U.S. patent application number 14/323811 was filed with the patent office on 2015-01-22 for stable anthrax vaccine formulations.
This patent application is currently assigned to EMERGENT BIOSOLUTIONS INC.. The applicant listed for this patent is EMERGENT BIOSOLUTIONS INC.. Invention is credited to Elizabet KAISHEVA.
Application Number | 20150023998 14/323811 |
Document ID | / |
Family ID | 42153468 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150023998 |
Kind Code |
A1 |
KAISHEVA; Elizabet |
January 22, 2015 |
Stable Anthrax Vaccine Formulations
Abstract
Formulations of anthrax protective antigen are provided that are
stable in storage for prolonged periods. Methods of using the
formulations to prepare vaccine are also provided. Vaccines
comprising the formulations are useful, for example, to protect
against anthrax infection.
Inventors: |
KAISHEVA; Elizabet;
(Belmont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMERGENT BIOSOLUTIONS INC. |
Rockville |
MD |
US |
|
|
Assignee: |
EMERGENT BIOSOLUTIONS INC.
Rockville
MD
|
Family ID: |
42153468 |
Appl. No.: |
14/323811 |
Filed: |
July 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13056378 |
Jun 8, 2011 |
8778359 |
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PCT/US2009/052279 |
Jul 30, 2009 |
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14323811 |
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61202090 |
Jan 28, 2009 |
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61084833 |
Jul 30, 2008 |
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Current U.S.
Class: |
424/190.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61P 37/04 20180101; A61K 2039/622 20130101; A61P 31/04 20180101;
A61K 39/07 20130101; A61K 2039/55505 20130101 |
Class at
Publication: |
424/190.1 |
International
Class: |
A61K 39/07 20060101
A61K039/07; A61K 47/18 20060101 A61K047/18 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention may have been made with government support in
the form of Department of Health and Human Services Contract Award
Number HHSO10020050001C awarded to VaxGen, Inc. on Nov. 4, 2004 and
National Institutes of Allergy and Infectious Disease Contract
Award Number N01-A1-30053 award to VaxGen, Inc. on Sep. 30, 2003.
The US Government may have certain rights in this invention.
Claims
1-16. (canceled)
17. A stable vaccine for stimulating an immune response to a
Bacillus anthracis protective antigen comprising: a) a purified B.
anthracis protective antigen protein; and b) a glycine formulation
buffer; and c) a pharmaceutically acceptable adjuvant.
18. The vaccine of claim 17, wherein said glycine formulation
buffer comprises about 50 mM to about 500 mM glycine.
19. The vaccine of claim 18, wherein said glycine formulation
buffer comprises about 250 mM glycine, about 25 mM sodium phosphate
and about 0.01% polysorbate 80.
20. The vaccine of claim 17, wherein said glycine formulation
buffer further comprises proline and/or alanine
21. The vaccine of claim 17, wherein said glycine formulation
buffer is at about pH 6.2-8.0.
22. The vaccine of claim 17, wherein said glycine formulation
buffer is at about pH 7.0.
23. The vaccine of claim 17, wherein said glycine formulation
buffer is at about pH 7.4.
24. (canceled)
25. The vaccine of claim 17, wherein said adjuvant is selected from
the group consisting of alhydrogel, CpG, an immunostimulatory
sequence (ISS) and calcium phosphate.
26. The vaccine of claim 25, wherein said adjuvant is
Alhydrogel.
27. The vaccine of claim 26, wherein said vaccine comprises about
750 .mu.g aluminum.
28. The vaccine of claim 17, wherein said purified B. anthracis
protective antigen protein is produced from an asporogenic B.
anthracis bacterium.
29. The vaccine of claim 28, wherein said asporogenic B. anthracis
bacterium is a .DELTA.Sterne-1(pPA102)CR4 strain of bacteria.
30. The vaccine of claim 17, wherein said purified B. anthracis
protective antigen protein comprises amino acids 30-735 of SEQ ID
NO: 1.
31. The vaccine of claim 17, wherein said purified B. anthracis
protective antigen protein comprises SEQ ID NO: 1.
32. The vaccine of claim 17, wherein said vaccine comprises at
least about 25 .mu.g purified B. anthracis protective antigen
protein.
33. (canceled)
34. (canceled)
35. The vaccine of claim 17, wherein said vaccine comprises at
least about 25 .mu.g purified B. anthracis protective antigen
protein and about 750 .mu.g aluminum.
36-45. (canceled)
46. A method of preventing or treating an anthrax disease
comprising administering a pharmaceutically effective amount of the
vaccine of claim 17 to a subject.
47. The method of claim 46, wherein said anthrax disease is
inhalation anthrax.
48. A method of inducing an immune response in a subject comprising
administering to the subject a vaccine of claim 17.
49. A method of vaccinating a subject against anthrax comprising
administering to the subject a vaccine of claim 17.
50. The vaccine of claim 17, wherein the vaccine is a liquid
solution or suspension.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of priority of U.S.
provisional application Nos. 61/084,833, filed Jul. 30, 2008 and
61/202,090, filed Jan. 28, 2009, the entire disclosures of which
are incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to stable formulations of
anthrax vaccines, methods of preparing stable formulations, and
methods of using those formulations.
BACKGROUND OF THE INVENTION
[0004] Anthrax is a well-known infectious disease caused by a
Gram-positive bacterium, Bacillus anthracis (B. anthracis). Among
the three types of anthrax infection (cutaneous, gastrointestinal,
and inhalation), cutaneous anthrax is the most common and is
relatively easily treatable with various antibiotics. The other two
types of anthrax infections are rare, but usually fatal even with
aggressive anti-microbial therapy.
[0005] The major virulence factor, anthrax toxin, is composed of
three proteins: protective antigen (PA, 83 kilo Dalton, kDa), edema
factor (EF, 89 kDa), and lethal factor (LF, 90 kDa). The toxin
components act in the binary combinations of PA+EF (edema toxin),
and PA+LF (lethal toxin). PA is a cell receptor-binding protein and
delivers the other two proteins (EF and LF) into the cytosol of
infected cells.
[0006] The most effective known method for preventing anthrax is
vaccination. The current and only FDA-approved anthrax vaccine in
the United States (produced by Emergent BioSolutions Inc. under the
trademark BioThrax.RTM. Anthrax Vaccine Adsorbed) is produced from
a sterile cell-free filtrate from an avirulent B. anthracis
V770-NP1-R strain. The licensed anthrax vaccine is also called
Anthrax Vaccine Adsorbed (or AVA). The vaccine primarily consists
of PA, and aluminum hydroxide is used as an adjuvant. The vaccine
was developed during the 1950s and 1960s and is licensed by the FDA
to Emergent BioSolutions Inc. The vaccine is safe, showing less
than 0.06% systemic reactions. The ability of the vaccine to elicit
an immune response in humans is well-documented. The BioThrax.RTM.
Anthrax Vaccine Adsorbed vaccine is currently licensed for six
doses over 18 months followed by annual boosts.
[0007] Although the BioThrax.RTM. Anthrax Vaccine Adsorbed vaccine
is effective and safe, new immunogenic compositions for preparing a
vaccine that protects a subject against a lethal B. anthracis
infection using recombinant technologies are under development.
Recombinant vaccine protein components could allow the use of new
types of adjuvants that could elicit enhanced or more diverse
immune responses. Because protective antigen (PA) is the common
factor required for both the actions of LF and EF, it is often used
to prepare vaccines for anthrax. Recombinant PA (rPA), however,
does not elicit a strong protective response against the disease
and there have also been issues with its stability. For example,
the FDA in November 2006 placed a clinical trial using VaxGen's
rPA102 vaccine on hold because of stability issues with the vaccine
formulation. Accordingly, there is a need for a rPA anthrax vaccine
that has improved stability.
SUMMARY OF THE INVENTION
[0008] The present invention provides vaccine formulations that
exhibit improve stability. In one embodiment, the disclosure
provides formulations of PA (e.g., rPA) that have improved storage
characteristics. The formulations vary in their exact composition,
but share in common that they provide improved PA stability, as can
be measured, for instance, by one or more assays set forth in the
disclosure. Those formulations may be used in the preparation of
vaccines that provide protection from anthrax infection. In another
embodiment, the formulations of the invention can be used for the
preparation of vaccines for treatment of an anthrax infection
(i.e., administered to a subject post-exposure).
[0009] Thus, in one embodiment, the invention provides a stable
vaccine for the prevention or treatment of a Bacillus anthracis
infection or related condition comprising: a) a B. anthracis
protective antigen protein; and b) a proline formulation buffer. In
general, the proline formulation buffer comprises about 50 mM to
about 500 mM proline. In some embodiments, the proline formulation
buffer further comprises about 10 to about 250 mM NaCl. In one
particular embodiment, the proline formulation buffer comprises
about 150 mM proline, about 100 mM NaCl, about 25 mM sodium
phosphate and about 0.01% polysorbate 80.
[0010] In another embodiment, the proline formulation buffer
further comprises glycine and/or alanine. For example, the proline
formulation buffer may comprise about 100 mM proline, about 50 mM
glycine, about 100 mM NaCl, about 25 mM sodium phosphate and about
0.01% polysorbate 80.
[0011] In each embodiment, the proline formulation buffer is at
about pH 6.2-8.0. In some embodiments, the proline formulation
buffer is at about pH 7.0. In other embodiments, the proline
formulation buffer is at about pH 7.4.
[0012] The invention also provides a stable vaccine for the
prevention or treatment of a Bacillus anthracis infection or
related condition comprising: a) a B. anthracis protective antigen
protein; and b) an alanine formulation buffer. In general, the
alanine formulation buffer comprises about 50 to 500 mM alanine. In
certain embodiments, the alanine formulation buffer comprises about
220 mM alanine, about 25 mM sodium phosphate and about 0.01%
polysorbate 80.
[0013] In another embodiment, the alanine formulation buffer
further comprises glycine and/or proline.
[0014] In each embodiment, the alanine formulation buffer is at
about pH 6.2-8.0. In certain embodiments, the alanine formulation
buffer is at about pH 7.0. In other embodiments, the alanine
formulation buffer is at about pH 7.4.
[0015] The invention also provides a stable vaccine for the
prevention or treatment of a Bacillus anthracis infection or
related condition comprising: a) a B. anthracis protective antigen
protein; and b) a glycine formulation buffer. In general, the
glycine formulation buffer comprises about 50 mM to about 500 mM
glycine. In certain embodiments, the glycine formulation buffer
comprises about 250 mM glycine, about 25 mM sodium phosphate and
about 0.01% polysorbate 80.
