3. At the 20% power setting, the power output was determined to
be 108 Æ 7 W, measured using a K type thermocouple
immersed in a polypropylene beaker containing 500 mL of
18.2 MΩ H 2 O. The power at 100% was determined to be
608 Æ 21 W.
4. The dry weight was calculated for each sample by subtracting
its moisture content (obtained by Karl Fisher titration) from
the average of mass of a 40 μL of sample (obtained by weighing
ten replicate samples). Dry weights for ten replicates were used
to determine an average dry weight for 40 μL of 1.1 M trehalose in Tris-EDTA buffer.
5. HEPES-buffered minimum essential medium (Gibco Laboratories, Grand Island NY) supplemented with 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, 100 IU/mL penicillin,
100 IU/mL streptomycin, and 4 mg/mL bovine serum albumin (Sigma-Aldrich, St. Louis, MO).
6. Samples dried for 30 min yielded moisture contents that corresponded to a T g of 26.3
C, a moisture content that should
provide for stable storage at room temperature (22–24
C).
7. The dry weight of the DS was determined through bakeout
method to be 7.01% of the mass of a sample. Dry weight was
adjusted to include the mass of the protein based on its concentration to determine the total drying weight.
8. The concentration of protein in drying solution was verified
using the absorption of light at 280 nm with a microplate
spectrophotometer (Bio-Tek Synergy HT).
References
1. Roy I, Gupta MN (2004) Freeze-drying of
proteins: some emerging concerns. Biotechnol
Appl Biochem 39:165–177
2. Fonseca F, Cenard S, Passot S (2015) Freezedrying of lactic acid bacteria. In: Wolkers WF,
Oldenhof H (eds) Methods in cryopreservation and freeze-drying, Methods in molecular
biology. Springer, New York, pp 477–488
3. Wang S, Goecke T, Meixner C, Haverich A,
Hilfiker A, Wolkers WF (2012) Freeze-dried
heart valve scaffolds. Tissue Eng Part C Methods 18:517–525
4. Wang S, Oldenhof H, Goecke T, Ramm R,
Harder M, Haverich A, Hilfiker A, Wolkers
WF (2015) Sucrose diffusion in decellularized
heart valves for freeze-drying. Tissue Eng Part
C Methods 21:922–931
5. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J
Pharm 203:1–60
6. Graves-Herring JE, Wildt DE, Comizzoli P
(2013) Retention of structure and function of
the cat germinal vesicle after air-drying and
storage at suprazero temperature. Biol Reprod
88:139
7. Feng HY, Wu LJ, Xu A, Hu BR, Hei TK, Yu
ZL (2004) Survival of mammalian cells under
high vacuum condition for ion bombardment.
Cryobiology 49:241–249
8. Millqvist-Fureby A, Malmsten M, Bergenstahl
B (1999) Spray-drying of trypsin – surface
characterisation and activity preservation. Int J
Pharm 188:243–253
9. Chakraborty N, Biswas D, Parker W, Moyer P,
Elliott GD (2008) A role for microwave processing in the dry preservation of mammalian
cells. Biotechnol Bioeng 100:782–796
10. Iglesias HA, Chirife J, Buera MP (1997)
Adsorption isotherm of amorphous trehalose.
J Sci Food Agr 75:183–186
Drying Technology for Preservation of Biologics
219
be 108 Æ 7 W, measured using a K type thermocouple
immersed in a polypropylene beaker containing 500 mL of
18.2 MΩ H 2 O. The power at 100% was determined to be
608 Æ 21 W.
4. The dry weight was calculated for each sample by subtracting
its moisture content (obtained by Karl Fisher titration) from
the average of mass of a 40 μL of sample (obtained by weighing
ten replicate samples). Dry weights for ten replicates were used
to determine an average dry weight for 40 μL of 1.1 M trehalose in Tris-EDTA buffer.
5. HEPES-buffered minimum essential medium (Gibco Laboratories, Grand Island NY) supplemented with 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, 100 IU/mL penicillin,
100 IU/mL streptomycin, and 4 mg/mL bovine serum albumin (Sigma-Aldrich, St. Louis, MO).
6. Samples dried for 30 min yielded moisture contents that corresponded to a T g of 26.3
C, a moisture content that should
provide for stable storage at room temperature (22–24
C).
7. The dry weight of the DS was determined through bakeout
method to be 7.01% of the mass of a sample. Dry weight was
adjusted to include the mass of the protein based on its concentration to determine the total drying weight.
8. The concentration of protein in drying solution was verified
using the absorption of light at 280 nm with a microplate
spectrophotometer (Bio-Tek Synergy HT).
References
1. Roy I, Gupta MN (2004) Freeze-drying of
proteins: some emerging concerns. Biotechnol
Appl Biochem 39:165–177
2. Fonseca F, Cenard S, Passot S (2015) Freezedrying of lactic acid bacteria. In: Wolkers WF,
Oldenhof H (eds) Methods in cryopreservation and freeze-drying, Methods in molecular
biology. Springer, New York, pp 477–488
3. Wang S, Goecke T, Meixner C, Haverich A,
Hilfiker A, Wolkers WF (2012) Freeze-dried
heart valve scaffolds. Tissue Eng Part C Methods 18:517–525
4. Wang S, Oldenhof H, Goecke T, Ramm R,
Harder M, Haverich A, Hilfiker A, Wolkers
WF (2015) Sucrose diffusion in decellularized
heart valves for freeze-drying. Tissue Eng Part
C Methods 21:922–931
5. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J
Pharm 203:1–60
6. Graves-Herring JE, Wildt DE, Comizzoli P
(2013) Retention of structure and function of
the cat germinal vesicle after air-drying and
storage at suprazero temperature. Biol Reprod
88:139
7. Feng HY, Wu LJ, Xu A, Hu BR, Hei TK, Yu
ZL (2004) Survival of mammalian cells under
high vacuum condition for ion bombardment.
Cryobiology 49:241–249
8. Millqvist-Fureby A, Malmsten M, Bergenstahl
B (1999) Spray-drying of trypsin – surface
characterisation and activity preservation. Int J
Pharm 188:243–253
9. Chakraborty N, Biswas D, Parker W, Moyer P,
Elliott GD (2008) A role for microwave processing in the dry preservation of mammalian
cells. Biotechnol Bioeng 100:782–796
10. Iglesias HA, Chirife J, Buera MP (1997)
Adsorption isotherm of amorphous trehalose.
J Sci Food Agr 75:183–186
Drying Technology for Preservation of Biologics
219