2. The end freezing temperature can be derived from studies of
the critical glass transition by DSC or the collapse temperature
by freeze-drying microscopy experiments.
3. The sample temperature should be measured during the entire
freezing and drying process (see Note 9).
4. Make sure the length of the freezing is sufficient to ensure that
all samples are completely frozen (see Note 10).
3.6 Primary Drying
(Sublimation Drying)
Water can be classified as free water that is freezable at low temperatures and bound water that cannot be frozen. Primary drying
refers sublimation of frozen “free water” directly to vapor.
1. For proteins, the shelf temperature can be set between À40 and
À80
C according to the specifications of the freeze dryer.
2. Set the temperature during sublimation lower than the maximum allowable temperature Tmax1 of proteins as shown in
Fig. 1 (see Note 11).
3. The vacuum inside the freeze dryer usually maintains at
10–100 μbar [11].
4. The primary drying time ranges from several hours to several
days dependent on the drying system and solvent being
removed.
5. During primary drying, heat can be provided to the samples
through conduction or radiation (see Note 12).
3.7 Secondary
Drying (Desorption
Drying)
After primary drying, when most of the water is removed, the
secondary drying process can start (see Note 13).
1. During the secondary drying process, the sample temperature,
Tw2, should be less than the maximum allowable temperature,
Tmax2 (see Fig. 1).
2. The maximum allowable temperature, Tmax2, is dependent on
the type of protein (see Note 14).
3. The vacuum pressure should be kept the same as during primary drying.
4. The secondary drying process ends when the remaining moisture final (RMF) of the proteins is less than 5% (see Note 15).
3.8 ConditioningPacking and Storage
Sealing and packing should be conducted by breaking vacuum in a
dry environment (see Note 16). The packing and sealing should be
carried out either in the vacuum chamber or in a chamber with an
inert gas (nitrogen or argon). For freeze-dried proteins in bottles or
vials, sealing may be carried out in the drying chamber, with rubber
stoppers as shown in Fig. 2. Bulk products in ampoules can be taken
out of the drying chamber through a vacuum channel, delivered to
a vacuum chamber (or a chamber with sufficient inert gas), and
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Baolin Liu and Xinli Zhou
the critical glass transition by DSC or the collapse temperature
by freeze-drying microscopy experiments.
3. The sample temperature should be measured during the entire
freezing and drying process (see Note 9).
4. Make sure the length of the freezing is sufficient to ensure that
all samples are completely frozen (see Note 10).
3.6 Primary Drying
(Sublimation Drying)
Water can be classified as free water that is freezable at low temperatures and bound water that cannot be frozen. Primary drying
refers sublimation of frozen “free water” directly to vapor.
1. For proteins, the shelf temperature can be set between À40 and
À80
C according to the specifications of the freeze dryer.
2. Set the temperature during sublimation lower than the maximum allowable temperature Tmax1 of proteins as shown in
Fig. 1 (see Note 11).
3. The vacuum inside the freeze dryer usually maintains at
10–100 μbar [11].
4. The primary drying time ranges from several hours to several
days dependent on the drying system and solvent being
removed.
5. During primary drying, heat can be provided to the samples
through conduction or radiation (see Note 12).
3.7 Secondary
Drying (Desorption
Drying)
After primary drying, when most of the water is removed, the
secondary drying process can start (see Note 13).
1. During the secondary drying process, the sample temperature,
Tw2, should be less than the maximum allowable temperature,
Tmax2 (see Fig. 1).
2. The maximum allowable temperature, Tmax2, is dependent on
the type of protein (see Note 14).
3. The vacuum pressure should be kept the same as during primary drying.
4. The secondary drying process ends when the remaining moisture final (RMF) of the proteins is less than 5% (see Note 15).
3.8 ConditioningPacking and Storage
Sealing and packing should be conducted by breaking vacuum in a
dry environment (see Note 16). The packing and sealing should be
carried out either in the vacuum chamber or in a chamber with an
inert gas (nitrogen or argon). For freeze-dried proteins in bottles or
vials, sealing may be carried out in the drying chamber, with rubber
stoppers as shown in Fig. 2. Bulk products in ampoules can be taken
out of the drying chamber through a vacuum channel, delivered to
a vacuum chamber (or a chamber with sufficient inert gas), and
696
Baolin Liu and Xinli Zhou