The physical/chemical function between the additives is
mutual. It is important to maintain a suitable amount of additives in the formulation. It is preferable if one type of additive
fulfills several protective functions simultaneously. For example, several sugars can be used as cryoprotectant as well as
lyoprotectant. In addition, many buffer solutions or salts cannot be used because they may change the pH during the
freezing process or decrease the glass transition temperature
of the formula.
5. Additives can be classified as lyoprotectant, emulsifier, bulking
agent, antioxidant, and buffer agent. Formulations of lyoprotectants for specific proteins are listed in Tables 1, 2, 3, 4, 8,
and 9.
6. Glassware should be washed without detergents and heated/
autoclaved to sterilize. Containers used should be capable of
withstanding quick freezing such as the immersion in liquid
nitrogen or freezing within a freezer at À70
C or lower
without cracking. The containers must be capable to withstand
exposure to deep vacuum conditions.
7. Not only the free water in the formulation should be frozen to
form crystalline ice completely, but also the other constitutes of
the formulation should be solidified, to form non-crystalline
solid (glassy solid). Rapid freezing rates are recommended,
especially if the target protein rapidly loses activity in the liquid
state or if the selected formulation results in marked pH shifts
during freezing and denaturation. However, rapid freezing
may result in small ice crystals and longer drying times. If
rapid freezing is necessary, an annealing step may be needed
to correct the effects [12].
8. The cooling rate and the solidification process are also important factors. In general, rapid cooling would lead to formation
of partial glassy state and prevent excessive dehydration of the
material during cooling. However, if the cooling rate is too fast
it may cause fracture. The variation in temperature during the
process is called “thermal history.” The thermal history during
the cooling process is important because it affects the thermal
properties of the formulation. It has been found that annealing
affects the glass transition temperature Tg.
9. Insert a thermocouple into the samples to monitor the freezing
and drying process. It should be noted, however, that the
thermocouple will affect the nucleation point.
10. The material should be completely solidified after cooling. The
solidified proteins contain both crystalline solid and
non-crystalline hard reticulation structure solid. The cooling
and solidification process is an extremely important process,
which is often underestimated. The end temperature of the
698
Baolin Liu and Xinli Zhou
mutual. It is important to maintain a suitable amount of additives in the formulation. It is preferable if one type of additive
fulfills several protective functions simultaneously. For example, several sugars can be used as cryoprotectant as well as
lyoprotectant. In addition, many buffer solutions or salts cannot be used because they may change the pH during the
freezing process or decrease the glass transition temperature
of the formula.
5. Additives can be classified as lyoprotectant, emulsifier, bulking
agent, antioxidant, and buffer agent. Formulations of lyoprotectants for specific proteins are listed in Tables 1, 2, 3, 4, 8,
and 9.
6. Glassware should be washed without detergents and heated/
autoclaved to sterilize. Containers used should be capable of
withstanding quick freezing such as the immersion in liquid
nitrogen or freezing within a freezer at À70
C or lower
without cracking. The containers must be capable to withstand
exposure to deep vacuum conditions.
7. Not only the free water in the formulation should be frozen to
form crystalline ice completely, but also the other constitutes of
the formulation should be solidified, to form non-crystalline
solid (glassy solid). Rapid freezing rates are recommended,
especially if the target protein rapidly loses activity in the liquid
state or if the selected formulation results in marked pH shifts
during freezing and denaturation. However, rapid freezing
may result in small ice crystals and longer drying times. If
rapid freezing is necessary, an annealing step may be needed
to correct the effects [12].
8. The cooling rate and the solidification process are also important factors. In general, rapid cooling would lead to formation
of partial glassy state and prevent excessive dehydration of the
material during cooling. However, if the cooling rate is too fast
it may cause fracture. The variation in temperature during the
process is called “thermal history.” The thermal history during
the cooling process is important because it affects the thermal
properties of the formulation. It has been found that annealing
affects the glass transition temperature Tg.
9. Insert a thermocouple into the samples to monitor the freezing
and drying process. It should be noted, however, that the
thermocouple will affect the nucleation point.
10. The material should be completely solidified after cooling. The
solidified proteins contain both crystalline solid and
non-crystalline hard reticulation structure solid. The cooling
and solidification process is an extremely important process,
which is often underestimated. The end temperature of the
698
Baolin Liu and Xinli Zhou