U.S. patent application number 14/941323 was filed with the patent office on 2016-05-26 for dry solid aluminum adjuvant-containing vaccines and related methods thereof.
The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Zhengrong Cui, Xinran Li, Robert O. Williams, III.
Application Number | 20160144023 14/941323 |
Document ID | / |
Family ID | 51899025 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160144023 |
Kind Code |
A1 |
Cui; Zhengrong ; et
al. |
May 26, 2016 |
DRY SOLID ALUMINUM ADJUVANT-CONTAINING VACCINES AND RELATED METHODS
THEREOF
Abstract
Described herein are dry vaccine compositions and methods of
freezing aluminum-containing vaccines such that when converted into
a dried powder, the dry vaccine can be readily reconstituted to
form a stable liquid vaccine without significant loss of
activity.
Inventors: |
Cui; Zhengrong; (Austin,
TX) ; Williams, III; Robert O.; (Austin, TX) ;
Li; Xinran; (Waltham, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Family ID: |
51899025 |
Appl. No.: |
14/941323 |
Filed: |
November 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2014/038475 |
May 16, 2014 |
|
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14941323 |
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61824181 |
May 16, 2013 |
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Current U.S.
Class: |
424/490 ;
424/184.1; 424/239.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/466 20180101; A61K 9/14 20130101; A61K 39/39 20130101; A61K
39/0005 20130101; Y02A 50/39 20180101; A61K 39/08 20130101; A61K
2039/55505 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 9/14 20060101 A61K009/14; A61K 39/08 20060101
A61K039/08; A61K 39/00 20060101 A61K039/00 |
Claims
1. A dry vaccine comprising: an antigenic protein and an aluminum
adjuvant, wherein at least 75% of said antigenic protein is
adsorbed to said aluminum adjuvant.
2. (canceled)
3. (canceled)
4. The dry vaccine of claim 1, wherein said aluminum adjuvant is
aluminum hydroxide, aluminum phosphate, potassium phosphate, or
potassium aluminum sulfate.
5. (canceled)
6. (canceled)
7. The dry vaccine of claim 1, comprising less than 2% water.
8. The dry vaccine of claim 1, wherein at least 80% of said
antigenic protein is adsorbed to said aluminum adjuvant.
9. (canceled)
10. (canceled)
11. The dry vaccine of claim 1, further comprising an
excipient.
12-16. (canceled)
17. A method for preparing a vaccine thin film comprising: applying
a liquid vaccine to a freezing surface; allowing said liquid
vaccine to disperse and freeze on said freezing surface thereby
forming a vaccine thin film.
18. The method of claim 17, wherein said liquid vaccine comprises
an aluminum adjuvant.
19. The method of claim 18, wherein said aluminum adjuvant
comprises aluminum hydroxide, aluminum phosphate, aluminum sulfate,
or aluminum potassium sulfate.
20. The method of claim 17, wherein said liquid vaccine comprises
about 0.5% to 5% (wt/vol) of an aluminum adjuvant/liquid
vaccine.
21. The method of claim 17, wherein said liquid vaccine comprises
an excipient.
22. The method of claim 17, wherein said liquid vaccine comprises
about 0.5% to 5% (wt/vol) of an excipient/liquid vaccine.
23-28. (canceled)
29. The method of claim 17, further comprising removing the solvent
from the vaccine thin film to form a dry vaccine.
30. The method of claim 29, wherein said dry vaccine comprises an
antigenic protein and an aluminum adjuvant, wherein at least 75% of
said antigenic protein is adsorbed to said aluminum adjuvant.
31.-36. (canceled)
37. The method of claim 30, wherein said dry vaccine comprises an
excipient.
38-40. (canceled)
41. The method of claim 29, wherein said removing of the solvent
comprises lyophilization.
42. The method of claim 29, further comprising solvating said dry
vaccine thereby forming a reconstituted liquid vaccine.
43-45. (canceled)
46. The method of claim 42, wherein the level of antigenic protein
adsorbed to said aluminum adjuvant of said reconstituted liquid
vaccine is at least 90% of the level of antigenic protein adsorbed
to said aluminum adjuvant of said liquid vaccine.
47. The method of claim 42, wherein said reconstituted liquid
vaccine comprises particles, wherein said particles comprise said
antigenic protein adsorbed to said aluminum adjuvant.
48. The method of claim 47 wherein said particles have an average
diameter of between 10 nm and 5 .mu.m.
49-53. (canceled)
54. A method of treating a disease in a patient in need of such
treatment, said method comprising administering a therapeutically
effective amount of a solvated dry vaccine of claim 1 to said
patient, wherein said disease is diphtheria, tetanus, pertussis,
influenza, pneumonia, otitis media, bacteremia, meningitis,
hepatitis, cirrhosis, anthrax poisoning, rabies, warts,
poliomyelitis, Japanese encephalitis, or cancer.
55. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/824,181, filed May 16, 2013, which is
incorporated herein by reference in its entirety and for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The invention generally relates to vaccine compositions.
More particularly, the invention relates to vaccine powders
produced from aqueous vaccine compositions.
[0003] Aluminum-containing compounds such as aluminum hydroxide and
aluminum phosphate have been used as human vaccine adjuvants for
decades. Many currently commercially available vaccines, such as
diphtheria-tetanus-pertussis vaccine, Hepatitis A vaccines,
Hepatitis B vaccines, Pneumococcal conjugate vaccines, anthrax
vaccines, and Rabies vaccines, contain aluminum-containing
adjuvants. However, a major limiting factor with these vaccines is
that they cannot be frozen (i.e., the vaccines must remain stored
as a refrigerated liquid dispersion from manufacturing through to
administration to patients), because freezing of the dispersion
causes irreversible coagulation that damages the vaccines (e.g.,
loss in potency and stability). Aluminum-containing vaccines are
formulated as liquid suspensions and are required to be kept
refrigerated at 2-8.degree. C. during transport and storage.
Vaccines that have been inadvertently exposed to freezing
conditions before being administered to patients must be discarded,
causing significant product waste and limited utility. This is
significant considering that this cold-chain storage alone accounts
for up to 80% of the financial cost of vaccination, and
complicating matters further, an estimated 75-100% of the vaccine
shipments are actually exposed to freezing temperatures, resulting
in costly waste and the loss of nearly half of all global vaccine
supplies (WHO data). It is evident that having aluminum-containing
vaccines in a solid form that can be easily reconstituted just
prior to injection would be hugely beneficial to our health care
system today. It is also evident that administration of the solid
form of the aluminum-containing vaccines, without having to
reconstitute into a liquid suspension, for example for
administration by inhalation, would be hugely beneficial as
well.
[0004] There are several main aluminum-containing adjuvants,
aluminum hydroxide, aluminum phosphate, and aluminum potassium
sulfate. Aluminum hydroxide adjuvant is composed of small primary
fibers with an average calculated dimension of
4.5.times.2.2.times.10 nm, whereas the primary particles of
aluminum phosphate adjuvant are around 50 nm. In solutions,
however, the size of the primary particles of both aluminum
hydroxide and aluminum phosphate becomes 1-20 .mu.m as a result of
aggregation. Aggregation is typically irreversible. Due to their
favorable safety profile, aluminum-containing adjuvants have been
widely used in human vaccines for decades. Recently, there had been
extensive efforts in identifying the relationship between the size
of particulate vaccine carriers and their adjuvant activities.
Although it remains controversial as to what particle size is
associated with the most potent adjuvant activity, it is clear now
that the size of particulate vaccine carriers significantly affects
their adjuvant activities.
[0005] Methods of making dry vaccines that retain particle size and
immunogenicity upon reconstitution would be useful and would
address the deficiencies that current exist in the field. Provided
herein are methods and compositions addressing these and other
needs in the art.
BRIEF SUMMARY OF THE INVENTION
[0006] In an aspect is provided a dry vaccine including an
antigenic protein and an aluminum adjuvant, wherein at least 75% of
the antigenic protein is adsorbed to the aluminum adjuvant.
[0007] In an aspect is provided a pharmaceutical composition
including a pharmaceutically acceptable excipient and any of the
compositions (e.g. vaccines) described herein (including
embodiment).
[0008] In an aspect is provided a method for preparing a vaccine
thin film including: applying a liquid vaccine to a freezing
surface; allowing the liquid vaccine to disperse and freeze on the
freezing surface thereby forming a vaccine thin film.
[0009] In an aspect is provided a method of treating a disease in a
patient in need of such treatment, the method including
administering a therapeutically effective amount of a solvated dry
vaccine as described herein (e.g. in an aspect, embodiment,
example, table, figure, or claims) (e.g. a reconstituted liquid
vaccine as described herein) to the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A-D. Microscopic images of OVA-adsorbed aluminum
hydroxide particles before freeze-drying (FIG. 1A) and after high
speed thin-film freeze-drying and reconstitution (FIG. 1B), slow
freezing at -20.degree. C., drying and reconstitution (FIG. 1C),
and slow freezing at -80.degree. C., drying and reconstitution
(FIG. 1D).
[0011] FIG. 2. The fraction of free (unbound) OVA as determined
from the intensity of the protein bands on the SDS-PAGE gel.
[0012] FIG. 3. In the thermogram of the lyophilized OVA-adsorbed
aluminum hydroxide powder, a glass transition temperature (Tg) of
about 120.degree. C. was observed.
[0013] FIG. 4A-C. Anti-OVA IgG levels in mice that were immunized
with the lyophilized and reconstituted OVA-adsorbed aluminum
hydroxide were not different from that in mice that were immunized
the freshly prepared OVA-adsorbed aluminum hydroxide particles;
female BALB/c mice (18-20 g, n=5) were subcutaneously injected with
OVA-adsorbed aluminum hydroxide particles, before or after
lyophilization and reconstitution, on days 0, 14 and 28 with 5
.mu.g (FIG. 4A), 10 .mu.g (FIG. 4B), or 20 .mu.g (FIG. 4C) of OVA
per mouse.
[0014] FIG. 5. Lyophilized OVA-adsorbed aluminum hydroxide
particles have a rough surface and are in irregular shapes.
[0015] FIG. 6A-B. FIG. 6A shows the images of OVA-adsorbed aluminum
hydroxide particles lyophilized with different concentrations of
trehalose. FIG. 6B shows the sizes of the reconstituted
OVA-adsorbed aluminum hydroxide powders lyophilized with various
concentrations of trehalose.
[0016] FIG. 7A-D. FIGS. 7A and 7B show photos of lyophilized
OVA-adsorbed aluminum hydroxide and OVA-adsorbed aluminum phosphate
using thin-film freezing, respectively. FIGS. 7C and 7D show
microscopic images of lyophilized OVA-adsorbed aluminum hydroxide
and OVA-adsorbed aluminum phosphate after reconstitution in water.
Shown in insets in FIG. 7C and FIG. 7D are the particle sizing
results from the laser diffraction instrument.
[0017] FIG. 8A-B. FIGS. 8A-B show the microscopic images of the
physical mixture of OVA-adsorbed Alhydrogel and the OVA-adsorbed
Alhydrogel dry powder after reconstitution, respectively.
[0018] FIG. 9A-C. FIG. 9A. Original vaccine. FIG. 9B. Vaccine after
TFF with 2% trehalose. FIG. 9C. Vaccine after TFF with 3%
trehalose.
[0019] FIG. 10A-D. FIG. 10A depicts the particle sizes (open bar)
and zeta potentials ( ) of aluminum hydroxide nanoparticles (NPs)
and microparticles (MPs). FIG. 10B the aluminum hydroxide
nanoparticles were stable when stored at 4.degree. C. for a month,
whereas the microparticles were slightly less stable. The X-ray
powder patterns of aluminum hydroxide particles are presented in
FIGS. 10C and 10D. FIG. 10C the nanoparticles were completely
amorphous. FIG. 10D the microparticles were mostly crystalline
Al(OH).sub.3, although the large peak in the left showed that some
amorphous AlO(OH) materials existed as well.
[0020] FIG. 11A-E. FIG. 11A Sizes (open bar) and zeta potentials (
) of the aluminum hydroxide nanoparticles and microparticles after
the adsorption of OVA protein at a 1:2 ratio (OVA vs. particle,
w/w). FIG. 11B Fractions of free OVA when a fixed amount of OVA was
mixed with an increasing amount of the aluminum hydroxide
nanoparticles or microparticles. FIGS. 11C and 11D depict SEM
pictures of OVA-adsorbed aluminum hydroxide nanoparticles (OVA-NPs)
and OVA-adsorbed aluminum hydroxide microparticles (OVA-MPs). FIG.
11E depicts a TEM picture of OVA-NPs.
[0021] FIG. 12A-B. FIG. 12A The OVA-adsorbed aluminum hydroxide
nanoparticles were successfully lyophilized with trehalose (2%) as
a lyoprotectant. FIG. 12B In a short-term 28-day study, the size of
the lyophilized, OVA-adsorbed aluminum hydroxide nanoparticles did
not change when stored as a lyophilized powder at 4.degree. C.
[0022] FIG. 13A-B. FIG. 13A The anti-OVA IgG level in mice that
were immunized with the OVA-adsorbed aluminum hydroxide
nanoparticles was significantly higher than that in mice that were
immunized with OVA alone or OVA-adsorbed microparticles at 100-fold
dilution. FIG. 13B. 31 days after tumor cell injection, tumors were
detected only in one of the 5 mice that were immunized with the
OVA-adsorbed aluminum hydroxide nanoparticles.
[0023] FIG. 14A-E. FIG. 14A, open bars The mean diameters of the
resultant PA-adsorbed aluminum hydroxide nanoparticles and
microparticles were 204.+-.25 nm and 7.1.+-.3.4 .mu.m,
respectively. FIG. 14B Mice were then immunized with the
PA-adsorbed aluminum hydroxide nanoparticles or microparticles on
days 0 and 14. One week after the first dose, anti-PA IgG was not
detectable in any mice. One week after the second dose, significant
anti-PA IgG responses were detected in mice that were immunized
with the PA-adsorbed aluminum hydroxide nanoparticles or
microparticles, although the levels of the anti-PA IgG response
were not different. FIG. 14C However, 4 weeks after the second
immunization, the anti-PA IgG levels in mice that were immunized
with the PA-adsorbed aluminum hydroxide nanoparticles were
significantly higher than that in mice that were immunized with the
PA-adsorbed aluminum hydroxide microparticles. FIG. 14D Anti-PA
IgG1 levels 4 weeks after the second immunization are shown. FIG.
14E The kinetics of the anti-PA IgG levels within 4 weeks is shown
in. Significant higher anti-PA IgG1 level was detected in mice
immunized with PA-adsorbed aluminum hydroxide nanoparticles as
compared to in mice immunized with PA-adsorbed aluminum hydroxide
microparticles. Anti-IgE level was not detected 4 weeks after
immunization with PA-adsorbed aluminum hydroxide nanoparticles or
microparticles.
[0024] FIG. 15 More OVA was internalized when adsorbed on the
aluminum hydroxide nanoparticles than when adsorbed on the aluminum
hydroxide microparticles.
[0025] FIG. 16A-C. Green fluorescence signal, an indication of the
location of the OVA protein, was detected only inside cells that
were incubated with OVA-adsorbed aluminum hydroxide nanoparticles,
not in cells that were incubated with OVA-adsorbed aluminum
hydroxide microparticles.
[0026] FIG. 17A-D. Microparticles and nanoparticles both induced
local cutaneous inflammation in the injection sites when examined
40 days after the last dose, but the inflammation induced by the
PA-adsorbed microparticles was much more severe, as shown by a
greater number of accumulations of neutrophils around the injection
sites and the pronounced epidermal hyperplasia.
[0027] FIG. 18A-B. The Engerix-B human hepatitis B vaccine from
GlaxoSmithKline contains aluminum hydroxide as an adjuvant.
Trehalose was added into the vaccine suspension to reach a final
concentration of 2% (w/v). The vaccine was then subjected to
thin-film freeze-drying (TFFD) as mentioned previously. The
moisture content in the TFFD powder was 1.15%. Shown in FIG. 18A is
a representative image of fresh Engerix-B vaccine under an Olympus
BX60 microscope. FIG. 18B is a representative image of the
Engerix-B vaccine after reconstitution from the TFFD powder. The
particle size of the Engerix-B after it was subjected to TFFD and
reconstitution was 3.29.+-.0.15 .mu.m, and particle size of the
fresh vaccine was 5.64.+-.0.01 .mu.m, as determined using a
Sympatec Helos laser diffraction instrument (Sympatec GmbH,
Germany) equipped with a R3 lens. Clearly, the human hepatitis B
vaccine Engerix-B can be converted into a dry powder using the TFFD
method.
[0028] FIG. 19A-C. Concentrated tetanus toxoid (TT vaccine)
contains aluminum potassium sulfate as an adjuvant. Trehalose was
added into 1-ml vial of TT vaccine to reach a final concentration
of 2% (w/v). The vaccine was then subjected to TFFD as mentioned
previously. Three vials of the dried TT vaccine powder and three
vials of fresh TT vaccine in 2% (w/v) of trehalose were frozen in
-20.degree. C. for 8 h and then thawed at 4.degree. C. for 16 h.
The freezing-and-thawing was repeated for three cycles. After the
third cycle, the dry TT vaccine powder was reconstituted and
examined under a microscope. FIG. 19A is a representative image of
the fresh TT vaccine under a microscope. Shown in FIG. 19B is a
representative image of the TT vaccine reconstituted from dry
powder after the powder was subjected to three cycles of
freezing-and-thawing. Shown in FIG. 19C is a representative image
of the fresh TT vaccine after 3 cycles of freezing-and-thawing.
Clearly, repeated freezing-and-thawing of the fresh TT vaccine
caused significant aggregation, while the dry TT vaccine powder is
not sensitive to freezing anymore.
[0029] FIG. 20A-C. Concentrated tetanus toxoid (TT) vaccine
contains aluminum potassium sulfate as an adjuvant. Trehalose was
added into 1-ml vial of the TT vaccine to reach a final
concentration of 2% (w/v). The vaccine was then subjected to TFFD.
The dry TT vaccine powder was reconstituted and stored at 4.degree.
C. for 6 days. FIG. 20A is a representative image of the fresh TT
vaccine. Shown in FIG. 20B and FIG. 20C are representative images
of the TT vaccine 0 and 6 days after reconstitution, respectively.
The TT vaccine does not have to be used immediately after it is
reconstituted from a dry powder.
[0030] FIG. 21A-B. Alhydrogel.TM. (25 ml, 10 mg Al.sup.3+/ml) was
added into a 50 ml tube, followed by addition of 25 ml of OVA
solution (1 mg/ml) at an OVA to Al.sup.3+ weight ratio of 1:10.
Trehalose was added to a final concentration of 2% (w/v). The
sample was subjected to TFFD, and the dried powder was quickly
transferred to a sealed container and stored in a desiccator at
room temperature. Ten months later, the dry powder was
reconstituted and observed under a microscope. Shown in FIG. 21A is
a representative image of the OVA/Alhydrogel vaccine powder after
10 months of storage at room temperature. The particle size of the
vaccine after reconstitution was 3.78.+-.0.94 .mu.m. A
representative image of freshly prepared OVA/Alhydrogel vaccine is
shown in FIG. 21B. It appears that there was not any significant
aggregation after the dry powder was stored at room temperature for
10 months.
[0031] FIG. 22A-D. Representative microscopy images of OVA-adsorbed
aluminum hydroxide (before FIG. 22A) and after lyophilization (FIG.
22B-D) with 2% trehalose (w/v)). In FIG. 22B-D, the method of
freezing was TFF, shelf-freezing at -20.degree. C., and
shelf-freezing at -80.degree. C., respectively.
[0032] FIG. 23A-B. TFFD of OVA-adsorbed aluminum hydroxide in
various concentrations of trehalose. FIG. 23A. Particle sizes of
OVA-adsorbed aluminum hydroxide reconstituted from powders that
were lyophilized using various concentrations of trehalose. FIG.
23B. Representative images of dried OVA-adsorbed aluminum hydroxide
powders prepared with 1%, 2%, or 3% (w/v) trehalose.
[0033] FIG. 24A-D. Characterization of OVA-adsorbed aluminum
hydroxide powder prepared with TFFD. FIG. 24A. The binding
efficiency of OVA to aluminum hydroxide before and after TFFD
(inset, OVA protein band in SDS-PAGE gel). FIG. 24B. DSC curves of
OVA-adsorbed aluminum hydroxide dry powder, OVA, trehalose, and
aluminum hydroxide alone. FIG. 24C. A representative SEM image
OVA-adsorbed aluminum hydroxide dry powder. FIG. 24D. A
representative SEM image of the freshly prepared OVA-adsorbed
aluminum hydroxide.
[0034] FIG. 25A-C. Anti-OVA IgG levels in mice immunized with
OVA-adsorbed aluminum hydroxide, before and after TFFD. Female
BALB/c mice (n=5) were s.c. injected with OVA-adsorbed aluminum
hydroxide, before or after lyophilization and reconstitution, on
days 0, 14 and 28 with 5 .mu.g (FIG. 25A), 10 .mu.g (FIG. 25B), or
20 .mu.g (FIG. 25C) of OVA per mouse. The ratio of OVA to aluminum
was 1 to 10. Sterile PBS and OVA alone (10 .mu.g) in PBS were used
as controls. Total anti-OVA IgG levels in serum samples were
measured 16 days after the third dose.
[0035] FIG. 26A-C. TFFD of OVA adjuvanted with aluminum phosphate
or Alhydrogel and its stability at room temperature. FIG. 26A A
representative microscopic image of OVA-adsorbed aluminum
phosphate. FIG. 26B-C Representative images of OVA-adsorbed
Alhydrogel.RTM. reconstituted immediately after TFFD (FIG. 26B) or
after 10 months of storage at room temperature (FIG. 26C),
respectively.
[0036] FIG. 27A-G. TFFD of tetanus toxoid vaccine and Engerix-B.
FIG. 27A. A representative microscopy image of the original TT
vaccine after dilution in 2% (w/v) of trehalose. FIG. 27B. A
representative microscopy image of the TT vaccine after TFFD and
reconstitution in a phosphate buffer. FIG. 27C. Intrinsic
tryptophan fluorescence spectra of TT vaccine before and after
TFFD. FIG. 27D. Anti-tetanus toxin IgG levels in serum samples of
mice immunized with TT vaccine before and after TFFD. Female BALB/c
mice (n=5) were s.c. injected with TT vaccine, before or after TFFD
and reconstitution, on days 0, 14 and 28 with 3.75 Lf of tetanus
toxoid per mouse per injection. Sterile PBS and original TT vaccine
diluted in sterile PBS or 2% trehalose were used as controls. Total
anti-tetanus toxin IgG levels in serum samples were measured 16
days after the third dose. FIG. 27E. A representative image of the
fresh TT vaccine after 3 cycles of freeze-and-thaw. FIG. 27F. A
representative images of TT vaccine reconstituted from dry powder
after the powder was subjected to three cycles of freeze-and-thaw.
FIG. 27G-H. Representative images of fresh Engerix-B vaccine (FIG.
27G) and Engerix-B after reconstitution from TFFD powder (FIG.
27H).
DETAILED DESCRIPTION
[0037] Described herein is a new method to freeze
aluminum-containing vaccines with a very low percentage
cryoprotectant(s) such that when converted into a dried powder, the
solid can be readily reconstituted to form a stable dispersion
without significant loss of stability or activity. The solid form
of the vaccines may now be transported and stored in a wide range
of temperatures without concern of accidental exposure to freezing
conditions. In addition, the solid form of the vaccine may also be
stored at room temperature, which will potentially decrease the
costs of vaccines.
[0038] Some aluminum salts, such as aluminum hydroxide and aluminum
phosphate, have been widely used as human vaccine adjuvants for
decades. The primary particles of aluminum hydroxide and aluminum
phosphate are in the nanometer-scale. However, when dispersed in an
aqueous solution, the primary particles aggregate to form larger
microparticles of 1-20 .mu.m [S. L. Hem, H. Hogenesch, Expert
review of vaccines, 6 (2007) 685-698; I. Z. Romero Mendez et al.,
Vaccine, 25 (2007) 825-833]. Thus, a vaccine that is prepared by
mixing an antigen with an aluminum salt is physically a suspension
of aluminum salt particles with antigens adsorbed on them. Three
mechanisms are frequently cited to explain the mechanisms
underlying the adjuvant activity of aluminum salts [I. Z. Romero
Mendez et al., Vaccine, 25 (2007) 825-833; H. HogenEsch, Vaccine,
20 Suppl 3 (2002) S34-39; L. S. Jones et al., The Journal of
biological chemistry, 280 (2005) 13406-13414; J. W. Mannhalter et
al., Clinical and experimental immunology, 61 (1985) 143-151; M.
Ulanova et al., Infection and immunity, 69 (2001) 1151-1159]: i)
for decades, it was thought to be the depot effect. Aluminum salts
form an antigen depot at the injection site, from where the
antigens are slowly released [A. T. Glenny et al., J. Pathol.
Bacteriol, 34 (1931) 267-275]; ii) Aluminum salts induce
inflammation, thus recruiting and activating antigen-presenting
cells that capture antigens [E. Tritto et al., Vaccine, 27 (2009)
3331-3334]; iii) The adsorption of soluble antigens on aluminum
salt particles makes them readily taken up by antigen-presenting
cells [E. Tritto et al., Vaccine, 27 (2009) 3331-3334]. Finally,
recent data showed that the molecular target for the
pro-inflammatory activity of aluminum salts is the NOD-like
receptor protein 3 (NLRP3) (or NALP3) [E. Tritto et al., Vaccine,
27 (2009) 3331-3334; S. C. Eisenbarth et al., Nature, 453 (2008)
1122-1126; L. Franchi et al., Eur J Immunol, 38 (2008) 2085-2089;
M. Kool et al., Journal of immunology, 181 (2008) 3755-3759; V.
Hornung et al., Nat Immunol, 9 (2008) 847-856].
[0039] Thin-film freezing (TFF) has been recently studied in the
biopharmaceutical field for preparing submicron protein particles
[J. D. Engstrom et al., Pharmaceutical research, 25 (2008)
1334-1346]. In TFF process, a liquid (e.g., solution) is spread out
on a cryogenic substrate to form a thin film in less than one
second. The resultant frozen film is then dried by lyophilization.
For example, Engstrom et al. produced dried protein powders with a
diameter of 300 nm using TFF, and the enzyme activity of the
proteins was fully preserved [J. D. Engstrom et al., Pharmaceutical
research, 25 (2008) 1334-1346]. In the present study, the
feasibility of freeze drying vaccines that are adjuvanted with
aluminum salts using high speed TFF was tested. Ovalbumin (OVA) was
initially used as a model protein antigen, and it was adsorbed onto
aluminum hydroxide or aluminum phosphate and lyophilized after thin
film freezing. In addition, a commercially available veterinary
tetanus toxoid vaccine (tetanus antitoxin concentrated/purified,
Colorado Serum Company) and a human hepatitis B vaccine (Engerix-B,
GlaxoSmithKline Biologics) were also prepared using the TFF
method.
A. DEFINITIONS
[0040] The abbreviations used herein have their conventional
meaning within the chemical and biological arts. Description of
compounds of the present invention is limited by principles of
chemical bonding known to those skilled in the art. Accordingly,
where a group may be substituted by one or more of a number of
substituents, such substitutions are selected so as to comply with
principles of chemical bonding and to give compounds which are not
inherently unstable and/or would be known to one of ordinary skill
in the art as likely to be unstable under ambient conditions, such
as aqueous, neutral, and several known physiological
conditions.
[0041] The terms "a" or "an," as used in herein means one or more.
In addition, the phrase "substituted with a[n]," as used herein,
means the specified group may be substituted with one or more of
any or all of the named substituents.
[0042] The terms "treating" or "treatment" refers to any indicia of
success in the treatment or amelioration of an injury, disease,
pathology or condition, including any objective or subjective
parameter such as abatement; remission; diminishing of symptoms or
making the injury, pathology or condition more tolerable to the
patient; slowing in the rate of degeneration or decline; making the
final point of degeneration less debilitating; improving a
patient's physical or mental well-being. The treatment or
amelioration of symptoms can be based on objective or subjective
parameters; including the results of a physical examination,
neuropsychiatric exams, and/or a psychiatric evaluation. For
example, the certain methods presented herein successfully treat a
disease associated with (e.g. caused by) an infectious agent (e.g.
bacterium or virus). The term "treating" and conjugations thereof,
include prevention of an injury, pathology, condition, or disease.
The term "preventing" or "prevention" refers to any indicia of
success in protecting a subject or patient (e.g. a subject or
patient at risk of developing a disease or condition) from
developing, contracting, or having a disease or condition (e.g. an
infectious disease or diseases associated with an infectious
agent), including preventing one or more symptoms of a disease or
condition or diminishing the occurrence, severity, or duration of
any symptoms of a disease or condition following administration of
a prophylactic or preventative composition as described herein.
[0043] An "effective amount" is an amount sufficient for a
composition (e.g. compound, vaccine, drug) to accomplish a stated
purpose relative to the absence of the composition (e.g. compound,
vaccine, drug) (e.g. achieve the effect for which it is
administered, treat a disease (e.g. reverse or prevent or reduce
severity), reduce spread of an infectious disease or agent, reduce
one or more symptoms of a disease or condition). An example of an
"effective amount" is an amount sufficient to contribute to the
treatment, prevention, or reduction of a symptom or symptoms of a
disease, which could also be referred to as a "therapeutically
effective amount." A "reduction" of a symptom or symptoms (and
grammatical equivalents of this phrase) means decreasing of the
severity or frequency of the symptom(s), or elimination of the
symptom(s). A "prophylactically effective amount" of a composition
(vaccine) is an amount of a composition that, when administered to
a subject, will have the intended prophylactic effect, e.g.,
preventing or delaying the onset (or reoccurrence) of an injury,
disease (e.g. infectious disease), pathology or condition, or
reducing the likelihood of the onset (or reoccurrence) of an
injury, disease, pathology, or condition, or their symptoms. The
full prophylactic effect does not necessarily occur by
administration of one dose, and may occur only after administration
of a series of doses (e.g. prime-boost). Thus, a prophylactically
effective amount may be administered in one or more
administrations. The exact amounts will depend on the purpose of
the treatment, and will be ascertainable by one skilled in the art
using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage
Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); Pickar, Dosage Calculations
(1999); and Remington: The Science and Practice of Pharmacy, 20th
Edition, 2003, Gennaro, Ed., Lippincott, Williams &
Wilkins).
[0044] "Control" or "control experiment" is used in accordance with
its plain ordinary meaning and refers to an experiment in which the
subjects or reagents of the experiment are treated as in a parallel
experiment except for omission of a procedure, reagent, or variable
of the experiment. In some instances, the control is used as a
standard of comparison in evaluating experimental effects. In some
embodiments, a control is the measurement of infection or one or
more symptoms of infection in the absence of a composition (e.g.
vaccine) as described herein (including embodiments).
[0045] "Contacting" is used in accordance with its plain ordinary
meaning and refers to the process of allowing at least two distinct
species (e.g. compositions, vaccines, bacterium, virus,
biomolecules, or cells) to become sufficiently proximal to react,
interact or physically touch. It should be appreciated; however,
the resulting reaction product can be produced directly from a
reaction between the added reagents or from an intermediate from
one or more of the added reagents which can be produced in the
reaction mixture.
