X-Message-Number: 12368
Date: Sat, 4 Sep 1999 21:16:50 -0700 (PDT)
From: Doug Skrecky <>
Subject: long-term storage at -20 C

  Kerwin BA.  Heller MC.  Levin SH.  Randolph TW.
  Department of Protein Science, Baxter Hemoglobin Therapeutics Inc., Boulder,
  Colorado 80301, USA. 
  Effects of Tween 80 and sucrose on acute short-term stability and
  long-term storage at -20 degrees C of a
  recombinant hemoglobin.
  Journal of Pharmaceutical Sciences.  87(9):1062-8, 1998 Sep.
  The addition of low levels of surfactant polyoxyethylene 20 sorbitan
  monooleate, Tween 80, to recombinant hemoglobin in phosphate-buffered saline
  minimized the level of protein aggregation during acute freeze-thaw studies.
  Addition of sucrose alone to the phosphate-buffered saline formulation, up to
  0.5 M, provided minimal protection against freeze-thaw induced aggregation.
  In contrast to the acute stability studies, long-term
  storage at -20 degrees C induced aggregation and
  methemoglobin formation in those formulations containing only Tween 80 in
  phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the
  formulation prevented formation of aggregates and severely arrested
  methemoglobin formation during the long-term -20 degrees C
  storage. Specific binding of Tween 80 to the hemoglobin was
  not observed using 16-doxyl stearic acid partitioning techniques with
  electron paramagnetic resonance. Minor structural changes to the protein
  secondary structure during freezing in the absence and presence of Tween 80
  were observed with Fourier transform infrared spectroscopy. The alterations
  were partially prevented by addition of the sucrose. It is likely that the
  Tween 80 severely reduced protein aggregation during the acute stability
  studies by preventing the hemoglobin from reaching the air-liquid interface
  or the liquid-surface interfaces. The reduction in methemoglobin formation
  and aggregation observed during long-term
  storage can be accounted for on the premise that the sucrose
  reduced localized unfolding of the protein in a manner similar to the
  preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982,
  Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute
  formulation screening studies, albeit useful, may not necessarily predict
  protein stability during long-term storage.

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