[0016] In another embodiment, the glycine formulation buffer
further comprises proline and/or alanine.
[0017] In each embodiment, the glycine formulation buffer is at
about pH 6.2-8.0. In certain embodiments, the glycine formulation
buffer is at about pH 7.0. In other embodiments, the glycine
formulation buffer is at about pH 7.4.
[0018] When the formulation is prepared as a vaccine, the
formulation generally further comprises a pharmaceutically
acceptable adjuvant. Adjuvants may be chosen from alhydrogel,
ImmunoStimulatory Sequences (ISS, CpG), or calcium phosphate. In
many embodiments, the adjuvant is Alhydrogel.
[0019] The source of the protective antigen may vary. Thus, in some
embodiments, the B. anthracis protective antigen protein is
produced from an asporogenic B. anthracis bacterium. In some
embodiments, the asporogenic B. anthracis bacterium is a
.DELTA.Sterne-1(pPA102) CR4 strain of bacteria.
[0020] In many embodiments, the B. anthracis protective antigen
protein comprises SEQ ID NO: 1. In some embodiments, however, the
B. anthracis protective antigen protein comprises a deletion of
residues 162-167, a substitution of isoleucine for serine at
residue 168, a deletion of residues 304-317, and a substitution of
glycine for serine at residue 319 of SEQ ID NO: 1.
[0021] As mentioned, the formulation and the resulting vaccines are
stable. Thus, in some embodiments the vaccine is stable at
temperatures below 25.degree. C. for at least 6 months. In other
embodiments, the vaccine is stable at temperatures below 25.degree.
C. for at least 1 year. In still other embodiments, it is stable at
temperatures below 25.degree. C. for at least 1.5 years. And in yet
other embodiments, the formulations and vaccine are stable at
temperatures below 25.degree. C. for at least 2 years.
[0022] The formulations and vaccines are also stable at lower
temperatures. For example, in many embodiments they are stable at
about 2-8.degree. C. for at least 6 months. Often, they are stable
at about 2-8.degree. C. for at least 1 year. They may also be
stable at about 2-8.degree. C. for at least 1.5 years, or even at
least 2 years.
[0023] The present invention includes methods of preventing and
treating an anthrax infection comprising administering to a subject
a pharmaceutically effective amount of one of the vaccines of the
invention. In another embodiment, the invention includes methods of
inducing an immune response in a subject comprising administering
to the subject a vaccine of the invention.
[0024] The present invention includes assays developed that are
useful for determining the stability of a vaccine composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. Results from the FCIA assay demonstrate a strong
positive correlation between the exposure of the native epitope
(bound by 14B7) and the ability to elute rPA102 from Alhydrogel.
Results also show a strong negative correlation between the
exposure of a non-native epitope (bound by 15F7) and an inability
to elute rPA102 from Alhydrogel.
[0026] FIG. 2. A strong correlation exists between native
elutability and the ED.sub.50 generated by the rabbit ELISA.
[0027] FIG. 3. Rabbit serum anti-PA IgG levels strongly correlated
with TNA activity.
[0028] FIG. 4. Biochemical activity measurements (MLA assay)
indicate that the protein in the phosphate spike formulation loses
conformation and activity at a 10-fold greater rate at 25.degree.
C. relative to protein stored at 2-8.degree. C.
[0029] FIG. 5. The new formulations demonstrated improved stability
at 25.degree. C. based on results from the MLA assay.
[0030] FIG. 6. The new formulations demonstrated improved stability
at 25.degree. C. based on the relative availability of neutralizing
vs. non-neutralizing epitopes using the FICA assay.
[0031] FIG. 7. The rPA102 formulated using the new formulations
demonstrate increased stability relative to the phosphate spiked
formulation. The figure is a western blot using anti-rPA as the
primary antibody.
[0032] FIG. 8. rPA102 in the proline and phosphate spike
formulations and stored at 25.degree. C. had substantially more
degradation than rPA in the alanine- and glycine-based
formulations.
[0033] FIG. 9. Results from the MLA assay indicate that rPA102 is
most stable in the alanine and glycine formulations and least
stable in the proline and phosphate spike formulations. All
percentages are normalized to an rPA reference standard.
[0034] FIG. 10. While the alanine and glycine based formulations
have the highest 3B6 to 15F7 ratio at 25.degree. C., all four
formulations are similar when stored at 2-8.degree. C. for 12
months.
[0035] FIG. 11. The Alanine-based formulation retains the highest
overall level of protein folding for the formulations tested.
[0036] FIG. 12. AVA incubated at 48-55.degree. C. for 5 days
(thermally degraded) has substantially lower levels of potency
compared to AVA stored under normal conditions of 2-8.degree.
C.
[0037] FIG. 13. Study design summary for experiment 1.
[0038] FIG. 14. Study design summary for experiment 2.
[0039] FIG. 15. Anti-PA IgG concentrations after rPA102
immunization (1/4 dilution) with fresh and aged vaccines in mouse
sera. Anti-PA IgG concentrations after rPA102 immunization (1/8
dilution) with fresh and aged vaccines in mouse sera.
[0040] FIG. 16. Anti-PA IgG concentrations after rPA102
immunization (1/8 dilution) with fresh and aged vaccines in mouse
sera.
[0041] FIG. 17. TNA (ED.sub.50) titers after rPA102 immunization
(1/4 dilution) with fresh and aged vaccines in mouse sera.
[0042] FIG. 18. TNA (ED.sub.50) titers after rPA102 immunization
(1/8 dilution) with fresh and aged vaccines in mouse sera.
[0043] FIG. 19. Anti-PA IgG concentrations after rPA102
immunization (1/4 and 1/8 dilutions) with fresh and aged vaccines
in mouse sera.
[0044] FIG. 20. TNA (ED50) titers after rPA102 immunization (1/4
and 1/8 dilutions) with fresh and aged vaccines in mouse sera
collected on day 21.
[0045] FIG. 21. Formulations to be tested for anti-microbial
effectiveness.
[0046] FIG. 22. Comparability study between the multi-dose
formulation containing preservative and the single-dose formulation
without preservative.
[0047] FIG. 23. Stability plan and testing for formulations
containing preservatives.
[0048] FIG. 24. Anti-PA IgG concentrations in guinea pig sera after
immunization with rPA102 vaccine candidates.
[0049] FIG. 25. TNA titers in guinea pig sera after two
immunizations with rPA102 vaccine candidates.
[0050] FIG. 26. Anti-PA IgG concentrations in rabbit sera after two
immunizations with rPA102 vaccine candidates.
[0051] FIGS. 27A and 27B. TNA titers of rabbit sera after two
immunizations with rPA102 vaccine candidates or BioThrax. FIG. 27A
presents the ED.sub.50. FIG. 27B presents the NF.sub.50.
DETAILED DESCRIPTION
[0052] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited
herein, including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated
by reference in their entirety for any purpose. In the event that
one or more of the incorporated documents or portions of documents
defines a term that contradicts that term's definition in the
application, the definition that appears in this application
controls.
[0053] The use of the singular includes the plural unless
specifically stated otherwise. The word "a" or "an" means "at least
one" unless specifically stated otherwise. The use of "or" means
"and/or" unless stated otherwise. The meaning of the phrase "at
least one" is equivalent to the meaning of the phrase "one or
more." Furthermore, the use of the term "including," as well as
other forms, such as "includes" and "included," is not limiting.
Also, terms such as "element" or "component" encompass both
elements or components comprising one unit and elements or
components comprising more than one unit unless specifically stated
otherwise.
I. DEFINITIONS
[0054] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0055] Protective antigen (PA)--the component of anthrax toxin
(approx 83 kDa) that contains the receptor-binding and
translocation domains. One example of a full length PA amino acid
sequence is:
TABLE-US-00001 (SEQ ID NO: 1)
EVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSEL
ENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKA
SNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDN
LQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKR
TFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPE
ARHPLVAAYPIVHVDMENTILSKNEDQSTQNTDSQTRTISKNTSTSRTHT
SEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETM
GLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQ
LSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRL
DTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNL
VERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEF
DENFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDR
NNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE
GLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYA
VTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.
[0056] SEQ ID NO: 1 is the amino acid sequence of rPA102 which is
expressed from plasmid pPA102. During secretion of rPA102 from B.
anthracis .DELTA.Sterne-1(pPA102)CR4 into the extracellular space,
the first 29 amino acids (the signal peptide) are removed yielding
the mature rPA protein of 735 amino acids (82,674 Da). The mature
rPA sequence is underlined.
[0057] The rPA102 amino acid sequence is but one example of one
particular anthrax protein within the scope of the invention.
Additional amino acid sequences of PA proteins, including native
proteins, from various strains of anthrax are known in the art and
include, for example, GenBank Accession Nos: NP.sub.--652920.1,
ZP.sub.--02937261.1, ZP.sub.--02900013.1, ZP.sub.--02880951.1 which
are incorporated by reference. Various fragments, mutations, and
modifications in PA to reduce its toxicity or to improve its
expression characteristics are also known, such as those described
elsewhere in the specification, as are various fusion proteins.
Those fragments, mutants, and fusion proteins are included in the
term "PA" unless the context or text clearly indicates that those
forms are excluded. Where indicated, PA fragments, mutants, and
fusion proteins (whether with full length PA or a PA fragment) are
those that elicit an antisera that is active in the toxin
neutralization assay (TNA).
[0058] Stable Vaccine: the formulations of the present invention
are stable compared to a control PA formulation in which the buffer
consists of 20 mM sodium phosphate/20 mM Tris/150 mM sodium
chloride and 0.01% polysorbate 80 (PS80), pH 7.4. With the
exception of the buffer, the control PA formulation is similar or
identical to the stable vaccine (i.e., PA and other components of
the vaccine are the same). As used herein, "stable" or "stability"
can be measured using any one or more of the assays described
herein, including the working examples, as well as assays known in
the art that are used to measure activity, potency and/or peptide
degradation. A stable vaccine as used herein is a vaccine that
exhibits no or little decrease in activity and/or potency and/or
degradation over time. In one embodiment, a stable vaccine exhibits
substantially less of a decrease in activity and/or potency and/or
degradation over time compared to a control PA formulation. By
"substantially less" it is meant that there is at least a 1 fold, 2
fold, 3 fold, 4 fold, 5 fold or 10 fold or more difference in
activity and/or potency and/or degradation between the stable
vaccine and control PA formulation.