[0046] The term "contacting" may include allowing two species to
react, interact, or physically touch, wherein the two species may
be a composition (e.g. vaccine) as described herein and a cell,
virus, virus particle, protein, enzyme, or patient. In some
embodiments contacting includes allowing a composition described
herein to interact with a protein or enzyme that is involved in a
signaling pathway. In some embodiments contacting includes allowing
a composition described herein to interact with a component of a
subject's immune system involved in developing immunity to a
component of the composition.
[0047] As defined herein, the term "inhibition", "inhibit",
"inhibiting" and the like in reference to a protein-inhibitor or
interaction means negatively affecting (e.g. decreasing) the
activity or function of the protein. In some embodiments inhibition
refers to reduction of a disease or symptoms of disease. In some
embodiments inhibition refers to reduction of the growth,
proliferation, or spread of an infectious agent (e.g. bacterium or
virus). In some embodiments inhibition refers to preventing the
infection of a subject by an infectious agent (e.g. bacterium or
virus). In some embodiments, inhibition refers to a reduction in
the activity of a signal transduction pathway or signaling pathway.
Thus, inhibition includes, at least in part, partially or totally
blocking stimulation, decreasing, preventing, or delaying
activation, or inactivating, desensitizing, or down-regulating the
signaling pathway or enzymatic activity or the amount of a
protein.
[0048] The term "modulator" refers to a composition that increases
or decreases the level of a target (e.g. molecule, cell, bacterium,
virus particle, protein) or the function of a target or the
physical state of the target.
[0049] The term "modulate" is used in accordance with its plain
ordinary meaning and refers to the act of changing or varying one
or more properties. "Modulation" refers to the process of changing
or varying one or more properties. For example, as applied to the
effects of a modulator on a target, to modulate means to change by
increasing or decreasing a property or function of the target or
the amount of the target.
[0050] "Patient" or "subject in need thereof" refers to a living
organism suffering from or prone to a disease or condition that can
be treated by administration of a composition (e.g. vaccine or
pharmaceutical composition) as provided herein. Non-limiting
examples include humans, other mammals, bovines, rats, mice, dogs,
monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
In some embodiments, a patient is human. In some embodiments, a
patient or subject in need thereof, refers to a living organism
(e.g. human) at risk of developing, contracting, or having a
disease or condition associated with an infectious agent (e.g.
bacterium or virus).
[0051] "Disease" or "condition" refer to a state of being or health
status of a patient or subject capable of being treated with the
compositions (e.g. vaccines) or methods provided herein. In some
embodiments, the disease is a disease related to (e.g. caused by)
an infectious agent (e.g. bacterium or virus).
[0052] "Pharmaceutically acceptable excipient" and
"pharmaceutically acceptable carrier" refer to a substance that
aids the administration of an active agent to or absorption by a
subject and can be included in the compositions of the present
invention without causing a significant adverse toxicological
effect on the patient. Non-limiting examples of pharmaceutically
acceptable excipients include water, NaCl, normal saline solutions,
lactated Ringer's, normal sucrose, normal glucose, binders,
fillers, disintegrants, lubricants, coatings, sweeteners, flavors,
salt solutions (such as Ringer's solution), alcohols, oils,
gelatins, carbohydrates such as lactose, amylose or starch, fatty
acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and
colors, and the like. Such preparations can be sterilized and, if
desired, mixed with auxiliary agents such as lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, and/or aromatic
substances and the like that do not deleteriously react with the
compounds of the invention. One of skill in the art will recognize
that other pharmaceutical excipients are useful in the present
invention. In embodiments, an excipient is a salt, sugar
(saccharide), buffer, detergent, polymer, amino acid, or
preservative. In embodiments, the excipient is disodium edetate,
sodium chloride, sodium citrate, sodium succinate, sodium
hydroxide, Sodium glucoheptonate, sodium acetyltryptophanate,
sodium bicarbonate, sodium caprylate, sodium pertechnetate, sodium
acetate, sodium dodecyl sulfate, ammonium citrate, calcium
chloride, calcium, potassium chloride, potassium sodium tartarate,
zinc oxide, zinc, stannous chloride, magnesium sulfate, magnesium
stearate, titanium dioxide, DL-lactic/glycolic acids, asparagine,
L-arginine, arginine hydrochloride, adenine, histidine, glycine,
glutamine, glutathione, imidazole, protamine, protamine sulfate,
phosphoric acid, Tri-n-butyl phosphate, ascorbic acid, cysteine
hydrochloride, hydrochloric acid, hydrogen citrate, trisodium
citrate, guanidine hydrochloride, mannitol, lactose, sucrose,
agarose, sorbitol, maltose, trehalose, surfactants, polysorbate 80,
polysorbate 20, poloxamer 188, sorbitan monooleate, triton n101,
m-cresol, benyl alcohol, ethanolamine, glycerin,
phosphorylethanolamine, tromethamine, 2-phenyloxyethanol,
chlorobutanol, dimethylsulfoxide, N-methyl-2-pyrrolidone,
propyleneglycol, polyoxyl 35 castor oil, methyl hydroxybenzoate,
tromethamine, corn oil-mono-di-triglycerides, poloxyl 40
hydrogenated castor oil, tocopherol, n-acetyltryptophan,
octa-fluoropropane, castor oil, polyoxyethylated oleic glycerides,
polyoxytethylated castor oil, phenol, glyclyglycine, thimerosal,
parabens, gelatin, Formaldehyde, Dulbecco's modified eagles medium,
hydrocortisone, neomycin, Von Willebrand factor, gluteraldehyde,
benzethonium chloride, white petroleum, p-aminopheyl-p-anisate,
monosodium glutamate, beta-propiolactone, acetate, citrate,
glutamate, glycinate, histidine, Lactate, Maleate, phosphate,
succinate, tartrate, tris, carbomer 1342 (copolymer of acrylic acid
and a long chain alkyl methacrylate cross-linked with allyl ethers
of pentaerythritol), glucose star polymer, silicone polymer,
polydimethylsiloxane, polyethylene glycol, polyvinylpyrrolidone,
carboxymethylcellulose, poly(glycolic acid),
poly(lactic-co-glycolic acid), polylactic acid, dextran 40, or
poloxamer.
[0053] The term "preparation" is intended to include the
formulation of the active compound with encapsulating material as a
carrier providing a capsule in which the active component with or
without other carriers, is surrounded by a carrier, which is thus
in association with it.
[0054] As used herein, the term "administering" means oral
administration, administration as a suppository, topical contact,
intravenous, intraperitoneal, intramuscular, intralesional,
intrathecal, intranasal, intradermal, mucosal, intrarectal,
intravaginal, topical, transcutaneous, or subcutaneous
administration. Administration is by any route, including
parenteral and transmucosal (e.g., buccal, sublingual, palatal,
gingival, nasal, vaginal, rectal, or transdermal). Parenteral
administration includes, e.g., intravenous, intramuscular,
intra-arteriole, intradermal, subcutaneous, intraperitoneal,
intraventricular, and intracranial. Other modes of delivery
include, but are not limited to, the use of liposomal formulations,
intravenous infusion, transdermal patches, etc. By "co-administer"
it is meant that a composition described herein is administered at
the same time, just prior to, or just after the administration of
one or more additional therapies, for example infection therapies
such as antiviral drugs or a vaccine (e.g different vaccine). The
compositions (e.g. vaccines) of the invention can be administered
alone or can be coadministered to the patient. Coadministration is
meant to include simultaneous or sequential administration of the
compounds individually or in combination (more than one
composition) and includes vaccine administration in a prime-boost
method. Thus, the preparations can also be combined, when desired,
with other active substances (e.g. to reduce metabolic degradation,
increase immune response (e.g. adjuvant)). The compositions of the
present invention can be delivered by transdermally, by a topical
route, transcutaneously, formulated as solutions, suspensions,
emulsions, gels, creams, ointments, pastes, jellies, paints,
powders, and aerosols.
[0055] The term "administer (or administering) a vaccine" means
administering a composition that prevents or treats an infection in
a subject. Administration may include, without being limited by
mechanism, allowing sufficient time for the vaccine to induce an
immune response in the subject or to reduce one or more symptoms of
a disease.
[0056] The terms "peptide," "polypeptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. An "antigenic protein" is a protein that may be included
in a vaccine as an antigen. In embodiments, an antigenic protein
may be an antigenic protein conjugated to a sugar (i.e. saccharide)
(e.g. monosaccharide, disaccharide, polysaccharide) "antigenic
protein saccharide conjugate". In embodiments, an antigenic protein
may be an antigenic protein that is not conjugated to a sugar
(saccharide).
[0057] The term "peptidyl" and "peptidyl moiety" means a monovalent
peptide.
[0058] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs. Naturally
occurring amino acids are those encoded by the genetic code, as
well as those amino acids that are later modified, e.g.,
hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, i.e., an
.alpha.-carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid. An oligomer
comprising amino acid mimetics is a peptidomimetic. A
peptidomimetic moiety is a monovalent peptidomimetic.
[0059] The term "isolated" refers to a nucleic acid,
polynucleotide, polypeptide, protein, or other component that is
partially or completely separated from components with which it is
normally associated (other proteins, nucleic acids, cells, etc.).
In some embodiments, an isolated polypeptide or protein is a
recombinant polypeptide or protein.
[0060] The terms "dose" and "dosage" are used interchangeably
herein. A dose refers to the amount of active ingredient given to
an individual at each administration. For the present methods and
compositions provided herein, the dose may generally refer to the
amount of disease treatment. The dose will vary depending on a
number of factors, including the range of normal doses for a given
therapy, frequency of administration; size and tolerance of the
individual; severity of the condition; risk of side effects; and
the route of administration. One of skill will recognize that the
dose can be modified depending on the above factors or based on
therapeutic progress. The term "dosage form" refers to the
particular format of the pharmaceutical or pharmaceutical
composition, and depends on the route of administration. For
example, a dosage form can be in a liquid form for nebulization,
e.g., for inhalants, in a tablet or liquid, e.g., for oral
delivery, or a saline solution, e.g., for injection.
[0061] The term "adjuvant" is used in accordance with its plain
ordinary meaning within Immunology and refers to a substance that
is commonly used as a component of a vaccine. Adjuvants may
increase an antigen specific immune response in a subject when
administered to the subject with one or more specific antigens as
part of a vaccine. In some embodiments, an adjuvant accelerates an
immune response to an antigen. In some embodiments, an adjuvant
prolongs an immune response to an antigen. In some embodiments, an
adjuvant enhances an immune response to an antigen. In some
embodiments, an adjuvant is an aluminum adjuvant.
[0062] Vaccine compositions typically include an adjuvant,
regardless of the nature of the agent. An adjuvant stimulates the
immune system and increases the response of the immune system to
the agent present in the vaccine. Most adjuvants used in vaccines
in the United States are aluminum salts. Examples of aluminum salts
include, but are not limited to: aluminum phosphate, aluminum
hydroxide, aluminum sulfate, and aluminum potassium sulfate.
[0063] The term "aluminum adjuvant" refers to an adjuvant including
aluminum. In some embodiments, an aluminum adjuvant includes
aluminum hydroxide. In some embodiments, an aluminum adjuvant is
aluminum hydroxide. In some embodiments, an aluminum adjuvant
includes aluminum phosphate. In some embodiments, an aluminum
adjuvant is aluminum phosphate. In some embodiments, an aluminum
adjuvant includes potassium aluminum sulfate. In some embodiments,
an aluminum adjuvant is potassium aluminum sulfate. In some
embodiments, an aluminum adjuvant includes aluminum sulfate. In
some embodiments, an aluminum adjuvant is aluminum sulfate. In some
embodiments, an aluminum adjuvant is aluminum hydroxide adjuvant.
In some embodiments, an aluminum adjuvant is aluminum phosphate
adjuvant. In some embodiments, an aluminum adjuvant is potassium
aluminum sulfate adjuvant. In some embodiments, an aluminum
adjuvant is Alum. In some embodiments, an aluminum adjuvant is CAS
no. 21645-51-2. In some embodiments, an aluminum adjuvant is
aluminum hydroxide gel. In some embodiments, an aluminum adjuvant
is aluminum hydroxide gel in the form of a white gelatinous
precipitate. In some embodiments, an aluminum adjuvant is CAS no.
7784-30-7. In some embodiments, an aluminum adjuvant is aluminum
phosphate gel. In some embodiments, an aluminum adjuvant is
aluminum phosphate gel in the form of a white gelatinous
precipitate. In some embodiments, an aluminum adjuvant is Imject
Alum Adjuvant.TM. In some embodiments, an aluminum adjuvant is
aluminum hydroxide without magnesium hydroxide. In some
embodiments, an aluminum adjuvant is Alhydrogel.TM.. In some
embodiments, an aluminum adjuvant is Adju-Phos.TM.. In some
embodiments, an aluminum adjuvant is AdjuPhos.TM.. In some
embodiments, an aluminum adjuvant is amorphous aluminum hydroxide
and not crystalline aluminum hydroxide. In some embodiments, an
aluminum adjuvant includes amorphous aluminum and not crystalline
aluminum. In some embodiments, aluminum adjuvant is crystalline
aluminum hydroxide and not amorphous aluminum hydroxide. In some
embodiments, an aluminum adjuvant includes crystalline aluminum and
not amorphous aluminum. In some embodiments, an aluminum adjuvant
includes crystalline aluminum oxyhydroxide. In some embodiments, an
aluminum adjuvant is crystalline aluminum oxyhydroxide. In some
embodiments, an aluminum adjuvant includes amorphous aluminum
hydroxyphosphate. In some embodiments, an aluminum adjuvant is
amorphous aluminum hydroxyphosphate. In some embodiments, an
aluminum adjuvant includes aluminum oxyhydroxide and not aluminum
hydroxycarbonate. In some embodiments, an aluminum adjuvant is
aluminum oxyhydroxide and not aluminum hydroxycarbonate. In some
embodiments, an aluminum adjuvant includes aluminum oxyhydroxide
and not magnesium hydroxide. In some embodiments, an aluminum
adjuvant is aluminum oxyhydroxide and not magnesium hydroxide. In
some embodiments, an aluminum adjuvant does not include amorphous
aluminum hydroxide in which some hydroxyls are replaced by sulfate
anions. In some embodiments, an aluminum adjuvant includes aluminum
oxyhydroxide in a Boehmite-like pattern. In some embodiments, an
aluminum adjuvant is aluminum oxyhydroxide in a Boehmite-like
pattern. In some embodiments of an aluminum adjuvant described
above, the description is of the aluminum adjuvant prior to
inclusion in a vaccine. In some embodiments, an aluminum adjuvant
is an aluminum containing adjuvant approved by the FDA for
administration to humans. In some embodiments, an aluminum adjuvant
is an aluminum hydroxide adjuvant approved for administration to
humans by the FDA. In some embodiments, an aluminum adjuvant is an
aluminum phosphate adjuvant approved for administration to humans
by the FDA.
[0064] The term "aluminum hydroxide adjuvant" as used herein refers
to the aluminum hydroxide adjuvant that includes aluminum hydroxide
and is currently used in human vaccines (e.g. marketed and/or
approved human vaccines, such as FDA approved human vaccines). In
some embodiments, "aluminum hydroxide adjuvant" as used herein
refers to the aluminum hydroxide adjuvant that is currently used in
human vaccines (e.g. marketed and/or approved human vaccines, such
as FDA approved human vaccines) and is used in accordance with the
use of that term in Hem S. L., Vaccine 23(2007) 4985-4986. In some
embodiments, an aluminum hydroxide adjuvant includes CAS no.
21645-51-2. In some embodiments, an aluminum hydroxide adjuvant is
aluminum hydroxide gel. In some embodiments, an aluminum hydroxide
adjuvant is aluminum hydroxide gel in the form of a white
gelatinous precipitate. In some embodiments, an aluminum hydroxide
adjuvant includes aluminum hydroxide and does not include magnesium
hydroxide. In some embodiments, an aluminum hydroxide adjuvant is
Alhydrogel.TM.. In some embodiments, an aluminum hydroxide adjuvant
includes crystalline aluminum hydroxide and not amorphous aluminum
hydroxide. In some embodiments, an aluminum hydroxide adjuvant
includes crystalline aluminum and not amorphous aluminum. In some
embodiments, an aluminum hydroxide adjuvant includes crystalline
aluminum oxyhydroxide. In some embodiments, an aluminum hydroxide
is crystalline aluminum oxyhydroxide. In some embodiments, an
aluminum hydroxide adjuvant includes aluminum oxyhydroxide and not
aluminum hydroxycarbonate. In some embodiments, an aluminum
hydroxide adjuvant is aluminum oxyhydroxide and not aluminum
hydroxycarbonate. In some embodiments, an aluminum hydroxide
adjuvant does not include amorphous aluminum hydroxide in which
some hydroxyls are replaced by sulfate anions. In some embodiments,
aluminum hydroxide adjuvant includes aluminum oxyhydroxide in a
Boehmite-like pattern. In some embodiments of an aluminum hydroxide
adjuvant described above, the description is of the aluminum
hydroxide adjuvant prior to inclusion in a vaccine.
[0065] The term "aluminum phosphate adjuvant" as used herein refers
to the aluminum phosphate adjuvant that includes aluminum phosphate
and is currently used in human vaccines (e.g. marketed and/or
approved human vaccines, such as FDA approved human vaccines). In
some embodiments, "aluminum phosphate adjuvant" as used herein
refers to the aluminum phosphate adjuvant that is currently used in
human vaccines (e.g. marketed and/or approved human vaccines, such
as FDA approved human vaccines) and is used in accordance with the
use of that term in Hem S. L., Vaccine 23(2007) 4985-4986. In some
embodiments, an aluminum phosphate adjuvant includes CAS no.
7784-30-7. In some embodiments, an aluminum phosphate adjuvant is
aluminum phosphate gel. In some embodiments, an aluminum phosphate
adjuvant is aluminum phosphate gel in the form of a white
gelatinous precipitate. In some embodiments, an aluminum phosphate
adjuvant is Adju-Phos.TM.. In some embodiments, an aluminum
phosphate adjuvant is AdjuPhos.TM.. In some embodiments, an
aluminum phosphate adjuvant includes amorphous aluminum
hydroxyphosphate. In some embodiments of an aluminum phosphate
adjuvant described above, the description is of the aluminum
phosphate adjuvant prior to inclusion in a vaccine.
[0066] The terms "bind", "bound", "binding", and other verb forms
thereof are used in accordance with their plain ordinary meaning
within Enzymology and Biochemistry and refer to the formation of
one or more interactions or contacts between two compositions that
may optionally interact. Binding may be intermolecular or
intramolecular.
[0067] The term "potassium aluminum sulfate adjuvant" refers to an
adjuvant that includes potassium aluminum sulfate. The term
"aluminum sulfate adjuvant" refers to an adjuvant that includes
aluminum sulfate.
[0068] The term "vaccine" is used according to its plain ordinary
meaning within medicine and Immunology and refers to a composition
including an antigenic component (e.g. antigenic protein) for
administration to a subject (e.g. human), which elicits an immune
response to the antigenic component (e.g. antigentic protein). In
some embodiments a vaccine is a therapeutic. In some embodiments, a
vaccine is prophylactic. In some embodiments a vaccine includes one
or more adjuvants (e.g. aluminum adjuvant). A liquid vaccine is a
vaccine in liquid form, which may be for example a solution,
suspension, emulsion, or dispersion or the antigenic component
(e.g. antigenic protein) of the vaccine and may optionally include
other components. A dry vaccine is a vaccine comprising 5% or less
of water.
[0069] A vaccine is a preparation employed to improve immunity to a
particular disease. Vaccines include an agent, which is used to
induce a response from the immune system of the subject. Various
agents that are typically used in a vaccine include, but are not
limited to: killed, but previously virulent, micro-organisms; live,
attenuated microorganisms; inactivated toxic compounds that are
produced by microorganism that cause an illness; protein subunits
of microorganisms; and conjugates. Examples of vaccines that may be
converted into a powder vaccine according to the methods described
herein include, but are not limited to: influenza vaccine, cholera
vaccine, bubonic plague vaccine, polio vaccine, Hepatitis A
vaccine, rabies vaccine, yellow fever vaccine, measles vaccine,
rubella vaccine, mumps vaccine, typhoid vaccine, tuberculosis
vaccine, tetanus vaccine, diphtheria vaccine,
diphtheria-tetanus-pertussis vaccine, Hepatitis B vaccine, human
papillomavirus (HPV) vaccine, Pneumococcal conjugate vaccines,
influenza vaccine, botulism vaccine, polio vaccine, and anthrax
vaccines.
[0070] The term "prime-boost" or "prime boost" as applied to a
methodology of administering vaccines is used according to its
plain ordinary meaning in Virology and Immunology and refers to a
method of vaccine administration in which a first dose of a vaccine
or vaccine component is administered to a subject or patient to
begin the administration (prime) and at a later time (e.g. hours,
days, weeks, months later) a second vaccine is administered to the
same patient or subject (boost). The first and second vaccines may
be the same or different but are intended to both elicit an immune
response useful in treating or preventing the same disease or
condition. In some embodiments the prime is one or more viral
proteins or portions thereof and the boost is one or more viral
proteins or portions thereof.
[0071] The term "associated" or "associated with" as used herein to
describe a disease (e.g. a virus associated disease or bacteria
associated disease) means that the disease is caused by, or a
symptom of the disease is caused by, what is described as disease
associated or what is described as associated with the disease. As
used herein, what is described as being associated with a disease,
if a causative agent, could be a target for treatment of the
disease.
[0072] The term "vaccinate", or additional verb forms thereof,
refers to administering a vaccine to a subject (e.g. human) and
eliciting an antigen specific immune response, wherein the antigen
(e.g. antigenic protein) is included in the vaccine. The term
"vaccinate" may also refer to eliciting an antigen specific immune
response against an administered antigen (e.g. antigenic protein).
In some embodiments, vaccinate is to provide prophylaxis against a
disease or infectious agent.
[0073] The term "portion" refers to a subset of a whole, which may
also be the whole. In some embodiments, a portion is about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. In some
embodiments, a portion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100%. Unless indicated otherwise, the term "about" in
the context of a numeric value indicates the nominal value.+-.10%
of the nominal value. In some embodiments, "about" may be the
nominal value.
B. COMPOSITIONS
[0074] In an aspect is provided a dry vaccine including an
antigenic protein and an aluminum adjuvant, wherein at least 75% of
the antigenic protein is adsorbed to the aluminum adjuvant.
[0075] In embodiments, at least 60% of the antigenic protein is not
denatured. In embodiments, at least 70% of the antigenic protein is
not denatured. In embodiments, at least 80% of the antigenic
protein is not denatured. In embodiments, at least 90% of the
antigenic protein is not denatured. In embodiments, at least 95% of
the antigenic protein is not denatured. In embodiments, at least
60% of the antigenic protein is in a conformationally native state.
In embodiments, at least 70% of the antigenic protein is in a
conformationally native state. In embodiments, at least 80% of the
antigenic protein is in a conformationally native state. In
embodiments, at least 90% of the antigenic protein is in a
conformationally native state. In embodiments, at least 95% of the
antigenic protein is in a conformationally native state. A
"conformationally native state" is a folded conformation
corresponding to an operative or functional protein. A "denatured"
protein is a protein having a conformation differing from the
folded active or functional conformation of the protein, wherein
the denatured protein has a reduced level of activity or function.
In embodiments, the antigentic protein is an unconjugated antigenic
protein. In embodiments, the antigenic protein is an antigenic
protein sugar (saccharide) conjugate. In embodiments, the sugar
(saccharide) is a monosaccharide. In embodiments, the sugar
(saccharide) is a disaccharide. In embodiments, the sugar
(saccharide) is a polysaccharide.
[0076] In embodiments, the aluminum adjuvant includes aluminum
hydroxide. In embodiments, the aluminum adjuvant includes aluminum
phosphate. In embodiments, the aluminum adjuvant includes potassium
aluminum sulfate. In embodiments, the aluminum adjuvant is aluminum
hydroxide. In embodiments, the aluminum adjuvant is aluminum
phosphate. In embodiments, the aluminum adjuvant is potassium
aluminum sulfate. In embodiments, the aluminum adjuvant is aluminum
sulfate. In embodiments, the dry vaccine includes between 0.5 and
5% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes between 0.5 and 4% (wt/wt) of the aluminum
adjuvant. In embodiments, the dry vaccine includes between 0.5 and
3% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes between 0.5 and 2% (wt/wt) of the aluminum
adjuvant. In embodiments, the dry vaccine includes between 0.75 and
2% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes between 1 and 2% (wt/wt) of the aluminum adjuvant.
In embodiments, the dry vaccine includes about 0.08, 0.09, 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes at least 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the
aluminum adjuvant. In embodiments, the dry vaccine includes less
than 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum adjuvant. In
embodiments, the dry vaccine includes 0.08, 0.09, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%
(wt/wt) of the aluminum adjuvant. In embodiments, the dry vaccine
includes between 0.08 and 1% (wt/wt) of the aluminum adjuvant.
[0077] In embodiments, the dry vaccine includes less than 5% water.
In embodiments, the dry vaccine includes less than 4% water. In
embodiments, the dry vaccine includes less than 3% water. In
embodiments, the dry vaccine includes less than 2% water. In
embodiments, the dry vaccine includes less than 1% water. In
embodiments, the dry vaccine includes less than 5% water (wt/wt).
In embodiments, the dry vaccine includes less than 4% water
(wt/wt). In embodiments, the dry vaccine includes less than 3%
water (wt/wt). In embodiments, the dry vaccine includes less than
2% water (wt/wt). In embodiments, the dry vaccine includes less
than 1% water (wt/wt). In embodiments, the dry vaccine includes
about 5% water. In embodiments, the dry vaccine includes about 4%
water. In embodiments, the dry vaccine includes about 3% water. In
embodiments, the dry vaccine includes about 2% water. In
embodiments, the dry vaccine includes about 1% water. In
embodiments, the dry vaccine includes about 5% water (wt/wt). In
embodiments, the dry vaccine includes about 4% water (wt/wt). In
embodiments, the dry vaccine includes about 3% water (wt/wt). In
embodiments, the dry vaccine includes about 2% water (wt/wt). In
embodiments, the dry vaccine includes about 1% water (wt/wt). In
embodiments, the dry vaccine includes less than 5% water (v/v). In
embodiments, the dry vaccine includes less than 4% water (v/v). In
embodiments, the dry vaccine includes less than 3% water (v/v). In
embodiments, the dry vaccine includes less than 2% water (v/v). In
embodiments, the dry vaccine includes less than 1% water (v/v). In
embodiments, the dry vaccine includes about 5% water (v/v). In
embodiments, the dry vaccine includes about 4% water (v/v). In
embodiments, the dry vaccine includes about 3% water (v/v). In
embodiments, the dry vaccine includes about 2% water (v/v). In
embodiments, the dry vaccine includes about 1% water (v/v).
[0078] In embodiments, at least 75% of the antigenic protein is
adsorbed to the aluminum adjuvant. In embodiments, at least 80% of
the antigenic protein is adsorbed to the aluminum adjuvant. In
embodiments, at least 85% of the antigenic protein is adsorbed to
the aluminum adjuvant. In embodiments, at least 90% of the
antigenic protein is adsorbed to the aluminum adjuvant. In
embodiments, at least 92% of the antigenic protein is adsorbed to
the aluminum adjuvant. In embodiments, at least 95% of the
antigenic protein is adsorbed to the aluminum adjuvant. In
embodiments, at least 98% of the antigenic protein is adsorbed to
the aluminum adjuvant. In embodiments, at least 99% of the
antigenic protein is adsorbed to the aluminum adjuvant. In
embodiments, about 75% of the antigenic protein is adsorbed to the
aluminum adjuvant. In embodiments, about 80% of the antigenic
protein is adsorbed to the aluminum adjuvant. In embodiments, about
85% of the antigenic protein is adsorbed to the aluminum adjuvant.
In embodiments, about 90% of the antigenic protein is adsorbed to
the aluminum adjuvant. In embodiments, about 92% of the antigenic
protein is adsorbed to the aluminum adjuvant. In embodiments, about
95% of the antigenic protein is adsorbed to the aluminum adjuvant.
In embodiments, about 98% of the antigenic protein is adsorbed to
the aluminum adjuvant. In embodiments, about 99% of the antigenic
protein is adsorbed to the aluminum adjuvant.
[0079] In embodiments, the dry vaccine includes an excipient. In
embodiments, the dry vaccine includes a plurality of different
excipients. In embodiments, the excipient is a salt, sugar
(saccharide), buffer, detergent, polymer, amino acid, or
preservative. In embodiments, the excipient is disodium edetate,
sodium chloride, sodium citrate, sodium succinate, sodium
hydroxide, Sodium glucoheptonate, sodium acetyltryptophanate,
sodium bicarbonate, sodium caprylate, sodium pertechnetate, sodium
acetate, sodium dodecyl sulfate, ammonium citrate, calcium
chloride, calcium, potassium chloride, potassium sodium tartarate,
zinc oxide, zinc, stannous chloride, magnesium sulfate, magnesium
stearate, titanium dioxide, DL-lactic/glycolic acids, asparagine,
L-arginine, arginine hydrochloride, adenine, histidine, glycine,
glutamine, glutathione, imidazole, protamine, protamine sulfate,
phosphoric acid, Tri-n-butyl phosphate, ascorbic acid, cysteine
hydrochloride, hydrochloric acid, hydrogen citrate, trisodium
citrate, guanidine hydrochloride, mannitol, lactose, sucrose,
agarose, sorbitol, maltose, trehalose, surfactants, polysorbate 80,
polysorbate 20, poloxamer 188, sorbitan monooleate, triton n101,
m-cresol, benyl alcohol, ethanolamine, glycerin,
phosphorylethanolamine, tromethamine, 2-phenyloxyethanol,
chlorobutanol, dimethylsulfoxide, N-methyl-2-pyrrolidone,
propyleneglycol, polyoxyl 35 castor oil, methyl hydroxybenzoate,
tromethamine, corn oil-mono-di-triglycerides, poloxyl 40
hydrogenated castor oil, tocopherol, n-acetyltryptophan,
octa-fluoropropane, castor oil, polyoxyethylated oleic glycerides,
polyoxytethylated castor oil, phenol, glyclyglycine, thimerosal,
parabens, gelatin, Formaldehyde, Dulbecco's modified eagles medium,
hydrocortisone, neomycin, Von Willebrand factor, gluteraldehyde,
benzethonium chloride, white petroleum, p-aminopheyl-p-anisate,
monosodium glutamate, beta-propiolactone, acetate, citrate,
glutamate, glycinate, histidine, Lactate, Maleate, phosphate,
succinate, tartrate, tris, carbomer 1342 (copolymer of acrylic acid
and a long chain alkyl methacrylate cross-linked with allyl ethers
of pentaerythritol), glucose star polymer, silicone polymer,
polydimethylsiloxane, polyethylene glycol, polyvinylpyrrolidone,
carboxymethylcellulose, poly(glycolic acid),
poly(lactic-co-glycolic acid), polylactic acid, dextran 40, or
poloxamer. In embodiments, the excipient is trehalose. In
embodiments, the dry vaccine includes less than 5% wt/wt of the
excipient. In embodiments, the dry vaccine includes less than 4%
wt/wt of the excipient. In embodiments, the dry vaccine includes
less than 3% wt/wt of the excipient. In embodiments, the dry
vaccine includes less than 2% wt/wt of the excipient. In
embodiments, the dry vaccine includes less than 1% wt/wt of the
excipient. In embodiments, the dry vaccine includes less than 0.5%
wt/wt of the excipient. In embodiments, the dry vaccine includes
about 5% wt/wt of the excipient. In embodiments, the dry vaccine
includes about 4% wt/wt of the excipient. In embodiments, the dry
vaccine includes about 3% wt/wt of the excipient. In embodiments,
the dry vaccine includes about 2% wt/wt of the excipient. In
embodiments, the dry vaccine includes about 1% wt/wt of the
excipient. In embodiments, the dry vaccine includes about 0.5%
wt/wt of the excipient.
