maintain activity of the protein [14]. Moisture content measurements are necessary to predict storage stability of the dried
protein formulation. Water contents can be determined using
the colorimetric Karl Fischer method or by thermogravimetric
analysis [15].
16. Avoiding contact with oxygen or water vapor in the air
increases storage stability of freeze-dried products. When the
product is needed, rehydration in water is necessary in most
cases.
17. The recovery and functional activity of the freeze-dried proteins should be assessed after the process. The appearance and
residual moisture content should also be assessed. Visually
inspect all vials in the batch and assess the appearance of the
freeze-dried samples. Heterogeneity should be documented to
see whether it correlates with a particular location in the freeze
dryer. Poor appearance may indicate suboptimal drying.
18. Some freeze-dried proteins can be stored at room temperature,
whereas other proteins need to be stored at 4
C or À18
C.
Accelerated aging studies at elevated temperatures and humidities can be used to estimate storage stability of the freeze-dried
protein. The trends in activity loss can be analyzed using
Arrhenius or other kinetic models and used to predict stability
at the storage temperature [16]. The recovered biological
activity should be measured against controls taken before
lyophilization, to determine the formulation that gives the
best activity after rehydration.
References
1. Passot S, Fonseca F, Alarcon-Lorca M (2005)
Physical characterisation of formulations for
the development of two stable freeze-dried
proteins during both dried and liquid storage.
Eur J Pharm Biopharm 60:335–348
2. Ma X, Wang W, Bouffard R (2001) Characterization of murine monoclonal antibody to
tumor necrosis factor (TNF-MAb) formulation
for freeze-drying cycle development. Pharm
Res 18:196–202
3. Nail SL, Her LM, Proffitt CPB (1994) An
improved microscope stage for direct observation of freezing and freeze drying. Pharm Res
11:1098–1100
4. Carpenter JF, Chang BS (1996) Lyophilization
of protein pharmaceuticals. In: Avis KE, Wu
VL (eds) Biotechnology and biopharmaceutical manufacturing, processing and preservation. Interpharm Press, Buffalo Grove, IL, pp
199–264
5. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J
Pharm 203:1–60
6. Chang LL, Pikal MJ (2009) Mechanisms of
protein stabilization in the solid state. J
Pharm Sci 98:2886–2908
7. Hua TC (2006) New technology of freezedrying. Science Press, Beijing
8. Hua TC, Liu BL, Zhang H (2010) Freezedrying of pharmaceutical and food products.
Woodhead Publishing Limited, Sawston
9. Lim JY, Kim NA, Lim DG, Kim KH, Choi DH,
Jeong SH (2016) Process cycle development of
freeze drying for therapeutic proteins with stability evaluation. J Pharm Invest 46:519–536
10. Lipi€ ainen T, Peltoniemi M, Sarkhel S,
Yrjo ¨nen T, Vuorela H, Urtti A, Juppo A
(2015) Formulation and stability of cytokine
therapeutics. J Pharm Sci 104:307–326
Freeze-Drying of Proteins
701
protein formulation. Water contents can be determined using
the colorimetric Karl Fischer method or by thermogravimetric
analysis [15].
16. Avoiding contact with oxygen or water vapor in the air
increases storage stability of freeze-dried products. When the
product is needed, rehydration in water is necessary in most
cases.
17. The recovery and functional activity of the freeze-dried proteins should be assessed after the process. The appearance and
residual moisture content should also be assessed. Visually
inspect all vials in the batch and assess the appearance of the
freeze-dried samples. Heterogeneity should be documented to
see whether it correlates with a particular location in the freeze
dryer. Poor appearance may indicate suboptimal drying.
18. Some freeze-dried proteins can be stored at room temperature,
whereas other proteins need to be stored at 4
C or À18
C.
Accelerated aging studies at elevated temperatures and humidities can be used to estimate storage stability of the freeze-dried
protein. The trends in activity loss can be analyzed using
Arrhenius or other kinetic models and used to predict stability
at the storage temperature [16]. The recovered biological
activity should be measured against controls taken before
lyophilization, to determine the formulation that gives the
best activity after rehydration.
References
1. Passot S, Fonseca F, Alarcon-Lorca M (2005)
Physical characterisation of formulations for
the development of two stable freeze-dried
proteins during both dried and liquid storage.
Eur J Pharm Biopharm 60:335–348
2. Ma X, Wang W, Bouffard R (2001) Characterization of murine monoclonal antibody to
tumor necrosis factor (TNF-MAb) formulation
for freeze-drying cycle development. Pharm
Res 18:196–202
3. Nail SL, Her LM, Proffitt CPB (1994) An
improved microscope stage for direct observation of freezing and freeze drying. Pharm Res
11:1098–1100
4. Carpenter JF, Chang BS (1996) Lyophilization
of protein pharmaceuticals. In: Avis KE, Wu
VL (eds) Biotechnology and biopharmaceutical manufacturing, processing and preservation. Interpharm Press, Buffalo Grove, IL, pp
199–264
5. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J
Pharm 203:1–60
6. Chang LL, Pikal MJ (2009) Mechanisms of
protein stabilization in the solid state. J
Pharm Sci 98:2886–2908
7. Hua TC (2006) New technology of freezedrying. Science Press, Beijing
8. Hua TC, Liu BL, Zhang H (2010) Freezedrying of pharmaceutical and food products.
Woodhead Publishing Limited, Sawston
9. Lim JY, Kim NA, Lim DG, Kim KH, Choi DH,
Jeong SH (2016) Process cycle development of
freeze drying for therapeutic proteins with stability evaluation. J Pharm Invest 46:519–536
10. Lipi€ ainen T, Peltoniemi M, Sarkhel S,
Yrjo ¨nen T, Vuorela H, Urtti A, Juppo A
(2015) Formulation and stability of cytokine
therapeutics. J Pharm Sci 104:307–326
Freeze-Drying of Proteins
701