[0059] Storage: refers to placement of a PA formulation at a
specified temperature (+/- at most 5.degree. C.) for a specified
period of time. Storage generally starts at least within 6 hours
following the initial preparation of the formulation, unless
otherwise indicated by the context or clearly specified.
[0060] Control Formulation: A formulation of PA from Bacillus
anthracis in 20 mM sodium phosphate/20 mM Tris/150 mM sodium
chloride and 0.01% polysorbate PS80, pH 7.4 The concentration of PA
will be the same (or within +/-5%) as that of the comparison
formulation. The source (e.g., recombinant or non-recombinant
material, production lot, etc.) of PA will also be the same as the
source of PA in the comparison formulation.
[0061] Formulation Buffer: An amino acid buffer comprising alanine,
glycine and/or proline that stabilizes an rPA vaccine. Although the
term "buffer" is used herein, the term should be understood to be
equivalent to the term "excipient" when used to describe the
stabilizing properties of the amino acids on a rPA vaccine.
II. FORMULATIONS OF STABLE PROTECTIVE ANTIGEN
[0062] The invention provides formulations that improve the
stability of Bacillus anthracis protective antigen (PA) during
storage. Improvement in the storage characteristics of PA can be
measured, for instance, either by evaluating the extent of protein
degradation, the retention of functional activity, or the
percentage of protein in native conformation at different time
points and different storage temperatures. Measurements for
determining whether a PA formulation has improved storage
characteristics are made compared to a control formulation, such as
a PA formulation in which the buffer consists of 20 mM sodium
phosphate/20 mM Tris/150 mM sodium chloride and 0.01% polysorbate
80, pH 7.4.
[0063] In one embodiment, the formulation buffer of the invention
comprises one or more free amino acids. In other words, in one
embodiment of the invention, the PA vaccines of the invention
comprise one or more amino acid excipients. Often, the amino acid
is chosen from alanine, glycine, proline, or combinations thereof.
In some embodiments, the amino acid is alanine. In other
embodiments, it is glycine. In still other embodiments, the amino
acid is proline. And in yet other embodiments, the formulation
comprises a combination of glycine and proline, or glycine and
alanine, or proline and alanine. In certain embodiments of the
invention, the formulation comprises a single free amino acid
chosen from alanine, glycine, proline. That is, although the
formulation may comprise additional ingredients, the amino acids in
the formulation consist of either alanine, glycine, or proline.
[0064] The amino acid of the formulation is usually present in the
range of about 50 mM to about 500 mM. In some embodiments, the
amino acid is present in the formulation at about 50 mM to about
400 mM, or about 50 mM to about 300 MM, or about 50 mM to about 200
mM, or about 50 mM to about 100 mM. In other embodiments, it is
present at about 100 mm to about 500 mM, at about 100 mM to about
400 mM, at about 100 mM to about 300 mM, or at about 100 mM to
about 200 mM. In still other embodiments, the amino acid is present
in the formulation at about 200 mm to about 500 mM, at about 200 mM
to about 400 mM, or at about 300 mM to about 300 mM. In yet other
embodiments, it is present at about 300 mM to about 500 mM, or
about 300 mM to about 400 mM, or even at about 400 mM to about 500
mM. Of course, it is also possible for the amino acid to be present
in the formulation at about 50 mM, about 100 mM, about 150 mM,
about 200 mM, about 210 mM, about 220 mM, about 230 MM, about 240
mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about
290 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or
at about 500 mM.
[0065] The pH of the formulation may also vary. In general, it is
between about pH 6.2 to about pH 8.0. In some embodiments, the pH
is about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about
7.2, about 7.4, about 7.6, about 7.8, or about 8.0. Of course, the
pH may also be within a range of values. Thus, in some embodiments
the pH is between about 6.2 and about 8.0, between about 6.2 and
7.8, between about 6.2 and 7.6, between about 6.2 and 7.4, between
about 6.2 and 7.2, between about 6.2 and 7.0, between about 6.2 and
6.8, between about 6.2 and about 6.6, or between about 6.2 and 6.4.
In other embodiments, the pH is between 6.4 and about 8.0, between
about 6.4 and 7.8, between about 6.4 and 7.6, between about 6.4 and
7.4, between about 6.4 and 7.2, between about 6.4 and 7.0, between
about 6.4 and 6.8, or between about 6.4 and about 6.6. In still
other embodiments, the pH is between about 6.6 and about 8.0,
between about 6.6 and 7.8, between about 6.6 and 7.6, between about
6.6 and 7.4, between about 6.6 and 7.2, between about 6.6 and 7.0,
or between about 6.6 and 6.8. In yet other embodiments, it is
between about 6.8 and about 8.0, between about 6.8 and 7.8, between
about 6.8 and 7.6, between about 6.8 and 7.4, between about 6.8 and
7.2, or between about 6.8 and 7.0. In still other embodiments, it
is between about 7.0 and about 8.0, between about 7.0 and 7.8,
between about 7.0 and 7.6, between about 7.0 and 7.4, between about
7.0 and 7.2, between about 7.2 and 8.0, between about 7.2 and 7.8,
between about 7.2 and about 7.6, between about 7.2 and 7.4, between
about 7.4 and about 8.0, about 7.4 and about 7.6, or between about
7.6 and about 8.0.
[0066] In some embodiments, the formulation further comprises one
or more additional ingredients that are not free amino acids. For
example, the formulation may include one or more salts, such as
sodium chloride, sodium phosphate, or a combination thereof. In
general, each salt is present in the formulation at about 10 mM to
about 200 mM. Thus, in some embodiments, any salt that is present
is present at about 10 mM to about 200 mM, about 20 mM to about 200
mM, about 25 mM to about 200 mM, at about 30 mM to about 200 mM, at
about 40 mM to about 200 mM, at about 50 mM to about 200 mM, at
about 75 mM to about 200 mM, at about 100 mM to about 200 mM, at
about 125 mM to about 200 mM, at about 150 mM to about 200 mM, or
at about 175 mM to about 200 mM. In other embodiments, any salt
that is present is present at about 10 mM to about 175 mM, about 20
mM to about 175 mM, about 25 mM to about 175 mM, at about 30 mM to
about 175 mM, at about 40 mM to about 175 mM, at about 50 mM to
about 175 mM, at about 75 mM to about 175 mM, at about 100 mM to
about 175 mM, at about 125 mM to about 175 mM, or at about 150 mM
to about 175 mM. In still other embodiments, any salt that is
present is present at about 10 mM to about 150 mM, about 20 mM to
about 150 mM, about 25 mM to about 150 mM, at about 30 mM to about
150 mM, at about 40 mM to about 150 mM, at about 50 mM to about 150
mM, at about 75 mM to about 150 mM, at about 100 mM to about 150
mM, or at about 125 mM to about 150 mM. In yet other embodiments,
any salt that is present is present at about 10 mM to about 125 mM,
about 20 mM to about 125 mM, about 25 mM to about 125 mM, at about
30 mM to about 125 mM, at about 40 mM to about 125 mM, at about 50
mM to about 125 mM, at about 75 mM to about 125 mM, or at about 100
mM to about 125 mM. In some embodiments, any salt that is present
is present at about 10 mM to about 100 mM, about 20 mM to about 100
mM, about 25 mM to about 100 mM, at about 30 mM to about 100 mM, at
about 40 mM to about 100 mM, at about 50 mM to about 100 mM, or at
about 75 mM to about 100 mM. In yet other embodiments, any salt
that is present is present at about 10 mM to about 75 mM, about 20
mM to about 75 mM, about 25 mM to about 75 mM, at about 30 mM to
about 75 mM, at about 40 mM to about 75 mM, or at about 50 mM to
about 75 mM. In still other embodiments, any salt that is present
is present at about 10 mM to about 50 mM, about 20 mM to about 50
mM, about 25 mM to about 50 mM, at about 30 mM to about 50 mM, or
at about 40 mM to about 50 mM. In other embodiments, any salt that
is present is present at about 10 mM to about 40 mM, about 20 mM to
about 40 mM, about 25 mM to about 40 mM, at about 30 mM to about 40
mM, at about 10 mM to about 30 mM, at about 20 mM to about 30, at
about 25 mM to about 30 mM, at about 10 mM to about 25 mM, at about
20 mM to about 25 mM, or at about 10 mM to about 20 mM. In
particular embodiments, the sodium chloride is present in the
formulation at about 100 mM. In particular embodiments, the sodium
phosphate is present in the formulation at about 25 mM.
[0067] In one embodiment of the invention, the vaccine composition
comprises at least about 25 .mu.g PA. In another embodiment of the
invention, the vaccine comprises at least 50 .mu.g PA. In yet
another embodiment, the vaccine comprises at least 75 .mu.g PA.
[0068] Formulations of the invention are stable in that their
characteristics change little over a given period of time at a
defined temperature. In general, formulations of the invention are
stable for at least about a month. In some embodiments, the
formulations are stable for at least about 6 weeks, at least about
2 months, at least about 4 months, at least about 6 months, at
least about 8 months, at least about 10 months, at least about 12
months (1 year), at least about 14 months, at least about 16
months, at least about 18 months (1.5 years), at least about 20
months, at least about 22 months, at least about 24 months (2
years), at least about 26 months, at least about 28 months, at
least about 30 months, at least about 32 months, at least about 34
months, at least about 36 months (3 years), at least about 38
months, at least about 40 months, at least about 42 months, at
least about 44 months, at least about 46 months or at least about
48 months (4 years).
[0069] The temperatures over which a formulation is stable are
generally below about 30.degree. C. In some embodiments, the
formulation's stability is in reference to a temperature below
about 25.degree. C., about 20.degree. C., about 15.degree. C.,
about 10.degree. C., about 8.degree. C., about 5.degree. C., about
4.degree. C., or about 2.degree. C. Thus, in some embodiments, the
temperature is in the range of about 25.degree. C. to about
2.degree. C., about 20.degree. C. to about 2.degree. C., about
15.degree. C. to about 2.degree. C., about 10.degree. C. to about
2.degree. C., about 8.degree. C. to about 2.degree. C., or about
5.degree. C. to about 2.degree. C. In other embodiments, the
temperature is in the range of about 25.degree. C. to about
5.degree. C., about 20.degree. C. to about 5.degree. C., about
15.degree. C. to about 5.degree. C., about 10.degree. C. to about
5.degree. C., or about 8.degree. C. to about 5.degree. C. In still
other embodiments, the temperature is in the range of about
25.degree. C. to about 8.degree. C., about 20.degree. C. to about
8.degree. C., about 15.degree. C. to about 8.degree. C., or about
10.degree. C. to about 8.degree. C. In yet other embodiments, the
temperature is in the range of about 25.degree. C. to about
10.degree. C., about 20.degree. C. to about 10.degree. C., about
15.degree. C. to about 10.degree. C., about 25.degree. C. to about
15.degree. C., about 20.degree. C. to about 15.degree. C., or about
25.degree. C. to about 20.degree. C.
