Solute crystallization in mannitol-glycine systems - implications on protein stabilization in freeze-dried formulations

Solute crystallization in mannitol-glycine systems - implications on protein stabilization in freeze-dried formulations
25 Aug 2003
Received: 17 March 2003; Revised: 9 May 2003; Accepted: 29 May 2003
Abira Pyne, Koustuv Chatterjee, Raj Suryanarayanan *
Journal of Pharmaceutical Sciences
Volume 92, Issue 11 , Pages 2272 - 2283
Wiley InterScience
College of Pharmacy, 308 Harvard St. S.E., University of Minnesota, Minneapolis, Minnesota 55455

email: Raj Suryanarayanan (surya001@tc.umn.edu)
*Correspondence to Raj Suryanarayanan, College of Pharmacy, 308 Harvard St. S.E., University of Minnesota, Minneapolis, Minnesota 55455. Telephone: 612-624-9626; Fax: 612-626-2125
Keywords
mannitol ? glycine ? sodium phosphate buffers ? lyoprotectants ? freeze drying ? crystallization
Abstract
The use of mannitol in combination with glycine has resulted in stable freeze-dried protein formulations. Our objectives were to (1) study solute crystallization in ternary systems containing mannitol, glycine, and water during all the stages of freeze drying as a function of processing conditions and formulation variables; (2) investigate the effect of sodium phosphate buffer salts on the crystallization of both mannitol and glycine and vice versa; and (3) investigate the effects of these excipients in a freeze-dried lactate dehydrogenase (LDH) formulation. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze-drying process in situ in the sample chamber of the diffractometer. LDH activity after freeze drying was determined spectrophotometrically. In frozen aqueous solutions containing mannitol and glycine, each solute influenced the extent of crystallization of the other. The solutes crystallized as -mannitol and -glycine during primary drying. Glycine had a stronger tendency to crystallize, while it was easier to influence mannitol crystallization. The buffer salts inhibited the crystallization of mannitol and glycine. However, in some cases, during primary drying, glycine crystallization was followed by that of disodium hydrogen phosphate dodecahydrate. The latter underwent dehydration forming an amorphous anhydrate. It was possible to correlate the extent of crystallization of mannitol and glycine in the lyophile with the retention of protein activity. An increase in buffer concentration decreased the crystallinity of mannitol and glycine. This translated to increased retention of protein activity. ? 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2272-2283, 2003
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