[0080] In embodiments, the dry vaccine includes particles, wherein
the particles include the antigenic protein adsorbed to the
aluminum adjuvant. In embodiments, the dry vaccine is prepared from
a liquid vaccine.
[0081] In an embodiment, a powder (e.g. dry) vaccine, which retains
its efficacy, may be made from a vaccine composition. The method
includes obtaining a liquid (e.g. aqueous) vaccine composition. The
vaccine composition includes an agent that resembles a
disease-causing microorganism or a compound associated with the
disease-causing microorganism (e.g. antigenic protein). The vaccine
composition also includes an adjuvant (e.g. aluminum adjuvant). The
vaccine composition is frozen to obtain a frozen vaccine
composition (e.g. vaccine thin film). Water is removed from the
frozen vaccine composition to form a powder (e.g. dry) vaccine that
includes the agent or compound (e.g. antigenic protein) and the
adjuvant (e.g. aluminum adjuvant).
[0082] A cryoprotectant may be added to the vaccine composition to
protect the organisms or agents present in the composition (either
live or dead) from damage during the freezing process. Examples of
cryoprotectants include dimethyl sulfoxide, glycerol,
monosaccharides, and polysaccharides (e.g., trehalose). A
cryoprotectant may be present in amounts up to about 5% by
weight.
[0083] Additionally, the solid form of the vaccine is expected to
be advantageous over vaccine dispersion (i.e., suspension) for
stockpiling vaccines that are critical to national security and
public health. For example, botulism is a life-threatening disease
caused by botulinum neurotoxins (BoNTs), which are produced by one
of the seven structurally similar Clostridium botulinum serotypes,
designated A to G. Each of the toxins is immunologically distinct,
except that serotypes C and D share significant cross-homology.
BoNTs are the most poisonous substances known in nature. A single
gram of crystalline toxin, evenly dispersed and inhaled, would kill
more than one million people. Previously, an investigational
pentavalent botulism toxoid (PBT) vaccine aiming to protect against
BoNT serotypes A-E had been available. However, as of November
2011, the PBT vaccine has been discontinued by the Centers for
Disease Control and Prevention (CDC), based on "an assessment of
the available data, which indicate a decline in immunogenicity of
some of the toxin serotypes". Since the investigative PBT vaccine
was the only botulism vaccine available in the U.S.,
discontinuation of it has significant national security
implications.
[0084] In another embodiment, an aqueous vaccine composition may be
composed of an agent and an aluminum adjuvant that form particles
having a particle size of less than about 200 nm (e.g. less than
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200 nm). In some embodiments, aluminum
hydroxide or aluminum phosphate particles having a diameter of less
than 200 nm (e.g. less than 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm) may be
used as adjuvants in a vaccine composition. The vaccine composition
may be formed by mixing the agent of the vaccine with the aluminum
adjuvant particles in water. The aqueous vaccine composition may be
used to vaccinate a subject against the disease related to the
agent. In some embodiments, the aqueous vaccine composition can be
converted to a vaccine powder, as described above, for storage, for
use as an inhalant, or use in other delivery modes.
[0085] In embodiments, a dry vaccine is the dry vaccine described
herein, including in embodiments, examples, tables, figures, and
claims. In embodiments, a dry vaccine is a dry vaccine made by a
method described herein, including in aspects, embodiments,
examples, tables, figures, and claims. Provided herein is a
reconstituted liquid vaccine comprising a dry vaccine as described
herein (including in an aspect, embodiment, example, table, figure,
or claim) or a dry vaccine prepared using a method as described
herein (including in an aspect, embodiment, example, table, figure,
or claim) and a solvent (e.g. water, buffer, solution, liquid
including an excipient).
[0086] Provided in another aspect is a pharmaceutical composition
including a pharmaceutically acceptable excipient and any of the
compositions (e.g. vaccines) described herein (including
embodiment).
[0087] The compositions described herein (including embodiments and
examples) can be administered alone or can be coadministered to the
patient. Coadministration is meant to include simultaneous or
sequential administration of the compositions individually or in
combination (more than one composition). Thus, the preparations can
also be combined, when desired, with other active substances (e.g.
to reduce metabolic degradation, increase immune response (e.g.
adjuvants)). An example of coadministration of vaccine compositions
is a prime-boost method of administration.
[0088] Pharmaceutical compositions provided by the present
invention include compositions wherein the active ingredient (e.g.
compositions described herein, including embodiments) is contained
in a therapeutically or prophylactically effective amount, i.e., in
an amount effective to achieve its intended purpose. The actual
amount effective for a particular application will depend, inter
alia, on the condition being treated. When administered in methods
to treat a disease, such compositions will contain an amount of
active ingredient effective to achieve the desired result, e.g.,
prevent infection, and/or reducing, eliminating, or slowing the
progression of disease symptoms. Determination of a therapeutically
or prophylactically effective amount of a composition of the
invention is well within the capabilities of those skilled in the
art, especially in light of the detailed disclosure herein.
C. METHODS
[0089] In an aspect is provided a method for preparing a vaccine
thin film including: applying a liquid vaccine to a freezing
surface; allowing the liquid vaccine to disperse and freeze on the
freezing surface thereby forming a vaccine thin film. The liquid
vaccine includes aluminum (e.g. aluminum adjuvant).
[0090] In embodiments, the aluminum adjuvant includes aluminum
hydroxide. In embodiments, the aluminum adjuvant includes aluminum
phosphate. In embodiments, the aluminum adjuvant includes potassium
aluminum sulfate. In embodiments, the aluminum adjuvant is aluminum
hydroxide. In embodiments, the aluminum adjuvant is aluminum
phosphate. In embodiments, the aluminum adjuvant is potassium
aluminum sulfate. In embodiments, the aluminum adjuvant includes
aluminum sulfate. In embodiments, the aluminum adjuvant is aluminum
sulfate. In embodiments, the liquid vaccine includes between 0.5
and 5% (wt/wt) of the aluminum adjuvant. In embodiments, the liquid
vaccine includes between 0.5 and 4% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes between 0.5
and 3% (wt/wt) of the aluminum adjuvant. In embodiments, the liquid
vaccine includes between 0.5 and 2% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes between 0.75
and 2% (wt/wt) of the aluminum adjuvant. In embodiments, the liquid
vaccine includes between 1 and 2% (wt/wt) of the aluminum adjuvant.
In embodiments, the liquid vaccine includes about 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%
(wt/wt) of the aluminum adjuvant. In embodiments, the liquid
vaccine includes at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes less than
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/wt) of the aluminum adjuvant. In embodiments, the
liquid vaccine includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes between 0.5
and 5% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between 0.5 and 4%
(wt/vol) of the aluminum adjuvant/liquid vaccine. In embodiments,
the liquid vaccine includes between 0.5 and 3% (wt/vol) of the
aluminum adjuvant/liquid vaccine. In embodiments, the liquid
vaccine includes between 0.5 and 2% (wt/vol) of the aluminum
adjuvant/liquid vaccine. In embodiments, the liquid vaccine
includes between 0.75 and 2% (wt/vol) of the aluminum
adjuvant/liquid vaccine. In embodiments, the liquid vaccine
includes between 1 and 2% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes about 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes at least 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes less than
0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between 0.08 and 1%
(wt/vol) of the aluminum adjuvant/liquid vaccine. In embodiments,
the liquid vaccine includes between about 0.5 and about 5% (wt/wt)
of the aluminum adjuvant. In embodiments, the liquid vaccine
includes between about 0.5 and about 4% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes between about
0.5 and about 3% (wt/wt) of the aluminum adjuvant. In embodiments,
the liquid vaccine includes between about 0.5 and about 2% (wt/wt)
of the aluminum adjuvant. In embodiments, the liquid vaccine
includes between about 0.75 and about 2% (wt/wt) of the aluminum
adjuvant. In embodiments, the liquid vaccine includes between about
1 and about 2% (wt/wt) of the aluminum adjuvant. In embodiments,
the liquid vaccine includes about about 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of
the aluminum adjuvant. In embodiments, the liquid vaccine includes
at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum adjuvant. In
embodiments, the liquid vaccine includes less than about 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10% (wt/wt) of the aluminum adjuvant. In embodiments, the liquid
vaccine includes about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum adjuvant.
In embodiments, the liquid vaccine includes between about 0.5 and
about 5% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between about 0.5 and
about 4% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between about 0.5 and
about 3% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between about 0.5 and
about 2% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between about 0.75 and
about 2% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes between about 1 and about
2% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes about about 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes at least about 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid vaccine. In
embodiments, the liquid vaccine includes less than about 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes about 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10% (wt/vol) of the aluminum adjuvant/liquid
vaccine. In embodiments, the liquid vaccine includes between about
0.08 and about 1% (wt/vol) of the aluminum adjuvant/liquid
vaccine.
[0091] In embodiments, the liquid vaccine includes a ratio of
antigenic protein to aluminum adjuvant (wt/wt) of about 1:10. In
embodiments, the liquid vaccine includes a ratio of antigenic
protein to aluminum adjuvant (wt/wt) of about 1:9. In embodiments,
the liquid vaccine includes a ratio of antigenic protein to
aluminum adjuvant (wt/wt) of about 1:8. In embodiments, the liquid
vaccine includes a ratio of antigenic protein to aluminum adjuvant
(wt/wt) of about 1:7. In embodiments, the liquid vaccine includes a
ratio of antigenic protein to aluminum adjuvant (wt/wt) of about
1:6. In embodiments, the liquid vaccine includes a ratio of
antigenic protein to aluminum adjuvant (wt/wt) of about 1:5. In
embodiments, the liquid vaccine includes a ratio of antigenic
protein to aluminum adjuvant (wt/wt) of about 1:4. In embodiments,
the liquid vaccine includes a ratio of antigenic protein to
aluminum adjuvant (wt/wt) of about 1:3. In embodiments, the liquid
vaccine includes a ratio of antigenic protein to aluminum adjuvant
(wt/wt) of about 1:2. In embodiments, the liquid vaccine includes a
ratio of antigenic protein to aluminum adjuvant (wt/wt) of about
1:1. In embodiments, the liquid vaccine includes a ratio of
antigenic protein to aluminum adjuvant (wt/wt) of less than 1:10.
In embodiments, the liquid vaccine includes a ratio of antigenic
protein to aluminum adjuvant (wt/wt) of 1:10. In embodiments, the
liquid vaccine includes a ratio of antigenic protein to aluminum
adjuvant (wt/wt) of 1:9. In embodiments, the liquid vaccine
includes a ratio of antigenic protein to aluminum adjuvant (wt/wt)
of 1:8. In embodiments, the liquid vaccine includes a ratio of
antigenic protein to aluminum adjuvant (wt/wt) of 1:7. In
embodiments, the liquid vaccine includes a ratio of antigenic
protein to aluminum adjuvant (wt/wt) of 1:6. In embodiments, the
liquid vaccine includes a ratio of antigenic protein to aluminum
adjuvant (wt/wt) of 1:5. In embodiments, the liquid vaccine
includes a ratio of antigenic protein to aluminum adjuvant (wt/wt)
of 1:4. In embodiments, the liquid vaccine includes a ratio of
antigenic protein to aluminum adjuvant (wt/wt) of 1:3. In
embodiments, the liquid vaccine includes a ratio of antigenic
protein to aluminum adjuvant (wt/wt) of 1:2. In embodiments, the
liquid vaccine includes a ratio of antigenic protein to aluminum
adjuvant (wt/wt) of 1:1.
[0092] In embodiments, the liquid vaccine includes an excipient. In
embodiments, the liquid vaccine includes a plurality of different
excipients. In embodiments, the excipient is a salt, sugar
(saccharide), buffer, detergent, polymer, amino acid, or
preservative. In embodiments, the excipient is disodium edetate,
sodium chloride, sodium citrate, sodium succinate, sodium
hydroxide, Sodium glucoheptonate, sodium acetyltryptophanate,
sodium bicarbonate, sodium caprylate, sodium pertechnetate, sodium
acetate, sodium dodecyl sulfate, ammonium citrate, calcium
chloride, calcium, potassium chloride, potassium sodium tartarate,
zinc oxide, zinc, stannous chloride, magnesium sulfate, magnesium
stearate, titanium dioxide, DL-lactic/glycolic acids, asparagine,
L-arginine, arginine hydrochloride, adenine, histidine, glycine,
glutamine, glutathione, imidazole, protamine, protamine sulfate,
phosphoric acid, Tri-n-butyl phosphate, ascorbic acid, cysteine
hydrochloride, hydrochloric acid, hydrogen citrate, trisodium
citrate, guanidine hydrochloride, mannitol, lactose, sucrose,
agarose, sorbitol, maltose, trehalose, polysorbate 80, polysorbate
20, poloxamer 188, sorbitan monooleate, triton n101, m-cresol,
benyl alcohol, ethanolamine, glycerin, phosphorylethanolamine,
tromethamine, 2-phenyloxyethanol, chlorobutanol, dimethylsulfoxide,
N-methyl-2-pyrrolidone, propyleneglycol, polyoxyl 35 castor oil,
methyl hydroxybenzoate, tromethamine, corn
oil-mono-di-triglycerides, poloxyl 40 hydrogenated castor oil,
tocopherol, n-acetyltryptophan, octa-fluoropropane, castor oil,
polyoxyethylated oleic glycerides, polyoxytethylated castor oil,
phenol, glyclyglycine, thimerosal, parabens, gelatin, Formaldehyde,
Dulbecco's modified eagles medium, hydrocortisone, neomycin, Von
Willebrand factor, gluteraldehyde, benzethonium chloride, white
petroleum, p-aminopheyl-p-anisate, monosodium glutamate,
beta-propiolactone, acetate, citrate, glutamate, glycinate,
histidine, Lactate, Maleate, phosphate, succinate, tartrate, tris,
carbomer 1342 (copolymer of acrylic acid and a long chain alkyl
methacrylate cross-linked with allyl ethers of pentaerythritol),
glucose star polymer, silicone polymer, polydimethylsiloxane,
polyethylene glycol, polyvinylpyrrolidone, carboxymethylcellulose,
poly(glycolic acid), poly(lactic-co-glycolic acid), polylactic
acid, dextran 40, or poloxamer. In embodiments, the excipient is
trehalose. In embodiments, the liquid vaccine includes less than 5%
wt/vol of the excipient/liquid vaccine. In embodiments, the liquid
vaccine includes less than 4% wt/vol of the excipient/liquid
vaccine. In embodiments, the liquid vaccine includes less than 3%
wt/vol of the excipient/liquid vaccine. In embodiments, the liquid
vaccine includes less than 2% wt/vol of the excipient/liquid
vaccine. In embodiments, the liquid vaccine includes less than 1%
wt/vol of the excipient/liquid vaccine. In embodiments, the liquid
vaccine includes less than 0.5% wt/vol of the excipient/liquid
vaccine. In embodiments, the liquid vaccine includes about 5%
wt/vol of the excipient/liquid vaccine. In embodiments, the liquid
vaccine includes about 4% wt/vol of the excipient/liquid vaccine.
In embodiments, the liquid vaccine includes about 3% wt/vol of the
excipient/liquid vaccine. In embodiments, the liquid vaccine
includes about 2% wt/vol of the excipient/liquid vaccine. In
embodiments, the liquid vaccine includes about 1% wt/vol of the
excipient/liquid vaccine. In embodiments, the liquid vaccine
includes about 0.5% wt/vol of the excipient/liquid vaccine. In
embodiments, the liquid vaccine includes about 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/vol)
of the excipient/liquid vaccine. In embodiments, the liquid vaccine
includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10% (wt/vol) of the excipient/liquid vaccine. In
embodiments, the liquid vaccine includes less than 5% of the
excipient. In embodiments, the liquid vaccine includes less than 4%
of the excipient. In embodiments, the liquid vaccine includes less
than 3% of the excipient. In embodiments, the liquid vaccine
includes less than 2% of the excipient. In embodiments, the liquid
vaccine includes less than 1% of the excipient. In embodiments, the
liquid vaccine includes less than 0.5% of the excipient. In
embodiments, the liquid vaccine includes about 5% of the excipient.
In embodiments, the liquid vaccine includes about 4% of the
excipient. In embodiments, the liquid vaccine includes about 3% of
the excipient. In embodiments, the liquid vaccine includes about 2%
of the excipient. In embodiments, the liquid vaccine includes about
1% of the excipient. In embodiments, the liquid vaccine includes
about 0.5% of the excipient. In embodiments, the liquid vaccine
includes about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10% of the excipient. In embodiments, the
liquid vaccine includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of the excipient.
[0093] In embodiments, the applying includes spraying or dripping
droplets of the liquid vaccine. In embodiments, the vapor-liquid
interface of the droplets is less than 500 cm.sup.-1 area/volume.
In embodiments, the vapor-liquid interface of the droplets is less
than 400 cm.sup.-1 area/volume. In embodiments, the vapor-liquid
interface of the droplets is less than 300 cm.sup.-1 area/volume.
In embodiments, the vapor-liquid interface of the droplets is less
than 200 cm.sup.-1 area/volume. In embodiments, the vapor-liquid
interface of the droplets is less than 100 cm.sup.-1 area/volume.
In embodiments, the vapor-liquid interface of the droplets is less
than 50 cm.sup.-1 area/volume. In embodiments, the vapor-liquid
interface of the droplets is less than 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,
350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, or 500 cm.sup.-1 area/volume.
[0094] In embodiments, the method further includes contacting the
droplets with a freezing surface having a temperature below the
freezing temperature of the liquid vaccine (e.g. 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100 degrees Celsius below the
freezing temperature). In embodiments, the method further includes
contacting the droplets with a freezing surface having a
temperature differential of at least 30.degree. C. between the
droplets and the surface. In embodiments, the temperature
differential is at least 40.degree. C. between the droplets and the
surface. In embodiments, the temperature differential is at least
50.degree. C. between the droplets and the surface. In embodiments,
the temperature differential is at least 60.degree. C. between the
droplets and the surface. In embodiments, the temperature
differential is at least 70.degree. C. between the droplets and the
surface. In embodiments, the temperature differential is at least
80.degree. C. between the droplets and the surface. In embodiments,
the temperature differential is at least 90.degree. C. between the
droplets and the surface. In embodiments, the temperature
differential between the droplets and the surface is at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 degrees
Celsius.
[0095] In embodiments, the vaccine thin film has a thickness of
less than 500 micrometers. In embodiments, the vaccine thin film
has a thickness of less than 400 micrometers. In embodiments, the
vaccine thin film has a thickness of less than 300 micrometers. In
embodiments, the vaccine thin film has a thickness of less than 200
micrometers. In embodiments, the vaccine thin film has a thickness
of less than 100 micrometers. In embodiments, the vaccine thin film
has a thickness of less than 50 micrometers. In embodiments, the
vaccine thin film has a thickness of less than 10, 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,
460, 470, 480, 490, or 500 micrometers. In embodiments, the vaccine
thin film has a thickness of about 500 micrometers. In embodiments,
the vaccine thin film has a thickness of about 400 micrometers. In
embodiments, the vaccine thin film has a thickness of about 300
micrometers.
[0096] In embodiments, the vaccine thin film has a thickness of
about 200 micrometers. In embodiments, the vaccine thin film has a
thickness of about 100 micrometers. In embodiments, the vaccine
thin film has a thickness of about 50 micrometers. In embodiments,
the vaccine thin film has a thickness of about 10, 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,
460, 470, 480, 490, or 500 micrometers.
[0097] In embodiments, the vaccine thin film has a surface area to
volume ratio of between 25 and 500 cm.sup.-1. In embodiments, the
vaccine thin film has a surface area to volume ratio of between 25
and 400 cm.sup.-1. In embodiments, the vaccine thin film has a
surface area to volume ratio of between 25 and 300 cm.sup.-1. In
embodiments, the vaccine thin film has a surface area to volume
ratio of between 25 and 200 cm.sup.-1. In embodiments, the vaccine
thin film has a surface area to volume ratio of between 25 and 100
cm.sup.-1. In embodiments, the vaccine thin film has a surface area
to volume ratio of between 100 and 500 cm.sup.-1. In embodiments,
the vaccine thin film has a surface area to volume ratio of between
200 and 500 cm.sup.-1. In embodiments, the vaccine thin film has a
surface area to volume ratio of between 300 and 500 cm.sup.-1. In
embodiments, the vaccine thin film has a surface area to volume
ratio of between 400 and 500 cm.sup.-1. In embodiments, the vaccine
thin film has a surface area to volume ratio of between 100 and 400
cm.sup.-1. In embodiments, the vaccine thin film has a surface area
to volume ratio of between 200 and 300 cm.sup.-1. In embodiments,
the vaccine thin film has a surface area to volume ratio of about
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,
420, 430, 440, 450, 460, 470, 480, 490, or 500 cm.sup.-1. In
embodiments, the vaccine thin film has a surface area to volume
ratio of between about 25 and about 500 cm.sup.-1. In embodiments,
the vaccine thin film has a surface area to volume ratio of between
about 25 and about 400 cm.sup.-1. In embodiments, the vaccine thin
film has a surface area to volume ratio of between about 25 and
about 300 cm.sup.-1. In embodiments, the vaccine thin film has a
surface area to volume ratio of between about 25 and about 200
cm.sup.-1. In embodiments, the vaccine thin film has a surface area
to volume ratio of between about 25 and about 100 cm.sup.-1. In
embodiments, the vaccine thin film has a surface area to volume
ratio of between about 100 and about 500 cm.sup.-1. In embodiments,
the vaccine thin film has a surface area to volume ratio of between
about 200 and about 500 cm.sup.-1. In embodiments, the vaccine thin
film has a surface area to volume ratio of between about 300 and
about 500 cm.sup.-1. In embodiments, the vaccine thin film has a
surface area to volume ratio of between about 400 and about 500
cm.sup.-1. In embodiments, the vaccine thin film has a surface area
to volume ratio of between about 100 and about 400 cm.sup.-1. In
embodiments, the vaccine thin film has a surface area to volume
ratio of between about 200 and about 300 cm.sup.-1.
[0098] In embodiments, the freezing rate of the droplets is between
about 10 K/second and about 10.sup.5 K/second. In embodiments, the
freezing rate of the droplets is between about 10 K/second and
about 10.sup.4 K/second. In embodiments, the freezing rate of the
droplets is between about 10 K/second and about 10.sup.3 K/second.
In embodiments, the freezing rate of the droplets is between about
10.sup.2 K/second and about 10.sup.3 K/second. In embodiments, the
freezing rate of the droplets is between about 50 K/second and
about 5.times.10.sup.2 K/second. In embodiments, the freezing rate
of the droplets is between 10 K/second and 10.sup.5 K/second. In
embodiments, the freezing rate of the droplets is between 10
K/second and 10.sup.4 K/second. In embodiments, the freezing rate
of the droplets is between 10 K/second and 10.sup.3 K/second. In
embodiments, the freezing rate of the droplets is between 10.sup.2
K/second and 10.sup.3 K/second. In embodiments, the freezing rate
of the droplets is between 50 K/second and 5.times.10.sup.2
K/second. In embodiments, the freezing rate of the droplets is
about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,
570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,
700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,
830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,
960, 970, 980, 990, or 1000 K/second. In embodiments, the freezing
rate of the droplets is 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,
530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,
660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,
790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,
920, 930, 940, 950, 960, 970, 980, 990, or 1000 K/second. In
embodiments, each of the droplets freezes upon contact with the
freezing surface in less than about 50, 75, 100, 125, 150, 175,
200, 250, 500, 1,000, or 2,000 milliseconds. In embodiments, each
of the droplets freezes upon contact with the freezing surface in
less than 50, 75, 100, 125, 150, 175, 200, 250, 500, 1,000, or
2,000 milliseconds.
[0099] In embodiments, the droplets have an average diameter
between about 0.1 and about 5 mm, between about 20 and about 24
degrees Celsius. In embodiments, the droplets have an average
diameter between about 2 and about 4 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between about 1 and about 4 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between about 2 and about 3 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between about 1 and about 3 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between about 1 and about 2 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between about 3 and about 4 mm, between about 20 and about
24 degrees Celsius. In embodiments, the droplets have an average
diameter between 0.1 and 5 mm, between 20 and 24 degrees Celsius.
In embodiments, the droplets have an average diameter between 2 and
4 mm, between 20 and 24 degrees Celsius.
[0100] In embodiments, the droplets have an average diameter
between 1 and 4 mm, between 20 and 24 degrees Celsius. In
embodiments, the droplets have an average diameter between 2 and 3
mm, between 20 and 24 degrees Celsius. In embodiments, the droplets
have an average diameter between 1 and 3 mm, between 20 and 24
degrees Celsius. In embodiments, the droplets have an average
diameter between 1 and 2 mm, between 20 and 24 degrees Celsius. In
embodiments, the droplets have an average diameter between 3 and 4
mm, between 20 and 24 degrees Celsius.
[0101] In embodiments, the step of spraying or dripping droplets is
repeated to overlay one or more additional vaccine thin films on
top of the vaccine thin film. In embodiments, the step of spraying
or dripping droplets is repeated to overlay 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100 additional vaccine thin films on top
of the first vaccine thin film.
[0102] In embodiments, the method further includes removing the
solvent (e.g. water or liquid) from the vaccine thin film to form a
dry vaccine.
[0103] In embodiments, is a method of making a dry vaccine from a
vaccine thin film (e.g. including a vaccine thin film made using a
method as described herein), including removing the solvent (e.g.
water or liquid) from the vaccine thin film to form a dry vaccine.
In embodiments of the methods described herein, the dry vaccine is
a dry vaccine as described herein, including in an aspect,
embodiment, example, table, figure, or claim. In embodiments, a
method of making a vaccine thin film or a method of making dry
vaccine is used to make a dry vaccine as described herein,
including in an aspect, embodiment, example, table, figure, or
claim.
[0104] In embodiments, the removing of the solvent includes
lyophilization. In embodiments, the removing of the solvent
includes lyophilization at temperatures of 20 degrees Celsius or
less. In embodiments, the removing of the solvent includes
lyophilization at temperatures of 25 degrees Celsius or less. In
embodiments, the solvent includes lyophilization at temperatures of
40 degrees Celsius or less. In embodiments, the removing of the
solvent includes lyophilization at temperatures of 50 degrees
Celsius or less. In embodiments, the removing of the solvent
includes lyophilization at temperatures of about 20 degrees Celsius
or less. In embodiments, the removing of the solvent includes
lyophilization at temperatures of about 25 degrees Celsius or less.
In embodiments, the solvent includes lyophilization at temperatures
of about 40 degrees Celsius or less. In embodiments, the removing
of the solvent includes lyophilization at temperatures of about 50
degrees Celsius or less.
[0105] In embodiments, the dry vaccine includes between about 0.5
and about 5% (wt/wt) of the aluminum adjuvant. In embodiments, the
dry vaccine includes between about 0.5 and about 4% (wt/wt) of the
aluminum adjuvant. In embodiments, the dry vaccine includes between
about 0.5 and about 3% (wt/wt) of the aluminum adjuvant. In
embodiments, the dry vaccine includes between about 0.5 and about
2% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes between about 0.75 and about 2% (wt/wt) of the
aluminum adjuvant. In embodiments, the dry vaccine includes between
about 1 and about 2% (wt/wt) of the aluminum adjuvant. In
embodiments, the dry vaccine includes between 0.5 and 5% (wt/wt) of
the aluminum adjuvant. In embodiments, the dry vaccine includes
between 0.5 and 4% (wt/wt) of the aluminum adjuvant. In
embodiments, the dry vaccine includes between 0.5 and 3% (wt/wt) of
the aluminum adjuvant. In embodiments, the dry vaccine includes
between 0.5 and 2% (wt/wt) of the aluminum adjuvant. In
embodiments, the dry vaccine includes between 0.75 and 2% (wt/wt)
of the aluminum adjuvant. In embodiments, the dry vaccine includes
between 1 and 2% (wt/wt) of the aluminum adjuvant. In embodiments,
the dry vaccine includes about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum
adjuvant. In embodiments, the dry vaccine includes at least 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10% (wt/wt) of the aluminum adjuvant. In embodiments, the dry
vaccine includes less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the aluminum
adjuvant. In embodiments, the dry vaccine includes at least about
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/wt) of the aluminum adjuvant. In embodiments, the
dry vaccine includes less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/wt) of the
aluminum adjuvant. In embodiments, the dry vaccine includes 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10% (wt/wt) of the aluminum adjuvant.
[0106] In embodiments, the method further includes solvating the
dry vaccine thereby forming a reconstituted liquid vaccine. A
reconstituted liquid vaccine may also be called a solvated dry
vaccine.
[0107] In embodiments, is a method of making a reconstituted liquid
vaccine from a dry vaccine (e.g. including a dry vaccine made using
a method as described herein), including solvating a dry vaccine
and thereby forming a reconstituted liquid vaccine. In embodiments
of the methods described herein, the dry vaccine is a dry vaccine
as described herein, including in an aspect, embodiment, example,
table, figure, or claim. In embodiments, a method of making a
vaccine thin film, a method of making a dry vaccine, or a method of
reconstituting a liquid vaccine is used to make a reconstituted
liquid vaccine as described herein, including in an aspect,
embodiment, example, table, figure, or claim.
[0108] In embodiments, the immunogenicity of the reconstituted
liquid vaccine is at least 60% the immunogenicity of the liquid
vaccine (prior to forming the dry vaccine from the liquid vaccine).
In embodiments, the immunogenicity of the reconstituted liquid
vaccine is at least 70% the immunogenicity of the liquid vaccine
(prior to forming the dry vaccine from the liquid vaccine). In
embodiments, the immunogenicity of the reconstituted liquid vaccine
is at least 80% the immunogenicity of the liquid vaccine (prior to
forming the dry vaccine from the liquid vaccine). In embodiments,
the immunogenicity of the reconstituted liquid vaccine is at least
90% the immunogenicity of the liquid vaccine (prior to forming the
dry vaccine from the liquid vaccine). In embodiments, the
immunogenicity of the reconstituted liquid vaccine is at least 95%
the immunogenicity of the liquid vaccine (prior to forming the dry
vaccine from the liquid vaccine). In embodiments, the
immunogenicity of the reconstituted liquid vaccine is at least
about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% the immunogenicity of the
liquid vaccine (prior to forming the dry vaccine from the liquid
vaccine). In embodiments, the immunogenicity of the reconstituted
liquid vaccine is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% the
immunogenicity of the liquid vaccine (prior to forming the dry
vaccine from the liquid vaccine).
