehalose to partially or completely crystallize into hydrate forms may lead to the material acting * Corresponding author. Tel/Fax: 44 (0)28 90 272129. E-mail: duncan.
[email protected].
as a “sink” for excess water in the frozen or freeze-dried product, thereby protecting the protein from hydrolysis while also serving to maintain a high glass transition (Tg) within the remaining amorphous matrix via removal of plasticizing water.1 However, it should be noted the generation of hydrate forms on storage of an amorphous freeze-dried product is unacceptable from a pharmaceutical regulatory viewpoint, irrespective of the effect on protein stability, as it is essential that the physical structure of a formulation remains essentially unchanged over the shelf life of a product. On this basis, it is essential to have an understanding of the interconversion of physical forms of the trehalose, both to assist understanding of the mechanisms of cryoprotection and to enable prediction of solid state changes that are likely to occur on product storage. Given the importance of understanding the recrystallization of trehalose from the amorphous state, it is perhaps surprising that comparatively few studies have addressed this as a specific issue. The crystalline state of trehalose and associated interconversions have been discussed by numerous authors,5-12 although the precise number of polymorphic and pseudopolymorphic modifications that can exist is still uncertain. A significant proportion of work published on the crystalline forms of trehalose has concentrated on transitions occurring during the heating of the dihydrate, leading to dehydration and possibly recrystallization into one of a number of possible crystalline forms, depending on the experimental and sample conditions used. The literature is somewhat contradictory in terms of the number of crystalline forms that may be generated, almost certainly because the behavior of the system appears to be highly
10.1021/jp0262822 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/14/2003
Crystallization of R,R-Trehalose from the Amorphous State dependent on the experimental conditions used. There is general agreement on the existence of a stable dihydrate (commonly denoted Th) and anhydrous form (Tβ). However Sussich et al.8 have suggested that a second anhydrous form may be generated via gentle dehydration of the dihydrate (i.e., using low heating rates and allowing water to escape). This form (denoted by the authors as TR) effectively retains the molecular conformation and unit cell characteristics of the dihydrate and may be easily rehydrated on exposure to water due to the comparatively open crystalline structure of this material. On rapid heating the more stable and less hygroscopic Tβ anhydrous form may be generated. Sussich et al.9 have also suggested that trehalose may also exist in a form designated Tγ, generated by cold crystallization of partially dehydrated dihydrate at ca. 115 °C. Interestingly, these authors suggest that the form is a mixture of the dihydrate and anhydrous (Tβ) materials, possibly comprising an anhydrous shell around a dihydrate core on a particulate basis. The authors also stress that the form may only be generated at heating rates between 12 and 40 °C/min. Macdonald and Johari,13 however, have suggested that there are only two true crystal forms of trehalose, the Tβ anhydrous and a single dihydrate; these authors acknowledge the existence of the Tγ form but do not consider this to be a distinct phase. A further important consideration is the generation of an amorphous phase on heating the dihydrate. Taylor and York5 have suggested that the amorphous phase may be an intermediate in the conversion of the dihydrate to the anhydrous material depending on the particle size of the sample, with smaller fractions forming an intermediate amorphous phase followed by recrystallization while larger particles undergo a solid-solid transition to the hydrate. There has been additional debate regarding the existence of a further anhydrous form (designated Form II), prepared by leaving the dihydrate at 323 K for 48 h under vacuum.14 Nagase et al.15 have also referred to a further anhydrate as the Tκ form, suggesting that this may be equivalent to the TR form reported by Sussich et al.8 As stated earlier, the recrystallization of the amorphous material has not been widely studied, despite the suggestion by Aldous et al.1 that this process may be intrinsically associated with cryoprotection. In this investigation we have attempted to delineate the recrystallization behavior of amorphous trehalose by focusing on two experimental variables that we believe may have been responsible for some of the confusion in the literature. In the first instance, we have studied the effect of DSC pan type on the thermal response of both trehalose dihydrate and amorphous trehalose. Previous studies in the literature on the effect of pan type have focused on transitions occurring in the crystalline dihydrate form (e.g., ref 5), but to date this has not been studied in the context of amorphous material. Second we have investigated the effects of water content on the recrystallization behavior of the amorphous material. By using DSC, TGA, and powder XRD in conjunction, we intend to both identify the forms adopted by the amorphous trehalose and to understand how different experimental conditions may influence the recrystallization process, with concomitant implications for understanding the stability of freeze-dried trehalose products.
J. Phys. Chem. B, Vol. 107, No. 27, 2003 6615 mM were freeze-dried in 7 mL serum vials with a fill volume of 0.5 mL. Vials were cooled to -50 °C and held for 2 h before proceeding with the primary drying at shelf temperatures from -20 °C to +30 °C and the chamber pressure at 500 mTorr for 6 h followed by secondary drying at 60 °C and 800 mTorr for a further 8 h. Vials were stoppered under a dry nitrogen atmosphere. The amorphous nature of the material was confirmed using XRD. After freeze-drying, the samples were found to contain 1.4 ( 0.7% w/w water. The vials were stored unstoppered at 12% RH for up to 5 days to obtain materials with water contents up to 3% w/w, over 33% RH for up to 7 days to obtain samples with water contents up to 6% w/w. Four vials were prepared under each set of conditions and samples taken from each vial for analysis; tabulated data represent averages of the four samples. Preparation of Tβ and Tγ Forms. The Tβ form was prepared by heating a sample of amorphous trehalose at low initial water content (
