change, such as ionic strength and relative composition of
solutes due to selective crystallization. These changes may
potentially destabilize a protein. Generally, lowering the temperature reduces the rate of chemical reactions. However,
chemical reactions may actually accelerate in a partially frozen
aqueous solution due to increased solute concentration.
3. Formation of ice-water interface. Freezing a protein solution
generates an ice-water interface. Proteins can be adsorbed to
the interface, loosening the native folding of proteins and
resulting in surface-induced denaturation. Rapid (quench)
cooling generates a large ice-water interface, while a smaller
interface is induced by slow cooling.
4. pH changes during freezing. Many proteins are stable only in a
narrow pH range. At extreme pHs, increased electrostatic
repulsion between charged groups in proteins tend to cause
protein unfolding or denaturation. Freezing a buffered protein
solution may selectively crystallize one buffering species, causing pH changes. Na 2 HPO 4 crystallizes more readily than
NaH 2 PO 4 . Because of this, a sodium phosphate buffer at
pH 7 has a molar [NaH 2 PO 4 ]:[Na 2 HPO 4 ] ratio of 0.72, but
this ratio increases to 57 at the ternary eutectic temperature
during freezing. This can lead to a significant pH drop during
freezing, which then denatures pH-sensitive proteins.
5. Phase separation during freezing. Freezing polymer solutions
may cause phase separation due to polymers’ altered solubilities
at low temperatures and create a large excess of interface,
denaturing the protein. Freezing-induced phase separation
can easily occur in a solution containing two incompatible
polymers such as dextran and ficoll. Several strategies have
been proposed to mitigate or prevent phase separation-induced
protein denaturation during freezing. These include use of
alternative salts, adjustment of the relative composition of
polymers to avoid or to rapidly pass over a temperature region
where the system may result in liquid-liquid phase separation,
and chemical modification of the protein such as pegylation.
6. Dehydration stresses. A fully hydrated protein has a monolayer of
water covering the protein surface, which is termed the hydration shell. Lyophilization removes part of the hydration shell.
Removal of the hydration shell may disrupt the native state of a
protein and cause denaturation. A hydrated protein, when
exposed to a water-poor environment during dehydration,
tends to transfer protons to ionized carboxyl groups and thus
abolishes as many charges as possible in the protein. The
decreased charge density may facilitate protein-protein hydrophobic interaction, causing protein aggregation.
Freeze-Drying of Proteins
685
solutes due to selective crystallization. These changes may
potentially destabilize a protein. Generally, lowering the temperature reduces the rate of chemical reactions. However,
chemical reactions may actually accelerate in a partially frozen
aqueous solution due to increased solute concentration.
3. Formation of ice-water interface. Freezing a protein solution
generates an ice-water interface. Proteins can be adsorbed to
the interface, loosening the native folding of proteins and
resulting in surface-induced denaturation. Rapid (quench)
cooling generates a large ice-water interface, while a smaller
interface is induced by slow cooling.
4. pH changes during freezing. Many proteins are stable only in a
narrow pH range. At extreme pHs, increased electrostatic
repulsion between charged groups in proteins tend to cause
protein unfolding or denaturation. Freezing a buffered protein
solution may selectively crystallize one buffering species, causing pH changes. Na 2 HPO 4 crystallizes more readily than
NaH 2 PO 4 . Because of this, a sodium phosphate buffer at
pH 7 has a molar [NaH 2 PO 4 ]:[Na 2 HPO 4 ] ratio of 0.72, but
this ratio increases to 57 at the ternary eutectic temperature
during freezing. This can lead to a significant pH drop during
freezing, which then denatures pH-sensitive proteins.
5. Phase separation during freezing. Freezing polymer solutions
may cause phase separation due to polymers’ altered solubilities
at low temperatures and create a large excess of interface,
denaturing the protein. Freezing-induced phase separation
can easily occur in a solution containing two incompatible
polymers such as dextran and ficoll. Several strategies have
been proposed to mitigate or prevent phase separation-induced
protein denaturation during freezing. These include use of
alternative salts, adjustment of the relative composition of
polymers to avoid or to rapidly pass over a temperature region
where the system may result in liquid-liquid phase separation,
and chemical modification of the protein such as pegylation.
6. Dehydration stresses. A fully hydrated protein has a monolayer of
water covering the protein surface, which is termed the hydration shell. Lyophilization removes part of the hydration shell.
Removal of the hydration shell may disrupt the native state of a
protein and cause denaturation. A hydrated protein, when
exposed to a water-poor environment during dehydration,
tends to transfer protons to ionized carboxyl groups and thus
abolishes as many charges as possible in the protein. The
decreased charge density may facilitate protein-protein hydrophobic interaction, causing protein aggregation.
Freeze-Drying of Proteins
685