[0109] In embodiments, the level of antigenic protein adsorbed to
the aluminum adjuvant of the reconstituted liquid vaccine is at
least 60% of the level of antigenic protein adsorbed to the
aluminum adjuvant of the liquid vaccine (prior to forming the dry
vaccine from the liquid vaccine). In embodiments, the level of
antigenic protein adsorbed to the aluminum adjuvant of the
reconstituted liquid vaccine is at least 70% of the level of
antigenic protein adsorbed to the aluminum adjuvant of the liquid
vaccine (prior to forming the dry vaccine from the liquid vaccine).
In embodiments, the level of antigenic protein adsorbed to the
aluminum adjuvant of the reconstituted liquid vaccine is at least
80% of the level of antigenic protein adsorbed to the aluminum
adjuvant of the liquid vaccine (prior to forming the dry vaccine
from the liquid vaccine). In embodiments, the level of antigenic
protein adsorbed to the aluminum adjuvant of the reconstituted
liquid vaccine is at least 90% of the level of antigenic protein
adsorbed to the aluminum adjuvant of the liquid vaccine (prior to
forming the dry vaccine from the liquid vaccine). In embodiments,
the level of antigenic protein adsorbed to the aluminum adjuvant of
the reconstituted liquid vaccine is at least 95% of the level of
antigenic protein adsorbed to the aluminum adjuvant of the liquid
vaccine (prior to forming the dry vaccine from the liquid vaccine).
In embodiments, the level of antigenic protein adsorbed to the
aluminum adjuvant of the reconstituted liquid vaccine is at least
99% of the level of antigenic protein adsorbed to the aluminum
adjuvant of the liquid vaccine (prior to forming the dry vaccine
from the liquid vaccine). In embodiments, the level of antigenic
protein adsorbed to the aluminum adjuvant of the reconstituted
liquid vaccine is at least about 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
of the level of antigenic protein adsorbed to the aluminum adjuvant
of the liquid vaccine (prior to forming the dry vaccine from the
liquid vaccine). In embodiments, the level of antigenic protein
adsorbed to the aluminum adjuvant of the reconstituted liquid
vaccine is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the level of
antigenic protein adsorbed to the aluminum adjuvant of the liquid
vaccine (prior to forming the dry vaccine from the liquid
vaccine).
[0110] In embodiments, the reconstituted liquid vaccine includes
particles, wherein the particles include the antigenic protein
adsorbed to the aluminum adjuvant. In embodiments, the particles
have an average diameter of between about 10 nm and about 2 .mu.m.
In embodiments, the particles have an average diameter of between
about 20 nm and about 2 .mu.m. In embodiments, the particles have
an average diameter of between about 50 nm and about 2 .mu.m. In
embodiments, the particles have an average diameter of between
about 100 nm and about 2 .mu.m. In embodiments, the particles have
an average diameter of between about 200 nm and about 2 .mu.m. In
embodiments, the particles have an average diameter of between
about 500 nm and about 2 .mu.m. In embodiments, the particles have
an average diameter of between about 1 .mu.m and about 2 .mu.m. In
embodiments, the particles have an average diameter of between
about 10 nm and about 1 .mu.m. In embodiments, the particles have
an average diameter of between about 10 nm and about 500 nm. In
embodiments, the particles have an average diameter of between
about 10 nm and about 200 nm. In embodiments, the particles have an
average diameter of between about 10 nm and about 200 nm. In
embodiments, the particles have an average diameter of between
about 10 nm and about 100 nm. In embodiments, the particles have an
average diameter of between about 10 nm and about 50 nm. In
embodiments, the particles have an average diameter of between
about 10 nm and about 20 nm. In embodiments, the particles have an
average diameter of between about 20 nm and about 1 .mu.m. In
embodiments, the particles have an average diameter of between
about 50 nm and about 500 nm. In embodiments, the particles have an
average diameter of between about 100 nm and about 500 nm. In
embodiments, the particles have an average diameter of between
about 100 nm and about 200 nm. In embodiments, the reconstituted
liquid vaccine includes particles, wherein the particles include
the antigenic protein adsorbed to the aluminum adjuvant. In
embodiments, the particles have an average diameter of between 10
nm and 2 .mu.m. In embodiments, the particles have an average
diameter of between 20 nm and 2 .mu.m. In embodiments, the
particles have an average diameter of between 50 nm and 2 .mu.m. In
embodiments, the particles have an average diameter of between 100
nm and 2 .mu.m. In embodiments, the particles have an average
diameter of between 200 nm and 2 .mu.m. In embodiments, the
particles have an average diameter of between 500 nm and 2 .mu.m.
In embodiments, the particles have an average diameter of between 1
.mu.m and 2 .mu.m. In embodiments, the particles have an average
diameter of between 10 nm and 1 .mu.m. In embodiments, the
particles have an average diameter of between 10 nm and 500 nm. In
embodiments, the particles have an average diameter of between 10
nm and 200 nm. In embodiments, the particles have an average
diameter of between 10 nm and 200 nm. In embodiments, the particles
have an average diameter of between 10 nm and 100 nm. In
embodiments, the particles have an average diameter of between 10
nm and 50 nm. In embodiments, the particles have an average
diameter of between 10 nm and 20 nm. In embodiments, the particles
have an average diameter of between 20 nm and 1 .mu.m. In
embodiments, the particles have an average diameter of between 50
nm and 500 nm. In embodiments, the particles have an average
diameter of between 100 nm and 500 nm. In embodiments, the
particles have an average diameter of between 100 nm and 200 nm. In
embodiments, the particles are non-crystalline. In embodiments, the
particles are amorphous.
[0111] In embodiments, the particles have an average diameter of
between about 1 .mu.m and about 50 .mu.m. In embodiments, the
particles have an average diameter of between about 10 .mu.m and
about 50 .mu.m. In embodiments, the particles have an average
diameter of between about 20 .mu.m and about 50 .mu.m. In
embodiments, the particles have an average diameter of between
about 30 .mu.m and about 50 .mu.m. In embodiments, the particles
have an average diameter of between about 40 .mu.m and about 50
.mu.m. In embodiments, the particles have an average diameter of
between about 10 .mu.m and about 40 .mu.m. In embodiments, the
particles have an average diameter of between about 10 .mu.m and
about 30 .mu.m. In embodiments, the particles have an average
diameter of between about 10 .mu.m and about 20 .mu.m. In
embodiments, the particles have an average diameter of between
about 1 .mu.m and about 10 .mu.m. In embodiments, the particles
have an average diameter of between 1 .mu.m and 50 .mu.m. In
embodiments, the particles have an average diameter of between 10
.mu.m and 50 .mu.m. In embodiments, the particles have an average
diameter of between 20 .mu.m and 50 .mu.m. In embodiments, the
particles have an average diameter of between 30 .mu.m and 50
.mu.m. In embodiments, the particles have an average diameter of
between 40 .mu.m and 50 .mu.m. In embodiments, the particles have
an average diameter of between 10 .mu.m and 40 .mu.m. In
embodiments, the particles have an average diameter of between 10
.mu.m and 30 p.m. In embodiments, the particles have an average
diameter of between 10 .mu.m and 20 .mu.m. In embodiments, the
particles have an average diameter of between 1 .mu.m and 10 .mu.m.
In embodiments, the particles have an average diameter of about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 .mu.m. In
embodiments, the particles have an average diameter of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 .mu.m.
[0112] In embodiments, the reconstituted liquid vaccine includes
particles including antigenic protein adsorbed to the aluminum
adjuvant of the same average diameter as the liquid vaccine (prior
to forming the dry vaccine from the liquid vaccine) particles
including antigenic protein adsorbed to the aluminum adjuvant. In
embodiments, the reconstituted liquid vaccine includes particles
including antigenic protein adsorbed to the aluminum adjuvant
having an average diameter within 5% of the average diameter of
particles including the antigenic protein adsorbed to the aluminum
adjuvant in the liquid vaccine (prior to forming the dry vaccine
from the liquid vaccine). In embodiments, the reconstituted liquid
vaccine includes particles including antigenic protein adsorbed to
the aluminum adjuvant having an average diameter within 10% of the
average diameter of particles including the antigenic protein
adsorbed to the aluminum adjuvant in the liquid vaccine (prior to
forming the dry vaccine from the liquid vaccine). In embodiments,
the reconstituted liquid vaccine includes particles including
antigenic protein adsorbed to the aluminum adjuvant having an
average diameter within 20% of the average diameter of particles
including the antigenic protein adsorbed to the aluminum adjuvant
in the liquid vaccine (prior to forming the dry vaccine from the
liquid vaccine). In embodiments, the reconstituted liquid vaccine
includes particles including antigenic protein adsorbed to the
aluminum adjuvant having an average diameter within 30% of the
average diameter of particles including the antigenic protein
adsorbed to the aluminum adjuvant in the liquid vaccine (prior to
forming the dry vaccine from the liquid vaccine). In embodiments,
the reconstituted liquid vaccine includes particles including
antigenic protein adsorbed to the aluminum adjuvant having an
average diameter within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30% of the average diameter of particles including the
antigenic protein adsorbed to the aluminum adjuvant in the liquid
vaccine (prior to forming the dry vaccine from the liquid vaccine).
In embodiments, the reconstituted liquid vaccine includes particles
including antigenic protein adsorbed to the aluminum adjuvant
having an average diameter within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30% of the average diameter of particles including the
antigenic protein adsorbed to the aluminum adjuvant in the liquid
vaccine (prior to forming the dry vaccine from the liquid
vaccine).
[0113] In embodiments, the solvating of the dry vaccine is at least
one day after preparing the dry vaccine from the liquid vaccine
(e.g. the dry vaccine is stored for at least one day). In
embodiments, the solvating of the dry vaccine is at least two days
after preparing the dry vaccine from the liquid vaccine (e.g. the
dry vaccine is stored for at least two days). In embodiments, the
solvating of the dry vaccine is at least three days after preparing
the dry vaccine from the liquid vaccine (e.g. the dry vaccine is
stored for at least three days). In embodiments, the solvating of
the dry vaccine is at least one week after preparing the dry
vaccine from the liquid vaccine (e.g. the dry vaccine is stored for
at least one week). In embodiments, the solvating of the dry
vaccine is at least two weeks after preparing the dry vaccine from
the liquid vaccine (e.g. the dry vaccine is stored for at least two
weeks). In embodiments, the solvating of the dry vaccine is at
least one month after preparing the dry vaccine from the liquid
vaccine (e.g. the dry vaccine is stored for at least one month). In
embodiments, the solvating of the dry vaccine is at least two
months after preparing the dry vaccine from the liquid vaccine
(e.g. the dry vaccine is stored for at least two months). In
embodiments, the solvating of the dry vaccine is at least three
months after preparing the dry vaccine from the liquid vaccine
(e.g. the dry vaccine is stored for at least three months). In
embodiments, the solvating of the dry vaccine is at least six
months after preparing the dry vaccine from the liquid vaccine
(e.g. the dry vaccine is stored for at least six months). In
embodiments, the solvating of the dry vaccine is at least one year
after preparing the dry vaccine from the liquid vaccine (e.g. the
dry vaccine is stored for at least one year). In embodiments, the
solvating of the dry vaccine is at least two years after preparing
the dry vaccine from the liquid vaccine (e.g. the dry vaccine is
stored for at least two years). In embodiments, the solvating of
the dry vaccine is at least three years after preparing the dry
vaccine from the liquid vaccine (e.g. the dry vaccine is stored for
at least three years). In embodiments, the solvating of the dry
vaccine is at least five years after preparing the dry vaccine from
the liquid vaccine (e.g. the dry vaccine is stored for at least
five years). In embodiments, the solvating of the dry vaccine is at
least ten years after preparing the dry vaccine from the liquid
vaccine (e.g. the dry vaccine is stored for at least ten
years).
[0114] In embodiments, prior to the solvating of the dry vaccine,
the dry vaccine is stored at about 4 degrees Celsius for at least
99% of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at less than 4 degrees Celsius
for at least 99% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at less
than 0 degrees Celsius for at least 99% of the time. In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at less than -20 degrees Celsius for at least 99%
of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at about -20 degrees Celsius for
at least 99% of the time. In embodiments, prior to the solvating of
the dry vaccine, the dry vaccine is stored at less than -80 degrees
Celsius for at least 99% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at about
-80 degrees Celsius for at least 99% of the time. In embodiments,
prior to the solvating of the dry vaccine, the dry vaccine is
stored at ambient temperatures (e.g. room temperature). In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at between 20 and 24 degrees Celsius for at least
99% of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at between 4 and 24 degrees
Celsius for at least 99% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at between
0 and 24 degrees Celsius for at least 99% of the time. In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at between 4 and 40 degrees Celsius for at least
99% of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at between 0 and 40 degrees
Celsius for at least 99% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at about 4
degrees Celsius for at least 90% of the time. In embodiments, prior
to the solvating of the dry vaccine, the dry vaccine is stored at
less than 4 degrees Celsius for at least 90% of the time. In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at less than 0 degrees Celsius for at least 90%
of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at less than -20 degrees Celsius
for at least 90% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at between
20 and 24 degrees Celsius for at least 90% of the time. In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at between 4 and 24 degrees Celsius for at least
90% of the time. In embodiments, prior to the solvating of the dry
vaccine, the dry vaccine is stored at between 0 and 24 degrees
Celsius for at least 90% of the time. In embodiments, prior to the
solvating of the dry vaccine, the dry vaccine is stored at between
4 and 40 degrees Celsius for at least 90% of the time. In
embodiments, prior to the solvating of the dry vaccine, the dry
vaccine is stored at between 0 and 40 degrees Celsius for at least
90% of the time.
[0115] In embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine remains homogeneous. As used in
reference to the status of a reconstituted liquid vaccine, the term
"homogenous" refers to a lack of a significant amount of
aggregation and/or precipitation forming, such that the
reconstituted liquid vaccine does not include solid matter that is
not evenly dispersed (e.g. solid matter visible to the naked eye,
solid matter that settles in the liquid, solid matter that was not
apparent in a liquid vaccine prior to formation of the dry vaccine
and reconstitution, precipitate that was not present in the liquid
vaccine prior to formation of the dry vaccine). A homogenous
reconstituted liquid sample may include particles of antigenic
protein adsorbed to aluminum adjuvant (e.g. that are suspended or
dispersed in the reconstituted liquid vaccine). In embodiments,
upon solvating the dry vaccine the resulting reconstituted liquid
vaccine remains homogeneous for at least one day. In embodiments,
upon solvating the dry vaccine the resulting reconstituted liquid
vaccine remains homogeneous for at least two days. In embodiments,
upon solvating the dry vaccine the resulting reconstituted liquid
vaccine remains homogeneous for at least three days. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine remains homogeneous for at least one
week. In embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine remains homogeneous for at least two
weeks. In embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine remains homogeneous for at least one
month. In embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine remains homogeneous for at least three
months. In embodiments, upon solvating the dry vaccine the
resulting reconstituted liquid vaccine remains homogeneous for at
least six months. In embodiments, upon solvating the dry vaccine
the resulting reconstituted liquid vaccine remains homogeneous for
at least one year. In embodiments, upon solvating the dry vaccine
the resulting reconstituted liquid vaccine does not form a
precipitate (e.g. solid matter visible to the naked eye, solid
matter that settles in the liquid, solid matter that was not
apparent in a liquid vaccine prior to formation of the dry vaccine
and reconstitution, precipitate that was not present in the liquid
vaccine prior to formation of the dry vaccine). In embodiments,
upon solvating the dry vaccine the resulting reconstituted liquid
vaccine does not form a precipitate for at least one day. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate for at
least two days. In embodiments, upon solvating the dry vaccine the
resulting reconstituted liquid vaccine does not form a precipitate
for at least three days. In embodiments, upon solvating the dry
vaccine the resulting reconstituted liquid vaccine does not form a
precipitate for at least one week. In embodiments, upon solvating
the dry vaccine the resulting reconstituted liquid vaccine does not
form a precipitate for at least two weeks. In embodiments, upon
solvating the dry vaccine the resulting reconstituted liquid
vaccine does not form a precipitate for at least one month. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate for at
least three months. In embodiments, upon solvating the dry vaccine
the resulting reconstituted liquid vaccine does not form a
precipitate for at least six months. In embodiments, upon solvating
the dry vaccine the resulting reconstituted liquid vaccine does not
form a precipitate for at least one year. In embodiments, the
precipitate includes particles having an average diameter greater
than 50 .mu.m. In embodiments, the precipitate includes particles
having an average diameter greater than 100 p.m. In embodiments,
the precipitate includes particles having an average diameter
greater than 200 .mu.m. In embodiments, the precipitate includes
particles having an average diameter greater than 300 .mu.m. In
embodiments, the precipitate includes particles having an average
diameter greater than 400 .mu.m. In embodiments, the precipitate
includes particles having an average diameter greater than 500
.mu.m. In embodiments, the precipitate includes particles having an
average diameter greater than 600 .mu.m. In embodiments, the
precipitate includes particles having an average diameter greater
than 700 .mu.m. In embodiments, the precipitate includes particles
having an average diameter greater than 800 .mu.m. In embodiments,
the precipitate includes particles having an average diameter
greater than 900 .mu.m. In embodiments, the precipitate includes
particles having an average diameter greater than 1000 .mu.m. In
embodiments, the precipitate includes particles having an average
diameter greater than about 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,
530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,
660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,
790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,
920, 930, 940, 950, 960, 970, 980, 990, or 1000 .mu.m. In
embodiments, the precipitate includes particles having an average
diameter of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,
420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,
550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,
680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,
810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,
940, 950, 960, 970, 980, 990, or 1000 .mu.m. In embodiments, the
precipitate includes particles having an average diameter greater
than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,
570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,
700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,
830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,
960, 970, 980, 990, or 1000 .mu.m. In embodiments, the precipitate
(that is not formed) includes particles having an average diameter
of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,
320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,
450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,
580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700,
710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,
840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,
970, 980, 990, or 1000 .mu.m. In embodiments, the precipitate (that
is not formed) includes at least 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of
the total antigenic protein absorbed to an aluminum adjuvant in the
reconstituted liquid vaccine. In embodiments, upon solvating the
dry vaccine the resulting reconstituted liquid vaccine does not
form a precipitate including more than about 1% of the total
antigenic protein in the reconstituted liquid vaccine. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate including
more than about 2% of the total antigenic protein in the
reconstituted liquid vaccine. In embodiments, upon solvating the
dry vaccine the resulting reconstituted liquid vaccine does not
form a precipitate including more than about 3% of the total
antigenic protein in the reconstituted liquid vaccine. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate including
more than about 4% of the total antigenic protein in the
reconstituted liquid vaccine. In embodiments, upon solvating the
dry vaccine the resulting reconstituted liquid vaccine does not
form a precipitate including more than about 5% of the total
antigenic protein in the reconstituted liquid vaccine. In
embodiments, upon solvating the dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate including
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20% of the total antigenic protein in the
reconstituted liquid vaccine. In embodiments the precipitate
includes irreversible aggregates of antigenic protein and/or
aluminum adjuvant.
[0116] In embodiments, the liquid vaccine includes a commercially
available vaccine. In embodiments, the liquid vaccine is a
commercially available vaccine. In embodiments, the liquid vaccine
has received market approval from the US FDA or the corresponding
authority in another country. In embodiments, the liquid vaccine is
a vaccine for the treatment of diphtheria, tetanus, pertussis,
influenza, pneumonia, otitis media, bacteremia, meningitis,
hepatitis, cirrhosis, anthrax poisoning, botulism, rabies, warts,
poliomyelitis, Japanese encephalitis, or cancer. In embodiments,
the liquid vaccine is a vaccine for the treatment of infection by
Clostridium tetani, Clostridium botulinum, Streptococcus pneumonia,
Hepatitis A, Hepatitis B, Haemophilus influenza, Corynebacterium
diphtheria, Bordetella pertussis, Human papillomavirus, Bacillus
anthracis, Rabies virus, Japanese encephalitis virus, or
Poliovirus. In embodiments, the liquid vaccine includes a
commercially available vaccine and another component not included
in the commercially available vaccine (e.g. an excipient (e.g.
trehalose)).
[0117] In an aspect is provided a method of treating a disease in a
patient in need of such treatment, the method including
administering a therapeutically effective amount of a solvated dry
vaccine as described herein (e.g. in an aspect, embodiment,
example, table, figure, or claims) (e.g. a reconstituted liquid
vaccine as described herein) to the patient.
[0118] In embodiments, the disease is diphtheria, tetanus,
pertussis, influenza, pneumonia, otitis media, bacteremia,
meningitis, hepatitis, cirrhosis, anthrax poisoning, rabies, warts,
poliomyelitis, Japanese encephalitis, or cancer. In embodiments,
the disease is caused by an infectious agent. In embodiments, the
infectious agent is a bacterium. In embodiments, the infectious
agent is a virus. In embodiments, the infectious agent is
Clostridium tetani, Clostridium botulinum, Streptococcus pneumonia,
Hepatitis A, Hepatitis B, Haemophilus influenza, Corynebacterium
diphtheria, Bordetella pertussis, Human papillomavirus, Bacillus
anthracis, Rabies virus, Japanese encephalitis virus, or
Poliovirus.
[0119] In an aspect is provided a method of treating a disease in a
patient in need of such treatment, the method including
administering a therapeutically effective amount of dry vaccine as
described herein (e.g. in an aspect, embodiment, example, table,
figure, or claims) (e.g. a reconstituted liquid vaccine as
described herein) to the patient.
[0120] In embodiments, the disease is diphtheria, tetanus,
pertussis, influenza, pneumonia, otitis media, bacteremia,
meningitis, hepatitis, cirrhosis, anthrax poisoning, botulism,
rabies, warts, poliomyelitis, Japanese encephalitis, or cancer. In
embodiments, the disease is caused by an infectious agent. In
embodiments, the infectious agent is a bacterium. In embodiments,
the infectious agent is a virus. In embodiments, the infectious
agent is Clostridium tetani, Clostridium botulinum, Streptococcus
pneumonia, Hepatitis A, Hepatitis B, Haemophilus influenza,
Corynebacterium diphtheria, Bordetella pertussis, Human
papillomavirus, Bacillus anthracis, Rabies virus, Japanese
encephalitis virus, or Poliovirus.
[0121] In embodiments, the dry vaccine is administered by
inhalation, intradermally, orally, or vaginally. In embodiments,
the dry vaccine is administered through the nasal mucosa,
bronchoalveolar mucosa, or gastrointestinal mucosa.
[0122] In embodiments, the method is a method described herein,
including in an aspect, embodiment, example, table, figure, or
claim. Provided herein is a method of preparing a dry vaccine
including a method of preparing a vaccine thin film as described
herein (including in an aspect, embodiment, example, table, figure,
or claim) and a method of removing a solvent from a vaccine thin
film as described herein (including in an aspect, embodiment,
example, table, figure, or claim). Provided herein is a method of
preparing a reconstituted dry vaccine including a method of
preparing a dry vaccine as described herein (including in an
aspect, embodiment, example, table, figure, or claim), a method of
preparing a vaccine thin film as described herein (including in an
aspect, embodiment, example, table, figure, or claim) and a method
of removing a solvent from a vaccine thin film as described herein
(including in an aspect, embodiment, example, table, figure, or
claim).
[0123] In embodiments, to form a powder vaccine, an aqueous vaccine
composition is first frozen to form a frozen vaccine composition,
then the frozen water is removed to form the vaccine powder. A fast
freezing process is used to form the frozen vaccine composition. A
fast freezing process, as used herein, is a process that can freeze
a thin film of liquid (less than about 500 microns) in a time of
less than or equal to one second. Examples of fast freezing
processes that may be used include thin film freezing (TFF), spray
freeze-drying (SFD), or spray freezing into liquids (SFL). In the
TFF process liquid droplets fall from a given height and impact,
spread, and freeze on a cooled solid substrate. Typically, the
substrate is a metal drum that is cooled to below 250.degree. K, or
below 200.degree. K or below 150.degree. K. On impact the droplets
that are deformed into thin films freeze in a time of between about
70 ms and 1000 ms. The frozen thin films may be removed from the
substrate by a stainless steel blade mounted along the rotating
drum surface. The frozen thin films are collected in liquid
nitrogen to maintain in the frozen state. Further details regarding
thin film freezing processes may be found in the paper to Engstrom
et al. "Formation of Stable Submicron Protein Particles by Thin
Film Freezing" Pharmaceutical Research, Vol. 25, No. 6, June 2008,
1334-1346, which is incorporated herein by reference.
[0124] Water (e.g. frozen water) is removed from the frozen vaccine
composition to produce a vaccine powder. Water (e.g. frozen water)
may be removed by a lyophilization process or a freeze-drying
process. Water may also be removed by an atmospheric freeze-drying
process.
[0125] The resulting vaccine powder can be readily reconstituted to
form a stable dispersion without significant loss of stability or
activity. The vaccine powder may be transported and stored in a
wide range of temperatures without concern of accidental exposure
to freezing conditions. In addition, the vaccine powder may also be
stored at room temperature, which will potentially decrease the
costs of vaccines. In fact, it is generally less costly to
transport dry solid powder than liquid.
[0126] Currently human vaccines (e.g. marketed and/or approved
human vaccines, such as FDA approved human vaccines) that have
aluminum-containing adjuvant are all administered by
needle-syringe-based injections. It would be beneficial to patients
and the healthcare system if the vaccines were administered
non-invasively without hypodermic needles. Our dried
aluminum-containing vaccine powder can potentially be administered
by an alternative route such as, but not limited to, inhalation as
a dried powder, intradermally using a solid jet injection device
(e.g., powder jet injector), orally in tablets or capsules,
buccally in buccal tablets or films, or vaginally using a special
vaginal drug delivery device. The above-mentioned routes of
administration are not only more convenient and friendly to
patients, but more importantly they can enable the induction of
mucosal immune responses. Functional antibodies in the mucosal
secretion (e.g., nasal mucus, bronchoalveolar mucus, or the
gastrointestinal mucus) of a host can effectively neutralize
pathogens or toxins even before they enter the host.
[0127] Described herein are compositions and methods for preparing
a vaccine thin film or a dry vaccine by spraying or dripping
droplets of a liquid vaccine (e.g. aluminum adjuvant containing)
such that the antigenic protein adsorbed to the aluminum adjuvant
in the liquid vaccine (e.g. aluminum adjuvant containing) is
exposed to an vapor-liquid interface of less than 500 cm.sup.-1
area/volume (e.g. less than 50, 100, 150, 200, 250, 300, 400) and
contacting the droplet with a freezing surface having a temperature
lower than the freezing temperature of the liquid vaccine (e.g.
aluminum adjuvant containing) (e.g. has a temperature differential
of at least 30.degree. C. between the droplet and the surface),
wherein the surface freezes the droplet into a thin film with a
thickness of less than 500 micrometers (e.g. less than 450, 400,
350, 300, 250, 200, 150, 100, or 50 micrometers) and a surface area
to volume between 25 to 500 cm.sup.-1. In embodiments, the method
may further include the step of removing the liquid (e.g. solvent,
water) from the frozen material to form a dry vaccine (e.g.
particles). In embodiments, the droplets freeze upon contact with
the surface in less than 50, 75, 100, 125, 150, 175, 200, 250, 500,
1,000 or 2,000 milliseconds. In embodiments, the droplets freeze
upon contact with the surface in less than 50 or 150 milliseconds.
In embodiments, the droplet has a diameter between 2 and 5 mm at
room temperature. In embodiments, the droplet forms a thin film on
the freezing surface of between 50 and 500 micrometers in
thickness. In embodiments, the droplets have a cooling rate of
between 50-250 K/s. In embodiments, the particles of the dry
vaccine, after liquid (e.g. solvent or water) removal, have a
surface area of at least 10, 15, 25, 50, 75, 100, 125, 150 or 200
m.sup.2/gr (e.g. surface area of 10, 15, 25, 50, 75, 100, 125, 150
or 200 m.sup.2/gr). Minimizing gas-liquid interface can improve
protein stability by limiting the amount of protein that can adsorb
to the interface.
[0128] In embodiments, the droplets may be delivered to the cold or
freezing surface in a variety of manners and configurations. In
embodiments, the droplets may be delivered in parallel, in series,
at the center, middle or periphery or a platen, platter, plate,
roller, conveyor surface. In embodiments, the freezing or cold
surface may be a roller, a belt, a solid surface, circular,
cylindrical, conical, oval and the like that permit for the droplet
to freeze. For a continuous process a belt, platen, plate or roller
may be particularly useful. In embodiments, the frozen droplets may
form beads, strings, films or lines of frozen liquid vaccine. In
embodiments, the effective ingredient is removed from the surface
with a scraper, wire, ultrasound or other mechanical separator
prior to the lyophilization process. Once the material is removed
from the surface of the belt, platen, roller or plate the surface
is free to receive additional material.
[0129] In embodiments, the surface is cooled by a cryogenic solid,
a cryogenic gas, a cryogenic liquid or a heat transfer fluid
capable of reaching cryogenic temperatures or temperatures below
the freezing point of the liquid vaccine (e.g. at least 30.degree.
C. less than the temperature of the droplet). In embodiments, the
liquid vaccine further includes one or more excipients selected
from sugars, phospholipids, surfactants, polymeric surfactants,
vesicles, polymers, including copolymers and homopolymers and
biopolymers, dispersion aids, and serum albumin. In embodiments,
aggregation of the antigenic protein is less than 3% of the total
antigenic protein in the vaccine (e.g. irreversible aggregation).
In embodiments, the temperature differential between the droplet
and the surface is at least 50.degree. C. In embodiments, the
excipients or stabilizers that can be included in the liquid
vaccines that are to be frozen as described herein include:
cryoprotectants, lyoprotectants, surfactants, fillers, stabilizers,
polymers, protease inhibitors, antioxidants and absorption
enhancers. Specific nonlimiting examples of excipients that may be
included in the vaccines described herein include: sucrose,
trehaolose, Span 80, Tween 80, Brij 35, Brij 98, Pluronic,
sucroester 7, sucroester 11, sucroester 15, sodium lauryl sulfate,
oleic acid, laureth-9, laureth-8, lauric acid, vitamin E TPGS,
Gelucire 50/13, Gelucire 53/10, Labrafil, dipalmitoyl phosphadityl
choline, glycolic acid and salts, deoxycholic acid and salts,
sodium fusidate, cyclodextrins, polyethylene glycols, labrasol,
polyvinyl alcohols, polyvinyl pyrrolidones and tyloxapol.
[0130] In embodiments, the method may further include the step of
removing the liquid (e.g. solvent or water) from the frozen liquid
vaccine to form a dry vaccine. In embodiments, the solvent further
includes at least one or more excipient or stabilizers selected
from, e.g., sugars, phospholipids, surfactants, polymeric
surfactants, vesicles, polymers, including copolymers and
homopolymers and biopolymers, dispersion aids, and serum albumin.
In embodiments, the temperature differential between the solvent
and the surface is at least 50.degree. C.