[0070] Formulations of the invention may further comprise a
solubilizing agent such as a nonionic detergent. Such detergents
include, but are not limited to polysorbate 80 (Tween.RTM. 80),
TritonX100 and polysorbate 20.
III. STABILITY ASSAYS
[0071] The invention provides formulations that improve the
stability of Bacillus anthracis protective antigen (PA) during
storage. Improvement in the storage characteristics of PA can be
measured either by evaluating the extent of protein degradation,
the retention of functional activity, or the percentage of protein
in native conformation at different time points and different
storage temperatures. Measurements for determining whether a PA
formulation has improved storage characteristics are made compared
to a control formulation, such as a PA formulation in which the
buffer consists of 20 mM sodium phosphate/20 mM Tris/150 mM sodium
chloride and 0.01% polysorbate 80, pH 7.4. Various assays for
measuring stability exist including, but not limited to, various
assays described herein.
[0072] In order to properly evaluate the protein and understand the
degradation pathways, several additional assays were developed.
These assays include:
[0073] Native Elutability (NE). This assay is used to estimate the
fraction of PA that can be recovered from the adjuvant under
non-denaturing conditions. Results from the native elutability
assay are reported as relative to the native elutability for each
specific formulation at time=0. A decrease in NE is interpreted as
due either to conformational changes in PA or to deamidation,
resulting in increased binding to the adjuvant.
[0074] Biochemical Activity. This assay is based on the macrophage
lysis assay (MLA) and is used as a functional cell based bioassay
that measures the cytotoxicity of lethal toxin. Lethal toxin is
formed by mixing lethal factor with either reference PA or the test
PA. The assay requires the elution of active PA from the adjuvant.
A decrease in activity is interpreted as a change in the native
conformation of the PA eluted from the adjuvant.
[0075] Flow Cytometric Immunoassay (FCIA). This assay measures the
exposure of different PA epitopes. The FCIA assay is based on the
binding of monoclonal antibodies to specific epitopes. The test
antibodies include 14B7, which blocks cell-receptor binding and
indicates that PA is in its native conformation. Antibody 15F7 has
been shown to bind epitopes of unfolded PA and therefore can detect
non-native conformation. The assay can be performed while the PA is
bound to an adjuvant. A decrease in the ratio of signal from the
14B7 antibody relative to signal from the 15F7 antibody is
interpreted as a change in the PA native conformation.
[0076] Peptide map analysis. RP-HPLC/ESI-MS provides information on
deamidation of the PA.
[0077] Front Faced Fluorescence (FFF). This assay measures the
intrinsic tryptophan fluorescence and provides information on
protein tertiary structure changes. Changes in tryptophan exposure
to the environment are interpreted as conformational changes of the
PA, either free or bound to an adjuvant.
[0078] Additional details and yet other assays are described in the
Examples section.
IV. SOURCES OF PROTECTIVE ANTIGEN
[0079] The invention provides methods of preparing stable
formulations of protective antigen from Bacillus anthracis. In
general, the formulations are more stable in storage than is a
control formulation using the same source of PA.
[0080] Methods of producing PA for inclusion in the formulations of
the invention are known in the art and are described, for example
in U.S. Pat. No. 7,201,912, to Park and Giri, U.S. Pat. No.
6,387,665 to Ivins et al., U.S. Pat. No. 6,316,006 to Worsham et
al., and U.S. Pat. No. 7,261,900 to Leppla et al., each of which is
incorporated by reference in its entirety. For example, as
described in U.S. Pat. No. 7,201,912, pBP103 is an expression
vector for full-length, wild-type rPA. The PA sequence from pBP103
is identical to that of wild-type PA.
[0081] The present invention includes formulations comprising PA
expressed in B. anthracis, including expression in both sporulating
and non-sporulating strains of B. anthracis. For instance, the PA
can be derived from non-sporulating B. anthracis strain
.DELTA.Sterne-1 (pPA102)CR4 (i.e., rPA102). See, for instance, U.S.
Pat. No. 6,316,006 and U.S. Pat. No. 6,387,665, both to Ivins et
al., each of which is herein incorporated by reference in its
entirety.
[0082] The formulations of the invention may also include B.
anthracis PA expressed by a heterologous organism. For instance,
the invention includes PA expressed in E. coli.
[0083] In addition, various PA fragments, mutants, and fusion
proteins have also been described and can be used in the current
formulations. For example, PA may be modified to lack a functional
binding site, thereby preventing PA from binding to either Anthrax
Toxin Receptor (ATR) (see Bradley, K. A., et al (2001)) to which
native PA binds, or to native LF. By way of example, a modification
made within or near to amino acid residues 315-735 or within or
near to residues 596-735 of Domain 4 may render PA incapable of
binding to ATR. Alternatively (or in addition), the PA furin
cleavage site "RKKR" (SEQ ID NO: 2), which in most full length PA
sequences is found at or around residues 163-168, may be
inactivated by deletion, insertion, or substitution within or near
to the furin cleavage site. For example, all of the furin cleavage
site residues of native PA may be deleted. Other mutant PAs include
those in which the dipeptide Phe-Phe has been modified to render
the PA resistant to chemotrypsin. A PA fragment or PA fusion
protein may also be a PA mutant.
[0084] Specific examples of PA fragments include those in U.S. Pat.
No. 7,201,912, for example, PA64 expressed by pBP111, PA47
expressed by pBP113, PA27 expressed by pBP115. Some of those
fragments also include mutations to, for example, eliminate the
furin cleavage site RKKR (SEQ ID NO: 2) or the chemotrypsin
sensitive site formed by the dipeptide sequence Phe-Phe (FF). In
addition, fragments may include one or two additional amino acids
at the N-terminus. Examples of fusion proteins involving PA include
those in U.S. Pat. No. 7,201,912, for example the PA-LF fusion
proteins expressed by plasmids pBP107, pBP108, and pBP109. The
invention also includes formulations comprising a HIS-tag PA. When
a fragment, mutant, or fusion protein is used, however, it is
generally desirable that the fragment, mutant, or fusion protein
elicit protective immunity to a challenge with an LD.sub.50 of
anthrax spores of the Ames strain in one or more of mice, guinea
pigs, or rabbits.
[0085] Although PA from a recombinant source is generally
preferred, formulations prepared from non-recombinant sources can
also be used and the stability of such preparations improved by the
formulations of the invention.
[0086] Methods of expression B. anthracis proteins, including PA
(as well as fragments, mutants, and fusion proteins), are known and
include those described in U.S. Pat. No. 7,201,912, which is
incorporated by reference in its entirety.
V. VACCINES
[0087] Formulations of the invention can be used to elicit
antibodies to protective antigen, which may provide protection from
infection with anthrax. Thus, one embodiment of the invention is a
vaccine comprising one or more of the formulations comprising
PA.
[0088] When the formulations are used as a vaccine, they typically,
although not always, further comprise one or more adjuvants.
Examples of adjuvants include, but are not limited to, aluminum
(e.g., Alhydrogel), ImmunoStimulatory Sequences (ISS, CpG), and
calcium phosphate. For aluminum hydroxide, the protein formulation
is added to the adjuvant at the desired ratio (e.g., 175 .mu.g PA
per 1500 .mu.g aluminum). In some embodiments, the vaccine
comprises approximately 200 .mu.g/mL rPA102 and approximately 0.5
mg/mL (for example, between 0.43 and 0.58 mg/mL) aluminum (e.g.,
Alhydrogel). In further embodiments, the vaccine comprises
approximately 250 .mu.g rPA per 250 to 100 .mu.g aluminum (e.g.,
Alhydrogel). For ISS, protein samples are generally used at a final
protein concentration 50 .mu.g/ml. Other non-limiting examples of
adjuvants include but are not limited to: CGP7909 (see U.S. Pat.
No. 7,223,741, which is herein incorporated by reference in its
entirety), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE), and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion.
[0089] Typically, vaccines are prepared as injectables, either as
liquid solutions or suspensions. Of course, solid forms suitable
for solution in, or suspension in, liquid prior to injection may
also be prepared. The preparation may also be emulsified, or the
peptide encapsulated in liposomes or microcapsules.
[0090] Vaccine administration is generally by conventional routes,
for instance, intravenous, subcutaneous, intraperitoneal, or
mucosal routes. The administration may be by parenteral injection,
for example, a subcutaneous or intramuscular injection.
[0091] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
prophylactically and/or therapeutically effective. The quantity to
be administered, which is generally in the range of 5 .mu.g to 250
.mu.g of antigen per dose, depends on the subject to be treated,
capacity of the subject's immune system to synthesize antibodies,
and the degree of protection desired. In one embodiment, the
vaccine comprises at least about 10 .mu.g PA, 25 .mu.g PA, 50 .mu.g
PA, 75 .mu.g PA, 100 .mu.g PA, 125 .mu.g PA, 150 .mu.g PA, 200
.mu.g PA, or 225 .mu.g PA. Precise amounts of active ingredient
required to be administered may depend on the judgment of the
practitioner and may be particular to each subject.
[0092] The vaccine may be given in a single dose schedule, or
optionally in a multiple dose schedule. The vaccine composition may
be administered, for instance, in a 0.5 mL dose. For pre-exposure
prophylaxis, a multiple dose schedule is one in which a primary
course of vaccination may be with 1-6 separate doses, followed by
other doses given at subsequent time intervals required to maintain
and or reinforce the immune response, for example, at 1-4 months
for a second dose, and if needed, a subsequent dose(s) after
several months.
[0093] For post-exposure prophylaxis, the vaccine may also be
administered according to a multiple dose regimen. For instance, in
one embodiment, the vaccine is administered in 3 doses at times 0,
2 and 4 weeks post exposure. The dosage regimen will also, at least
in part, be determined by the need of the individual and be
dependent upon the judgment of the practitioner.
