3.2.5 Moisture Content
The moisture content in the dry powder is measured using a Karl
Fisher Titrator Aquapal III from CSC Scientific Company (Fairfax,
VA) [24].
3.2.6 Antigen-Binding
Efficiency
Free, unbound antigens before and after the vaccine is subjected to
TFFD and reconstitution can be measured by centrifuging the
vaccine for 5 min (4500 Â rcf) and measuring the protein content
in the supernatant [24]. The integrity of the antigen can be determined using SDS-PAGE after it is desorbed from the aluminum
salts [6, 32].
4 Notes
The following notes are listed to provide further description of the
exemplary adjuvanted vaccine dry powder and methods. The reader
can find supporting details described in the notes from the following references [20, 24–29].
1. The TFF apparatus requires a mechanical stirrer with the flexibility of adjusting the speed of the system. A Heidolph mechanical stirrer (Model RZR2041) connected to speed controller
(Model GS6013G) is used in our apparatus. Any other compatible model could be used to rotate the cylinder.
2. The cylinder surface temperature should be confirmed to be
around À40 to À50
C before starting to drop the vaccine
liquid onto the cylinder.
3. The rotating cylinder speed should be adjusted to avoid any
overlap between droplets over the cylinder. For our apparatus
model, a rotating speed of 5–7 rpm is optimal to achieve one
frozen film at a time.
4. Trehalose at a final concentration of 2% (w/v) has proven to be
sufficient for a successful TFFD. Its concentration may need to
be increased or optimized if a different vaccine is used.
5. The liquid nitrogen should be added to the reservoir to keep
the thin films frozen during and after the TFF process. Otherwise, the thin films will melt, and this could lead to a failure in
the entire TFF process.
6. Different designs may be used to apply the liquid.
7. The collected frozen films should be transferred to a À80
C
freezer if they are not lyophilized immediately.
8. The dry powder is hygroscopic and fluffy. Therefore, the lyophilized powder should be stored immediately in a desiccator
to reduce moisture absorption.
9. The neck and mouth of the silanized glass vial should not be
immersed in the liquid nitrogen. Yet the bottom of the vial
496
Riyad F. Alzhrani et al.
The moisture content in the dry powder is measured using a Karl
Fisher Titrator Aquapal III from CSC Scientific Company (Fairfax,
VA) [24].
3.2.6 Antigen-Binding
Efficiency
Free, unbound antigens before and after the vaccine is subjected to
TFFD and reconstitution can be measured by centrifuging the
vaccine for 5 min (4500 Â rcf) and measuring the protein content
in the supernatant [24]. The integrity of the antigen can be determined using SDS-PAGE after it is desorbed from the aluminum
salts [6, 32].
4 Notes
The following notes are listed to provide further description of the
exemplary adjuvanted vaccine dry powder and methods. The reader
can find supporting details described in the notes from the following references [20, 24–29].
1. The TFF apparatus requires a mechanical stirrer with the flexibility of adjusting the speed of the system. A Heidolph mechanical stirrer (Model RZR2041) connected to speed controller
(Model GS6013G) is used in our apparatus. Any other compatible model could be used to rotate the cylinder.
2. The cylinder surface temperature should be confirmed to be
around À40 to À50
C before starting to drop the vaccine
liquid onto the cylinder.
3. The rotating cylinder speed should be adjusted to avoid any
overlap between droplets over the cylinder. For our apparatus
model, a rotating speed of 5–7 rpm is optimal to achieve one
frozen film at a time.
4. Trehalose at a final concentration of 2% (w/v) has proven to be
sufficient for a successful TFFD. Its concentration may need to
be increased or optimized if a different vaccine is used.
5. The liquid nitrogen should be added to the reservoir to keep
the thin films frozen during and after the TFF process. Otherwise, the thin films will melt, and this could lead to a failure in
the entire TFF process.
6. Different designs may be used to apply the liquid.
7. The collected frozen films should be transferred to a À80
C
freezer if they are not lyophilized immediately.
8. The dry powder is hygroscopic and fluffy. Therefore, the lyophilized powder should be stored immediately in a desiccator
to reduce moisture absorption.
9. The neck and mouth of the silanized glass vial should not be
immersed in the liquid nitrogen. Yet the bottom of the vial
496
Riyad F. Alzhrani et al.