[0131] In embodiments, the resulting powder can be redispersed into
a suitable aqueous medium such as saline, buffered saline, water,
buffered aqueous media, solutions of amino acids, solutions of
vitamins, solutions of carbohydrates, or the like, as well as
combinations of any two or more thereof, to obtain a suspension
that can be administered to mammals (e.g. humans).
[0132] In embodiments, is described a single-step, single-vial
method for preparing a vaccine thin film or dry vaccine by reducing
the temperature of a vial wherein the vial has a temperature below
the freezing temperature of a liquid vaccine (e.g. a temperature
differential of at least 30.degree. C. between the liquid vaccine
and the vial) and spraying or dripping droplets of a liquid vaccine
directly into the vial such that the antigenic protein of the
liquid vaccine is exposed to a vapor-liquid interface of less than
500 cm.sup.-1 area/volume, wherein the surface freezes the droplet
into a thin film with a thickness of less than 500 micrometers and
a surface area to volume between 25 to 500 cm.sup.-1. In
embodiments, the droplets freeze upon contact with the surface in
less than about 50, 75, 100, 125, 150, 175, 200, 250, 500, 1,000 or
2,000 milliseconds (e.g. in about 50, 75, 100, 125, 150, 175, 200,
250, 500, 1,000 or 2,000 milliseconds), and may freeze upon contact
with the surface in about 50 or 150 to 500 milliseconds. In
embodiments, a droplet has a diameter between 0.1 and 5 mm at room
temperature (e.g. a diameter between 2 and 4 mm at room
temperature). In embodiments, the droplet forms a thin film on the
surface of between 50 and 500 micrometers in thickness. In
embodiments, the droplets have a cooling rate of between 50-250
K/s. In embodiments, the vial may be cooled by a cryogenic solid, a
cryogenic gas, a cryogenic liquid, a freezing fluid, a freezing
gas, a freezing solid, a heat exchanger, or a heat transfer fluid
capable of reaching cryogenic temperatures or temperatures below
the freezing point of the liquid vaccine. In embodiments, the vial
may be rotated as the spraying or droplets are delivered to permit
the layering or one or more layers of the liquid vaccine. In
embodiments, the vial and the liquid vaccine are pre-sterilized
prior to spraying or dripping. In embodiments, the step of spraying
or dripping is repeated to overlay one or more thin films on top of
each other to fill the vial to any desired level up to totally
full.
D. ADDITIONAL EMBODIMENTS
[0133] 1p. A method of making a powder vaccine comprising:
obtaining an aqueous vaccine composition, the vaccine composition
comprising an agent that resembles a disease-causing microorganism
or a compound associated with the disease-causing microorganism and
an adjuvant; freezing the vaccine composition to obtain a frozen
vaccine composition; and converting the frozen vaccine composition
into a dry powder comprising the agent or compound and the
adjuvant. 2p. The method of embodiment 1p, wherein the adjuvant is
an aluminum-containing adjuvant. 3p. The method of embodiment 1p,
wherein the vaccine composition comprises a killed microorganism.
4p. The method of embodiment 1p, wherein the vaccine composition
comprises live, attenuated microorganisms. 5p. The method of
embodiment 1p, wherein the vaccine composition comprises a
bacterial toxin. 6p. The method of embodiment 1p, wherein the
vaccine composition comprises a protein subunit. 7p The method of
embodiment 1p, wherein the vaccine composition comprises a
conjugate. 8p. The method of embodiment 1p, wherein the vaccine
composition comprises a cryoprotectant. 9p. The method of
embodiment 1p, wherein freezing the vaccine composition comprises:
forming a droplet of the vaccine composition; applying the droplet
to a cooled surface, wherein the surface is at a temperature
sufficient to freeze the vaccine composition in a time of less than
or equal to about 1 second; removing the frozen vaccine composition
from the surface. 10p. The method of embodiment 9p, further
comprising removing water from the frozen vaccine composition to
create a vaccine powder. 11p. The method of embodiment 8p, wherein
removing the water is done by a lyophilization process. 12p. The
method of embodiment 8p, wherein removing the water is done by
freeze-drying process. 13p. A powder vaccine made by the process of
any one of embodiments 1p-12p, wherein the powder vaccine comprises
an agent that resembles a disease-causing microorganism or a
compound associated with the disease-causing microorganism and an
adjuvant. 14p. A method of administering a vaccine to a subject
comprising: obtaining a powder vaccine as described in embodiment
13p; and administering the powder vaccine to the subject. 15p. The
method of embodiment 14p, wherein the vaccine is administered by
inhalation of the powder vaccine. 16p. The method of embodiment
14p, wherein the vaccine is administered by: adding water to the
powder vaccine to create an aqueous vaccine composition comprising
the powder vaccine; and injecting the vaccine composition in the
subject. 17p. A vaccine composition comprising an agent that
resembles a disease-causing microorganism or a compound associated
with the disease-causing microorganism and an aluminum adjuvant
having an average particle size of less than 200 nm. 18p. A method
of making a reconstituted vaccine composition comprising: obtaining
an aqueous vaccine composition, the vaccine composition comprising
an agent that resembles a disease-causing microorganism or a
compound associated with the disease-causing microorganism and an
adjuvant; freezing the vaccine composition to obtain a frozen
vaccine composition; converting the frozen vaccine composition into
a dry powder comprising the agent or compound and the adjuvant;
adding an aqueous reconstitution agent to the powder vaccine to
create the reconstituted vaccine composition. 19p. The method of
embodiment 18p, wherein the aqueous reconstitution agent is water.
20p. The method of embodiment 18p, wherein the aqueous
reconstitution agent is a saline solution. 21p. The method of
embodiment 18p, wherein the aqueous reconstitution agent is an
aqueous buffer solution. 22p. The method of embodiment 18p, wherein
the reconstituted vaccine composition has a stability and
efficacy/activity that is substantially the same as the stability
and efficacy/activity of the aqueous vaccine composition. 23p. The
method of embodiment 18p, wherein the reconstituted vaccine
composition is suitable for injection. 24p. The method of
embodiment 18p, wherein the reconstituted vaccine composition is
suitable for inhalation. 1. A dry vaccine comprising: an antigenic
protein and an aluminum adjuvant, wherein at least 75% of said
antigenic protein is adsorbed to said aluminum adjuvant. 2. The dry
vaccine of embodiment 1, wherein at least 60% of said antigenic
protein is not denatured. 3. The dry vaccine of embodiment 1,
wherein at least 70% of said antigenic protein is not denatured. 4.
The dry vaccine of embodiment 1, wherein at least 80% of said
antigenic protein is not denatured. 5. The dry vaccine of
embodiment 1, wherein at least 90% of said antigenic protein is not
denatured. 6. The dry vaccine of embodiment 1, wherein at least 95%
of said antigenic protein is not denatured. 7. The dry vaccine of
one of embodiments 1 to 6, wherein said aluminum adjuvant is
aluminum hydroxide. 8. The dry vaccine of one of embodiments 1 to
6, wherein said aluminum adjuvant is aluminum phosphate. 9. The dry
vaccine of one of embodiments 1 to 6, wherein said aluminum
adjuvant is aluminum sulfate. 10. The dry vaccine of one of
embodiments 1 to 6, wherein said aluminum adjuvant is potassium
aluminum sulfate. 11. The dry vaccine of one of embodiments 1 to 10
comprising less than 4% water. 12. The dry vaccine of one of
embodiments 1 to 10 comprising less than 3% water. 13. The dry
vaccine of one of embodiments 1 to 10 comprising less than 2%
water. 14. The dry vaccine of one of embodiments 1 to 10 comprising
less than 1% water. 15. The dry vaccine of one of embodiments 1 to
14, wherein at least 80% of said antigenic protein is adsorbed to
said aluminum adjuvant. 16. The dry vaccine of one of embodiments 1
to 14, wherein at least 85% of said antigenic protein is adsorbed
to said aluminum adjuvant. 17. The dry vaccine of one of
embodiments 1 to 14, wherein at least 90% of said antigenic protein
is adsorbed to said aluminum adjuvant. 18. The dry vaccine of one
of embodiments 1 to 14, wherein at least 92% of said antigenic
protein is adsorbed to said aluminum adjuvant. 19. The dry vaccine
of one of embodiments 1 to 14, wherein at least 95% of said
antigenic protein is adsorbed to said aluminum adjuvant. 20. The
dry vaccine of one of embodiments 1 to 14, wherein at least 98% of
said antigenic protein is adsorbed to said aluminum adjuvant. 21.
The dry vaccine of one of embodiments 1 to 14, wherein at least 99%
of said antigenic protein is adsorbed to said aluminum adjuvant.
22. The dry vaccine of one of embodiments 1 to 21, further
comprising an excipient. 23. The dry vaccine of embodiment 22,
wherein said excipient is a salt, sugar, buffer, detergent,
polymer, amino acid, or preservative. 24. The dry vaccine of
embodiment 22, wherein said excipient is disodium edetate, sodium
chloride, sodium citrate, sodium succinate, sodium hydroxide,
Sodium glucoheptonate, sodium acetyltryptophanate, sodium
bicarbonate, sodium caprylate, sodium pertechnetate, sodium
acetate, sodium dodecyl sulfate, ammonium citrate, calcium
chloride, calcium, potassium chloride, potassium sodium tartarate,
zinc oxide, zinc, stannous chloride, magnesium sulfate, magnesium
stearate, titanium dioxide, DL-lactic/glycolic acids, asparagine,
L-arginine, arginine hydrochloride, adenine, histidine, glycine,
glutamine, glutathione, imidazole, protamine, protamine sulfate,
phosphoric acid, Tri-n-butyl phosphate, ascorbic acid, cysteine
hydrochloride, hydrochloric acid, hydrogen citrate, trisodium
citrate, guanidine hydrochloride, mannitol, lactose, sucrose,
agarose, sorbitol, maltose, trehalose, surfactants, polysorbate 80,
polysorbate 20, poloxamer 188, sorbitan monooleate, triton n101,
m-cresol, benyl alcohol, ethanolamine, glycerin,
phosphorylethanolamine, tromethamine, 2-phenyloxyethanol,
chlorobutanol, dimethylsulfoxide, N-methyl-2-pyrrolidone,
propyleneglycol, polyoxyl 35 castor oil, methyl hydroxybenzoate,
tromethamine, corn oil-mono-di-triglycerides, poloxyl 40
hydrogenated castor oil, tocopherol, n-acetyltryptophan,
octa-fluoropropane, castor oil, polyoxyethylated oleic glycerides,
polyoxytethylated castor oil, phenol, glyclyglycine, thimerosal,
parabens, gelatin, Formaldehyde, Dulbecco's modified eagles medium,
hydrocortisone, neomycin, Von Willebrand factor, gluteraldehyde,
benzethonium chloride, white petroleum, p-aminopheyl-p-anisate,
monosodium glutamate, beta-propiolactone, acetate, citrate,
glutamate, glycinate, histidine, Lactate, Maleate, phosphate,
succinate, tartrate, tris, carbomer 1342 (copolymer of acrylic acid
and a long chain alkyl methacrylate cross-linked with allyl ethers
of pentaerythritol), glucose star polymer, silicone polymer,
polydimethylsiloxane, polyethylene glycol, polyvinylpyrrolidone,
carboxymethylcellulose, poly(glycolic acid),
poly(lactic-co-glycolic acid), polylactic acid, dextran 40, or
poloxamer. 25. The dry vaccine of embodiment 22, wherein said
excipient is trehalose. 26. The dry vaccine of one of embodiments
22 to 25, comprising less than 5% wt/wt of said excipient. 27. The
dry vaccine of one of embodiments 22 to 25, comprising less than 4%
wt/wt of said excipient. 28. The dry vaccine of one of embodiments
22 to 25, comprising less than 3% wt/wt of said excipient. 29. The
dry vaccine of one of embodiments 22 to 25, comprising less than 2%
wt/wt of said excipient. 30. The dry vaccine of one of embodiments
22 to 25, comprising less than 1% wt/wt of said excipient. 31. The
dry vaccine of one of embodiments 22 to 25, comprising less than
0.5% wt/wt of said excipient. 32. The dry vaccine of one of
embodiments 1 to 31, comprising between 0.5 and 5% (wt/wt) of said
aluminum adjuvant. 33. The dry vaccine of one of embodiments 1 to
31, comprising between 0.5 and 3% (wt/wt) of said aluminum
adjuvant. 34. The dry vaccine of one of embodiments 1 to 31,
comprising between 0.5 and 2% (wt/wt) of said aluminum adjuvant.
35. The dry vaccine of one of embodiments 1 to 31, comprising
between 0.75 and 2% (wt/wt) of said aluminum adjuvant. 36. The dry
vaccine of one of embodiments 1 to 31, comprising between 1 and 2%
(wt/wt) of said aluminum adjuvant. 37. The dry vaccine of one of
embodiments 1 to 36 comprising particles, wherein said particles
comprise said antigenic protein adsorbed to said aluminum adjuvant.
38. The dry vaccine of one of embodiments 1 to 37, wherein said dry
vaccine is prepared from a liquid vaccine. 39. A method for
preparing a vaccine thin film comprising: applying a liquid vaccine
to a freezing surface; allowing said liquid vaccine to disperse and
freeze on said freezing surface thereby forming a vaccine thin
film. 40. The method of embodiment 39, wherein said liquid vaccine
comprises an aluminum adjuvant. 41. The method of embodiment 40,
wherein said aluminum adjuvant in said liquid vaccine comprises
aluminum hydroxide. 42. The method of embodiment 40, wherein said
aluminum adjuvant in said liquid vaccine comprises aluminum
phosphate. 43. The method of embodiment 40, wherein said aluminum
adjuvant in said liquid vaccine comprises aluminum sulfate. 44. The
method of embodiment 40, wherein said aluminum adjuvant in said
liquid vaccine comprises aluminum potassium sulfate. 45. The method
of one of embodiments 40 to 44, wherein said liquid vaccine
comprises about 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 46. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10% (wt/vol) of the aluminum adjuvant/liquid vaccine. 47. The
method of one of embodiments 40 to 44, wherein said liquid vaccine
comprises at least 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (wt/vol) of the
aluminum adjuvant/liquid vaccine. 48. The method of one of
embodiments 40 to 44, wherein said liquid vaccine comprises less
than 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 49. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between about 0.08 and
about 1% (wt/vol) of the aluminum adjuvant/liquid vaccine. 50. The
method of one of embodiments 40 to 44, wherein said liquid vaccine
comprises between about 0.5 and about 5% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 51. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between about 0.5 and
about 4% (wt/vol) of the aluminum adjuvant/liquid vaccine. 52. The
method of one of embodiments 40 to 44, wherein said liquid vaccine
comprises between about 0.5 and about 3% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 53. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between about 0.5 and
about 2% (wt/vol) of the aluminum adjuvant/liquid vaccine. 54. The
method of one of embodiments 40 to 44, wherein said liquid vaccine
comprises between about 0.5 and about 1% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 55. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between about 1 and about
2% (wt/vol) of the aluminum adjuvant/liquid vaccine. 56. The method
of one of embodiments 40 to 44, wherein said liquid vaccine
comprises between 0.08 and 1% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 57. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between 0.5 and 5%
(wt/vol) of the aluminum adjuvant/liquid vaccine. 58. The method of
one of embodiments 40 to 44, wherein said liquid vaccine comprises
between 0.5 and 4% (wt/vol) of the aluminum adjuvant/liquid
vaccine. 59. The method of one of embodiments 40 to 44, wherein
said liquid vaccine comprises between 0.5 and 3% (wt/vol) of the
aluminum adjuvant/liquid vaccine. 60. The method of one of
embodiments 40 to 44, wherein said liquid vaccine comprises between
0.5 and 2% (wt/vol) of the aluminum adjuvant/liquid vaccine. 61.
The method of one of embodiments 40 to 44, wherein said liquid
vaccine comprises between 0.5 and 1% (wt/vol) of the aluminum
adjuvant/liquid vaccine. 62. The method of one of embodiments 40 to
44, wherein said liquid vaccine comprises between 1 and 2% (wt/vol)
of the aluminum adjuvant/liquid vaccine. 63. The method of one of
embodiments 39 to 62, wherein said liquid vaccine comprises an
excipient. 64. The method of embodiment 63, wherein said excipient
in said liquid vaccine is a salt, sugar (saccharide), buffer,
detergent, polymer, amino acid, or preservative. 65. The method of
embodiment 63, wherein said excipient in said liquid vaccine is
disodium edetate, sodium chloride, sodium citrate, sodium
succinate, sodium hydroxide, Sodium glucoheptonate, sodium
acetyltryptophanate, sodium bicarbonate, sodium caprylate, sodium
pertechnetate, sodium acetate, sodium dodecyl sulfate, ammonium
citrate, calcium chloride, calcium, potassium chloride, potassium
sodium tartarate, zinc oxide, zinc, stannous chloride, magnesium
sulfate, magnesium stearate, titanium dioxide, DL-lactic/glycolic
acids, asparagine, L-arginine, arginine hydrochloride, adenine,
histidine, glycine, glutamine, glutathione, imidazole, protamine,
protamine sulfate, phosphoric acid, Tri-n-butyl phosphate, ascorbic
acid, cysteine hydrochloride, hydrochloric acid, hydrogen citrate,
trisodium citrate, guanidine hydrochloride, mannitol, lactose,
sucrose, agarose, sorbitol, maltose, trehalose, surfactants,
polysorbate 80, polysorbate 20, poloxamer 188, sorbitan monooleate,
triton n101, m-cresol, benyl alcohol, ethanolamine, glycerin,
phosphorylethanolamine, tromethamine, 2-phenyloxyethanol,
chlorobutanol, dimethylsulfoxide, N-methyl-2-pyrrolidone,
propyleneglycol, polyoxyl 35 castor oil, methyl hydroxybenzoate,
tromethamine, corn oil-mono-di-triglycerides, poloxyl 40
hydrogenated castor oil, tocopherol, n-acetyltryptophan,
octa-fluoropropane, castor oil, polyoxyethylated oleic glycerides,
polyoxytethylated castor oil, phenol, glyclyglycine, thimerosal,
parabens, gelatin, Formaldehyde, Dulbecco's modified eagles medium,
hydrocortisone, neomycin, Von Willebrand factor, gluteraldehyde,
benzethonium chloride, white petroleum, p-aminopheyl-p-anisate,
monosodium glutamate, beta-propiolactone, acetate, citrate,
glutamate, glycinate, histidine, Lactate, Maleate, phosphate,
succinate, tartrate, tris, carbomer 1342 (copolymer of acrylic acid
and a long chain alkyl methacrylate cross-linked with allyl ethers
of pentaerythritol), glucose star polymer, silicone polymer,
polydimethylsiloxane, polyethylene glycol, polyvinylpyrrolidone,
carboxymethylcellulose, poly(glycolic acid),
poly(lactic-co-glycolic acid), polylactic acid, dextran 40, or
poloxamer.
66. The method of embodiment 63, wherein said excipient in said
liquid vaccine is trehalose. 67. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises less than 5% wt/vol
of said excipient/liquid vaccine. 68. The method of one of
embodiments 63 to 66, wherein said liquid vaccine comprises less
than 4% wt/vol of said excipient/liquid vaccine. 69. The method of
one of embodiments 63 to 66, wherein said liquid vaccine comprises
less than 3% wt/vol of said excipient/liquid vaccine. 70. The
method of one of embodiments 63 to 66, wherein said liquid vaccine
comprises less than 2% wt/vol of said excipient/liquid vaccine. 71.
The method of one of embodiments 63 to 66, wherein said liquid
vaccine comprises less than 1% wt/vol of said excipient/liquid
vaccine. 72. The method of one of embodiments 63 to 66, wherein
said liquid vaccine comprises less than 0.5% wt/vol of said
excipient/liquid vaccine. 73. The method of one of embodiments 63
to 66, wherein said liquid vaccine comprises about 5% wt/vol of
said excipient/liquid vaccine. 74. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises about 4% wt/vol of
said excipient/liquid vaccine. 75. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises about 3% wt/vol of
said excipient/liquid vaccine. 76. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises about 2% wt/vol of
said excipient/liquid vaccine. 77. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises about 1% wt/vol of
said excipient/liquid vaccine. 78. The method of one of embodiments
63 to 66, wherein said liquid vaccine comprises about 0.5% wt/vol
of said excipient/liquid vaccine. 79. The method of one of
embodiments 63 to 66, wherein said liquid vaccine comprises about
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10% (wt/vol) of said excipient/liquid vaccine. 80. The
method of one of embodiments 63 to 66, wherein said liquid vaccine
comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10% (wt/vol) of said excipient/liquid vaccine.
81. The method of one of embodiments 39 to 80, wherein said
applying comprises spraying or dripping droplets of said liquid
vaccine. 82. The method of embodiment 81, wherein the vapor-liquid
interfaces of said droplets are less than 500 cm.sup.-1 area/volume
83. The method of one of embodiments 81 to 82, further comprising
contacting the droplets with a freezing surface having a
temperature differential of at least 30.degree. C. between the
droplets and the surface. 84. The method of one of embodiments 39
to 83, wherein the vaccine thin film has a thickness of less than
500 micrometers. 85. The method of one of embodiments 39 to 84,
wherein the vaccine thin film has a surface area to volume ratio of
between 25 and 500 cm.sup.-1. 86. The method of one of embodiments
81 to 85, wherein the freezing rate of said droplets is between 10
K/second and 10.sup.5 K/second. 87. The method of one of
embodiments 81 to 85, wherein the freezing rate of said droplets is
between 10 K/second and 10.sup.4 K/second. 88. The method of one of
embodiments 81 to 85, wherein the freezing rate of said droplets is
between 10 K/second and 10.sup.3 K/second. 89. The method of one of
embodiments 81 to 85, wherein the freezing rate of said droplets is
between 10.sup.2 K/second and 10.sup.3 K/second. 90. The method of
one of embodiments 81 to 85, wherein the freezing rate of said
droplets is between 50 K/second and 5.times.10.sup.2 K/second. 91.
The method of one of embodiments 81 to 90, wherein each of said
droplets freezes upon contact with the freezing surface in less
than 50, 75, 100, 125, 150, 175, 200, 250, 500, 1,000, or 2,000
milliseconds. 92. The method of one of embodiments 81 to 91,
wherein said droplets have an average diameter between 0.1 and 5
mm, between 20 and 24 degrees Celsius. 93. The method of one of
embodiments 81 to 91, wherein said droplets have an average
diameter between 2 and 4 mm, between 20 and 24 degrees Celsius. 94.
The method of one of embodiments 39 to 93, wherein said vaccine
thin film has a thickness of less than 250 micrometers. 95. The
method of one of embodiments 39 to 93, wherein said vaccine thin
film has a thickness of less than 100 micrometers. 96. The method
of one of embodiments 39 to 93, wherein said vaccine thin film has
a thickness of less than 50 micrometers. 97. The method of one of
embodiments 81 to 96, wherein said droplet vapor-liquid interface
is less than 250 cm.sup.-1 area/volume. 98. The method of one of
embodiments 81 to 96, wherein said droplet vapor-liquid interface
is less than 100 cm.sup.-1 area/volume. 99. The method of one of
embodiments 81 to 98, wherein the step of spraying or dripping
droplets is repeated to overlay one or more additional vaccine thin
films on top of the vaccine thin film. 100. The method of one of
embodiments 39 to 99, further comprising removing the solvent from
the vaccine thin film to form a dry vaccine. 101. The method of
embodiment 100, wherein said dry vaccine comprises an antigenic
protein and an aluminum adjuvant, wherein at least 75% of said
antigenic protein is adsorbed to said aluminum adjuvant. 102. The
method of embodiment 101, wherein at least 60% of said antigenic
protein is not denatured. 103. The method of embodiment 101,
wherein at least 70% of said antigenic protein is not denatured.
104. The method of embodiment 101, wherein at least 80% of said
antigenic protein is not denatured. 105. The method of embodiment
101, wherein at least 90% of said antigenic protein is not
denatured. 106. The method of embodiment 101, wherein at least 95%
of said antigenic protein is not denatured. 107. The method of one
of embodiments 101 to 106, wherein said dry vaccine comprises
between 0.5 and 5% (wt/wt) of said aluminum adjuvant. 108. The
method of one of embodiments 101 to 106, wherein said dry vaccine
comprises between 0.5 and 4% (wt/wt) of said aluminum adjuvant.
109. The method of one of embodiments 101 to 106, wherein said dry
vaccine comprises between 0.5 and 3% (wt/wt) of said aluminum
adjuvant. 110. The method of one of embodiments 101 to 106, wherein
said dry vaccine comprises between 0.5 and 2% (wt/wt) of said
aluminum adjuvant. 111. The method of one of embodiments 101 to
106, wherein said dry vaccine comprises between 0.75 and 2% (wt/wt)
of said aluminum adjuvant. 112. The method of one of embodiments
101 to 106, wherein said dry vaccine comprises between 1 and 2%
(wt/wt) of said aluminum adjuvant. 113. The method of one of
embodiments 101 to 112, wherein said aluminum adjuvant is aluminum
hydroxide. 114. The method of one of embodiments 101 to 112,
wherein said aluminum adjuvant is aluminum phosphate. 115. The
method of one of embodiments 101 to 112, wherein said aluminum
adjuvant is aluminum sulfate. 116. The method of one of embodiments
101 to 112, wherein said aluminum adjuvant is potassium aluminum
sulfate. 117. The method of one of embodiments 100 to 116 wherein
said dry vaccine comprises less than 4% water. 118. The method of
one of embodiments 100 to 116 wherein said dry vaccine comprises
less than 3% water. 119. The method of one of embodiments 100 to
116 wherein said dry vaccine comprises less than 2% water. 120. The
method of one of embodiments 100 to 116 wherein said dry vaccine
comprises less than 1% water. 121. The method of one of embodiments
101 to 120, wherein at least 80% of said antigenic protein is
adsorbed to said aluminum adjuvant. 122. The method of one of
embodiments 101 to 120, wherein at least 85% of said antigenic
protein is adsorbed to said aluminum adjuvant. 123. The method of
one of embodiments 101 to 120, wherein at least 90% of said
antigenic protein is adsorbed to said aluminum adjuvant. 124. The
method of one of embodiments 101 to 120, wherein at least 92% of
said antigenic protein is adsorbed to said aluminum adjuvant. 125.
The method of one of embodiments 101 to 120, wherein at least 95%
of said antigenic protein is adsorbed to said aluminum adjuvant.
126. The method of one of embodiments 101 to 120, wherein at least
98% of said antigenic protein is adsorbed to said aluminum
adjuvant. 127. The method of one of embodiments 101 to 120, wherein
at least 99% of said antigenic protein is adsorbed to said aluminum
adjuvant. 128. The method of one of embodiments 101 to 127, wherein
said dry vaccine comprises an excipient. 129. The method of
embodiment 128, wherein said excipient is a salt, sugar, buffer,
detergent, polymer, amino acid, or preservative. 130. The method of
embodiment 128, wherein said excipient is disodium edetate, sodium
chloride, sodium citrate, sodium succinate, sodium hydroxide,
Sodium glucoheptonate, sodium acetyltryptophanate, sodium
bicarbonate, sodium caprylate, sodium pertechnetate, sodium
acetate, sodium dodecyl sulfate, ammonium citrate, calcium
chloride, calcium, potassium chloride, potassium sodium tartarate,
zinc oxide, zinc, stannous chloride, magnesium sulfate, magnesium
stearate, titanium dioxide, DL-lactic/glycolic acids, asparagine,
L-arginine, arginine hydrochloride, adenine, histidine, glycine,
glutamine, glutathione, imidazole, protamine, protamine sulfate,
phosphoric acid, Tri-n-butyl phosphate, ascorbic acid, cysteine
hydrochloride, hydrochloric acid, hydrogen citrate, trisodium
citrate, guanidine hydrochloride, mannitol, lactose, sucrose,
agarose, sorbitol, maltose, trehalose, surfactants, polysorbate 80,
polysorbate 20, poloxamer 188, sorbitan monooleate, triton n101,
m-cresol, benyl alcohol, ethanolamine, glycerin,
phosphorylethanolamine, tromethamine, 2-phenyloxyethanol,
chlorobutanol, dimethylsulfoxide, N-methyl-2-pyrrolidone,
propyleneglycol, polyoxyl 35 castor oil, methyl hydroxybenzoate,
tromethamine, corn oil-mono-di-triglycerides, poloxyl 40
hydrogenated castor oil, tocopherol, n-acetyltryptophan,
octa-fluoropropane, castor oil, polyoxyethylated oleic glycerides,
polyoxytethylated castor oil, phenol, glyclyglycine, thimerosal,
parabens, gelatin, Formaldehyde, Dulbecco's modified eagles medium,
hydrocortisone, neomycin, Von Willebrand factor, gluteraldehyde,
benzethonium chloride, white petroleum, p-aminopheyl-p-anisate,
monosodium glutamate, beta-propiolactone, acetate, citrate,
glutamate, glycinate, histidine, Lactate, Maleate, phosphate,
succinate, tartrate, tris, carbomer 1342 (copolymer of acrylic acid
and a long chain alkyl methacrylate cross-linked with allyl ethers
of pentaerythritol), glucose star polymer, silicone polymer,
polydimethylsiloxane, polyethylene glycol, polyvinylpyrrolidone,
carboxymethylcellulose, poly(glycolic acid),
poly(lactic-co-glycolic acid), polylactic acid, dextran 40, or
poloxamer. 131. The method of embodiment 128, wherein said
excipient is trehalose. 132. The method of one of embodiments 128
to 131, wherein said dry vaccine comprises less than 5% wt/wt of
said excipient. 133. The method of one of embodiments 128 to 131,
wherein said dry vaccine comprises less than 4% wt/wt of said
excipient. 134. The method of one of embodiments 128 to 131,
wherein said dry vaccine comprises less than 3% wt/wt of said
excipient. 135. The method of one of embodiments 128 to 131,
wherein said dry vaccine comprises less than 2% wt/wt of said
excipient. 136. The method of one of embodiments 128 to 131,
wherein said dry vaccine comprises less than 1% wt/wt of said
excipient. 137. The method of one of embodiments 128 to 131,
wherein said dry vaccine comprises less than 0.5% wt/wt of said
excipient. 138. The method of one of embodiments 101 to 138 wherein
said dry vaccine comprises particles, wherein said particles
comprise said antigenic protein adsorbed to said aluminum adjuvant.
