3. Prepare an OVA antigen solution in a normal saline solution
and mix the OVA solution and the Alhydrogel
® suspension at a
desired rate while stirring.
4. Incubate the mixture for at least 20 min while stirring in a
cold room.
5. Adjust the pH of the final vaccine formulation, if needed.
6. Add trehalose powder directly to the vaccine formulation to
reach a concentration of 2% (w/v) (see Note 4).
3.1.2 Preparation
of Commercially Available
Vaccines, Gardasil
®
and Engerix-B
®
Gardasil
® is an FDA-approved vaccine for the prevention of diseases caused by the human papillomavirus virus (HPV). Gardasil4
® is composed mainly of quadrivalent capsid L1 proteins (HPV-6,
11, 16 and 18) adsorbed onto amorphous aluminum hydroxyphosphate sulfate (225 μg of aluminum salt per a 500 μL dose) [30].
1. Dilute the vaccine tenfold in a normal saline solution.
2. Mix the vaccine gently by pipetting up and down.
3. Add trehalose powder directly to the diluted vaccine formulation to reach a concentration of 2% (w/v).
Engerix-B
® is a human vaccine against hepatitis B virus. The
vaccine is a suspension of hepatitis B virus surface antigen (HBsAg)
adsorbed onto aluminum hydroxide (500 μg per 1 mL dose) [31].
1. Shake the vaccine vials gently.
2. Add trehalose powder directly to the vaccine to reach a concentration of 2% (w/v).
3.2 Preparation
of Aluminum
Salt-Adjuvanted
Vaccine Dry Powder
The TFFD process can be divided into two steps, thin-film freezing
and drying. The liquid vaccine droplets are converted to frozen thin
films initially followed by drying via lyophilization [29]. The application of the TFFD have been largely demonstrated using the TFF
apparatus (Fig. 2a) [24–29], but a method of TFF in a single vial
(Fig. 2b) has also been developed [25].
3.2.1 Thin-Film Freezing
Process Using the TFF
Apparatus
1. Place a stainless-steel reservoir containing liquid nitrogen
below the rotating cryogenic cylinder (see Note 5).
2. Monitor the cylinder temperature (see Note 2) and adjust the
speed at which the cylinder rotates to be 5–10 rotations per
minute (rpm) (see Note 3).
3. Load vaccine formulation in an applicator (see Note 6) and
apply dropwise to the rotating cryogenically cooled cylinder
surface. The stainless-steel blade mounted along the surface of
the rotating cylinder will facilitate the removal of the thin films,
which will then drop into the stainless-steel reservoir below.
494
Riyad F. Alzhrani et al.
and mix the OVA solution and the Alhydrogel
® suspension at a
desired rate while stirring.
4. Incubate the mixture for at least 20 min while stirring in a
cold room.
5. Adjust the pH of the final vaccine formulation, if needed.
6. Add trehalose powder directly to the vaccine formulation to
reach a concentration of 2% (w/v) (see Note 4).
3.1.2 Preparation
of Commercially Available
Vaccines, Gardasil
®
and Engerix-B
®
Gardasil
® is an FDA-approved vaccine for the prevention of diseases caused by the human papillomavirus virus (HPV). Gardasil4
® is composed mainly of quadrivalent capsid L1 proteins (HPV-6,
11, 16 and 18) adsorbed onto amorphous aluminum hydroxyphosphate sulfate (225 μg of aluminum salt per a 500 μL dose) [30].
1. Dilute the vaccine tenfold in a normal saline solution.
2. Mix the vaccine gently by pipetting up and down.
3. Add trehalose powder directly to the diluted vaccine formulation to reach a concentration of 2% (w/v).
Engerix-B
® is a human vaccine against hepatitis B virus. The
vaccine is a suspension of hepatitis B virus surface antigen (HBsAg)
adsorbed onto aluminum hydroxide (500 μg per 1 mL dose) [31].
1. Shake the vaccine vials gently.
2. Add trehalose powder directly to the vaccine to reach a concentration of 2% (w/v).
3.2 Preparation
of Aluminum
Salt-Adjuvanted
Vaccine Dry Powder
The TFFD process can be divided into two steps, thin-film freezing
and drying. The liquid vaccine droplets are converted to frozen thin
films initially followed by drying via lyophilization [29]. The application of the TFFD have been largely demonstrated using the TFF
apparatus (Fig. 2a) [24–29], but a method of TFF in a single vial
(Fig. 2b) has also been developed [25].
3.2.1 Thin-Film Freezing
Process Using the TFF
Apparatus
1. Place a stainless-steel reservoir containing liquid nitrogen
below the rotating cryogenic cylinder (see Note 5).
2. Monitor the cylinder temperature (see Note 2) and adjust the
speed at which the cylinder rotates to be 5–10 rotations per
minute (rpm) (see Note 3).
3. Load vaccine formulation in an applicator (see Note 6) and
apply dropwise to the rotating cryogenically cooled cylinder
surface. The stainless-steel blade mounted along the surface of
the rotating cylinder will facilitate the removal of the thin films,
which will then drop into the stainless-steel reservoir below.
494
Riyad F. Alzhrani et al.