[0094] In addition, the vaccine containing the immunogenic
antigen(s) may be administered in conjunction with other
immunoregulatory agents, for example, immunoglobulins, antibiotics,
interleukins (e.g., IL-2, IL-12), and/or cytokines (e.g., IFN-beta,
IFN-alpha).
[0095] In one embodiment, the vaccine is administered to a subject
post-exposure to anthrax. In this embodiment, the vaccine may be
administered in conjunction with an antibiotic. Antibiotics that
may be administered with the vaccine include, but are not limited
to, penicillin, doxycycline and ciprofloxacin.
[0096] The formulations of the invention may be further modified to
provide other formulations that are suitable for other modes of
administration include microcapsules, suppositories and, in some
cases, oral formulations or formulations suitable for distribution
as aerosols. For suppositories, traditional binders and carriers
may include, for example, polyalkylene glycols or triglycerides;
such suppositories may be formed from mixtures containing the
active ingredient in the range of 0.5% to 10%, preferably
1%-2%.
[0097] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0098] The invention includes methods of treating (post-exposure
prophylaxis) or preventing (pre-exposure prophylaxis) an anthrax
infection comprising administering to a subject a pharmaceutically
effective amount of a vaccine of the invention. In one embodiment,
the anthrax infection is the result of inhaling anthrax (i.e.,
inhalation anthrax). As used herein, a pharmaceutically effective
amount of a vaccine is an amount that induces an immune response.
In one embodiment, a pharmaceutically effective amount of a vaccine
is an amount comprising at least 25 .mu.g PA. As used herein, a
subject is a mammal such as a human.
[0099] The invention also provides methods of stimulating an immune
response in a subject by administering the to subject an amount of
a vaccine of the invention sufficient to stimulate an immune
response. In some embodiments, immune stimulation is measured by an
increased protective effect compared to a vaccine comprising, for
instance, the same peptide in a buffer solution comprising 20 mM
sodium phosphate/20 mM Tris/150 mM sodium chloride and 001%
polysorbate 80, pH 7.4. In other embodiments, immune stimulation is
measured by increases in antibody titer that is specific for the
antigen in the vaccine. In still other embodiments, immune
stimulation is measured by an increased frequency in cytotoxic T
lymphocytes specific for the antigen in the vaccine.
[0100] The immunogenicity of the rPA formulations can be tested as
described in the various examples. For example, mice can be
immunized with, for example, 10 .mu.g, 20 .mu.g, or more of rPA
suspended in an adjuvant emulsion. Control mice are immunized with
saline emulsified in adjuvant for use as negative controls. The
mice are generally immunized, then bled at various intervals, e.g.,
day 0, day 21 and day 28 post-immunization. The serum is then
analyzed for the presence of specific antibody, e.g., by ELISA,
which can also be used to determine the titer of the antisera.
[0101] A mouse toxin-neutralizing antibody assay can also be used
to determine if the rPA formulations elicit protective antibodies.
In this assay, mice immunized with rPA are then challenged i.p.
with 2 lethal doses of lethal toxin (i.e., PA and lethal factor
(LF)). Four days after challenge, the mice are scored for
survivors.
[0102] The rPA formulations can also be used to prepare
compositions comprising neutralizing antibodies that immunoreact
with the anthrax toxin. The resulting antisera can be used for the
manufacture of a medicament for treating exposure to anthrax. In
one embodiment of the invention, the antibody composition comprises
a purified anti-PA antibody. By "purified," it is meant that the
antibody is substantially free of other biological material with
which it is naturally associated. Purified antibodies of the
invention are at least 60% weight pure, at least 70% weight pure,
at least 80% weight pure, at least 90% weight pure or at least 95%
weight pure. The antisera, or antibodies purified from the
antisera, can also be used as diagnostic agents to detect either PA
fragments or native protein.
[0103] The invention will be further clarified by the following
examples, which are intended to be purely exemplary of the
invention and in no way limiting.
VI. EXAMPLES
Example 1
rPA102 Drug Product and Stability Assays
[0104] Stability issues with the FDP formulations used in VaxGen's
initial phase 2 clinical trial led to the material produced for
clinical trial VAX023 being placed on clinical hold by the FDA.
This resulted in a decision to further examine the degradation
pathways for rPA102 and to re-evaluate alternative formulations for
the rPA102 vaccine.
[0105] In order to properly evaluate the protein and understand the
degradation pathways, several additional assays were developed.
These assays included:
[0106] Native Elutability (NE) which was used to estimate the
fraction of rPA102 which could be recovered from the Alhydrogel
under non-denaturing conditions; results from the native
elutability assay are reported as relative to the native
elutability for each specific formulation at time=0. A decrease in
NE is interpreted as due either to conformational changes in rPA102
or to deamidation, resulting in increased binding to the
Alhydrogel.
[0107] rPA102 Biochemical Activity assay based on the macrophage
lysis assay (MLA) is used as a functional cell based bioassay which
measures the cytotoxicity of lethal toxin. Lethal toxin is formed
by mixing lethal factor with either reference rPA or the test rPA.
The assay requires the elution of active rPA102 from the
Alhydrogel. A decrease in activity is interpreted as a change in
the native conformation of the rPA eluted from the Alhydrogel.
[0108] Flow Cytometric Immunoassay (FCIA) is used to measure the
exposure of different rPA102 epitopes. The FCIA assay is based on
the binding of monoclonal antibodies to specific epitopes. The test
antibodies include 14B7 that blocks rPA102 cell-receptor binding,
which indicates rPA102 is in its native conformation. Antibody 15F7
has been shown to bind epitopes of unfolded rPA102, and therefore
can detect non-native conformation. The assay is performed while
the antigen is bound to the Alhydrogel. A decrease in the ratio of
signal from the 14B7 antibody relative to signal from the 15F7
antibody is interpreted as a change in the native conformation of
the rPA102.
[0109] Peptide map analysis by RP-HPLC/ESI-MS provides information
on deamidation of the rPA102.
[0110] Front Faced Fluorescence (FFF) measures the intrinsic
tryptophan fluorescence and provides information on protein
tertiary structure changes. Changes in tryptophan exposure to the
environment are interpreted as conformational changes of the
rPA102, either free or bound to Alhydrogel.
[0111] These analytical methods were utilized in a series of
experiments designed to generate a better understanding of the
reasons for the loss of rPA102 potency in the initial phase 2 trial
and to develop more stable formulations. Data from the FCIA assay
(FIG. 1) demonstrated that rPA102 adsorbed to Alhydrogel undergoes
conformational changes as a function of incubation time and
incubation temperature. rPA102 in the native protein conformation
(based on the availability of the 14B7 epitope) positively
correlated with an increased ability to elute rPA102 from
Alhydrogel under non-denaturing conditions. rPA102 in the
non-native protein conformation (based on the availability of the
15F7 epitope) was found to have a negative correlation with the
ability of rPA102 to be eluted from Alhydrogel under non-denaturing
conditions. These data indicate that the FCIA method is capable of
assessing rPA in its native conformation. These conclusions were
confirmed by data generated during analysis using the NE and MLA
assays. Data from the peptide map analysis indicated an increase in
deamidation, potentially resulting in an increased negative charge
that could lead to the formation of unusually strong bonds between
the rPA102 molecule and the Alhydrogel.
[0112] Further analysis of the data shows a strong statistical
correlation (P<0.05) between the in vitro assays, such as the
native elutability assay and the in vivo ELISA-based rabbit potency
assay (FIG. 2). Other studies (data not shown) indicate that high
immunogenicity (low ED.sub.50) correlates with increased exposure
of the epitope recognized by the 14B7 antibody, high native
elutability, and low percent deamidation, all of which are markers
of rPA102 in the native confirmation. A statistically significant
correlation was also established for rPA102 protein activity as
measured by MLA and the rabbit potency assay. In addition, a strong
correlation was observed between the ELISA results and data from
the TNA assay generated using rabbit sera (FIG. 3). These
correlations provide further assurance of the relevance of results
obtained using the biochemical/biophysical stability assays.
[0113] Correlations were also established between degradation rates
at 2-8.degree. C. and at 25.degree. C., based primarily on the
extensive data from the phosphate spike formulation and the newly
developed in vitro assays (FIG. 4). rPA102 stored at 25.degree. C.
and at 2-8.degree. C. for up to 38 weeks was analyzed for the loss
of biochemical activity using the MLA assay. The data presented
demonstrates both the linear nature of the loss of rPA102
biochemical activity over time and the different rates of protein
degradation for the two temperatures. Based on this analysis,
phosphate spike stability results for one week at 25.degree. C.
correspond to approximately 3 weeks at 2-8.degree. C. Results from
the other assays support these conclusions.
Example 2
Formulation Development: Physico-Chemical Studies
[0114] A second series of formulation studies was carried out which
was more focused on the addition of excipients to the formulation
buffer including: glycine; alanine and proline; polysorbates and
other sugars; salts as well as variations in final pH. The
experiments were carried out using a statistical "design of
experiments" screening approach to evaluate both the individual and
combined effects of the various excipients. The results indicated
that proline (Pro), glycine (Gly) and alanine (Ala)-based
formulation buffers had a stabilizing effect on rPA bound to
Alhydrogel. An experiment was then performed to identify the
optimum excipient concentrations and pH in order to obtain maximum
protein stability. Based on the model simulations the following
promising formulation prototypes were identified:
[0115] Gly formulation: 250 mM Gly, 25 mM Na Phosphate buffer,
0.01% PS80, pH 7.0.
[0116] Ala formulation: 220 mM Ala, 25 mM Na Phosphate buffer,
0.01% PS80, pH 7.0.
[0117] Pro formulation: 150 mM Pro and 100 mM NaCl, 25 mM Na
Phosphate buffer, 0.01% PS80, pH 7.4.
[0118] Pro/Gly formulation: 50 mM Gly, 100 mM Pro, 100 mM NaCl, 25
mM Na Phosphate buffer, 0.01% PS80, pH 7.4.
