The ice and solute crystal structure resulting from sample
freeze has a major impact on subsequent freeze-drying behavior,
encouraging the sample to dry efficiently or with defects such as
melt or collapse depending on freezing rate used. The preferred ice
structure comprising large contiguous ice crystals is induced by
cooling the sample at a slow rate of ~0.1–0.5
C/min, which may
also induce the crystallization of solutes reluctant to crystallize
when faster rates of cooling are used. However, a slow rate of
cooling may exacerbate the development of a surface skin, which
inhibits sublimation efficiency, and can also inactivate a bioproduct
by prolonging exposure to the solute concentrate. Conversely, a
fast rate of cooling can result in the formation of numerous, small,
randomly orientated ice crystals embedded in an amorphous solute
matrix, which may be difficult to freeze-dry, although snap freezing
may be unavoidable for some products such as adjuvanted vaccines
or whole cells where ice crystal growth could physically rupture the
outer membrane or cell wall. Complicating the choice of freezing
regimes is the fact that the optimal cooling rate cannot be sustained
uniformly throughout the entire sample, particularly with increased
fill depths or volumes, where temperature gradients within a single
container will be more marked. Therefore, defining cooling rates
often requires a compromise in sample requirements.
2.2.3 Ice Structure
and Freeze Consolidation
A period of consolidation (defined as the hold or soak time) is
necessary at the end of the initial cooling step to ensure that all
the contents in the sample batch have frozen adequately, although
excessively long hold times may only serve to increase the overall
cycle time without offering any particular other advantage. Indeed,
it is a fallacy to assume that the ice structure induced remains
unchanged during this consolidation period, and an ice structure
comprising a large number of small ice crystals, induced by rapid
cooling, is thermodynamically less stable than an ice structure
comprising fewer, larger crystals. The thermodynamic equilibrium
can be maintained by Ostwald ripening of ice from small to large
crystals, a process termed grain growth. Although ice structure
changes take place randomly from one container to another (e.g.,
vial to vial), the hold period is a major factor in ice recrystallization,
resulting in significant variation in crystal structure; as a consequence, sublimation efficiency from sample to sample may vary by
a wider margin the longer the freezing hold period is employed.
As an alternative to increasing the length of the hold time to
encourage ice recrystallization, a more controlled and time-efficient
method of inducing recrystallization is to anneal the frozen sample
[10]. Essentially, annealing is achieved by:
1. Cooling the product to freeze the solvent (usually water) and
any readily crystallizable solutes.
2. Raising the product temperature during the freezing stage to
recrystallize ice from a small to a large ice crystal matrix
Principles of Freeze-Drying
107
freeze has a major impact on subsequent freeze-drying behavior,
encouraging the sample to dry efficiently or with defects such as
melt or collapse depending on freezing rate used. The preferred ice
structure comprising large contiguous ice crystals is induced by
cooling the sample at a slow rate of ~0.1–0.5
C/min, which may
also induce the crystallization of solutes reluctant to crystallize
when faster rates of cooling are used. However, a slow rate of
cooling may exacerbate the development of a surface skin, which
inhibits sublimation efficiency, and can also inactivate a bioproduct
by prolonging exposure to the solute concentrate. Conversely, a
fast rate of cooling can result in the formation of numerous, small,
randomly orientated ice crystals embedded in an amorphous solute
matrix, which may be difficult to freeze-dry, although snap freezing
may be unavoidable for some products such as adjuvanted vaccines
or whole cells where ice crystal growth could physically rupture the
outer membrane or cell wall. Complicating the choice of freezing
regimes is the fact that the optimal cooling rate cannot be sustained
uniformly throughout the entire sample, particularly with increased
fill depths or volumes, where temperature gradients within a single
container will be more marked. Therefore, defining cooling rates
often requires a compromise in sample requirements.
2.2.3 Ice Structure
and Freeze Consolidation
A period of consolidation (defined as the hold or soak time) is
necessary at the end of the initial cooling step to ensure that all
the contents in the sample batch have frozen adequately, although
excessively long hold times may only serve to increase the overall
cycle time without offering any particular other advantage. Indeed,
it is a fallacy to assume that the ice structure induced remains
unchanged during this consolidation period, and an ice structure
comprising a large number of small ice crystals, induced by rapid
cooling, is thermodynamically less stable than an ice structure
comprising fewer, larger crystals. The thermodynamic equilibrium
can be maintained by Ostwald ripening of ice from small to large
crystals, a process termed grain growth. Although ice structure
changes take place randomly from one container to another (e.g.,
vial to vial), the hold period is a major factor in ice recrystallization,
resulting in significant variation in crystal structure; as a consequence, sublimation efficiency from sample to sample may vary by
a wider margin the longer the freezing hold period is employed.
As an alternative to increasing the length of the hold time to
encourage ice recrystallization, a more controlled and time-efficient
method of inducing recrystallization is to anneal the frozen sample
[10]. Essentially, annealing is achieved by:
1. Cooling the product to freeze the solvent (usually water) and
any readily crystallizable solutes.
2. Raising the product temperature during the freezing stage to
recrystallize ice from a small to a large ice crystal matrix
Principles of Freeze-Drying
107