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Trehalose Crystallization During Freeze-Drying: 
Implications On Lyoprotection  
Prakash Sundaramurthi and Raj Suryanarayanan,* 
Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455, USA.  
AUTHOR EMAIL ADDRESS (surya001@umn.edu) 
Supplementary information:  
 
 
Page No. 
Materials and methods 
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Aqueous solubility of trehalose and sucrose as the function of 
temperature. 
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MATERIALS AND METHODS 
Materials 
Trehalose dihydrate (C12H22O11·2H2O; Sigma, purity >99%) was used as received. Appropriate 
amount of trehalose dihydrate was dissolved in degassed deionized water to yield a 4% w/v 
trehalose (C12H22O11) solution. The deionized water was degassed by stirring at 70°C for 5 
minutes, followed immediately by filtration (0.45 µm membrane filter). The water was allowed 
to cool to room temperature (RT) in a closed container.  
Methods 
X-ray diffractometry (XRD) 
A powder X-ray diffractometer (Model XDS 2000, Scintag; Bragg-Brentano focusing 
geometry) with a variable temperature stage (High-Tran Cooling System, Micristar, Model 
828D, R.G. Hansen & Associates; working temperature range: −190 to 300 °C) and a solid-state 
detector was used. The sample stage was cooled using liquid nitrogen while a vacuum pump was 
used to evacuate the chamber. 
About 1 ml of the trehalose solution was filled into a custom designed sample holder. The 
sample chamber was sealed using a stainless steel cover. The beryllium window on the cover 
enabled X-ray diffraction studies during the freeze-drying. Thus the entire freeze-drying cycle 
was simulated in the sample chamber of the diffractometer, and the phase transitions were 
continuously monitored. Typically, the solutions were cooled, at 0.5 °C/min, from RT to −40 °C, 
held for 15 minutes, and warmed at 0.5 °C/min to the annealing temperature of −18 °C. While 
the primary drying was conducted for 3 hours at −25 °C (~ 200 mTorr), the secondary drying 
was performed at −10 °C for 1 hour, and subsequent drying was conducted at 0 and at 10 °C, for 
30 minutes at each temperature. The sample was periodically exposed to Cu Kα radiation (45 kV 
x 40 mA) over an angular range of 5 to 40° 2θ with a step size of 0.05° and a dwell time of 1 
second.  
Selected experiments were performed at the synchrotron beamline 6-ID-B of the Midwest 
Universities Collaborative Team (MUCAT), at the Advanced Photon Source, Argonne National 


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Laboratory (Argonne, IL, USA). More details of the synchrotron XRD instrumental setup, 
experimental conditions, and data refinement are provided in our earlier publication.17 The XRD 
patterns were compared with the published data in the Powder Diffraction Files (PDF) of the 
International Centre for Diffraction Data (ICDD).16 
In an effort to accelerate solute crystallization, experiments were also preformed with 
seeding. The frozen solution was seeded with crystals of either succinic acid or trehalose 
dihydrate and annealed. 
 
 


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Aqueous solubility of trehalose and sucrose as the function of temperature. The data were taken from 
Miller et al., 1997.7 The horizontal line represents the trehalose concentration in the prelyophilization 
solution.   
-20
0
20
40
60
80
0
10
20
30
40
50
60
70
80
 Trehalose
 Sucrose
 Succinic a
Solubility, % w/w
Temperature, °C
No ice 
crystallization
4 % w/w solution
-20
0
20
40
60
80
0
10
20
30
40
50
60
70
80
 Trehalose
 Sucrose
 Succinic a
Solubility, % w/w
Temperature, °C
No ice 
crystallization
4 % w/w solution