139. The method of one of embodiments 100 to 138, wherein said
removing of the solvent comprises lyophilization. 140. The method
of one of embodiments 100 to 139, wherein said removing of the
solvent comprises lyophilization at temperatures of 20 degrees
Celsius or less. 141. The method of one of embodiments 100 to 139,
wherein said removing of the solvent comprises lyophilization at
temperatures of 25 degrees Celsius or less. 142. The method of one
of embodiments 100 to 139, wherein said removing of the solvent
comprises lyophilization at temperatures of 40 degrees Celsius or
less. 143. The method of one of embodiments 100 to 139, wherein
said removing of the solvent comprises lyophilization at
temperatures of 50 degrees Celsius or less. 144. The method of one
of embodiments 100 to 143, further comprising solvating said dry
vaccine thereby forming a reconstituted liquid vaccine. 145. The
method of embodiment 144, wherein the immunogenicity of said
reconstituted liquid vaccine is at least 60% the immunogenicity of
said liquid vaccine. 146. The method of embodiment 144, wherein the
immunogenicity of said reconstituted liquid vaccine is at least 70%
the immunogenicity of said liquid vaccine. 147. The method of
embodiment 144, wherein the immunogenicity of said reconstituted
liquid vaccine is at least 80% the immunogenicity of said liquid
vaccine. 148. The method of embodiment 144, wherein the
immunogenicity of said reconstituted liquid vaccine is at least 90%
the immunogenicity of said liquid vaccine. 149. The method of
embodiment 144, wherein the immunogenicity of said reconstituted
liquid vaccine is at least 95% the immunogenicity of said liquid
vaccine. 150. The method of one of embodiments 144 to 149, wherein
the level of antigenic protein adsorbed to said aluminum adjuvant
of said reconstituted liquid vaccine is at least 60% of the level
of antigenic protein adsorbed to said aluminum adjuvant of said
liquid vaccine. 151. The method of one of embodiments 144 to 149,
wherein the level of antigenic protein adsorbed to said aluminum
adjuvant of said reconstituted liquid vaccine is at least 70% of
the level of antigenic protein adsorbed to said aluminum adjuvant
of said liquid vaccine. 152. The method of one of embodiments 144
to 149, wherein the level of antigenic protein adsorbed to said
aluminum adjuvant of said reconstituted liquid vaccine is at least
80% of the level of antigenic protein adsorbed to said aluminum
adjuvant of said liquid vaccine. 153. The method of one of
embodiments 144 to 149, wherein the level of antigenic protein
adsorbed to said aluminum adjuvant of said reconstituted liquid
vaccine is at least 90% of the level of antigenic protein adsorbed
to said aluminum adjuvant of said liquid vaccine. 154. The method
of one of embodiments 144 to 149, wherein the level of antigenic
protein adsorbed to said aluminum adjuvant of said reconstituted
liquid vaccine is at least 95% of the level of antigenic protein
adsorbed to said aluminum adjuvant of said liquid vaccine. 155. The
method of one of embodiments 144 to 149, wherein the level of
antigenic protein adsorbed to said aluminum adjuvant of said
reconstituted liquid vaccine is at least 99% of the level of
antigenic protein adsorbed to said aluminum adjuvant of said liquid
vaccine.
156. The method of one of embodiments 144 to 155, wherein said
reconstituted liquid vaccine comprises particles, wherein said
particles comprise said antigenic protein adsorbed to said aluminum
adjuvant. 157. The method of embodiment 156 wherein said particles
have an average diameter of between 10 nm and 5 .mu.m. 158. The
method of embodiment 156 wherein said particles have an average
diameter of between 1 .mu.m and 5 .mu.m. 159. The method of
embodiment 156 wherein said particles have an average diameter of
between 2 .mu.m and 4 .mu.m. 160. The method of embodiment 156
wherein said particles have an average diameter of between 1 .mu.m
and 3 .mu.m. 161. The method of embodiment 156 wherein said
particles have an average diameter of between 10 nm and 2 .mu.m.
162. The method of embodiment 156 wherein said particles have an
average diameter of between 20 nm and 2 .mu.m. 163. The method of
embodiment 156 wherein said particles have an average diameter of
between 50 nm and 2 .mu.m. 164. The method of embodiment 156
wherein said particles have an average diameter of between 100 nm
and 2 .mu.m. 165. The method of embodiment 156 wherein said
particles have an average diameter of between 200 nm and 2 .mu.m.
166. The method of embodiment 156 wherein said particles have an
average diameter of between 500 nm and 2 .mu.m. 167. The method of
embodiment 156 wherein said particles have an average diameter of
between 1 .mu.m and 2 .mu.m. 168. The method of embodiment 156
wherein said particles have an average diameter of between 10 nm
and 1 .mu.m. 169. The method of embodiment 156 wherein said
particles have an average diameter of between 10 nm and 500 nm.
170. The method of embodiment 156 wherein said particles have an
average diameter of between 10 nm and 200 nm. 171. The method of
embodiment 156 wherein said particles have an average diameter of
between 10 nm and 200 nm. 172. The method of embodiment 156 wherein
said particles have an average diameter of between 10 nm and 100
nm. 173. The method of embodiment 156 wherein said particles have
an average diameter of between 10 nm and 50 nm. 174. The method of
embodiment 156 wherein said particles have an average diameter of
between 10 nm and 20 nm. 175. The method of embodiment 156 wherein
said particles have an average diameter of between 20 nm and 1
.mu.m. 176. The method of embodiment 156 wherein said particles
have an average diameter of between 50 nm and 500 nm. 177. The
method of embodiment 156 wherein said particles have an average
diameter of between 100 nm and 500 nm. 178. The method of
embodiment 156 wherein said particles have an average diameter of
between 100 nm and 200 nm. 179. The method of one of embodiments
144 to 178, wherein said reconstituted liquid vaccine comprises
particles comprising antigenic protein adsorbed to said aluminum
adjuvant of the same average diameter as the liquid vaccine
particles comprising antigenic protein adsorbed to said aluminum
adjuvant. 180. The method of one of embodiments 144 to 178, wherein
said reconstituted liquid vaccine comprises particles comprising
antigenic protein adsorbed to said aluminum adjuvant having an
average diameter within 5% of the average diameter of particles
comprising said antigenic protein adsorbed to said aluminum
adjuvant in said liquid vaccine. 181. The method of one of
embodiments 144 to 178, wherein said reconstituted liquid vaccine
comprises particles comprising antigenic protein adsorbed to said
aluminum adjuvant having an average diameter within 10% of the
average diameter of particles comprising said antigenic protein
adsorbed to said aluminum adjuvant in said liquid vaccine. 182. The
method of one of embodiments 144 to 178, wherein said reconstituted
liquid vaccine comprises particles comprising antigenic protein
adsorbed to said aluminum adjuvant having an average diameter
within 20% of the average diameter of particles comprising said
antigenic protein adsorbed to said aluminum adjuvant in said liquid
vaccine. 183. The method of one of embodiments 144 to 178, wherein
said reconstituted liquid vaccine comprises particles comprising
antigenic protein adsorbed to said aluminum adjuvant having an
average diameter within 30% of the average diameter of particles
comprising said antigenic protein adsorbed to said aluminum
adjuvant in said liquid vaccine. 184. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least one day after preparing said dry vaccine from said
liquid vaccine. 185. The method of one of embodiments 144 to 183,
wherein said solvating of said dry vaccine is at least two days
after preparing said dry vaccine from said liquid vaccine. 186. The
method of one of embodiments 144 to 183, wherein said solvating of
said dry vaccine is at least three days after preparing said dry
vaccine from said liquid vaccine. 187. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least one week after preparing said dry vaccine from said
liquid vaccine. 188. The method of one of embodiments 144 to 183,
wherein said solvating of said dry vaccine is at least two weeks
after preparing said dry vaccine from said liquid vaccine. 189. The
method of one of embodiments 144 to 183, wherein said solvating of
said dry vaccine is at least one month after preparing said dry
vaccine from said liquid vaccine. 190. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least two months after preparing said dry vaccine from said
liquid vaccine. 191. The method of one of embodiments 144 to 183,
wherein said solvating of said dry vaccine is at least three months
after preparing said dry vaccine from said liquid vaccine. 192. The
method of one of embodiments 144 to 183, wherein said solvating of
said dry vaccine is at least six months after preparing said dry
vaccine from said liquid vaccine. 193. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least six months after preparing said dry vaccine from said
liquid vaccine. 194. The method of one of embodiments 144 to 183,
wherein said solvating of said dry vaccine is at least six months
after preparing said dry vaccine from said liquid vaccine. 195. The
method of one of embodiments 144 to 183, wherein said solvating of
said dry vaccine is at least one year after preparing said dry
vaccine from said liquid vaccine. 196. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least two years after preparing said dry vaccine from said
liquid vaccine. 197. The method of one of embodiments 144 to 183,
wherein said solvating of said dry vaccine is at least three years
after preparing said dry vaccine from said liquid vaccine. 198. The
method of one of embodiments 144 to 183, wherein said solvating of
said dry vaccine is at least five years after preparing said dry
vaccine from said liquid vaccine. 199. The method of one of
embodiments 144 to 183, wherein said solvating of said dry vaccine
is at least ten years after preparing said dry vaccine from said
liquid vaccine. 200. The method of one of embodiments 144 to 199,
wherein prior to said solvating of said dry vaccine, said dry
vaccine is stored at about 4 degrees Celsius for at least 99% of
the time. 201. The method of one of embodiments 144 to 199, wherein
prior to said solvating of said dry vaccine, said dry vaccine is
stored at less than 4 degrees Celsius for at least 99% of the time.
202. The method of one of embodiments 144 to 199, wherein prior to
said solvating of said dry vaccine, said dry vaccine is stored at
less than 0 degrees Celsius for at least 99% of the time. 203. The
method of one of embodiments 144 to 199, wherein prior to said
solvating of said dry vaccine, said dry vaccine is stored at less
than -20 degrees Celsius for at least 99% of the time. 204. The
method of one of embodiments 144 to 199, wherein prior to said
solvating of said dry vaccine, said dry vaccine is stored at
between 20 and 24 degrees Celsius for at least 99% of the time.
205. The method of one of embodiments 144 to 199, wherein prior to
said solvating of said dry vaccine, said dry vaccine is stored at
between 4 and 24 degrees Celsius for at least 99% of the time. 206.
The method of one of embodiments 144 to 199, wherein prior to said
solvating of said dry vaccine, said dry vaccine is stored at
between 0 and 24 degrees Celsius for at least 99% of the time. 207.
The method of one of embodiments 144 to 199, wherein prior to said
solvating of said dry vaccine, said dry vaccine is stored at
between 4 and 40 degrees Celsius for at least 99% of the time. 208.
The method of one of embodiments 144 to 199, wherein prior to said
solvating of said dry vaccine, said dry vaccine is stored at
between 0 and 40 degrees Celsius for at least 99% of the time. 209.
The method of one of embodiments 144 to 208, wherein upon solvating
said dry vaccine the resulting reconstituted liquid vaccine remains
homogeneous. 210. The method of one of embodiments 144 to 208,
wherein upon solvating said dry vaccine the resulting reconstituted
liquid vaccine remains homogeneous for at least one day. 211. The
method of one of embodiments 144 to 208, wherein upon solvating
said dry vaccine the resulting reconstituted liquid vaccine remains
homogeneous for at least two days. 212. The method of one of
embodiments 144 to 208, wherein upon solvating said dry vaccine the
resulting reconstituted liquid vaccine remains homogeneous for at
least three days. 213. The method of one of embodiments 144 to 208,
wherein upon solvating said dry vaccine the resulting reconstituted
liquid vaccine remains homogeneous for at least one week. 214. The
method of one of embodiments 144 to 208, wherein upon solvating
said dry vaccine the resulting reconstituted liquid vaccine remains
homogeneous for at least two weeks. 215. The method of one of
embodiments 144 to 208, wherein upon solvating said dry vaccine the
resulting reconstituted liquid vaccine remains homogeneous for at
least one month. 216. The method of one of embodiments 144 to 208,
wherein upon solvating said dry vaccine the resulting reconstituted
liquid vaccine remains homogeneous for at least three months. 217.
The method of one of embodiments 144 to 208, wherein upon solvating
said dry vaccine the resulting reconstituted liquid vaccine remains
homogeneous for at least six months. 218. The method of one of
embodiments 144 to 208, wherein upon solvating said dry vaccine the
resulting reconstituted liquid vaccine remains homogeneous for at
least one year. 219. The method of one of embodiments 144 to 218,
wherein upon solvating said dry vaccine the resulting reconstituted
liquid vaccine does not form a precipitate. 220. The method of one
of embodiments 144 to 218, wherein upon solvating said dry vaccine
the resulting reconstituted liquid vaccine does not form a
precipitate for at least one day. 221. The method of one of
embodiments 144 to 218, wherein upon solvating said dry vaccine the
resulting reconstituted liquid vaccine does not form a precipitate
for at least two days. 222. The method of one of embodiments 144 to
218, wherein upon solvating said dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate for at
least three days. 223. The method of one of embodiments 144 to 218,
wherein upon solvating said dry vaccine the resulting reconstituted
liquid vaccine does not form a precipitate for at least one week.
224. The method of one of embodiments 144 to 218, wherein upon
solvating said dry vaccine the resulting reconstituted liquid
vaccine does not form a precipitate for at least two weeks. 225.
The method of one of embodiments 144 to 218, wherein upon solvating
said dry vaccine the resulting reconstituted liquid vaccine does
not form a precipitate for at least one month. 226. The method of
one of embodiments 144 to 218, wherein upon solvating said dry
vaccine the resulting reconstituted liquid vaccine does not form a
precipitate for at least three months. 227. The method of one of
embodiments 144 to 218, wherein upon solvating said dry vaccine the
resulting reconstituted liquid vaccine does not form a precipitate
for at least six months. 228. The method of one of embodiments 144
to 218, wherein upon solvating said dry vaccine the resulting
reconstituted liquid vaccine does not form a precipitate for at
least one year. 229. A method of treating a disease in a patient in
need of such treatment, said method comprising administering a
therapeutically effective amount of a solvated dry vaccine of one
of embodiments 1 to 38 to said patient, wherein said disease is
diphtheria, botulism, tetanus, pertussis, influenza, pneumonia,
otitis media, bacteremia, meningitis, hepatitis, cirrhosis, anthrax
poisoning, rabies, warts, poliomyelitis, Japanese encephalitis, or
cancer. 230. A method of treating a disease caused by an infectious
agent in a patient in need of such treatment, said method
comprising administering a therapeutically effective amount of a
solvated dry vaccine of one of embodiments 1 to 38 to said patient,
wherein said infectious agent is Clostridium tetani, Clostridium
botulinum, Streptococcus pneumonia, Hepatitis A, Hepatitis B,
Haemophilus influenza, Corynebacterium diphtheria, Bordetella
pertussis, Human papillomavirus, Bacillus anthracis, Rabies virus,
Japanese encephalitis virus, or Poliovirus.
E. EXAMPLES
[0134] In the present study, using ovalbumin as a model antigen
adsorbed onto aluminum hydroxide or aluminum phosphate, a
commercially available vaccine (e.g. tetanus toxoid vaccine
adjuvanted with aluminum potassium sulfate, a human hepatitis B
vaccine adjuvanted with aluminum hydroxide) it was shown that
vaccines containing a relatively high concentration of aluminum
salts (.about.1%, w/v) can be converted into a dry powder by
thin-film freezing followed by removal of the frozen solvent by
lyophilization while using low levels of trehalose (i.e., as low as
2% w/v) as an excipient. Importantly, the thin-film freeze-drying
process did not cause vaccine coagulation or aggregation and
preserved the immunological potency of the vaccines. Moreover,
repeated freezing-and-thawing of the dry vaccine powder did not
cause aggregation or coagulation. Thin-film freeze drying is a
viable platform technology to produce dry powder of vaccines that
contain aluminum salts.
1. Lyophilization of Ovalbumin (OVA)-Adsorbed Aluminum Hydroxide
Particles with 2% Trehalose (w/v) Under Different Freezing
Rates
[0135] OVA solution was initially mixed with aluminum hydroxide
particles in suspension. Trehalose as a cryoprotectant was added in
all samples at a final concentration of 2% (w/v). One sample of the
suspension was treated to a high-speed thin-film freezing process.
Briefly, the OVA-adsorbed aluminum hydroxide suspension was dropped
onto a pre-cooled cryogenic substrate. The frozen film-like solids
were collected in liquid nitrogen and dried using a VirTis
Advantage bench top tray lyophilizer. Samples were also treated by
using a slow freezing method. The OVA-adsorbed aluminum hydroxide
suspension was frozen on a shelf at -20.degree. C. or -80.degree.
C. overnight and then lyophilized in FreeZone plus 4.5 liter
cascade console freeze dry system (Labconco corporation, Kansas
city, MO). Particle size for all samples was determined using a
Sympatec Helos laser diffraction instrument (Sympatec GmbH,
Germany) equipped with a R3 lens. Images were taken using an
Olympus BX60 microscope (Olympus America, Inc., Center Valley,
Pa.).
[0136] Results are depicted in FIG. 1. Microscopic images of
OVA-adsorbed aluminum hydroxide particles before freeze-drying
(FIG. 1A) and after high speed thin-film freeze-drying and
reconstitution (FIG. 1B), slow freezing at -20.degree. C., drying
and reconstitution (FIG. 1C), and slow freezing at -80.degree. C.,
drying and reconstitution (FIG. 1D). As shown in FIG. 1A-B, the
high-speed thin-film freezing method did not cause significant
aggregation of the OVA-adsorbed aluminum hydroxide particles. The
lyophilized powder was easily reconstituted in water, normal
saline, or phosphate buffered saline (PBS). The size of freshly
prepared OVA-adsorbed aluminum hydroxide particles was 9.4.+-.1.7
.mu.m, which is not different from the size of the lyophilized
OVA-adsorbed aluminum hydroxide particles after reconstitution
(9.7.+-.2.5 .mu.m). However, when the OVA-adsorbed aluminum
hydroxide particles were lyophilized using lower freezing rates,
significant aggregations occurred (FIG. 1C-1D).
2. The Binding Efficiency of OVA to the Aluminum Hydroxide
Particles after Lyophilization
[0137] SDS-PAGE was used to determine the binding efficiency of the
OVA to aluminum hydroxide particles after lyophilization.
Initially, OVA was mixed with aluminum hydroxide particles at 1 to
10 ratio (OVA vs. Al.sup.3+, w/w) in a suspension with 2% (w/v) of
trehalose. The OVA-adsorbed aluminum hydroxide particles were
lyophilized using the thin-film freezing method, reconstituted in
water, and applied on SDS-PAGE gel. As a control, OVA alone (OVA)
or freshly prepared OVA-adsorbed aluminum hydroxide particles
without lyophilization (NON TFF) were also included. Samples were
mixed with Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25%
glycerol, 2% SDS, and 0.01% Bromophenol Blue) before applying to
7.5% Mini-PROTEAN.RTM. TGX.TM. precast polyacrylamide gels
(Bio-Rad). Precision plus protein standards were also run along
with the samples at 130 V for 1 h. The gel was then stained in a
Bio-Safe Coomassie blue staining solution and scanned using a Kodak
Image Station 440CF (Rochester, N.Y.).
[0138] FIG. 2 shows the fraction of free (unbound) OVA as
determined from the intensity of the protein bands on the SDS-PAGE
gel. As shown in FIG. 2, the percent of OVA remaining adsorbed to
the aluminum hydroxide particles after the lyophilization is
estimated to be 92%, indicating only 8% of protein was desorbed
from aluminum hydroxide particles after the thin-film freezing and
lyophilization. This 92% binding efficiency still meets the United
States Food and Drug Administration (FDA) requirement for
aluminum-containing vaccines. For example, 75% adsorption is the
minimum requirement for diphtheria toxoid and tetanus toxoid
antigens.
3. Thermal Analysis of OVA-Adsorbed Aluminum Hydroxide Particles
Dried after Thin-Film Freezing
[0139] Thermal analyses of lyophilized OVA-adsorbed aluminum
hydroxide powder and its three individual ingredients, OVA protein,
aluminum hydroxide and trehalose, were conducted using modulated
temperature DSC (Model 2920, TA Instruments, New Castle, Del.).
Four to seven mg of each sample was weighed into the aluminum pans
and crimped subsequently. An empty aluminum pan was used as a
reference. Samples were then heated at a ramp rate of 3.degree.
C./min from -30 to 300.degree. C. Data were analyzed using the TA
Universal Analysis 2000 software (TA Instruments, New Castle,
Del.).
[0140] Modulated DSC was carried out to study the thermal
properties of lyophilized OVA-adsorbed aluminum hydroxide particles
(OVA-Al(OH).sub.3 (TFF)). In the thermogram of the lyophilized
OVA-adsorbed aluminum hydroxide powder (FIG. 3), a glass transition
temperature (Tg) of about 120.degree. C. was observed.
4. Thin-Film Freeze-Drying of OVA-Adsorbed Aluminum Hydroxide
Particles Did not Affect the Immunogenicity of the OVA
[0141] Female BALB/c mice (18-20 g, n=5) were subcutaneously
injected with OVA-adsorbed aluminum hydroxide particles, before or
after lyophilization and reconstitution, on days 0, 14 and 28 with
5 .mu.g (FIG. 4A), 10 .mu.g (FIG. 4B), or 20 .mu.g (FIG. 4C) of OVA
per mouse. The ratio of OVA to aluminum was 1 to 10. Sterile PBS or
OVA (10 .mu.g) dissolved in PBS was used as controls. Total
anti-OVA IgG level in serum samples was measured 16 days after the
third dose using ELISA.
[0142] A major limitation of aluminum-containing vaccines is that
they cannot be frozen, because freezing of them causes irreversible
coagulation that may damage the vaccines and therefore decrease
their potency. We hypothesized that using the high speed thin-film
freezing method will prevent aggregation during the freeze-drying
process, and the resultant lyophilized vaccine powder will retain
its potency. Data in FIG. 4A-C clearly show that the anti-OVA IgG
levels in mice that were immunized with the lyophilized and
reconstituted OVA-adsorbed aluminum hydroxide were not different
from that in mice that were immunized the freshly prepared
OVA-adsorbed aluminum hydroxide particles.
5. Typical SEM Pictures of the Lyophilized OVA-Adsorbed Aluminum
Hydroxide Powder
[0143] The morphology of lyophilized OVA-adsorbed aluminum
hydroxide powder was examined using a Zeiss Supra 40 VP Scanning
Electron Microscope. One thin layer of lyophilized OVA-aluminum
hydroxide powder was deposited on the specimen stub using a double
stick carbon tape. The specimen stubs with samples were then placed
in the sputter coater chamber and coated with a very thin film of
lead (Pb) before SEM examination.
[0144] As shown in FIG. 5, the lyophilized OVA-adsorbed aluminum
hydroxide particles have a rough surface and are in irregular
shapes. After lyophilization, trehalose became a leaf-like shape.
The OVA-aluminum hydroxide particles are entrapped in the bulk
structure of the trehalose, preventing the coagulation of
OVA-adsorbed aluminum hydroxide particles. The rough surface,
irregular shape particles embedded in the trehalose bulk structure
are similar to the morphology of the freshly prepared OVA-adsorbed
aluminum hydroxide particles observed using SEM (shown in lower
right).
6. Lyophilization of OVA-Adsorbed Aluminum Hydroxide Particles
Using Thin-Film Freezing with Various Concentrations of
Trehalose
[0145] Lyophilization of OVA-adsorbed aluminum hydroxide particles
using thin-film freezing were carried out as described in
Experiment 1. Trehalose was used as a cryoprotectant during
freeze-drying process. Freshly prepared OVA-adsorbed aluminum
hydroxide particles were used as a negative control. The particles
sizes of physical mixture and TFF powder reconstitutions were
determined using a Sympatec Helos laser diffraction instrument
(Sympatec GmbH, Germany) equipped with a R3 lens.
[0146] FIG. 6A shows the images of OVA-adsorbed aluminum hydroxide
particles lyophilized with different concentrations of trehalose.
Shown in FIG. 6B are the sizes of the reconstituted OVA-adsorbed
aluminum hydroxide powders lyophilized with various concentrations
of trehalose. It appears that when the concentration of the
trehalose is increased, the extent of aggregation slightly
decreased.
7. The Lyophilization of OVA-Adsorbed Aluminum Phosphate
[0147] Aluminum hydroxide or aluminum phosphate particles in
suspension were added into a 50 ml tube, followed by the addition
of ovalbumin (OVA) protein solution at a weight ratio of 10:1
(Al.sup.3+ vs. OVA). Trehalose as a cryoprotectant was also added
to a final concentration 2%. The particles were dried after
thin-film freezing as mentioned in Experiment 1. The obtained dry
powders were stored in a desiccator at room temperature before use.
The morphology and size of the lyophilized OVA-adsorbed aluminum
hydroxide or OVA-adsorbed aluminum phosphate were examined after
reconstitution in water using an Olympus BX60 microscope (Olympus
America, Inc., Center Valley, Pa.) and an Sympatec Helos laser
diffraction instrument (Sympatec GmbH, Germany) equipped with a R3
lens.
[0148] FIGS. 7A and 7B show photos of lyophilized OVA-adsorbed
aluminum hydroxide and OVA-adsorbed aluminum phosphate using
thin-film freezing, respectively. FIGS. 7C and 7D show microscopic
images of lyophilized OVA-adsorbed aluminum hydroxide and
OVA-adsorbed aluminum phosphate after reconstitution in water. The
OVA-adsorbed aluminum phosphate particles can also be successfully
lyophilized using the thin-film freezing method. A light
white-colored amorphous lyophilized powder was obtained after
drying. The powder can be easily reconstituted in water, normal
saline or PBS. Shown in insets in FIG. 7C and FIG. 7D are the
particle sizing results from the laser diffraction instrument.
8. The Preparation of a Dry Powder of Ovalbumin Adsorbed on a
Commercial Alhydrogel
[0149] In the previous experiments, we prepared the aluminum
hydroxide suspension by dispersing the Dried Aluminum Hydroxide Gel
(Powder, U.S.P) from Spectrum Chemicals & Laboratory Products
in water. To test whether our method of preparing vaccines having
aluminum-containing adjuvants in the dry solid form that are
suitable for reconstitution is applicable when commercially
available aluminum hydroxide wet gel suspension is used, we used
the Alhydrogel 2%, a ready-to-use, sterile aluminum hydroxide wet
gel (colloidal) suspension from InvivoGen (San Diego, Calif.).
Initially, twenty-five milliliters of the Alhydrogel 2% were added
into a 50 ml tube, followed by the addition of 25 ml of ovalbumin
(OVA) protein solution to a final Al.sup.3+ to OVA weight ratio of
10:1. Trehalose was also added to a final concentration of 2%
(w/v). The particles were processed by thin-film freezing as
described herein, and the frozen liquid was removed using a VirTis
Advantage bench top tray lyophilizer. The morphology and size of
the physical mixture of OVA-adsorbed Alhydrogel with 2% trehalose
and the reconstituted dried OVA-adsorbed Alhydrogel with 2%
trehalose (in phosphate buffer) were examined using an Olympus BX60
microscope (Olympus America, Inc., Center Valley, Pa.).
[0150] FIGS. 8A-B show the microscopic images of the physical
mixture of OVA-adsorbed Alhydrogel and the OVA-adsorbed Alhydrogel
dry powder after reconstitution, respectively. Clearly, our method
can be used to prepare a dry powder of the OVA-adsorbed
Alhydrogel.
9. Materials
[0151] Dried aluminum hydroxide gel was from Spectrum (Gardena,
Calif.). Aluminum chloride hexahydrate, sodium hydroxide, OVA,
horse serum, Laemmli sample buffer, fluorescein-5(6)-isothiocyanate
(MC), sodium bicarbonate, sodium carbonate, phosphate-buffered
saline (PBS), 5-(and-6-)-carboxylfluorescein diacetate succinimidyl
ester (CFSE), and incomplete Freund's adjuvant (IFA) were from
Sigma-Aldrich (St. Louis, Mo.). Goat anti-mouse immunoglobulins
(IgG) were from Southern Biotechnology Associates, Inc.
(Birmingham, Ala.). Carbon-coated 400-mesh grids were from Electron
Microscopy Sciences (Hatfield, Pa.). Vectashield mounting medium
with 4',6-diamidino-2-phenylindole (DAPI) was from Vector
Laboratories, Inc. (Burlingame, Calif.). Bacillus anthracis rPA
protein was from List Biological Laboratories, Inc. (Campbell,
Calif.). Bio-Safe.TM. Coomassie blue staining solution and Bio-Rad
DC.TM. protein assay reagents were from Bio-Rad Laboratories
(Hercules, Calif.). GM-CSF was from R&D Systems, Inc.
(Minneapolis, Minn.). Tissue-Tek.RTM. O.C.T. compound medium was
from Sakura Finetek USA, Inc. (Torrance, Calif.). Cell culture
medium and fetal bovine serum (FBS) were from Invitrogen (Carlsbad,
Calif.). Alhydrogel.RTM. (2%, w/v) was from InvivoGen (San Diego,
Calif.). Tetanus antitoxin concentrated/purified (TT vaccine) was
from Colorado Serum Company. The TT vaccine contains aluminum
potassium sulfate. Mouse Anti-Tetanus Toxoid Ig's ELISA kit was
from Alpha Diagnostic International (San Antonio, Tex.). Human
hepatitis B vaccine Engerix-B from GlaxoSmithKline was purchased
from the University of Texas at Austin University Health Services.
Engerix-B contains aluminum hydroxide.
10. Mice and Cell Lines
[0152] Female BALB/c and C57BL/6 mice, 6-8 weeks of age, were from
Charles River Laboratories, Inc. (Wilmington, Mass.). The
OVA-expressing B16-OVA cell line was generously provided by Dr.
Edith M. Lord and Dr. John Frelinger (University of Rochester
Medical Center, Rochester, N.Y.) and cultured in RPMI1640 medium
supplemented with 5% FBS and 400 .mu.g/ml of G418 (Sigma). Mouse
J774A.1 macrophage cells (# TIB-67TM) were from the American Type
and Culture Collection (Manassas, Va.) and grown in DMEM medium
supplemented with 10% FBS, 100 Um' of penicillin and 100 .mu.g/ml
of streptomycin, all from Invitrogen (Carlsbad, Calif.). DC2.4
cells (a mouse dendritic cell line) (University of Massachusetts
Medical School, Worcester, Mass.) grown in RPMI1640 medium
supplemented with 10% FBS, 100 Um' of penicillin and 100 .mu.g/ml
of streptomycin.
11. Preparation of Aluminum Hydroxide Nanoparticles and
Microparticles
[0153] Aluminum hydroxide nanoparticles of less than 200 nm were
synthesized by reacting aluminum chloride with sodium hydroxide in
a solution. An equal volume of a 3.6 mg/ml AlCl.sub.3. 6H.sub.2O
solution and a 0.04 M NaOH solution were added into a glass vial,
and a small volume of 0.01 M NaOH was added to adjust the pH to
7.0. After 20 min of stirring at room temperature, the particle
suspension was sonicated for 15 min to break down the particle
size. A PD 10 desalting column (Amersham Biosciences, Piscataway,
N.J.) was then used to remove the sodium chloride in the
suspension, and the eluted fractions were analyzed for
nanoparticles by measuring the particle size using a Malvern
Zetasizer Nano ZS (Westborough, Mass.), and for aluminum content
using a Varian 710-ES ICP Optical Emission Spectrometer (OES) in
the Civil Architectural and Environmental Engineering Department at
the University of Texas at Austin. The fourth fraction with the
highest concentration of aluminum was used for further studies. The
endotoxin level in the nanoparticle preparation was not detectable
with a ToxinSensor.TM. chromogenic limulus amebocyte lysate
endotoxin assay kit from GenScript (Piscataway, N.J.). Aluminum
hydroxide microparticles were prepared by dispersing dried aluminum
hydroxide gel into sterile water, followed by vigorous vortexing
and 5 min of water bath sonication, if needed. The sizes of the
microparticles were determined using a Sympatec Helos laser
diffraction instrument (Sympatec GmbH, Germany) equipped with a R3
lens.