[0119] rPA102 was formulated using the four new formulation buffers
at a protein to Alhydrogel ratio of 175 .mu.g rPA102 per 1500 .mu.g
aluminum (85 .mu.g rPA and 750 .mu.g aluminum (Alhydrogel) per 0.5
mL dose). Other formulations are also possible, for example, those
comprising approximately 200 .mu.g/mL rPA102 and approximately 0.5
mg/mL aluminum. Phosphate spike Alhydrogel, previously determined
by VaxGen to be more stable than the formulations used in the phase
1 and phase 2 studies, was used as a control. The phosphate spike
formulation contains 20 mM sodium phosphate/20 mM Tris/150 mM
sodium chloride and 0.01% PS80, pH 7.4. These formulations were
placed on stability at 25.degree. C. for 3 months. The stability of
the formulation prototypes were evaluated using the assays selected
to determine the degradation pathways. The results from the
stability studies were reproducible and indicate significant
improvement in FDP stability compared to the phosphate spike
formulation. Improvements in stability of the rPA102 included:
[0120] Prototype formulations containing proline or alanine had
recoverable levels of rPA102 after 12 weeks incubation at
25.degree. C. which were similar to the level of recoverable rPA102
at time=0. Recoverable rPA102 levels from the phosphate spike
formulation were 20% of the time=0 value
[0121] The biochemical activity of rPA102 after 12 weeks, as
measured by MLA, for the proline-based formulation was 84%; while
after 12 weeks incubation at 25.degree. C. formulations containing
alanine and glycine (See FIG. 5) retained 80% and 60% MLA activity,
respectively. At the same time the phosphate spike formulation had
.about.40% rPA102 biochemical activity after only 1-week at
25.degree. C. and after 4 weeks had decreased to <10% rPA102
biochemical activity. Data from this assay was also reported as a
percentage relative to a time equals zero sample.
[0122] FCIA analysis of the phosphate spike formulation shows
significant level of rPA unfolding after one month incubation at
25.degree. C. as measured by epitope exposure (See FIG. 6). The
extent of rPA unfolding observed in formulation prototypes Ala, Pro
or Gly is substantially lower indicating that the rPA configuration
was closer to the native structure.
[0123] Peptide map analysis by RP-HPLC/ESI-MS indicates significant
decrease of the level of deamidation in the formulation prototype
containing Ala after 3 months incubation at 25.degree. C., while
the phosphate spike formulation is almost completely
deamidated.
[0124] These data, combined with the temperature correlations
described earlier, indicate that the projected real time stability
of the alanine based formulation at 2-8.degree. C. should be at
least two years.
[0125] The preliminary data supported further investigation of
three formulations: the alanine-based formulation; the
proline-based formulation and the glycine-based formulation. While
results varied from assay to assay, the alanine-based formulation
was consistently stable across all assays while the proline and
glycine-based formulations had a higher degree of variation in
stability from assay to assay.
[0126] We have continued formulation development by conducting a
series of physichochemical and animal studies to select the best
formulation to take rPA102 to licensure. rPA102 was formulated
using the alanine, proline, glycine, and phosphate spike
formulations and compared to material produced using the same
formulations at VaxGen in July 2007. All samples were stored at
2-8.degree. C. The phosphate spike formulation was included to
correlate results from earlier studies.
[0127] Results from degradation analysis using western blot
indicate that the rPA102 in the alanine and glycine formulations
stored for 11 months at 2-8.degree. C. were intact whereas the
proline and phosphate-spike formulations had degradation resulting
in peptide backbone breakage (FIG. 7). rPA102 in the amino acid
formulations stored at 25.degree. C. for 7 months and analyzed by
SDS-PAGE and western blot had similar degradation patterns to those
seen for samples stored at 2-8.degree. C. (FIG. 8). However, the
level of protein degradation was substantially lower than that seen
at 25.degree. C.
[0128] rPA102, eluted from Alhydrogel under non-denaturing
conditions showed similar results with the lowest relative rPA102
activity being the 11 month phosphate spike and proline
formulations. The highest MLA activity was seen in the freshly
prepared glycine and alanine formulations as shown in FIG. 9. All
MLA activity assay values were compared to an rPA reference
standard.
[0129] rPA102 was also shown to retain the native conformation
based on the relative abilities of the antibodies 3B6 and 15F7 to
bind to the rPA102 (FIG. 10). 3B6 is a replacement for 14B7, which
was no longer available. However; 3B6 binds to the same
neutralizing epitope as 14B7 and provides similar data on the
relative availability of the neutralizing epitope. 3B6 mAb binds to
exposed epitope in native rPA and 15F7 binds only to the unfolded
rPA102. Each mAB is conjugated to a different fluorescent dye. FCIA
assays measure binding of each mAB to rPA102. The
alanine-containing and glycine-containing formulations retained the
highest overall level of native rPA102 protein conformation
compared to the phosphate spike and proline-based formulations
after storage for 8 months at 25.degree. C.
[0130] Results from the front faced fluorescence assay that
measures fluorescence of exposed Trp residues (normally buried
inside the protein hydrophobic pockets) confirm that the
alanine-based formulation retained the highest overall level of
native rPA102 protein conformation as compared to the phosphate
spike, the proline, and the glycine-based formulations after
storage for 11 months at 2-8.degree. C. (FIG. 11).
Example 3
Preliminary Potency and Stability Studies
[0131] The stability and potency of the revised rPA102 formulations
were tested utilizing a mouse toxin-neutralizing antibody assay
(TNA assay) currently under development at Emergent. This assay
uses mice immunized with formulated rPA. Sera from the mice are
then tested both by ELISA and by the TNA assay. The TNA assay
correlates to protection in animal challenge studies (Pitt et al.
2001). In addition, preliminary data generated from BioThrax
stability studies indicates that the mouse TNA assay detects
differences between non-degraded and thermally degraded AVA (FIG.
12 on the next page). Antibody titers are measured using a standard
rPA-binding ELISA.
[0132] Two lots (fresh and aged 11 months) of each three rPA102
formulations were administered to mice: 1) proline; 2) alanine; and
3) glycine. CD-1 female mice (210), 6-8 weeks old and 20-25 g body
weight were used for the study. Mice were bled prior to vaccination
to determine pre-immune titers; post-vaccination bleeds were
performed on days 21 and 28. The rPA102 vaccine was administered IP
on day 0 of the study at dilutions of 1/4 [21.25 .mu.g rPA102 and
Alhydrogel (187.5 .mu.g aluminum) per dose] (FIG. 13) and 1/8 [8.82
.mu.g rPA102 and Alhydrogel (93.8 .mu.g aluminum) per dose] (FIG.
14). AVA at dilutions of 1/2 and 1/4 was used as a positive
control. The study design for each experiment is summarized in FIG.
13 and EBS 14.
[0133] Sera from the pre-inoculation and from days 21 and 28 post
immunization were analyzed utilizing the TNA assay and the anti-PA
IgG ELISA. TNA results are presented as ED.sub.50 and anti-PA IgG
values from the ELISA are expressed as .mu.g/mL relative to an
external reference standard.
[0134] All three rPA102 vaccine formulations induced robust immune
responses at both dilutions. There was no statistical difference
(P>0.05) in levels of anti-rPA102 IgG between the freshly
prepared vaccines and vaccines stored at 2-8.degree. C. for 11
months (FIGS. 15 and 16). The alanine-and glycine-based
formulations induced slightly higher levels of anti-PA IgG than the
proline-based formulation; however, these differences were not
statistically significant.
[0135] Results from the TNA assay were consistent with the ELISA
results. The TNA assay demonstrated production of functionally
active, anti-PA neutralizing antibodies in response to rPA102
vaccination (FIGS. 17 and 18). Sera from mice injected with the
aged rPA formulations demonstrated no difference in toxin
neutralizing activity relative to mice injected with the freshly
prepared rPA. Sera from animals injected with the alanine- or
glycine-based formulations had higher TNA titers than sera from
animals inoculated with the proline based formulation (P<0.05).
There was no statistical difference between TNA titers induced by
the alanine- and glycine-based formulations.
[0136] Anti-PA IgG concentrations did not vary significantly over
the two dose levels (1/4 and 1/8) for any rPA formulations tested
(FIG. 19). But the two dose levels for AVA (1/2 and 1/4) did
produce a dose dependent IgG response indicating that the assay was
operating properly and was in the linear range for AVA (FIG.
19.).
[0137] Results from the TNA assay demonstrated a dose-dependent
effect for the vaccine (FIG. 20). Although the dilution effect was
present for both the fresh and the aged rPA formulations, there was
no apparent decrease in rPA induced neutralizing activity over
time. The immunological data obtained from day 28 sera confirmed
the day 21 sera results.
[0138] Data from the mouse TNA assay demonstrate that all rPA102
vaccine formulations induce toxin neutralizing antibodies in the
mouse model. In addition, all three formulations are stable over 11
months at 2-8.degree. C. in that there was no decrease in potency
of the formulated rPA102. No statistically significant difference
in immunogenicity of the rPA102 vaccine in the alanine-based
formulation was observed after storage for 18 months at 2-8.degree.
C. (data not shown). Finally, both the alanine and glycine-based
formulations generate more toxin neutralizing antibodies than the
proline-based formulation.
[0139] While the data from the physicochemical assays showed
varying levels of protein activity, native conformation and
availability in the different formulations; when the results were
viewed as a whole, the alanine-based formulation is the most stable
and presents the highest combination of desired characteristics.
These data are supported by the results from the mouse ELISA and
TNA assay.
Example 4
Immunogenicity and Efficacy of rPA102 Vaccine Candidate
Formulations in Guinea Pigs
[0140] The immunogenicity and efficacy of the alanine-, proline-
and glycine-containing formulations of rPA102 vaccine were
evaluated in a guinea pig lethal anthrax challenge model.
Sixty-four guinea pigs were randomly assigned to 8 groups of 8
animals (equal number of males and females per group). rPA102
vaccine formulations containing the glycine, alanine and proline
formulation buffers as described in example 2 were evaluated.
Vaccine formulations contained 100 .mu.g rPA102 and Alhydrogel (750
.mu.g aluminum) per 0.5 mL dose. The source of the rPA102 was cGMP
BDS lot AN0030. Groups of guinea pigs were vaccinated once (on day
0) or twice (on days 0 and 12) subcutaneously (SC) with 0.5 mL of
the appropriate rPA102 vaccine formulation or with undiluted
BioThrax.RTM. anthrax vaccine adsorbed as a positive control. On
day 30, animals were challenged via the intradermal (ID) route with
200 LD.sub.50 of anthrax spores and observed for mortality for 10
days post-challenge. Serum samples were collected prior to the
first vaccination and on day 28, and the immune response was
evaluated using TNA and anti-PA IgG ELISA.
