title: Influence of crystallizing and non-crystallizing cosolutes on trehalose crystallization during freeze-drying.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Prakash
authors.initials: P
authors.lastname: Sundaramurthi
authors.forename: Raj
authors.initials: R
authors.lastname: Suryanarayanan
mesh_terms: D002152:Calorimetry, Differential Scanning; D003460:Crystallization; D005612:Freeze Drying; D014199:Trehalose / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D014199:Trehalose
references.citation: Pharm Res. 1999 Sep;16(9):1441-8
references.pmid: 10496663
references.citation: Pharm Res. 1993 Aug;10(8):1232-7
references.pmid: 8415413
references.citation: Pharm Res. 2009 Jul;26(7):1596-606
references.pmid: 19326191
references.citation: J Pharm Sci. 1995 Mar;84(3):318-23
references.pmid: 7616371
references.citation: Pharm Res. 2004 Feb;21(2):191-200
references.pmid: 15032301
references.citation: Pharm Res. 2001 Apr;18(4):474-9
references.pmid: 11451034
references.citation: J Pharm Sci. 2005 Apr;94(4):809-20
references.pmid: 15729705
references.citation: J Pharm Sci. 2004 May;93(5):1253-63
references.pmid: 15067701
references.citation: Pharm Res. 1996 Jan;13(1):146-50
references.pmid: 8668665
references.citation: Pharm Res. 2005 Nov;22(11):1978-85
references.pmid: 16132343
references.citation: J Phys Chem B. 2010 Apr 15;114(14):4915-23
references.pmid: 20302316
references.citation: Pharm Res. 2004 Jul;21(7):1167-76
references.pmid: 15290856
references.citation: Science. 1963 Sep 6;141(3584):921-3
references.pmid: 14043338
references.citation: J Pharm Sci. 2002 Apr;91(4):914-22
references.pmid: 11948529
references.citation: Pharm Res. 2001 Oct;18(10):1448-54
references.pmid: 11697471
references.citation: J Pharm Sci. 2001 Jul;90(7):872-87
references.pmid: 11458336
references.citation: J Pharm Sci. 2002 Sep;91(9):1969-79
references.pmid: 12210044
references.citation: Pharm Res. 2001 Jan;18(1):90-7
references.pmid: 11336359
references.citation: Pharm Res. 2010 Nov;27(11):2374-83
references.pmid: 20811935
references.citation: Pharm Res. 2007 Feb;24(2):370-6
references.pmid: 17177113
references.citation: Pharm Biotechnol. 2002;14:47-127
references.pmid: 12189730
references.citation: Pharm Res. 1997 Aug;14(8):969-75
references.pmid: 9279875
references.citation: PDA J Pharm Sci Technol. 1997 Mar-Apr;51(2):96-101
references.pmid: 9146041
references.citation: Acta Crystallogr B. 1970 Mar 31;26(3):290-9
references.pmid: 5467313
references.citation: Pharm Dev Technol. 2004;9(1):85-95
references.pmid: 15000469
references.citation: Pharm Res. 2004 May;21(5):867-74
references.pmid: 15180347
references.citation: J Pharm Sci. 2005 Apr;94(4):798-808
references.pmid: 15729706
references.citation: J Pharm Sci. 2003 Nov;92(11):2272-83
references.pmid: 14603512
references.citation: Pharm Res. 2007 Mar;24(3):593-604
references.pmid: 17245648
references.citation: J Pharm Sci. 1997 Nov;86(11):1198-203
references.pmid: 9383725
references.citation: J Pharm Sci. 1998 Aug;87(8):931-5
references.pmid: 9687336