7. 1% (w/v) Thimerosal [2-(ethylmercuriomercapto)benzoic acid
sodium salt] solution, if needed.
8. Trehalose (i.e., α-D-glucopyranosyl-α-D-glucopyranoside).
9. Liquid nitrogen.
2.2 Equipment
and Supplies
1. Syringes, pipettes, and needles (e.g., 17 G).
2. Trays and beakers.
3. Desiccators.
4. Silanized glass vials.
5. Centrifuge.
6. Cold room (2–8
C).
7. pH meter,
8. Freezer (À80
C).
9. VirTis Advantage bench top freeze dryer apparatus.
2.3 Thin-Film
Freezing (TFF)
Apparatus
A typical TFF apparatus (Fig. 2a) has a rotating stainless steel
cylinder filled with liquid nitrogen, and its surface temperature is
monitored via an external thermocouple (see Notes 1 and 2)
[28]. The rotating speed of the drum is adjusted to enable one
thin-film formation at a time (see Note 3) [28, 29]. It is recommended to have a stainless steel blade mounted along the drum
surface to facilitate removal of the frozen thin films and collecting
them in a reservoir below the rotating drum, filled with liquid
nitrogen [28, 29]. The TFF apparatus is operated in a room with
low relative humidity (at least <~15%) to avoid the condensation of
water vapor on the drum surface [29].
3 Methods
3.1 Aluminum
Salt-Adjuvanted
Vaccines
Aluminum salts such as amorphous aluminum hydroxyphosphate
sulfate, aluminum (oxy)hydroxide, and aluminum (hydroxy)phosphate are the most common salt types used as adjuvants in
human and veterinary vaccines [6]. Different aluminum salts have
different physicochemical properties that could directly affect their
adjuvanticity. Based on the antigens, different conditions (e.g., pH,
binding efficiency, salt concentration) should be carefully investigated before preparing aluminum salt-adjuvanted vaccines. Readers
are referred to other methods of preparing aluminum salt containing vaccines in reference [6]. However, vaccines prepared by in situ
adsorption of protein antigens (e.g., ovalbumin (OVA)) onto aluminum salt particles will be described below as an exemplary
method to prepare aluminum salt-adjuvanted vaccines for TFFD.
Additionally, we include a method of converting two commercially
available vaccines from liquid to dry powder using the TFFD
method.
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Riyad F. Alzhrani et al.
sodium salt] solution, if needed.
8. Trehalose (i.e., α-D-glucopyranosyl-α-D-glucopyranoside).
9. Liquid nitrogen.
2.2 Equipment
and Supplies
1. Syringes, pipettes, and needles (e.g., 17 G).
2. Trays and beakers.
3. Desiccators.
4. Silanized glass vials.
5. Centrifuge.
6. Cold room (2–8
C).
7. pH meter,
8. Freezer (À80
C).
9. VirTis Advantage bench top freeze dryer apparatus.
2.3 Thin-Film
Freezing (TFF)
Apparatus
A typical TFF apparatus (Fig. 2a) has a rotating stainless steel
cylinder filled with liquid nitrogen, and its surface temperature is
monitored via an external thermocouple (see Notes 1 and 2)
[28]. The rotating speed of the drum is adjusted to enable one
thin-film formation at a time (see Note 3) [28, 29]. It is recommended to have a stainless steel blade mounted along the drum
surface to facilitate removal of the frozen thin films and collecting
them in a reservoir below the rotating drum, filled with liquid
nitrogen [28, 29]. The TFF apparatus is operated in a room with
low relative humidity (at least <~15%) to avoid the condensation of
water vapor on the drum surface [29].
3 Methods
3.1 Aluminum
Salt-Adjuvanted
Vaccines
Aluminum salts such as amorphous aluminum hydroxyphosphate
sulfate, aluminum (oxy)hydroxide, and aluminum (hydroxy)phosphate are the most common salt types used as adjuvants in
human and veterinary vaccines [6]. Different aluminum salts have
different physicochemical properties that could directly affect their
adjuvanticity. Based on the antigens, different conditions (e.g., pH,
binding efficiency, salt concentration) should be carefully investigated before preparing aluminum salt-adjuvanted vaccines. Readers
are referred to other methods of preparing aluminum salt containing vaccines in reference [6]. However, vaccines prepared by in situ
adsorption of protein antigens (e.g., ovalbumin (OVA)) onto aluminum salt particles will be described below as an exemplary
method to prepare aluminum salt-adjuvanted vaccines for TFFD.
Additionally, we include a method of converting two commercially
available vaccines from liquid to dry powder using the TFFD
method.
492
Riyad F. Alzhrani et al.