[0141] Most rPA102 vaccine-immunized animals survived the anthrax
challenge except for one group in the Group 2 that was immunized
with a single dose of the proline-formulated vaccine. The highest
mortality rate (37.5%) was observed in Group 4 vaccinated with a
single dose of BioThrax where 3 animals died. All three rPA
formulations induced a robust humoral immune response, even after a
single immunization, as measured by anti-PA IgG ELISA and TNA. The
second immunization induced a substantial increase in both anti-PA
IgG and neutralizing antibody levels. The results are illustrated
in FIGS. 24 and 25.
TABLE-US-00002 TABLE 1 Guinea Pig Survival after Immunization with
rPA102 Vaccine Candidates or BioThrax Group Vaccination No.
Alive/No. Number Vaccine Schedule (Days) Dosed (%) 1 rPA102-ALA 0
8/8 2 rPA102-PRO 0 6/7.sup.a 3 rPA102-GLY 0 8/8 4 BioThrax 0 5/8 5
rPA102-ALA 0, 12 7/7.sup.a 6 rPA102-PRO 0, 12 8/8 7 rPA102-GLY 0,
12 8/8 8 BioThrax 0, 12 8/8 .sup.aOne animal in each of Group 2 and
Group 5 died prior to the initiation of the study.
Example 5
Immunogenicity and Efficacy of rPA102 Vaccine Candidate
Formulations in Rabbits
[0142] The immunogenicity and efficacy of the rPA102 vaccine
candidate formulations were also evaluated in the New Zealand White
(NZW) rabbit lethal aerosol B. anthracis spore challenge model.
[0143] Forty-eight NZW rabbits were randomly assigned to 6 groups
of 8 animals (equal number of males and females per group). Animals
were immunized on days 0 and 14 with different rPA102 formulations
and BioThrax, as a positive control. Four rPA102 vaccine
formulations, prepared in alanine (rPA-ALA), proline (rPA-PRO),
glycine (rPA-GLY), or phosphate spike buffer (rPA102-Pi) were used.
Each formulation was administered at 1:4 dilution of formulation
that contained 100 .mu.g rPA102/Alhydrogel with 750 .mu.g aluminum
per 0.5 mL dose. BioThrax was also diluted 1:4 prior to
administration. A negative control group of animals was
administered the Alhydrogel adjuvant only. Animals were challenged
with 200 LD.sub.50 of aerosolized B. anthracis spores (Ames strain;
LD.sub.50 of 1.05.times.10.sup.5 CFU) on day 28, and observed for
mortality for 14 days. Serum samples were collected prior to the
first immunization and on day 27. Immune response was assessed by
anti-PA IgG ELISA and TNA. The study design is outlined in Table
2.
TABLE-US-00003 TABLE 2 Design of Rabbit Immunogenicity and Efficacy
Study Aerosol Blood Vaccination Anthrax Collection Group No. of
Schedule Challenge Schedule Number Animals Vaccine (Days) (Day)
(Days) 1 8 rPA102-ALA 0, 14 28 -4, 27 2 8 rPA102-PRO 0, 14 28 -4,
27 3 8 rPA102-GLY 0, 14 28 -4, 27 4 8 BioThrax 0, 14 28 -4, 27 5 8
rPA102-Pi 0, 14 28 -4, 27 6 8 Alhydrogel 0, 14 28 -4, 27
[0144] All animals that were immunized with rPA102-ALA, rPA102-PRO,
rPA102-GLY, rPA102-Pi or BioThrax survived following lethal aerosol
challenge with B. anthracis spores, while all animals in the
adjuvant-only immunized group died (data not shown). All four
rPA102 formulations induced a robust humoral immune response, as
measured by anti-PA IgG ELISA and TNA assay (FIGS. 26 and 27).
These results are consistent with those observed in the
above-described guinea pig immunogenicity and efficacy study
(Example 4), and demonstrate that all four rPA102 formulations are
both immunogenic and protective against lethal challenge in the
rabbit model.
Example 6
Evaluation of Various rPA102 and Alhydrogel Concentrations with
Alanine
[0145] Following the identification of alanine as the most
effective stabilizing excipient, rPA102 compositions comprising
varying concentrations of rPA and Alhydrogel were evaluated.
Specifically, this study evaluated rPA102 vaccine formulations
(formulated with 20 mM Tris/0.9% NaCl (w/v), with 0.01% PS80 (w/v),
pH 7.4) with the amounts of rPA102 and Alhydrogel as provided in
Table 3 in an alanine buffer (220 mM alanine, 25 mM sodium
phosphate buffer, 0.01% PS80 (w/v), at pH 7.0), at both 2-8.degree.
C., and 25.degree. C.
TABLE-US-00004 TABLE 3 Tested rPA102 and aluminum amounts Amt.
rPA102 Amt. Aluminum 100 .mu.g rPA102 750 .mu.g Aluminum 75 .mu.g
rPA102 750 .mu.g Aluminum 50 .mu.g rPA102 750 .mu.g Aluminum 25
.mu.g rPA102 750 .mu.g Aluminum 100 .mu.g rPA102 250 .mu.g Aluminum
50 .mu.g rPA102 250 .mu.g Aluminum 25 .mu.g rPA102 250 .mu.g
Aluminum
[0146] The formulations were tested for appearance (visual), free
rPA102 (ELISA; expressed in .mu.g/mL and in % (w/v)), pH, relative
potency (mouse relative potency assay) and front faced fluorescence
(expressed as relative percent of rPA102 folded.
[0147] The available results of this ongoing study are presented in
Tables 4-7.
TABLE-US-00005 TABLE 4 Stability results for the Screening Study of
rPA102 Formulations with 750 .mu.g aluminum (2-8.degree. C.) 100
.mu.g rPA102/ 75 .mu.g rPA102/ 50 .mu.g rPA102/ 25 .mu.g rPA102/
Test 750 .mu.g Al 750 .mu.g Al 750 .mu.g Al 750 .mu.g Al Results, t
= 0 Appearance White suspension White suspension White White
suspension suspension Free rPA102, 9.6 7 2.7 0.9 .mu.g/mL (%)***
(9.6) (9.3) (5.4) (3.6) pH 6.95 7.02 6.95 7 Relative 2.97 2.43 2.24
2.07 Potency (mouse relative potency test) FFF (%) 96.6 108.4 96.6
114.2 Results (2-8.degree. C.), t = 1 month Appearance White
suspension White suspension White White suspension suspension Free
rPA102, 4.8 7.6 1.4 1.1 .mu.g/mL (%)*** (4.8) (10.1) (2.8) (4.4) pH
6.96 7.05 6.98 7.04 Relative 3.02 2.19 3.02 1.70 Potency (mouse
relative potency test) FFF (%) 104.9 109 107.6 116.4 Results
(2-8.degree. C.), t = 3 months Appearance White suspension White
suspension White White suspension suspension Free rPA102, 10 9.8 3
1.2 .mu.g/mL (%)*** (10) (13) (6) (4.8) pH 7.1 6.92 7.06 6.95
Relative 2.38 1.61 2.28 1.44 Potency (mouse relative potency test)
FFF (%) 106.9 106 110.5 112 Results (2-8.degree. C.), t = 6 months
Appearance White suspension * White * suspension Free rPA102, 10 *
4 * .mu.g/mL (%)*** (10) (8) pH 7.03 * 7.02 * Relative * ** 1.57 **
Potency (mouse relative potency test) FFF (%) 107 * 103 * * = Not
Performed at this Time Point ** = Results Not Available ***=
Percentage free rPA is a function of the concentration of the
unbound rPA divided by the concentration of rPA formulated with
Alhydrogel
TABLE-US-00006 TABLE 5 Stability Results for the Screening Study of
rPA102 Formulations with 250 .mu.g aluminum (2-8.degree. C.) 100
.mu.g rPA102/ 50 .mu.g rPA102/ 25 .mu.g rPA102/ Test 250 .mu.g Al
250 .mu.g Al 250 .mu.g Al Results, t = 0 Appearance White
suspension White suspension White suspension Free rPA102, 56 18 4
.mu.g/mL (%)*** (56) (36) (16) pH 6.86 6.92 6.96 Relative Potency
2.09 1.47 1.11 (mouse relative potency test) FFF (%) 96.6 96.6
109.4 Results (2-8.degree. C.), t = 1 month Appearance White
suspension White suspension White suspension Free rPA102, 30.4 8.1
4.4 .mu.g/mL (%)*** (30) (16) (17) pH 6.87 6.89 7.03 Relative
Potency 1.50 1.66 1.56 (mouse relative potency test) FFF (%) 102.4
102.4 109 Results (2-8.degree. C.), t = 3 months Appearance White
suspension White suspension White suspension Free rPA102, 42 13 4.9
.mu.g/mL (%)*** (42) (26) (19.6) pH 7.04 7.02 7.0 Relative Potency
2.23 1.75 * (mouse relative potency test) FFF (%) 104 104.8 107
Results (2-8.degree. C.), t = 6 months Appearance White suspension
White suspension * Free rPA102, 57 14 * .mu.g/mL (%)*** (57) (28)
pH 6.92 6.96 * Relative Potency * * ** (mouse relative potency
test) FFF (%) 102 103 * * = Not Performed at this Time Point ** =
Results Not Available ***= Percentage free rPA is a function of the
concentration of the unbound rPA divided by the concentration of
rPA formulated with Alhydrogel
TABLE-US-00007 TABLE 6 Stability results for the Screening Study of
rPA102 Formulations with 750 .mu.g aluminum (25.degree. C.) 100
.mu.g rPA102/ 75 .mu.g rPA102/ 50 .mu.g rPA102/ 25 .mu.g rPA102/
Test 750 .mu.g Al 750 .mu.g Al 750 .mu.g Al 750 .mu.g Al Results, t
= 0 Appearance White White White White suspension suspension
suspension suspension Free rPA102, .mu.g/mL 9.6 7 2.7 0.9 (%)***
(9.6) (9.3) (5.4) (3.6) pH 6.95 7.02 6.95 7 Relative Potency 2.97
2.43 2.24 2.07 (mouse relative potency test) FFF (%) 96.6 108.4
96.6 114.2 Results (25.degree. C.), t = 1 month Appearance White
White White White suspension suspension suspension suspension Free
rPA102, .mu.g/mL 4.6 7.5 1.5 1.1 (%)*** (4.6) (10) (3) (4.4) pH
7.02 7.04 7.01 7.02 Relative Potency 2.30 2.20 2.11 1.78 (mouse
relative potency test) FFF (%) 95.5 100 95.5 109 Results
(25.degree. C.), t = 3 months Appearance White White White White
suspension suspension suspension suspension Free rPA102, .mu.g/mL 7
7.6 3.5 1.1 (%)*** (7) (10) (7) (4.4) pH 7.1 7.02 7.09 7.08
Relative Potency 1.30 0.99 1.88 0.98 (mouse relative potency test)
FFF (%) 90 90 94.6 99.6 Results (25.degree. C.), t = 6 months
Appearance * * * * Free rPA102, .mu.g/mL * * * * (%)*** pH * * * *
Relative Potency * ** 1.15 * (mouse relative potency test) FFF (%)
* * * * * = Not Performed at this Time Point ** = Results Not
Available ***= Percentage free rPA is a function of the
concentration of the unbound rPA divided by the concentration of
rPA formulated with Alhydrogel
TABLE-US-00008 TABLE 7 Stability Results for the Screening Study of
rPA102 Formulations with 250 .mu.g aluminum (25.degree. C.) 100
.mu.g rPA102/ 50 .mu.g rPA102/ 25 .mu.g rPA102/ Test 250 .mu.g Al
250 .mu.g Al 250 .mu.g Al Results, t = 0 Appearance White
suspension White suspension White suspension Free rPA102, 56 18 4
.mu.g/mL (%)*** (56) (36) (16) pH 6.86 6.92 6.96 Relative Potency
2.09 1.47 1.11 (mouse relative potency test) FFF (%) 96.6% 96.6%
109.4% Results (25.degree. C.), t = 1 month Appearance White
suspension White suspension White suspension Free rPA102, 33 6.5 4
.mu.g/mL (%)*** (33) (13) (16) pH 6.93 6.95 6.97 Relative Potency
