freeze-dried products, (2) improving the appearance of the
freeze-dried products, (3) increasing the solubility of solutes,
and (4) preventing the freeze-died products from collapse or
overflow. Antioxidants, such as Vitamin D, Vitamin E, protein
hydrolysate, and sodium hyposulfite, are used to prevent oxidation during freeze-drying and storage. Buffer and chelating
agents, such as phosphoric acid, amino acids, and EDTA, regulate the pH value of the material and sequester ions.
2 Materials
2.1 Freeze-Drying
Systems
1. A freeze dryer (e.g., Virtis Advantage, Millrock, Tofflon or
similar unit).
2. Suitable containers for freeze-drying the product, e.g., roundbottomed glass flasks, and glass vials.
3. Freezing system, e.g., liquid nitrogen, a À80
C commercial
freezer.
2.2 Formulations for
Freeze-Drying of
Proteins
1. Selection of the optimal pH value: An optimum pH is needed
to keep a protein stable and soluble in solution (see Note 1).
2. Selection of buffer agents: Many buffering agents covering a
wide pH range are available for selection in formulating solid
proteins. These agents include acetate, citrate, glycine, histidine, phosphate, Tris, etc. (see Note 2).
3. Selection of bulking agents: Mannitol and glycine are two
typical bulking agents. Most amino acids are potential bulking
agents as they easily crystallize out (see Note 3).
4. Selection of cryoprotectants and lyoprotectants: A stabilizer
(s) for a solid protein product should be at least partially
amorphous and able to replace water, forming intimate hydrogen bonds with the protein.
5. Overall consideration of formulation excipients (see Note 4):
Several freeze-drying formulations for a variety of proteins and
their protective efficacy are shown in Table 5. Freeze-dried
protein drugs commercially available with their incorporated
excipients are shown in Table 6. Formulations of freeze-dried
cytokine products are shown in Table 7.
3 Methods
3.1 Preparation of
the Materials
For proteins, it is necessary to add some additives before freezedrying in order to keep their activity and get good quality of final
products. It is called “formulation” of the mixture of proteins and
Freeze-Drying of Proteins
689
freeze-dried products, (3) increasing the solubility of solutes,
and (4) preventing the freeze-died products from collapse or
overflow. Antioxidants, such as Vitamin D, Vitamin E, protein
hydrolysate, and sodium hyposulfite, are used to prevent oxidation during freeze-drying and storage. Buffer and chelating
agents, such as phosphoric acid, amino acids, and EDTA, regulate the pH value of the material and sequester ions.
2 Materials
2.1 Freeze-Drying
Systems
1. A freeze dryer (e.g., Virtis Advantage, Millrock, Tofflon or
similar unit).
2. Suitable containers for freeze-drying the product, e.g., roundbottomed glass flasks, and glass vials.
3. Freezing system, e.g., liquid nitrogen, a À80
C commercial
freezer.
2.2 Formulations for
Freeze-Drying of
Proteins
1. Selection of the optimal pH value: An optimum pH is needed
to keep a protein stable and soluble in solution (see Note 1).
2. Selection of buffer agents: Many buffering agents covering a
wide pH range are available for selection in formulating solid
proteins. These agents include acetate, citrate, glycine, histidine, phosphate, Tris, etc. (see Note 2).
3. Selection of bulking agents: Mannitol and glycine are two
typical bulking agents. Most amino acids are potential bulking
agents as they easily crystallize out (see Note 3).
4. Selection of cryoprotectants and lyoprotectants: A stabilizer
(s) for a solid protein product should be at least partially
amorphous and able to replace water, forming intimate hydrogen bonds with the protein.
5. Overall consideration of formulation excipients (see Note 4):
Several freeze-drying formulations for a variety of proteins and
their protective efficacy are shown in Table 5. Freeze-dried
protein drugs commercially available with their incorporated
excipients are shown in Table 6. Formulations of freeze-dried
cytokine products are shown in Table 7.
3 Methods
3.1 Preparation of
the Materials
For proteins, it is necessary to add some additives before freezedrying in order to keep their activity and get good quality of final
products. It is called “formulation” of the mixture of proteins and
Freeze-Drying of Proteins
689