2.5 Reconstituting
the Product
It is often supposed that because freeze-drying only removes water,
then all products will be fully active by rehydrating only with water.
This may not be the case and freeze-dried products often exhibit
enhanced activity when reconstituted in an isotonic medium, such
as saline, rather than water. Establishing the correct rehydration
method can be as critical as developing a suitable and robust cycle;
this has been demonstrated in the freeze-drying and reconstitution
of red blood cells, where it was found that reconstitution with
hypotonic solutions could lead to regions of localized temporary
hypotonicity during the reconstitution process, which led to erythrocyte lysis, despite the fact that the pre-lyophilized suspension had
itself been significantly hypertonic [26].
3 Thermoanalytical and Microscopic Methods
Probably the most impactful change in freeze-drying over the past
20 years has been the introduction of commercially available freezedrying microscopy technology and the development of modulated
differential scanning calorimetry, which improved dramatically the
ability to measure glass transition and enthalpy relaxation behavior
of amorphous formulations. These technologies have transformed
how freeze-drying cycle and lyo formulation development have
been undertaken [11].
3.1 Freeze-Drying
Microscopy (FDM)
The principle of measuring the thermal properties of material under
the microscope was developed in the 1960s [27] but has only
become commercially available in the 2000s. Using a few microliters of sample mounted on glass slides or quartz crucibles, the
programmable cycle design can identify the freezing point, and
then the solidification of the freeze-dried matrix. Once a vacuum
is applied the rate of sublimation can be measured on small quantities of freeze-dried material under given conditions of vacuum
and temperature and the impact of modifying the freeze-drying
cycle can be evaluated [28]. In Fig. 1a, the FDM profile of 5%
trehalose is given, showing good drying at À37
C but collapse at
À33
C. The freeze-drying collapse temperature can be determined
for any given formulation over the course of a few experiments in
1–2 days. In Fig. 2a, the FDM images for a 5% sucrose 20 mM
histidine 0.01% Tween 20 formulation are given with a collapse
temperature around À35
C, and in Fig. 3a, the FDM profile of a
20 mg/mL heparin (a complex polysaccharide) sample is shown,
with collapse beginning around À21
C. Once robust data are
obtained from such studies, these conditions can then be transferred into the freeze-drying cycle for a given trial, together with
knowledge of the heat transfer properties of the selected containers;
this can result in a first freeze-drying cycle that has a far greater
chance not only of success at small scale but also in being robust,
transferable and scalable.
Principles of Freeze-Drying
117
the Product
It is often supposed that because freeze-drying only removes water,
then all products will be fully active by rehydrating only with water.
This may not be the case and freeze-dried products often exhibit
enhanced activity when reconstituted in an isotonic medium, such
as saline, rather than water. Establishing the correct rehydration
method can be as critical as developing a suitable and robust cycle;
this has been demonstrated in the freeze-drying and reconstitution
of red blood cells, where it was found that reconstitution with
hypotonic solutions could lead to regions of localized temporary
hypotonicity during the reconstitution process, which led to erythrocyte lysis, despite the fact that the pre-lyophilized suspension had
itself been significantly hypertonic [26].
3 Thermoanalytical and Microscopic Methods
Probably the most impactful change in freeze-drying over the past
20 years has been the introduction of commercially available freezedrying microscopy technology and the development of modulated
differential scanning calorimetry, which improved dramatically the
ability to measure glass transition and enthalpy relaxation behavior
of amorphous formulations. These technologies have transformed
how freeze-drying cycle and lyo formulation development have
been undertaken [11].
3.1 Freeze-Drying
Microscopy (FDM)
The principle of measuring the thermal properties of material under
the microscope was developed in the 1960s [27] but has only
become commercially available in the 2000s. Using a few microliters of sample mounted on glass slides or quartz crucibles, the
programmable cycle design can identify the freezing point, and
then the solidification of the freeze-dried matrix. Once a vacuum
is applied the rate of sublimation can be measured on small quantities of freeze-dried material under given conditions of vacuum
and temperature and the impact of modifying the freeze-drying
cycle can be evaluated [28]. In Fig. 1a, the FDM profile of 5%
trehalose is given, showing good drying at À37
C but collapse at
À33
C. The freeze-drying collapse temperature can be determined
for any given formulation over the course of a few experiments in
1–2 days. In Fig. 2a, the FDM images for a 5% sucrose 20 mM
histidine 0.01% Tween 20 formulation are given with a collapse
temperature around À35
C, and in Fig. 3a, the FDM profile of a
20 mg/mL heparin (a complex polysaccharide) sample is shown,
with collapse beginning around À21
C. Once robust data are
obtained from such studies, these conditions can then be transferred into the freeze-drying cycle for a given trial, together with
knowledge of the heat transfer properties of the selected containers;
this can result in a first freeze-drying cycle that has a far greater
chance not only of success at small scale but also in being robust,
transferable and scalable.
Principles of Freeze-Drying
117