1.90 1.83 1.18 (mouse relative potency test) FFF (%) 95.5% 93.4%
101.2% Results (25.degree. C.), t = 3 months Appearance White
suspension White suspension White suspension Free rPA102, 44 12 3.2
.mu.g/mL (%)*** (44) (24) pH 7.06 6.98 6.96 Relative Potency 1.10
1.20 0.63 (mouse relative potency test) FFF (%) 86.7% 89.% 93%
Results (25.degree. C.), t = 6 months Appearance * * * Free rPA102,
* * * .mu.g/mL (%)*** pH * * * Relative Potency * 0.87 ** (mouse
relative potency test) FFF (%) * * * * = Not Performed at this Time
Point ** = Results Not Available ***= Percentage free rPA is a
function of the concentration of the unbound rPA divided by the
concentration of rPA formulated with Alhydrogel
[0148] Relative potency is assessed by a mouse Relative Potency
test. The mouse Relative Potency test utilizes three components:
mouse vaccination and serum collection, serum testing by the Toxin
Neutralization Assay and analysis of data to assign the test
vaccine potency. Based on available data collected for five
reference vaccine samples stored at t=0, 1 and 3 months at
2-8.degree. C., the CV of the mouse relative potency test is
estimated to be approximately 15%. Mouse relative potency data for
the vaccine formulation containing 50 .mu.g rPA/750 .mu.g aluminum
indicates that this formulation is stable when stored for up to 6
months at 2-8.degree. C. and 25.degree. C. Available mouse relative
potency data for the remaining formulations containing 750 .mu.g
aluminum indicate that they are stable when stored for up to 3
months at 2-8.degree. C. (6 month stability data not yet
available). Formulations containing 100 .mu.g rPA exhibited an
approximate 50-60% decrease in relative potency when stored for 3
months at 25.degree. C.; these formulations also contained
relatively high levels of unadjuvanted ("free") rPA 0% w/v).
[0149] Formulations containing 250 .mu.g aluminum contained also
relatively high levels of free PA (16-57% w/v free rPA) compared to
formulations containing 750 .mu.g aluminum (3-10% w/v free rPA).
Formulations were selected to maximize stability and to minimize
free rPA to enhance manufacturing consistency (i.e., contain
<10% of unadjuvanted rPA). Based on these observations the
following formulations were selected: 25, 50 and 75 pg of rPA per
750 .mu.g aluminum.
Example 7
Selection of a Preservative
[0150] Since multi-dose vials require a preservative, further
developmental studies are required. We have identified two
preservatives, benzethonium chloride (phemerol) and
2-phenoxyethanol that are used in licensed multi-dose vaccines.
BioThrax contains benzethonium chloride, and therefore, we have
experience with this preservative. We will initially conduct
antimicrobial effectiveness testing on each of these two
preservatives in the rPA anthrax vaccine to determine what level of
each of the preservatives is required in the vaccine. Antimicrobial
effectiveness testing is followed by a formulation study, which is
used to down-select to one of the two preservatives. A stability
study is then completed to demonstrate that the rPA anthrax vaccine
containing the preservative is stable.
[0151] 2-Phenoxyethanol and benzethonium chloride are tested for
minimum acceptable levels which are defined as the ability of the
preservative to pass acceptance criteria for USP51. rPA will be
formulated to contain concentrations of either 2-phenoxyethanol or
benzethonium chloride in either the alanine buffer or the glycine
buffer, as described in FIG. 21. AME testing will be executed. In
addition, we will determine the lower limits of quantitation and
variability of the assays to measure preservative levels. This
study will allow us to determine the amounts of 2-phenoxyethanol
and benzethonium chloride to use in the formulation studies.
[0152] Formulation studies for rPA anthrax vaccine focus on two
primary areas: 1) the effect of each of the preservatives on the
binding isotherm between rPA and Alhydrogel, and 2) the effect of
each of the preservatives on the stability of the rPA102 under
accelerated degradation conditions. The initial studies focus on
determining if the addition of preservative alters the amount of
rPA that is bound per mg of aluminum.
[0153] After analysis of the binding isotherm in the presence of
preservative, a series of experiments will be performed to examine
stability of rPA under forced degradation conditions. Forced
degradation of the rPA in formulation buffer containing excipient
will be conducted at temperatures of 25 and 35.degree. C. All
formulations will be tested using release assays as well as by the
additional characterization assays described in FIG. 22.
[0154] The rPA in the vaccine will be analyzed using standard
physicochemical methods including front faced fluorescence, epitope
exposure, native elutability, relative levels of bound versus free
rPA, and physical structure of the rPA measured by SDS-PAGE and
peptide digest with detection by mass spectrometry (MS). Specific
deamidation sites on the rPA will be identified using peptide
digest followed by liquid chromatography-mass spectrometry (LC-MS),
while deamidated residues will be identified by mass
spectrometry-mass spectrometry (MS-MS).
[0155] The study on the stability of rPA under forced degradation
conditions will be performed over one month. Selection of a
preservative will be based on a combination of the results of the
initial antimicrobial effectiveness testing and the effects of each
preservative on the relative stability of the rPA. The baseline for
comparison of the effects of preservative on formulated rPA will
be, for example, a vaccine comprising approximately 200 .mu.g/mL
rPA102 and approximately 0.5 mg/mL aluminum hydroxide, formulated
using the alanine and glycine based buffers without
preservative.
[0156] A stability study will be performed using the preservative
selected from the prior two studies. The rPA anthrax vaccine will
be formulated in alanine with the selected preservative. The
formulations and testing schedule is presented in FIG. 23.
Sequence CWU 1
1
21735PRTArtificial SequenceSynthetic polypeptide 1Glu Val Lys Gln
Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser 1 5 10 15 Gln Gly
Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30
Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35
40 45 Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala
Ile 50 55 60 Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr
Thr Phe Ala 65 70 75 80 Thr Ser Ala Asp Asn His Val Thr Met Trp Val
Asp Asp Gln Glu Val 85 90 95 Ile Asn Lys Ala Ser Asn Ser Asn Lys
Ile Arg Leu Glu Lys Gly Arg 100 105 110 Leu Tyr Gln Ile Lys Ile Gln
Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125 Gly Leu Asp Phe Lys
Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140 Val Ile Ser
Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150 155 160
Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165
170 175 Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly
Tyr 180 185 190 Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro
Trp Ile Ser 195 200 205 Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr
Lys Ser Ser Pro Glu 210 215 220 Lys Trp Ser Thr Ala Ser Asp Pro Tyr
Ser Asp Phe Glu Lys Val Thr 225 230 235 240 Gly Arg Ile Asp Lys Asn
Val Ser Pro Glu Ala Arg His Pro Leu Val 245 250 255 Ala Ala Tyr Pro
Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser 260 265 270 Lys Asn
Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr 275 280 285
Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290
295 300 Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser
Val 305 310 315 320 Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val
Ala Ile Asp His 325 330 335 Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp
Ala Glu Thr Met Gly Leu 340 345 350 Asn Thr Ala Asp Thr Ala Arg Leu
Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365 Thr Gly Thr Ala Pro Ile
Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380 Leu Gly Lys Asn
Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln 385 390 395 400 Leu
Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410
415 Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile
420 425 430 Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys
Gln Leu 435 440 445 Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala
Thr Tyr Asn Phe 450 455 460 Glu Asn Gly Arg Val Arg Val Asp Thr Gly
Ser Asn Trp Ser Glu Val 465 470 475 480 Leu Pro Gln Ile Gln Glu Thr
Thr Ala Arg Ile Ile Phe Asn Gly Lys 485 490 495 Asp Leu Asn Leu Val
Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp 500 505 510 Pro Leu Glu
Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525 Ile
Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535
540 Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln
545 550 555 560 Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn
Ile Tyr Thr 565 570 575 Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met
Asn Ile Leu Ile Arg 580 585 590 Asp Lys Arg Phe His Tyr Asp Arg Asn
Asn Ile Ala Val Gly Ala Asp 595 600 605 Glu Ser Val Val Lys Glu Ala
His Arg Glu Val Ile Asn Ser Ser Thr 610 615 620 Glu Gly Leu Leu Leu
Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser 625 630 635 640 Gly Tyr
Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655
Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660
665 670 Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu
Tyr 675 680 685 Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val
Thr Lys Glu 690 695 700 Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp
Thr Ser Thr Asn Gly 705 710 715 720 Ile Lys Lys Ile Leu Ile Phe Ser
Lys Lys Gly Tyr Glu Ile Gly 725 730 735 24PRTArtificial
SequenceSynthetic peptide 2Arg Lys Lys Arg 1
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