Influence of crystallizing and non-crystallizing cosolutes on trehalose crystallization during freeze-drying

Prakash Sundaramurthi, Raj Suryanarayanan

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Purpose: To study the influence of crystallizing and non-crystallizing cosolutes on the crystallization behavior of trehalose in frozen solutions and to monitor the phase behavior of trehalose dihydrate and mannitol hemihydrate during drying. Methods: Trehalose (a lyoprotectant) and mannitol (a bulking agent) are widely used as excipients in freeze-dried formulations. Using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), the crystallization behavior of trehalose in the presence of (i) a crystallizing (mannitol), (ii) a non-crystallizing (sucrose) solute and (iii) a combination of mannitol and a model protein (lactose dehydrogenase, catalase, or lysozyme) was evaluated. By performing the entire freeze-drying cycle in the sample chamber of the XRD, the phase behavior of trehalose and mannitol were simultaneously monitored. Results: When an aqueous solution containing trehalose (4% w/v) and mannitol (2% w/v) was cooled to -40°C at 0.5°C/min, hexagonal ice was the only crystalline phase. However, upon warming the sample to the annealing temperature (-18°C), crystallization of mannitol hemihydrate was readily evident. After 3 h of annealing, the characteristic XRD peaks of trehalose dihydrate were also observed. The DSC heating curve of frozen and annealed solution showed two overlapping endotherms, attributed by XRD to the sequential melting of trehalose dihydrate - ice and mannitol hemihydrate - ice eutectics, followed by ice melting. While mannitol facilitated trehalose dihydrate crystallization, sucrose completely inhibited it. In the presence of protein (2 mg/ml), trehalose crystallization required a longer annealing time. When the freeze-drying was performed in the sample chamber of the diffractometer, drying induced the dehydration of trehalose dihydrate to amorphous anhydrate. However, the final lyophiles prepared in the laboratory lyophilizer contained trehalose dihydrate and mannitol hemihydrate. Conclusions: Using XRD and DSC, the sequential crystallization of ice, mannitol hemihydrate, and trehalose dihydrate was observed in frozen solutions. Mannitol, by readily crystallizing as a hemihydrate, accelerated trehalose dihydrate crystallization in frozen solutions. However, by remaining amorphous, sucrose completely inhibited trehalose dihydrate crystallization. Crystallization of the lyoprotectantt in the model protein formulations might have serious implications on protein stability.

Original languageEnglish (US)
Pages (from-to)2384-2393
Number of pages10
JournalPharmaceutical research
Issue number11
StatePublished - Nov 2010

Bibliographical note

Funding Information:
We thank Dr Len Thomas and Dr Steve Aubuchon from TA instruments for their comments. The XRD work was carried out at the College of Science and Engineering Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

Copyright 2011 Elsevier B.V., All rights reserved.


  • DSC
  • XRD
  • dehydration
  • mannitol hemihydrate
  • processing
  • protein
  • trehalose dihydrate


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