12. Adsorption of Protein Antigens on Aluminum Hydroxide
Particles
[0154] The adsorption of proteins (OVA or PA) on aluminum hydroxide
particles of different sizes was carried out by mixing particles in
suspension with proteins in solution. Briefly, a certain volume of
the protein solution was added into a tube (10 .mu.g OVA or 4 .mu.g
PA), followed by the addition of particles in suspension at a
weight ratio of 1:2 to 1:1 (OVA vs. particles) or 1:5 (PA vs.
particles). After 20 min of gentle stirring, the protein-particle
mixtures were stored at 4.degree. C. before use and, if needed,
freeze-dried before further use.
[0155] SDS-PAGE was used to determine the binding efficiency of the
OVA to aluminum hydroxide before and after TFFD. The OVA-adsorbed
aluminum hydroxide dry powder (OVA to Al.sup.3+ ratio (w/w), 1 to
10) was reconstituted in a phosphate buffer and applied on SDS-PAGE
gel. As a control, OVA alone and freshly prepared OVA-adsorbed
aluminum hydroxide suspension (with 2% trehalose, w/v) were also
included. Samples were mixed with a Laemmli sample buffer (62.5 mM
Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, and 0.01% Bromophenol Blue)
before applied to 7.5% Mini-PROTEAN.RTM. TGX.TM. precast
polyacrylamide gels (Bio-Rad). Precision plus protein standards
were also run along with the samples at 130 V for 1 h. The gel was
then stained in a Bio-Safe.TM. Coomassie blue staining solution and
scanned using a Kodak Image Station 440CF (Rochester, N.Y.). The
intensity of the protein bands in the gel was quantified using the
NIH ImageJ software, and the binding efficiency was calculated by
subtracting the percentage of unbound protein (band intensity from
vaccine dry powder or freshly prepared vaccine suspension) from the
total protein (band intensity of OVA alone).
13. Thin-Film Freeze Drying (TFFD
[0156] Three types of aluminum-containing compounds, dried aluminum
hydroxide gel (USP grade), 2% Alhydrogel.RTM., and aluminum
phosphate, were used to adsorb OVA as a model antigen. The
OVA-adsorbed aluminum hydroxide vaccine was prepared by mixing an
OVA solution with an aluminum hydroxide suspension in PBS (pH 7.4,
10 mM) to reach an OVA to Al.sup.3+ weight ratio of 1:10. The
vaccine contained 31.4 .mu.g/ml of OVA, 0.09% of aluminum
hydroxide, and 0-5% (w/v) of trehalose. The OVA-adsorbed aluminum
phosphate vaccine (31.4 .mu.g/ml of OVA, 0.142% (w/v) of aluminum
phosphate, and 2% (w/v) of trehalose) was prepared similarly. When
the 2% Alhydrogel.RTM. was used, Alhydrogel.RTM. (25 ml) was added
into a 50 ml tube, followed by the addition of 25 ml of an OVA
solution (1 mg/ml) at an OVA to Al.sup.3+ weight ratio of 1:10, and
1 g of trehalose to obtain a final formulation with 2% (w/v) of
trehalose, .about.1% (w/v) of Alhydrogel.RTM., and 0.5 mg/ml of
OVA. The samples were subjected to TFF and lyophilized as described
previously [J. D. Engstrom et al., Pharmaceutical Research, 25
(2008) 1334-1346; M. Zhang et al., European journal of
pharmaceutics and biopharmaceutics, 82 (2012) 534-544]. Briefly,
the aluminum-containing vaccine suspensions/dispersions were
dropped onto a pre-cooled rotating cryogenic steel surface to
formed thin films. The thin films were removed by a steel blade. In
order to avoid the overlap of two droplets, the speed at which the
vaccine suspension was dropped on the cryogenic substance was
controlled at 7 rpm. The frozen film-like solids were collected in
liquid nitrogen and dried using a VirTis Advantage bench top tray
lyophilizer (The VirTis Company, Inc. Gardiner, N.Y.).
Lyophilization was performed over 72 h at pressures less than 200
mTorr, while the shelf temperature was gradually ramped from
.about.40.degree. C. to 26.degree. C. After lyophilization, the
solid vaccine powder was quickly transferred to a sealed container
and stored in a desiccator at room temperature before further use
[A. B. Watts et al., Pharmaceutical research, 30 (2013) 813-825].
To preliminarily evaluate the stability of the TFFD powder, the
vaccine powder (OVA adjuvanted with Alhydrogel.RTM.) was
reconstituted after 10-month of storage (at room temperature) and
examined under a microscope (Olympus BX60 microscope, Olympus
America, Inc., Center Valley, Pa.).
[0157] To dry the TT vaccine, trehalose was added directly into the
TT vaccine, or after the TT vaccine was diluted 50-fold in a
phosphate buffered saline (PBS, pH 6.3, 10 mM) to adjust the final
concentration of trehalose to 2% (w/v). The vaccine was then
subjected to TFFD as mentioned above. To dry Engerix-B, trehalose
was added directly into the vaccine to obtain a formulation with 2%
(w/v) of trehalose, .about.20 mg/ml of HBsAg, and 0.144% (w/v) of
aluminum hydroxide, and the vaccine was then subjected to TFFD.
[0158] The morphology of the vaccines in suspension was examined
under an Olympus BX60 microscope. The sizes of particles in all
samples were determined using a Sympatec Helos laser diffraction
instrument (Sympatec GmbH, Germany) equipped with a R3 lens.
14. Shelf Freeze-Drying
[0159] An OVA-adsorbed aluminum hydroxide vaccine that contained 2%
of trehalose (w/v), 0.09% of aluminum hydroxide, and 31.4 .mu.g/ml
of OVA in PBS (pH 7.4, 10 mM) was frozen on the shelf of a
-20.degree. C. or -80.degree. C. freezer overnight and then
lyophilized using a VirTis Advantage bench top tray lyophilizer as
mentioned above. The dry powder was stored in a desiccator at room
temperature before use.
15. The Effect of the Concentration of Trehalose in Vaccine on
Thin-Film Freeze Drying
[0160] In order to investigate the effect of the concentration of
trehalose on TFFD of vaccines, various amounts of trehalose were
added into the OVA-adsorbed aluminum hydroxide in suspension (1:10,
OVA vs. Al.sup.3+, w/w) to prepare vaccine formulations that
contained 0%, 1%, 2%, 3%, 4%, and 5% of trehalose (w/v). The
suspensions were then subjected to TFFD as mentioned above.
16. Residual Moisture Content
[0161] Aliquots of methanol are dispensed through the septum of
scintillation vials to form a suspension concentration of 10-100
mg/mL. Vials are then placed in a bath sonicator (Mettler
Electronics) for 5 minutes at maximum power to insure complete
suspension of the dry vaccine. Moisture content is measured for a
200 .mu.L aliquot with an Aquatest 8 Karl-Fischer Titrator
(Photovolt Instruments). The moisture values are corrected with a
200 .mu.L methanol blank control.
17. Transmission Electron Microscopy (TEM)
[0162] The morphology and size of the OVA-adsorbed aluminum
hydroxide nanoparticles were examined using an FEI Tecnai
Transmission Electron Microscope in the Institute for Cellular and
Molecular Biology (ICMB) Microscopy and Imaging Facility at The
University of Texas at Austin. Carbon-coated 400-mesh grids were
activated for 1-2 min. One drop of the OVA-nanoparticle suspension
was deposited on the grids and incubated for 2 min at room
temperature. The grids were washed with water and dried for 1 min.
Extra water was removed using filter paper. The grids were then
stained with uranyl acetate for 2 min, washed with water, and
allowed to dry for 15 min before observation.
18. Scanning Electron Microscope (SEM)
[0163] The size and morphology of OVA-adsorbed aluminum hydroxide
nanoparticles and microparticles were also examined using a Zeiss
Supra 40 VP Scanning Electron Microscope in the ICMB Microscopy and
Imaging facility. One drop of aluminum hydroxide particle
suspension was deposited on the specimen stub using a double stick
carbon tape and allowed to dry overnight. The specimen stubs with
samples were then placed in the sputter coater chamber and coated
with a very thin film of iridium before SEM examination.
[0164] The morphology of the OVA-adsorbed aluminum hydroxide dry
powder and freshly prepared OVA-adsorbed aluminum hydroxide
suspension was examined using a Zeiss Supra 40 VP scanning electron
microscope in the ICMB Microscopy and Imaging Facility at The
University of Texas at Austin [W. T. Leach et al., Journal of
pharmaceutical sciences, 94 (2005) 56-69]. When preparing the TFFD
samples for SEM, one thin layer of the dried powder was deposited
on the specimen stub using a double stick carbon tape. For the
freshly prepared OVA-adsorbed aluminum hydroxide suspension, the
suspension was placed on the specimen stub and allowed to dry
overnight. The specimen stubs with samples were then placed in the
sputter coater chamber and coated with a very thin film of lead
(Pb) before examination.
19. Differential Scanning Calorimetry (DSC)
[0165] Thermal analysis of the OVA-adsorbed aluminum hydroxide dry
powder and its individual components, OVA, aluminum hydroxide, and
trehalose, were conducted using a modulated temperature DSC (Model
2920, TA Instruments, New Castle, Del.) [M. Zhang et al., European
journal of pharmaceutics and biopharmaceutics, 82 (2012) 534-544].
Four to seven milligrams of each sample was weighed into the
aluminum pans (PerkinElmer Instruments, Norwalk, Conn.), which were
crimped subsequently. An empty aluminum pan was used as a
reference. Samples were then heated at a ramp rate of 3.degree.
C./min from -30.degree. C. to 300.degree. C. Data were analyzed
using the TA Universal Analysis 2000 software (TA Instruments, New
Castle, Del.).
20. X-Ray Diffraction
[0166] The X-ray diffractograms of aluminum hydroxide particles
were obtained with a Scintag X1 theta-theta powder diffractometer
using Cu K-alpha radiation and a solid state Si(Li) detector in the
Texas Materials Institute X-Ray Facility in the Chemical
Engineering Department at the University of Texas at Austin.
21. Stability of Aluminum Hydroxide Particles
[0167] The stability of aluminum hydroxide particles in suspension
was initially examined before adsorption with proteins. The
particles in suspension were kept at 4.degree. C. for 30 days and
the sizes were measured on days 0 and 30. A short-term stability of
the OVA-adsorbed aluminum hydroxide particles was then carried out.
After the adsorption of OVA, the aluminum hydroxide particles of
different sizes were kept at 4.degree. C. for 48 h, and their sizes
were measured every 24 h. To evaluate whether the OVA-adsorbed
aluminum hydroxide nanoparticles can be lyophilized, the
nanoparticles adsorbed with OVA, or nanoparticles adsorbed with OVA
but suspended in 2% (w/v) of trehalose, were lyophilized using a
FreeZone plus 4.5 liter cascade console freeze dry system (Labconco
corporation, Kansas city, MO). The lyophilized powder was
reconstituted with de-ionized and filtered (0.2 nm) water. In order
to evaluate the stability of the lyophilized particles, the
lyophilized powder was stored at 4.degree. C. and reconstituted in
de-ionized and filtered water on days 0, 14 and 28 to measure the
particle size.
22. Repeated Freeze-Thawing of Thin-Film Freeze Dried Vaccine
Powder
[0168] The dried powder of TT vaccine was subjected to three cycles
of freezing (-20.degree. C. for 8 h) and thawing (4.degree. C. for
16 h), reconstituted, and examined under a microscope to detect
aggregation. As a control, fresh TT vaccine was also subjected to
the same three cycles of freezing and thawing and examined under a
microscope.
23. SDS-PAGE
[0169] SDS-PAGE assay was used to determine the extent to which the
protein antigen was bound onto the aluminum hydroxide particles.
Briefly, OVA (10 .mu.g) was mixed with various amount of aluminum
hydroxide particles in suspension (0, 1, 2, 5, 10, 20, 50, and 100
.mu.g). The OVA-particle mixtures were then lyophilized. The
resultant powders were reconstituted in de-ionized water and mixed
with Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol,
2% SDS, and 0.01% Bromophenol Blue). Electrophoresis was performed
with 7.5% Mini-PROTEAN.RTM. TGX.TM. precast polyacrylamide gels
(Bio-Rad). Precision plus protein standards were also run along
with the samples at 130 V for 1 h. The gels were then stained in a
Bio-Safe Coomassie blue staining solution and scanned using a Kodak
Image Station 440CF (Rochester, N.Y.).
24. Preparation of Bone Marrow Dendritic Cells
[0170] Bone marrow dendritic cells (BMDCs) were generated from bone
marrow precursors from C57BL/6 mice. Briefly, femur bones were
removed from C57BL/6 mice and purified from surrounding tissues.
The bones were left in 70% ethanol for 2 min for disinfection and
washed with sterile PBS. After both ends of femur bones were
removed, bone marrow was flushed out with PBS using a hypodermic
needle attached to syringe. After 3 washes with PBS, all leukocytes
obtained were transferred into a bacteriological petri dishes and
cultured with 10 ml of RPMI1640 medium supplemented with 10% FBS,
100 Um' of penicillin, 100 .mu.g/ml of streptomycin,
2-mercaptomethanol (50 .mu.M) and granulocyte-macrophage-colony
stimulating factor (GM-CSF) (100 .mu.g/ml). Cells were allowed to
grow at 37.degree. C. under 5% CO.sub.2 for 3 days, and another 10
ml of culture medium was added into the original dish. On day 6,
half of the supernatant was collected and centrifuged at 800 rpm
for 4 min. Cell pellet was re-suspended in culture medium and added
back into the original dish. Cells on days 7 or 8 were used for
further studies. In order to examine the purity, the cells were
stained with antibodies against CD11c (BD Pharmingen, San Diego,
Calif.) [22], and analyzed using a Guava EasyCyte 8HT
microcapillary flow cytometer (Millipore Corporation, Hayward,
Calif.). A high purity of 86.5% bone marrow dendritic cells was
obtained after 8 days in culture medium.
25. Uptake of the OVA-Adsorbed Particles by BMDCs, DC2.4 Cells and
J77A4.1 Cells in Culture
[0171] In vitro uptake studies were carried out using OVA that was
pre-labeled with FITC. BMDCs, DC2.4 or J77A4.1 cells (50,000
cells/well) were seeded into 24-well plates and allowed to grow
overnight at 37.degree. C., 5% CO.sub.2. FITC-labeled OVA-particles
were added into the cell culture and incubated at 37.degree. C.
under 5% CO.sub.2 or at 4.degree. C. After 3 or 6 h of incubation,
cells were washed with PBS (10 mM, pH 7.4) three times, lyzed with
Triton X-100 (0.17%, v/v) and then applied to a BioTek Synergy HT
microplate reader to measure the fluorescence intensity.
Endocytosis is inhibited at 4.degree. C. Therefore, a subtraction
of the fluorescence intensity of the cells incubated at 4.degree.
C. from the fluorescence intensity of the cells incubated at
37.degree. C., 5% CO.sub.2, allows us to estimate the amount of
FITC-OVA that was internalized.
26. Fluorescence Microscopy
[0172] DC2.4 cells (1.5.times.104) were plated on
poly-D-lysine-coated glass coverslips overnight. FITC-labeled
OVA-adsorbed particles were added and incubated with the cells for
30 to 60 min at 37.degree. C., 5% CO.sub.2. Cells were then washed
with PBS, fixed in 3% paraformaldehyde for 20 min at room
temperature, followed by three times of wash with PBS. Coverslips
were mounted on the slides using Vectashield mounting medium with
DAPI. Fluorescent images were acquired using an Olympus BX60
Biological Microscope (Center Valley, Pa.).
[0173] The TT vaccine was used in this study. The vaccine was dried
using TFFD and reconstituted in a phosphate buffer before
examination. Freshly diluted TT vaccine (in a phosphate buffer) was
used as a negative control. The final trehalose concentration in
both the samples was 2% (w/v). Fluorescence emission spectrum was
recorded using a PTI Quanmaster spectrofluorimeter (Photon
Technology International, Santa Clara, Calif.). An excitation
wavelength of 290 nm was employed, and the emission spectrum was
collected from 280 nm to 530 nm [G. Jiang et al., Journal of
pharmaceutical sciences, 95 (2006) 80-96].
27. Animal Studies
[0174] All animal studies were carried out following National
Institutes of Health guidelines for animal care and use. The animal
protocol was approved by the Institutional Animal Care and Use
Committee at The University of Texas at Austin. When OVA was used
as the antigen, female BALB/c mice (18-20 g) were immunized with
OVA-adsorbed aluminum hydroxide particles once a week for three
consecutive weeks by subcutaneous injection. The dose of the OVA
was 10 .mu.g per mouse per injection; 20 .mu.g per mouse per
injection for the particles. Sterile PBS or OVA (10 .mu.g)
dissolved in PBS was used as controls. Twenty seven days after the
first dose, mice were bled for antibody assay.
[0175] When the PA was used as the antigen, female BALB/c mice
(18-20 g) were immunized subcutaneously with PA-adsorbed aluminum
hydroxide particles on days 0 and 14. As negative controls, mice
were injected with sterile PBS or PA alone. The dose of PA was 4
.mu.g per mouse per injection, and the dose of the particles was 20
.mu.g per mouse per injection. Mice were bled 1 week and 1 month
after the second immunization for antibody assay.
[0176] All animal studies were carried out following the National
Research Council guide for the care and use of laboratory animals.
The animal protocol was approved by the Institutional Animal Care
and Use Committee at The University of Texas at Austin. Female
BALB/c mice, 6-8 weeks of age, were from Charles River
Laboratories, Inc. (Wilmington, Mass.). Mice (n=5) were
subcutaneously (s.c.) injected with OVA-adsorbed aluminum hydroxide
or the TT vaccine, freshly prepared or reconstituted from TFFD
powder. For the OVA-adsorbed aluminum hydroxide, mice were
immunized on days 0, 14 and 28 with 5 .mu.g, 10 .mu.g, or 20 .mu.g
of OVA per mouse. As controls, mice were injected with sterile PBS
or OVA alone (10 .mu.g) dissolved in PBS. For the TT vaccine, mice
were immunized on days 0, 14, and 28, and the dose of TT was 3.75
Lf (flocculation units) of tetanus toxoid per mouse per injection.
Sterile PBS and TT vaccine freshly diluted with 2% trehalose were
used as controls. Sixteen days after the third dose, mice were bled
for antibody assay. Total anti-OVA IgG or anti-TT IgG levels in
serum samples were measured using ELISA.
28. Enzyme-Linked Immunosorbent Assay (ELISA
[0177] ELISA was completed as previously described [B. R. Sloat et
al., Journal of controlled release, 141 (2010) 93-100]. EIA/RIA
flat bottom, medium-binding, polystyrene 96-well plates (Corning
Costar, Corning, N.Y.) were coated with 100 ng of OVA in 0.1 ml of
carbonate buffer (0.1 M, pH 9.6) overnight at 4.degree. C. After
washed with PBS/Tween 20 (10 mM, pH 7.4, 0.05% Tween 20), the
plates were blocked with 5% (v/v) horse serum in PBS/Tween 20 (for
mice immunized with OVA as an antigen) for 1 h at 37.degree. C.
Serum samples were diluted in 5% horse serum/PBS/Tween 20 (or 4%
BSA/PBS/Tween 20) and added to the plates after the removal of the
blocking solution. The plates were incubated for an additional 2 h
at 37.degree. C. The samples were removed, and the plates were
washed with PBS/Tween 20 five times. Horseradish peroxidase-labeled
goat-anti-mouse immunoglobulins (IgG, 5000-fold dilution) were
added as the secondary antibody into the plates, followed by 1 h of
incubation at 37.degree. C. The plates were washed five times with
PBS/Tween 20 again. After 30 min incubation with a
3,3',5,5'-Tetramethylbenzidine (TMB) solution at room temperature,
the reaction was stopped with sulfuric acid (0.2 M), and the
absorbance was read at 450 nm using a BioTek Synergy HT microplate
reader (Winooski, Vt.). Anti-TT IgG levels were determined using a
mouse Anti-Tetanus Toxoid Ig's ELISA Kit following the
manufacturer's instructions.
29. Tumor Prevention Assays
[0178] Female C57BL/6 mice (18-20 g) were immunized with
OVA-adsorbed particles, PBS, or OVA alone on days 0, 7, and 14 by
subcutaneous injection. The dose of OVA was 10 .mu.g per mouse per
injection, and the particles were 20 .mu.g. On day 21, B16-OVA
cells (50,000/mouse) were subcutaneously injected in the right
flank of the mice. Tumor growth was monitored daily, and tumor size
was measured using a caliper and calculated using the following
equation: tumor diameter=(L+W)/2.
30. Histological Examination
[0179] BALB/c mice were immunized with PA adsorbed aluminum
hydroxide particles on day 0 and 19. As negative controls, mice
were injected with sterile PBS or PA alone. On day 40, mice were
euthanized for histological examination. The hair on the injection
site was initially removed using Nair.RTM. lotion (Church and
Dwight Co, Princeton, N.J.). The skin at the injection sites,
including skin and muscle tissues, were removed and spread out on a
piece of index paper. The tissue and paper together were cut into a
1 cm.times.1 cm square and transferred to tissue cryomolds (25
mm.times.20 mm.times.5 mm, Sakura Finetek USA, Inc. Torrance,
Calif.). Any residual spaces in the cryomolds were filled with
Tissue-Tek.RTM. O.C.T. compound medium and fixed in the vapor of
liquid nitrogen for 10 min. After the O.C.T. compound medium was
frozen into a solid white color, the whole cryomoles were removed
and wrapped with aluminum foil. The prepared samples were stored at
-80.degree. C. for cryostate sectioning and staining with
Hematoxylin and eosin (H&E, Sigma, St. Louis, Mo.) in the
Histology and Tissue Analysis Core in the Dell Pediatric Research
Institute, University of Texas at Austin.
31. Statistics
[0180] Statistical analyses were conducted using analysis of
variance followed by Fischer's protected least significant
difference procedure. A p-value of <0.05 (two-tail) was
considered statistically significant.
32. Studies of the Effect of Aluminum Adjuvant Particle Size on
Efficacy of Vaccines
Synthesis and Characterization of Aluminum Hydroxide Particles
[0181] In order to evaluate the effect of the size of aluminum
hydroxide particles on their adjuvant activity, aluminum hydroxide
nanoparticles and microparticles with mean diameters of 112.+-.6.2
nm and 9.3.+-.2.2 .mu.m, respectively, were prepared. FIG. 10A
depicts the particle sizes (open bar) and zeta potentials ( ) of
aluminum hydroxide nanoparticles (NPs) and microparticles (MPs). At
neutral pH, the zeta potentials of both particles were positive
(FIG. 10A), but inversely correlated to their particle sizes. In
other words, the zeta potential of the aluminum hydroxide
microparticles was less positive than that of the aluminum
hydroxide nanoparticles (FIG. 10A). The positive charge of aluminum
hydroxide particles was likely due to the metallic hydroxyls on
their surface, which could accept protons and show a positive zeta
potential. Since the reduction of particle size increases the total
surface area of the particles, the aluminum hydroxide nanoparticles
are expected to have a relatively larger surface area than the
microparticles, and thus more metallic hydroxyl groups on their
surface, resulting in a more positive zeta potential. The aluminum
hydroxide nanoparticles were stable when stored at 4.degree. C. for
a month, whereas the microparticles were slightly less stable (FIG.
10B), likely because the zeta potential of the nanoparticles was
>30 mV, whereas the zeta potential of the microparticles was
<30 mV, at which the electrostatic repulsion is not strong
enough to prevent aggregation. The X-ray powder patterns of
aluminum hydroxide particles are presented in FIGS. 10C and 10D.
The nanoparticles were completely amorphous (FIG. 10C). The
microparticles were mostly crystalline Al(OH).sub.3 (FIG. 10D),
although the large peak in the left showed that some amorphous
AlO(OH) materials existed as well (FIG. 10D).
Characterization of OVA-Adsorbed Aluminum Hydroxide Particles
[0182] Shown in FIG. 11A are the sizes (open bar) and zeta
potentials ( ) of the aluminum hydroxide nanoparticles and
microparticles after the adsorption of OVA protein at a 1:2 ratio
(OVA vs. particle, w/w). The mean diameters of the OVA-adsorbed
nanoparticles and microparticles were 129.+-.20 nm and 9428.+-.1734
nm, respectively; and their zeta potentials were 16.+-.1.8 and
-23.+-.1.9, respectively. The sizes of both particles increased
after the adsorption of OVA. Since OVA is net negatively charged at
neutral pH (isoelectric point (pI), 4.7), after the adsorption of
OVA, the zeta potentials of the resultant nanoparticles became less
positive, and the zeta potential of microparticles even changed
from positive to negative (FIG. 11A).
[0183] Shown in FIG. 11B are the fractions of free OVA when a fixed
amount of OVA was mixed with an increasing amount of the aluminum
hydroxide nanoparticles or microparticles. As expected, the
fraction of unbound OVA decreased when the amount of aluminum
hydroxide particles added was increased. When the ratio of OVA to
nanoparticles was decreased to 1:2 and 1:5, the OVA protein bands
can no longer be detected on the SDS-PAGE, indicating that all OVA
protein were bound on the particles when OVA and particles were
mixed at 1:2 ratio or lower. The adsorption of the OVA to the
aluminum hydroxide microparticles was not as extensive as to the
nanoparticles. Only when the OVA and microparticles weight ratio
reached 1:5, the OVA protein bands were no longer detectable using
SDS-PAGE (FIG. 11B). The mechanisms of the adsorption of OVA to
aluminum hydroxide particles are likely two folds: (i) the
electrostatic interaction between OVA and aluminum hydroxide
particles because they have opposite net charges at neutral pH; and
(ii) ligand exchange as OVA protein contains up to two phosphate
groups, which could strongly bind to aluminum instead of a hydroxyl
group. The higher protein adsorption capacity of the aluminum
hydroxide nanoparticles is consistent with the larger total surface
area of the nanoparticles, which contain more binding sites for
protein adsorption. The smaller total surface area for the aluminum
hydroxide microparticles limited the amount of proteins that can be
adsorbed on them. Besides the effect of the surface area, the zeta
potential of the aluminum hydroxide particles may have also
contributed to the adsorption capacity. The zeta potential of the
aluminum hydroxide nanoparticles was more positively than that of
the microparticles (FIG. 10A). Therefore, the aluminum hydroxide
nanoparticles may have attracted more OVA proteins to their
surface.
[0184] FIGS. 11C and 11D depict SEM pictures of OVA-adsorbed
aluminum hydroxide nanoparticles (OVA-NPs) and OVA-adsorbed
aluminum hydroxide microparticles (OVA-MPs). FIG. 11E depicts a TEM
picture of OVA-NPs.
Stability of OVA-Adsorbed Aluminum Hydroxide Nanoparticles
[0185] The OVA-adsorbed aluminum hydroxide nanoparticles cannot be
stored as a suspension at 4.degree. C. for more than 24 h, because
the size of OVA-adsorbed nanoparticles was found increased by 7.2%
after 24 h storage and 22.3% after 48 h storage as compared to
their original size, respectively. As shown in FIG. 11A, after
adsorption of OVA, the zeta potential of the aluminum hydroxide
particles dropped into the range of -30 mV to +30 mV, in which the
electrostatic repulsion is too weak to prevent aggregation. In
addition, the small size of the nanoparticles favors aggregation to
minimize the free energy on the nanoparticle surface. Therefore, we
decided to lyophilize the OVA-adsorbed aluminum hydroxide
nanoparticles and to evaluate the nanoparticle stability when
stored as a lyophilized powder. The OVA-adsorbed aluminum hydroxide
nanoparticles were successfully lyophilized with trehalose (2%) as
a lyoprotectant (FIG. 12A). In a short-term 28-day study, the size
of the lyophilized, OVA-adsorbed aluminum hydroxide nanoparticles
did not change when stored as a lyophilized powder at 4.degree. C.
(FIG. 12B), indicating that storing the antigen-adsorbed aluminum
hydroxide nanoparticles as a lyophilized powder is a potentially
viable method to avoid aggregation during storage.
OVA-Adsorbed Small Aluminum Hydroxide Nanoparticles Induced a
Stronger OVA-Specific Antibody Immune Response than OVA-Adsorbed
Large Aluminum Hydroxide Microparticles.
[0186] Aluminum hydroxide particles with diameters in the range of
1-20 .mu.m have been widely used in human vaccines. Previous data
showed that nanoparticles with a mean diameter of around 200 nm
have a more potent adjuvant activity than larger particles. To test
whether small aluminum hydroxide nanoparticles of less than 200 nm
can help an antigen to induce a stronger immune response than
larger aluminum hydroxide microparticles, we compared the anti-OVA
immune responses induced by OVA-adsorbed on aluminum hydroxide
nanoparticles or microparticles. Data in FIG. 13A showed that the
anti-OVA IgG level in mice that were immunized with the
OVA-adsorbed aluminum hydroxide nanoparticles was significantly
higher than that in mice that were immunized the OVA alone or
OVA-adsorbed microparticles at 100-fold dilution (p<0.001,
OVA-NPs vs. OVA; p=0.018, OVA-NPs vs. OVA-MPs; p=0.05, OVA alone
vs. large OVA-MPs).
[0187] A tumor prevention study was carried out to evaluate the
capability of OVA-adsorbed aluminum hydroxide nanoparticles against
tumor growth. Twenty-one days after immunization with OVA-adsorbed
aluminum hydroxide nanoparticles or microparticles, mice were
challenged with the OVA-expressing B16-OVA tumor cells, and the
tumor growth was monitored. As shown in FIG. 13B, 31 days after
tumor cell injection, tumors were detected only in one of the 5
mice that were immunized with the OVA-adsorbed aluminum hydroxide
nanoparticles. In contrast, all mice immunized with the
OVA-adsorbed microparticles or with OVA alone developed tumors,
suggesting that the immune responses induced by OVA-adsorbed
aluminum hydroxide nanoparticles can inhibit tumor growth. The
antitumor activity was likely antibody-mediated.
PA-Adsorbed Aluminum Hydroxide Nanoparticles Induced a Stronger
PA-Specific Antibody Response than PA-Adsorbed Aluminum Hydroxide
Microparticles
[0188] The anthrax PA protein was used as a functional antigen in
this experiment. Anthrax is a toxin-mediated disease, and anthrax
toxin is consisted of three proteins, PA, lethal factor, and edema
factor. PA proteins form a heptamer on the surface of cells, from
which the edema factor and the lethal factor enter cells.
Therefore, the induction of anti-PA antibody responses is critical
and sufficient for a vaccine to prevent against anthrax. To further
evaluate the adjuvant activity of the aluminum hydroxide particles,
PA was absorbed on them at a particle to PA ratio of 5:1.
