4. Transfer the collected frozen films to the lyophilizer (see
Note 7).
5. Subject the thin films to lyophilization, collect the lyophilized
powder in sealed glass vials, and then store it in a desiccator at
room temperature before further analysis (see Note 8).
6. Characterize the powder (e.g., moisture content, particle size
distribution after reconstitution, antigen-binding efficacy,
immunogenicity).
3.2.2 TFF in a Single Vial
1. Immerse a salinized glass vial into liquid nitrogen to create a
cryogenically cooled surface in the inner bottom of the vial (see
Notes 9 and 10).
2. Using a pipette or syringe, add the vaccine formulation dropwise to the bottom of the vial so that the droplets, upon impact
of the surface, can rapidly freeze into thin films (see Notes 6
and 11).
3. Transfer the glass vial into a lyophilizer (see Note 7).
4. Subject the thin films to lyophilization, add a rubber stopper
and aluminum cap to the vial and store it in a desiccator at
room temperature before further analyses (see Note 8).
5. Characterize the powder.
3.2.3 Lyophilization
Drying is the next important step to obtain vaccine dry powder and
it could be achieved using lyophilization. Under certain pressure
and temperature, the water could be removed from the frozen-film
(sublimation), without going through the liquid phase. Any
remaining solvent might lead to loss of immunogenicity and/or
particle aggregation after reconstitution. To conduct the drying
using a lyophilizer, a VirTis Advantage bench top tray lyophilizer
or any other compatible tray lyophilizer can be used [24, 29].
1. Set the VirTis Advantage bench top tray lyophilizer shelf temperature at À40
C.
2. Prepare the collected reservoir or vials for the lyophilizer.
3. Start the lyophilization cycle: À40 to 25
C for 40 h (primary
drying), 20
C for 26 h (secondary drying), at a pressure
< 200 mTorr.
3.2.4 Particle Size
Distribution Measurement
The particle size of the antigen-adsorbed aluminum salt particles in
the vaccine is critical for the vaccine immunogenicity [2]. The
particle size distribution of the reconstituted vaccine may be
measured using a Sympatec Helos laser diffraction instrument
(Sympatec GmbH, Germany) equipped with an R3 lens [24].
Thin-Film Freeze-Drying Is a Viable Method to Convert Vaccines Containing. . .
495
Note 7).
5. Subject the thin films to lyophilization, collect the lyophilized
powder in sealed glass vials, and then store it in a desiccator at
room temperature before further analysis (see Note 8).
6. Characterize the powder (e.g., moisture content, particle size
distribution after reconstitution, antigen-binding efficacy,
immunogenicity).
3.2.2 TFF in a Single Vial
1. Immerse a salinized glass vial into liquid nitrogen to create a
cryogenically cooled surface in the inner bottom of the vial (see
Notes 9 and 10).
2. Using a pipette or syringe, add the vaccine formulation dropwise to the bottom of the vial so that the droplets, upon impact
of the surface, can rapidly freeze into thin films (see Notes 6
and 11).
3. Transfer the glass vial into a lyophilizer (see Note 7).
4. Subject the thin films to lyophilization, add a rubber stopper
and aluminum cap to the vial and store it in a desiccator at
room temperature before further analyses (see Note 8).
5. Characterize the powder.
3.2.3 Lyophilization
Drying is the next important step to obtain vaccine dry powder and
it could be achieved using lyophilization. Under certain pressure
and temperature, the water could be removed from the frozen-film
(sublimation), without going through the liquid phase. Any
remaining solvent might lead to loss of immunogenicity and/or
particle aggregation after reconstitution. To conduct the drying
using a lyophilizer, a VirTis Advantage bench top tray lyophilizer
or any other compatible tray lyophilizer can be used [24, 29].
1. Set the VirTis Advantage bench top tray lyophilizer shelf temperature at À40
C.
2. Prepare the collected reservoir or vials for the lyophilizer.
3. Start the lyophilization cycle: À40 to 25
C for 40 h (primary
drying), 20
C for 26 h (secondary drying), at a pressure
< 200 mTorr.
3.2.4 Particle Size
Distribution Measurement
The particle size of the antigen-adsorbed aluminum salt particles in
the vaccine is critical for the vaccine immunogenicity [2]. The
particle size distribution of the reconstituted vaccine may be
measured using a Sympatec Helos laser diffraction instrument
(Sympatec GmbH, Germany) equipped with an R3 lens [24].
Thin-Film Freeze-Drying Is a Viable Method to Convert Vaccines Containing. . .
495