the process of choice for improving the shelf life of proteins, the
process itself and the subsequent long-term storage generate several stresses responsible for protein denaturation. Even in the solid
state, chemical and physical degradation such as deamidation, oxidation, and aggregation can occur during the freeze-drying process, as well as during distribution and use. Stability problems
occurring during processing or storage are usually addressed
through process and formulation optimization.
In order to design an optimum freeze-drying process, process
development scientists need to know the critical properties of the
formulation and how to apply this information to design the process. The critical formulation properties include the collapse temperature of the formulation, the stability of the protein, and the
properties of the excipients used. Many approaches have been
explored to determine the thermal properties of formulations for
freeze-drying, including differential thermal analysis (DTA), differential scanning calorimetry (DSC) [1], electrical resistance analysis
(ERA) [2], and freeze-drying microscopy (FDM) [3].
To develop a protein formulation that minimizes protein
unfolding during freezing and drying, it is crucial that the specific
conditions (e.g., pH and specific stabilizing ligands) for optimum
protein stability be established and that the appropriate nonspecific
stabilizing additives (i.e., those excipients that generally stabilize
any protein) be incorporated into the formulation. Other physical
factors such as the glass transition temperature and the residual
moisture content of the dried solid must also be optimized to
assure storage stability in the dry state [4]. Here we will describe
how to design formulations that protect proteins during both
freezing and drying and discuss the mechanisms by which additives
stabilize proteins.
We wish to emphasize that each protein has unique physicochemical characteristics, which often manifest themselves as specific
routes of chemical and physical degradation. Thus, there is a great
need to increase the fundamental understanding of the mechanisms
by which protein stabilizers act and to document, by case studies,
the applicability of the general rules to individual proteins.
1.2 Impact of FreezeDrying on Proteins
The freeze-drying process generates a variety of stresses to denature
proteins. These include [5, 6]:
1. Low-temperature stress. Studies on low-temperature denaturation of model proteins indicated a temperature of maximum
stability exists, and both high and low temperature can destabilize a protein. The possible mechanism may be the decreasing
solvophobic interaction in proteins which can reach a point
where protein stability reaches zero, causing cold denaturation.
2. Concentration effect. Freezing a protein solution rapidly
increases the concentration of all solutes due to ice formation.
Thus, all physical properties related to concentration may
684
Baolin Liu and Xinli Zhou
process itself and the subsequent long-term storage generate several stresses responsible for protein denaturation. Even in the solid
state, chemical and physical degradation such as deamidation, oxidation, and aggregation can occur during the freeze-drying process, as well as during distribution and use. Stability problems
occurring during processing or storage are usually addressed
through process and formulation optimization.
In order to design an optimum freeze-drying process, process
development scientists need to know the critical properties of the
formulation and how to apply this information to design the process. The critical formulation properties include the collapse temperature of the formulation, the stability of the protein, and the
properties of the excipients used. Many approaches have been
explored to determine the thermal properties of formulations for
freeze-drying, including differential thermal analysis (DTA), differential scanning calorimetry (DSC) [1], electrical resistance analysis
(ERA) [2], and freeze-drying microscopy (FDM) [3].
To develop a protein formulation that minimizes protein
unfolding during freezing and drying, it is crucial that the specific
conditions (e.g., pH and specific stabilizing ligands) for optimum
protein stability be established and that the appropriate nonspecific
stabilizing additives (i.e., those excipients that generally stabilize
any protein) be incorporated into the formulation. Other physical
factors such as the glass transition temperature and the residual
moisture content of the dried solid must also be optimized to
assure storage stability in the dry state [4]. Here we will describe
how to design formulations that protect proteins during both
freezing and drying and discuss the mechanisms by which additives
stabilize proteins.
We wish to emphasize that each protein has unique physicochemical characteristics, which often manifest themselves as specific
routes of chemical and physical degradation. Thus, there is a great
need to increase the fundamental understanding of the mechanisms
by which protein stabilizers act and to document, by case studies,
the applicability of the general rules to individual proteins.
1.2 Impact of FreezeDrying on Proteins
The freeze-drying process generates a variety of stresses to denature
proteins. These include [5, 6]:
1. Low-temperature stress. Studies on low-temperature denaturation of model proteins indicated a temperature of maximum
stability exists, and both high and low temperature can destabilize a protein. The possible mechanism may be the decreasing
solvophobic interaction in proteins which can reach a point
where protein stability reaches zero, causing cold denaturation.
2. Concentration effect. Freezing a protein solution rapidly
increases the concentration of all solutes due to ice formation.
Thus, all physical properties related to concentration may
684
Baolin Liu and Xinli Zhou