X-Message-Number: 9887
Date: Wed, 10 Jun 1998 21:45:15 -0700 (PDT)
From: Doug Skrecky <>
Subject: KCL helps in freeze-drying 

Authors
  Heller MC.  Carpenter JF.  Randolph TW.
Institution
  Department of Chemical Engineering, University of Colorado at Boulder
  80309-0424, USA.
Title
  Manipulation of lyophilization-induced phase separation: implications for
  pharmaceutical proteins.
Source
  Biotechnology Progress.  13(5):590-6, 1997 Sep-Oct.
Abstract
  Lyophilization, or freeze-drying, of
  pharmaceutical proteins is often the only processing method that provides
  requisite long-term product stability. Freezing and drying,
  however, can cause acute damage to proteins. To alleviate damage,
  formulations frequently include protein stabilizers (often polymers and/or
  sugars), as well as buffering salts and "inert" bulking agents. While great
  efforts are placed on developing a formulation and suitable lyophilization
  cycle, incompatibilities among components through freezing and
  drying have been almost completely ignored. We demonstrate
  that solutions of poly(ethylene glycol) (PEG) and dextran, initially below
  critical concentrations for phase separation, do indeed experience a
  liquid-liquid phase separation induced by freeze
  concentration during the lyophilization cycle. The separation is shown to
  evolve with annealing at -7 degrees C and can be effectively inhibited simply
  by replacing NaCl with KCl in the formulation buffer. In addition, we show
  that phase separation causes unfolding of a model protein, recombinant
  hemoglobin, when freeze-dried in the PEG/dextran system.
  When the phase separation is averted by switching to KCl, the protein
  structural damage is also avoided. Measurements of pH in the frozen solutions
  show that the structural damage is not a result of pH changes. We suggest
  that KCl forms a glass with rapid cooling which kinetically prevents the
  phase separation and thus the protein structural damage.

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