[0189] The mean diameters of the resultant PA-adsorbed aluminum
hydroxide nanoparticles and microparticles were 204.+-.25 nm and
7.1.+-.3.4 .mu.m, respectively (FIG. 14A, open bars). Mice were
then immunized with the PA-adsorbed aluminum hydroxide
nanoparticles or microparticles on days 0 and 14. One week after
the first dose, anti-PA IgG was not detectable in any mice. One
week after the second dose, significant anti-PA IgG responses were
detected in mice that were immunized with the PA-adsorbed aluminum
hydroxide nanoparticles or microparticles (FIG. 14B), although the
levels of the anti-PA IgG response were not different. However, 4
weeks after the second immunization, the anti-PA IgG levels in mice
that were immunized with the PA-adsorbed aluminum hydroxide
nanoparticles were significantly higher than that in mice that were
immunized with the PA-adsorbed aluminum hydroxide microparticles
(FIG. 14C). Anti-PA IgG1 levels 4 weeks after the second
immunization are shown in FIG. 14D. Significant higher anti-PA IgG1
level was detected in mice immunized with PA-adsorbed aluminum
hydroxide nanoparticles as compared to in mice immunized with
PA-adsorbed aluminum hydroxide microparticles. Anti-IgE level was
not detected 4 weeks after immunization with PA-adsorbed aluminum
hydroxide nanoparticles or microparticles (data not shown). The
kinetics of the anti-PA IgG levels within 4 weeks is shown in FIG.
14E. It is clear that during the 4-week period after the second
immunization, the anti-PA IgG level significantly increased in mice
that were immunized with the PA-adsorbed aluminum hydroxide
nanoparticles (p=0.005, week 1 vs. week 4), but significantly
decreased in mice that were immunized with the PA-adsorbed aluminum
hydroxide microparticles (p=0.005, week 1 vs. week 4).
Uptake of OVA-Adsorbed Aluminum Hydroxide Particles by BMDCS, DC2.4
and J774A.1 Cells in Culture
[0190] One important step for an antigen to induce an immune
response is its uptake by APCs. Therefore, we evaluated the extent
to which DCs and macrophages, two critical APCs, can take up OVA as
an antigen adsorbed on aluminum hydroxide particles of different
sizes. BMDCs, DC2.4, or J774A.1 cells in culture were incubated
with fluorescein-labeled OVA adsorbed on aluminum hydroxide
nanoparticles or microparticles for up to 6 h, and the % of OVA
internalized by the cells was determined. In all three cells, more
OVA was internalized when adsorbed on the aluminum hydroxide
nanoparticles than when adsorbed on the aluminum hydroxide
microparticles (FIG. 15). The fluorescence microscopic pictures in
FIG. 16A-C are also supportive of the data in FIG. 15, and may
explain why the aluminum hydroxide nanoparticles were more
effective than the microparticles in facilitating the uptake of OVA
by DC2.4 cells. Green fluorescence signal, an indication of the
location of the OVA protein, was detected only inside cells that
were incubated with OVA-adsorbed aluminum hydroxide nanoparticles,
not in cells that were incubated with OVA-adsorbed aluminum
hydroxide microparticles (FIG. 16A-C). In fact, for cells that were
incubated with the OVA-adsorbed aluminum hydroxide microparticles,
almost all fluorescence signals were extracellular (FIG. 16A-C),
and it seemed that some OVA-adsorbed aluminum hydroxide
microparticles were even larger than the cells (FIG. 16A-C), which
may explain why the aluminum hydroxide microparticles did not
facilitate the uptake of the OVA adsorbed on them (FIG. 15).
Previous data showed that antigens eluted from adjuvants are taken
up by DCs by macropinocytosis, while those remaining adsorbed are
internalized with adjuvant particles by phagocytosis. Because of
close to 100% of the OVA was adsorbed on the aluminum hydroxide
nanoparticles, it is likely that phagocytosis or endocytosis was
the predominant mechanism for the internalization of the OVA that
was adsorbed on the aluminum hydroxide nanoparticles. In contrast,
only less than 20% OVA was adsorbed onto the microparticles (at the
OVA to particle ratio of 1:2). The small percentage of OVA that was
internalized by DC2.4 cells incubated with the OVA-adsorbed
aluminum hydroxide microparticles was probably from the
macropinocytosis of the unbound OVA and OVA eluted from the
microparticles. It has been reported that DCs are able to
internalize particles with a diameter larger than that of cells.
However, we could not find any internalization of the OVA-adsorbed
aluminum hydroxide microparticles using fluorescence microscope. It
has also been reported that nanoparticles (200-600 nm) were more
efficiently taken up by macrophages in comparison to microparticles
(2-8 .mu.m). As shown in FIG. 15, the percentage of OVA
internalized by macrophages was significantly higher when adsorbed
on the aluminum hydroxide nanoparticles than that when adsorbed on
microparticles. Thus, we suspect that ability of the aluminum
hydroxide nanoparticles to more effectively facilitate the uptake
of the OVA adsorbed on them by APCs is related to their potent
adjuvant ability (FIGS. 13A-B to 14A-E).
[0191] Finally, a comparison of the internalization of the OVA by
the macrophages (J774A.1 cells) and the DCs (BMDCs and DC2.4 cells)
indicated that the % of OVA adsorbed on the aluminum hydroxide
microparticles that was internalized by the macrophages was
relatively higher than by the DCs (FIG. 15). This finding is in
agreement with a previous report that macrophages can take up
particles larger than 500 nm very effectively, whereas DCs are more
effective in taking up smaller nanoparticles (<200 nm).
Aluminum Hydroxide Nanoparticles Induced a Milder Local
Inflammation than Aluminum Hydroxide Microparticles
[0192] Aluminum adjuvants have been administered safely to humans
since 1932. Adverse reactions that have been reported with aluminum
containing vaccines are generally local reactions including
subcutaneous (s.c.) nodule, granulomatous inflammation, and sterile
abscesses. In order to evaluate the safety profile of aluminum
hydroxide nanoparticles, the injection sites were examined
histologically. As shown in FIG. 17A-D, microparticles and
nanoparticles both induced local cutaneous inflammation in the
injection sites when examined 40 days after the last dose, but the
inflammation induced by the PA-adsorbed microparticles was much
more severe, as shown by a greater number of accumulations of
neutrophils around the injection sites and the pronounced epidermal
hyperplasia. It appears that the aluminum hydroxide nanoparticles
have a more potent adjuvant activity than aluminum hydroxide
microparticles, but are less pre-inflammatory than the
microparticles.
33. Thin-Film Freeze Drying of OVA-Adsorbed Aluminum Hydroxide
[0193] In order to test whether the TFFD can be used to lyophilize
an aluminum hydroxide-adjuvanted, protein-based vaccine,
OVA-adsorbed aluminum hydroxide was suspended in 2% (w/v) of
trehalose and subjected to TFFD. A white powder was formed, which
can be readily reconstituted with water, PBS, or normal saline with
no or only minimal agitation. The moisture content in the powder
was 1-3%. The size of the particles in the reconstituted
OVA-adsorbed aluminum hydroxide was 9.7.+-.2.5 .mu.m, which is not
different from the size of the particles in freshly prepared
OVA-adsorbed aluminum hydroxide suspension (9.4.+-.1.7 .mu.m),
demonstrating that the OVA-adsorbed aluminum hydroxide suspension
can be successfully lyophilized into a dry powder form using TFFD
without significant effect on the size of the particles in the
vaccine suspension. The microscopic images in FIGS. 22A-B also show
that subjecting the OVA-adsorbed aluminum hydroxide to TFFD did not
cause significant aggregation. In contrast, when the same
OVA-adsorbed aluminum hydroxide suspension was slowly frozen by
placing it on a -80.degree. C. or -20.degree. C. shelf before
lyophilization, significant aggregations were detected (FIGS.
22C-D). As mentioned by Zapata et al., aluminum hydroxide gel could
form aggregates ranged from 65 to 160 .mu.m after just one
freeze-thaw cycle at -24.degree. C. [M. I. Zapata et al., Journal
of pharmaceutical sciences, 73 (1984) 3-8]. It is thought that the
reason of particle coagulation is due to the large water crystals
formed during the slow freezing process, which bring aluminum
hydroxide particles close enough to overcome repulsive forces and
cause aggregation, and the original aluminum hydroxide suspension
could not be reproduced upon coagulation [Y. F. Maa et al., Journal
of pharmaceutical sciences, 92 (2003) 319-332]. By increasing the
freezing rate, only smaller ice crystals are formed as a result of
a greater rate of nucleation, which are not strong enough to
overcome the repulsive forces between particles, and particle
aggregation is prevented consequently [Y. F. Maa et al., Journal of
pharmaceutical sciences, 92 (2003) 319-332]. In TFF process, a
solution or suspension is spread out on a cryogenic substrate to
form a thin film in less than one second (cooling rate, .about.100
K/s) [J. D. Engstrom et al., Pharmaceutical research, 25 (2008)
1334-1346], which may explain why there were not significant
aggregation after the OVA adsorbed on aluminum hydroxide was
subjected to TFFD. As mentioned early, it was reported previously
that higher cooling/freezing rates help minimize agglomeration of
vaccines adjuvanted with aluminum salts [Y. F. Maa et al., Journal
of pharmaceutical sciences, 92 (2003) 319-332; A. Clausi et al.,
Journal of pharmaceutical sciences, 97 (2008) 5252-5262].
34. Thin-Film Freeze Drying of OVA-Adsorbed Aluminum Hydroxide in
Various Concentrations of Trehalose
[0194] Certain sugars, such as trehalose, mannitol, dextran, and
sucrose, have been shown to be effective at maintaining protein
activity and stabilize aluminum salts in vaccine formulations
during freezing process [K. A. Overhoff et al., J. DRUG. DEL. SCI.
TECH, 19 (2009) 89-98; A. L. Clausi et al., Journal of
pharmaceutical sciences, 97 (2008) 2049-2061; L. Wolff et al.,
Colloids and Surfaces A: Physicochemical and Engineering Aspects.,
330 (2008) 116-126]. Trehalose forms fragile glass during freezing,
resulting in an increase on the viscosity, which limits the
mobility of protein molecules or aluminum salt particles and thus,
prevents coagulation [A. L. Clausi et al., Journal of
pharmaceutical sciences, 97 (2008) 2049-2061; W. Wang,
International journal of pharmaceutics, 203 (2000) 1-60]. The
formation of glass also resulted in a trehalose-containing phase
with maximum concentration that prevents the non-ice concentration
or pH-induced aggregation of aluminum salts during freezing [A. L.
Clausi et al., Journal of pharmaceutical sciences, 97 (2008)
2049-2061]. Randolph's group studied the effect of the
concentration of trehalose on spray freeze drying vaccines
containing aluminum hydroxide or aluminum phosphate, and claimed in
their patent that 5-20% (w/v) of trehalose was required to
successfully spray freeze dry vaccines adjuvanted with aluminum
salts [T. W. Randolph et al., W.I.P. Organization (Ed.), 2008]. To
determine the optimal concentration of trehalose needed to prevent
aggregation during TFFD, OVA-adsorbed aluminum hydroxide suspended
in various concentrations of trehalose (i.e., 0%, 1%, 2%, 3%, 4%,
5%, w/v) was subjected to TFFD. As shown in FIG. 23A, when the
OVA-adsorbed aluminum hydroxide suspension was subjected to TFFD in
the absence of trehalose, the size of particles after
reconstitution was significantly larger than that in the freshly
prepared OVA-adsorbed aluminum hydroxide suspension, indicating
that a cryoprotectant such as trehalose is needed to successfully
convert the OVA-adsorbed aluminum hydroxide into a powder by TFFD.
Trehalose at 1% (w/v) was not optimal (FIG. 1A); and at least 2% of
trehalose was used to successfully lyophilize the OVA-adsorbed
aluminum hydroxide into a powder following thin-film freezing (FIG.
22A). Shown in FIG. 23B are representative images of OVA-adsorbed
aluminum hydroxide that were subjected to TFFD with 1%, 2%, and 3%
(w/v) of trehalose, respectively. In the present study, trehalose
alone was used during the TFFD process. It is expected that other
cryoprotectants such as sucrose, glycine and other amino acids, and
polyvinylpyrrolidone may also help to prevent aggregation during
the TFFD process. Moreover, the concentration of trehalose needed
to successfully thin-film freeze dry OVA-adsorbed aluminum
hydroxide was only 2% (w/v). Trehalose at concentrations of above
7.5% is generally used when spray freeze dry vaccines adjuvanted
with aluminum salts [A. L. Clausi et al., Journal of pharmaceutical
sciences, 98 (2009) 114-121; A. Clausi et al., Journal of
pharmaceutical sciences, 97 (2008) 5252-5262; T. W. Randolph et
al., W.I.P. Organization (Ed.), 2008]. The particle size of the
lysozyme vaccines increased slightly following freeze drying and
reconstitution, as compared to the untreated lysozyme vaccines [A.
Clausi et al., Journal of pharmaceutical sciences, 97 (2008)
5252-5262]. Interestingly, in their lysozyme vaccines, only 10% of
the lysozyme was bound to aluminum salts [A. Clausi et al., Journal
of pharmaceutical sciences, 97 (2008) 5252-5262].
35. Characterization of Thin Film Freeze Dried Powder of
OVA-Adsorbed Aluminum Hydroxide
[0195] To understand the influence of the TFFD process on aluminum
hydroxide-adjuvanted vaccines, several studies were conducted to
characterize the dried powder of the OVA-adsorbed aluminum
hydroxide. Initially, a desorption of OVA from the aluminum
hydroxide after the OVA-adsorbed aluminum hydroxide was subjected
to TFFD was evaluated using SDS-PAGE. The intensity of the OVA band
on the SDS-PAGE gel image is inversely correlated to the level of
free unbounded OVA in the OVA-adsorbed aluminum hydroxide
preparation (FIG. 24A). At the OVA to Al.sup.3+ weight ratio of
1:10, all OVA were bound on the aluminum hydroxide (FIG. 24A, NON
TFF). After the OVA-adsorbed aluminum hydroxide was subjected to
TFFD and reconstitution, the percent of OVA that remained adsorbed
on the aluminum hydroxide was estimated to be 92% (FIG. 22A, TFF),
indicating that about 8% of the loosely bound OVA protein was
desorbed from aluminum hydroxide. This 92% binding efficiency still
meets the United States Food and Drug Administration (FDA)
requirement for vaccines adjuvanted with aluminum salts. For
example, 75% adsorption to aluminum salts is the minimum
requirement for diphtheria toxoid and tetanus toxoid antigens [L.
J. Braun, Interactions between antigen and adjuvant: Implications
for formulation, (2012)].
[0196] Modulated DSC was used to study the thermal properties of
the OVA-adsorbed aluminum hydroxide dry powder. The DSC thermogram
of the OVA-adsorbed aluminum hydroxide dry powder shows a glass
transition temperature (Tg) of about 120.degree. C. (FIG. 24B),
indicating that the OVA-adsorbed aluminum hydroxide particles
suspended in trehalose solution may have formed a glass after they
were subjected to TFFD [L. M. Crowe et al., Biophysical Journal, 71
(1996) 2087-2093]. The high Tg value of -120.degree. C. suggests
that the OVA-adsorbed aluminum hydroxide dry powder is highly
stable [J. Buitink et al., Biophysical Journal, 79 (2000)
1119-1123; W. Wang, International Journal of Pharmaceutics, 203
(2000) 1-60].
[0197] Shown in FIG. 24C is a representative SEM image of the
OVA-adsorbed aluminum hydroxide dry powder. It appears that the
OVA-adsorbed aluminum hydroxide particles, which have a rough
surface and irregular shape (FIG. 24D), are embedded in the bulk
structure of the trehalose (FIG. 24C inset). Therefore, it is
likely that the trehalose surrounding the OVA-adsorbed aluminum
hydroxide particles prevented the particles from interacting with
each other during the freeze drying process, and thus prevented
their aggregation.
36. The Immunogenicity of the OVA-Adsorbed Aluminum Hydroxide after
Thin-Film Freeze Drying
[0198] A major limitation of current aluminum salt-adjuvanted
vaccines is that the vaccine suspensions have to be kept at
2-8.degree. C. and may not be exposed to freezing conditions
intentionally or accidentally, because freezing causes irreversible
coagulation and aggregation that may damage the vaccines and
decrease their potency [H. HogenEsch, Vaccine, 20 Suppl 3 (2002)
S34-39]. As reported by Diminsky et al, the aggregation formed
during freezing often results in immunogenicity loss [D. Diminsky
et al., Vaccine, 18 (1999) 3-17]. To test whether the OVA-adsorbed
aluminum hydroxide after subjected to TFFD retains its
immunogenicity, the anti-OVA immune responses induced by
OVA-adsorbed aluminum hydroxide, freshly prepared or reconstituted
from TFDD powder were evaluated in a mouse model. As shown in FIG.
25, the anti-OVA IgG levels in mice that were immunized with
OVA-adsorbed aluminum hydroxide following TFFD and reconstitution
were not different from that in mice that were immunized the
freshly prepared OVA-adsorbed aluminum hydroxide, regardless of the
dose of OVA antigen used (i.e., 5, 10, or 20
.mu.g/mouse/injection). Clearly, the TFFD process not only avoided
the aggregation of the OVA-adsorbed aluminum hydroxide particles,
but also preserved the immunogenicity of the vaccine.
37. Thin-Film Freeze Drying of OVA-Adsorbed Aluminum Phosphate and
OVA-Adsorbed Alhydrogel.RTM.
[0199] Both aluminum hydroxide and aluminum phosphate are commonly
used in human vaccines. Therefore, we also tested whether a protein
antigen adjuvanted with aluminum phosphate can be successfully
lyophilized by TFFD using OVA as a model antigen. Moreover, in the
above studies, the aluminum hydroxide suspension was prepared in
our own laboratories by dispersing dried aluminum hydroxide gel
(USP grade) in water. Alhydrogel.RTM. (2%, w/v) is a commercially
available aluminum hydroxide wet gel suspended in normal saline.
Therefore, we also tested the feasibility of drying OVA-adsorbed
Alhydrogel.RTM. using TFFD. Both OVA-adsorbed aluminum phosphate
and OVA-adsorbed Alhydrogel.RTM. were successfully converted into
powders using TFFD. Both dried samples appeared as light
white-colored powder and were easily reconstituted in water, normal
saline, or PBS with no or minimum agitation. As shown in FIGS.
26A-B, no large aggregation was detected under microscope. The
particle size of OVA-adsorbed aluminum phosphate after subjected to
TFFD and reconstitution was 9.66.+-.2.52 .mu.m. The particle sizes
of OVA-adsorbed Alhydrogel.RTM. before and after TFFD and
reconstitution were 6.37.+-.0.02 .mu.m and 7.59.+-.0.22 .mu.m,
respectively. Clearly, the TFFD can be used to convert vaccines
adjuvanted with aluminum phosphate or with the commercially
available Alhydrogel.RTM. into a dry powder.
[0200] The dry OVA-Alhydrogel.RTM. powder was stored in a
desiccator at room temperature. Shown in FIG. 26C is a
representative microscopic image of the OVA-Alhydrogel.RTM. powder
reconstituted after about 10 months of storage at room temperature.
It appears that 10 months of storage of the OVA-Alhydrogel.RTM. dry
powder at room temperature did not lead to any significant
aggregation. It is likely that the amorphous glass of trehalose
with OVA-aluminum hydroxide particles embedded in helped prevent
the interaction of the particles, and thus their aggregation,
during the storage [D. Chen, D. Kristensen, Expert review of
vaccines, 8 (2009) 547-557; B. S. Chang et al., Archives of
biochemistry and biophysics, 331 (1996) 249-258]. Therefore, the
vaccine powder prepared with TFFD can be kept in a cold-chain
(2-8.degree. C.), but may also be stored at room temperature. A
comprehensive long-term stability test is underway to test the
feasibility of storing the vaccine powder at room temperature.
38. The Preparation of a Dry Powder of an Adjuvanted, Concentrated
Tetanus Toxoid Vaccine
[0201] In order to test whether our method of preparing vaccines
having aluminum-containing adjuvants in the dry solid form that are
suitable for reconstitution is applicable to currently marketed
vaccines, we used the Tetanus Toxoid Concentrated, Adjuvanted
Detoxified Toxin (Cat. #11411, 10.times.1 ml, 10.times.1 dose) from
Colorado Serum Company (Denver, Colo.). This tetanus toxoid
adsorbed vaccine is used for the vaccination of healthy domestic
animals. It is formulated by adsorbing detoxified tetanus toxin
(i.e., tetanus toxoid) on aluminum potassium sulfate (as an
adjuvant). Initially, we diluted 1 ml of the adjuvanted,
concentrated tetanus toxoid vaccine with sodium phosphate buffer
(pH 6.3) in a 50 ml tube, followed by the addition of trehalose (2%
or 3%, w/v). The vaccine was processed by thin-film freezing as
mentioned in Experiment 1, and the frozen liquid was removed using
a VirTis Advantage bench top tray lyophilizer. The obtained dry
powders were stored in a desiccator at room temperature before use.
The morphology and size of the adjuvanted, concentrated tetanus
toxoid vaccine and its reconstituted dried powders were examined
using an Olympus BX60 microscope (Olympus America, Inc., Center
Valley, Pa.) and an Sympatec Helos laser diffraction instrument
(Sympatec GmbH, Germany) equipped with a R3 lens.
[0202] FIG. 9A shows the microscopic image of original adjuvanted,
concentrated tetanus toxoid vaccine, which have particles of
irregular shapes with a particle diameter of 23.1.+-.2.1 .mu.m.
Reversible aggregation was observed in the original suspension.
FIGS. 9B-C show the microscopic images of the adjuvanted,
concentrated tetanus toxoid vaccine after dried with 2% or 3%
trehalose into powders and then reconstituted in sodium phosphate
buffer. Shown in the inset are the particle sizes determined using
the Sympatec Helos laser diffraction instrument. Apparently, our
drying process did not significantly increase the size of the
adjuvanted, concentrated tetanus toxoid vaccine.
39. Thin-Film Freeze Drying of Commercial Veterinary Tetanus Toxoid
Vaccine and Human Hepatitis B Vaccine
[0203] In order to further validate the applicability of the TFFD
in drying vaccines adjuvanted with aluminum salts, tetanus toxoid
concentrated, adjuvanted detoxified toxin, a veterinary TT vaccine,
and Engerix-B, a human hepatitis B vaccine, were subjected to TFFD.
The TT vaccine is formulated by precipitating detoxified tetanus
toxin with aluminum potassium sulfate in a phosphate buffer
containing phosphate, sulfate, and bicarbonate ions [O. H. D. T.,
Vaccine Adjuvants: Preparation methods and Research protocols,
Humana Press, 2000]. The final vaccine formulation is TT adjuvanted
with amorphous aluminum hydroxyl phosphate sulfate [O. H. D. T.,
Vaccine Adjuvants: Preparation methods and Research protocols,
Humana Press, 2000]. The TT vaccine concentrated was diluted, and
trehalose was added to a final concentration of 2% (w/v) before the
vaccine was subjected to TFFD. Shown in FIG. 27A and FIG. 27B are
representative microscopic images of the original TT vaccine after
dilution and the TT vaccine following TFFD and reconstitution,
respectively. The particles in the original vaccine have irregular
shape and an average diameter of 23.1.+-.2.1 .mu.m. The large
particles in FIG. 27A are likely due to reversible flocculation.
Large aggregates were not detected in the TT vaccine after TTFD and
reconstitution (FIG. 27B), and the average particle size of
reconstituted TT vaccine was 18.4.+-.0.2 .mu.m. Clearly, subjecting
the TT vaccine to TFFD (and reconstitution) did not cause any
significant aggregations.
[0204] To investigate whether the TFFD process significantly
altered the structure of the tetanus toxoid protein, the intrinsic
fluorescence spectra of the TT vaccine before and after it was
subjected to TFFD were acquired and compared. As shown in FIG. 27C,
the fluorescence spectrum of the TT vaccine after TFFD and
reconstitution only shifted slightly right (about 20 nm) when
compared to the freshly diluted TT vaccine. In addition, the
fluorescence intensity of the TT vaccine following TFFD and
reconstitution was also relatively lower, probably related at least
in part to antigen desorption during the TFFD process as shown in
FIG. 24A. In addition, freeze drying is known to perturb the
structure of proteins at any stage of the process, including
freezing, drying, and reconstitution [A. L. Clausi et al., Journal
of pharmaceutical sciences, 98 (2009) 114-121; T. Arakawa et al.,
Advanced drug delivery reviews, 46 (2001) 307-326; J. F. Carpenter
et al., Pharmaceutical research, 14 (1997) 969-975]. The TFFD may
have slightly altered the structure of the detoxified tetanus
toxoid. However, it is unclear how the TFFD have increased the
polarity of the environment surrounding the tryptophan residues in
the detoxified tetanus toxoid to induce a slight right shift in the
spectrum. Fortunately, when the immunogenicity of the TT vaccine
before and after the TFFD (and reconstitution) was tested and
compared in a mouse model, the anti-tetanus toxin IgG levels in all
the immunized groups were not significantly different (FIG. 27D),
demonstrating that the potency of the vaccine was preserved after
it was subjected to TFFD and reconstitution. It appears that the
slight protein structure change induced by the TFFD process did not
significantly change the immunogenicity of the antigen.
[0205] To test whether the TT vaccine after TFFD is still sensitive
to inadvertent freezing (and thawing), the dried TT vaccine powder
was subjected to three cycles of freeze-and-thaw, reconstituted,
and then examined under microscope. As a control, fresh TT vaccine
with 2% (w/v) of trehalose was also subjected to the same
freeze-and-thaw cycles. As shown in FIG. 27E, repeated
freezing-and-thawing of the TT vaccine in suspension caused
significant aggregation. However, subjecting the dried TT vaccine
powder to the same freezing-and-thawing cycles did not cause any
significant aggregation (FIG. 27F), demonstrating that the vaccine
powder prepared with TFFD is not sensitive to freezing conditions
anymore. It is noted that to prepare the TT vaccine powder that was
subjected to the repeated freeze-and-thaw cycles, the trehalose
concentration was adjusted to 2% (w/v) by adding trehalose powder
directly into the original TT vaccine, without further
dilution.
[0206] Engerix-B vaccine is a human hepatitis B vaccine, which
contains human hepatitis B virus surface antigen adjuvanted with
aluminum hydroxide. To further test the applicability of the TFFD
process in drying vaccines adjuvanted with aluminum salts,
trehalose was added into the Engerix-B vaccine to a final
concentration of 2% (w/v) without further dilution, and the
preparation was then subjected to TFFD. Shown in FIGS. 27G-H are
representative microscopic images of the Engerix-B vaccine before
(FIG. 27G) and after it was subjected to TFFD and reconstitution
(FIG. 27H). The particle size of the Engerix-B after it was
subjected to TFFD and reconstitution was 3.29.+-.0.15 .mu.m, and
particle size of the fresh Engerix-B vaccine was 5.64.+-.0.01
.mu.m. Clearly, the subjecting the Engerix-B vaccine to TFFD and
reconstition did not cause any significant aggregation. Therefore,
it is likely that the TFFD method can be used to convert any
vaccines that contain aluminum salts into dry powder.
40. The Preparation of a Dry Vaccine by Thin Film Freezing
[0207] Described herein is a method to produce stable dry vaccines.
The method is herein referred to as thin film freezing (TFF). In
TFF, liquid droplets fall from a given height and impact, spread,
and freeze on a cooled solid substrate. In embodiments, the droplet
falls from a given height, and impacts a spinning surface that has
a temperature of 223 K. As the droplet spreads out, a freezing
front is formed in advance of the unfrozen liquid. In embodiments,
the size of the completely frozen droplet is about 2-12 mm in
diameter (e.g. 2, 4, 6, 8, 10, or 12 mm), with a height of
approximately 50 to 500 .mu.m (e.g. 100, 200, 300, 400, or 500). In
embodiments, the liquid droplets (.about.2-4 mm in diameter) are
dispensed from a pipet above a cryogenically cooled metal surface.
In embodiments, upon impact, the droplets spread out into thin
films (.about.100-400 .mu.m) that freeze on time scales of 70 to
1000 ms, which corresponds to a cooling rate of .about.10.sup.2
K/s. Liquid vaccines are passed at a flow rate of 4 mL/min either
through a 17 gauge (1.1 mm ID, 1.5 mm OD) stainless steel syringe
needle producing 3.6 mm diameter droplets or through 3.9 mm ID, 6.4
mm OD stainless steel tubing producing 5.6 mm diameter droplets.
The droplets fall from a height of 10 cm above a rotating stainless
steel drum 17 cm long and 12 cm in diameter. The stainless steel
drum is hollow with 0.7 cm thick walls and is filled with dry ice
or liquid nitrogen to maintain drum surface temperatures of 223 K
or 133 K, respectively. Before each run, the surface temperature of
the drum is verified with a DiGi-Sense.RTM. Type K thermometer
using a 45.degree. angle surface probe thermocouple attachment
(Eutech Instruments). The drum rotates at approximately 12 rpm and
is powered by a Heidolph RZR2041 mechanical overhead stirrer
(ESSLAB) connected to a speed reducer. On impact the droplets
deform into thin films and freeze. The frozen thin films are
removed from the drum by a stainless steel blade mounted along the
rotating drum surface. The frozen thin films then fall 5 cm into a
400 mL Pyrex.RTM. beaker filled with liquid nitrogen. A Virtis
Advantage Lyophilizer (The Virtis Company, Inc.) is used to dry the
frozen thin films. The 400 mL beakers containing the frozen thin
films are covered with a single layer Kim-wipe. Primary drying is
carried out at -40.degree. C. for 36 hrs at 300 mTorr and secondary
drying at 25.degree. C. for 24 hrs at 100 mTorr. A 12 hour linear
ramp of the shelf temperature from -40.degree. C. to +25.degree. C.
is used at 100 mTorr.
[0208] In the present study, we synthesized aluminum hydroxide
nanoparticles with a mean diameter of 112 nm and showed that the
adjuvant activity of the aluminum hydroxide nanoparticles was more
potent than that of the traditional aluminum hydroxide
microparticles. The specific antibody responses induced by protein
antigens adsorbed on aluminum hydroxide nanoparticles were stronger
and more durable than that induced by the same amount of antigens
adsorbed on the traditional aluminum hydroxide microparticles. The
more potent adjuvant activity of the aluminum hydroxide
nanoparticles may be partially attributed to their ability to more
extensively bind to antigens and increase the uptake of the protein
antigens adsorbed on them by APCs. Moreover, the aluminum hydroxide
nanoparticles induced milder local inflammatory reactions in the
injection sites than the microparticles. Therefore, the new
aluminum hydroxide nanoparticles have the potential to be developed
in an effective adjuvant to develop new vaccines and to reformulate
existing vaccines.
[0209] Vaccines that are adjuvanted with aluminum salts, aluminum
hydroxide, aluminum phosphate, or aluminum potassium sulfate, can
be successfully converted from a liquid suspension into a dried
powder by thin-film freeze drying using a low concentration of
trehalose (2%, w/v) as an excipient, while maintaining the particle
size and the immunogenicity of the vaccines. It is expected that
this thin-film freeze drying method can be used to formulate new
vaccines or to reformulate existing vaccines that are adjuvanted
with aluminum salts into dry powder.
[0210] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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