Note: Descriptions are shown in the official language in which they were submitted.
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
VACCINE STABLE COMPOSITIONS
OF USE
FIELD OF THE INVENTION
The present invention relates generally to the field of protein storage,
especially following industrial scale preparation and where said proteins have
form part of a vaccine composition intended for administration to mammals,
especially human beings.
BACKGROUND OF THE INVENTION
Vaccines are commonly formulated using an adjuvant. A common
adjuvant-type is the aluminium based colloids, usually referred to as alum.
More specifically these are usually aluminium hydroxide (aluminium
oxyhydroxide) (also called alhydrogel) and aluminium phosphate (also called
adju-phos). For example, vaccines useful against organisms such as anthrax
(Bacillus anthracis) are commonly formulated with alhydrogel, which binds the
anthrax antigen used in such vaccines (so called subunit vaccines).
In the recent past, advances in vaccine therapy have made it possible
to produce a variety of proteins for use in vaccines, such as use of
recombinant Protective Antigen (rPA) from anthrax, using recombinant DNA
technology. Because of the relatively large size and complex nature of
proteins generally, the formulation of such proteins in vaccines poses some
unique problems. To remain antigenically active, a protein formulation must
preserve the conformational integrity of the protein representing the basic
antigenic structure in the vaccine. Degradation of proteins can involve
1
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
chemical instability (for example, where bond formation or cleavage results in
a new antigenic structure) or physical instability (for example, changes in
the
higher order structure of the protein). Physical instability can result from,
for
example, denaturation or aggregation of the protein(s) present in the vaccines
while chemical instability can result from chemical reactions that either
break
the amino acid chain of the protein or serve to modify one or more amino acid
side groups present on the protein (which groups may be essential for
immunogenic activity).
Lyophilization (commonly termed "freeze-drying") is a well known
technique for preserving proteins and operates by removing water from the
protein composition of interest. It is a process by which the material to be
dried is first frozen and then the frozen solvent (ice in the case of water)
is
removed by sublimation under high vacuum. Additives may be included in
pre-lyophilized formulations to enhance stability during the freeze-drying
process and/or to improve stability of the lyophilized product upon storage.
Unfortunately, such procedures have heretofore proved unreliable for the
lyophilization and storage of certain antigenic proteins useful in vaccines,
especially for anthrax vaccines, where stability of the lyophilized powder has
resulted in significant loss of activity following reconstitution.
The present invention solves at least some of the problems heretofore
encountered with lyophilization of protein formulations, especially of
formulations containing recombinant protective antigen (rPA) of anthrax
(Bacillus anthracis), by providing a formulation that retains antigenic
activity
and thus utility as a vaccine even with long storage in the lyophilized state,
so
that the reconstituted vaccine composition is still highly useful for
protection of
mammals, such as human being, against infection by anthrax bacilli.
BRIEF SUMMARY OF THE INVENTION
2
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
In one aspect, the present invention relates to a stable reconstituted
formulation or vaccine composition, which may be isotonic, comprising an
antigen or immunogen for use in a vaccine, preferably an anthrax antigen for
use in a vaccine against anthrax, more preferably anthrax Protective Antigen,
and most preferably a recombinant Protective Antigen (rPA), in an amount of
about 50 g/mL to about 400 g/mL, preferably about 200 g/ml, and a
pharmaceutically acceptable carrier, wherein this formulation has been
reconstituted from a lyophilized mixture or composition of the anthrax, or
other, antigen. In one embodiment, the reconstituted formulation may contain
trehalose, such as trehalose in an amount that provides lyoprotection of the
lyophilized mixture or composition.
In preferred embodiments of the reconstituted formulation, said
formulation contains a salt, such as phosphate or NaCI or both, a detergent,
such as Tween, and an adjuvant, such as alhydrogel (alum), chitosan, MPL or
CpG. In preferred embodiments, the adjuvant is CpG1018 or alhydrogel or,
especially preferred, both.
The vaccine according to the present invention may comprise
adjuvants additional to alum. Examples of such adjuvants are well known in
the art and include oligonucleotides, especially so-called "CpG"
oligonucleotides, especially phosphorothioate oligonucleotides, most
preferably oligonucleotides which target Toll Like Receptor 9 receptors. Other
additional adjuvants that may be present include saponins, PCPP polymer,
lipopolysaccharide derivatives, MPL [Monophosphoryl Lipid A], MDP muramyl
di-peptide (MDP) , t-MDP , Flagellin and flagellin-fusion proteins, IC31, OM-
174 and Leishmania elongation factor, ISCOMS, chitosan, SB-AS2, AS02,
SB-AS4, non-ionic block copolymers and SAF [Syntex Adjuvant Formulation].
When an additional adjuvant is employed, the additional
adjuvant is preferably incorporated with the stabilised sub-unit vaccine
although the additional adjuvant may alternatively be present in the separate
adjuvant and/or diluent composition.
3
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
In certain embodiments, the stable sub-unit vaccine composition or
formulation may also incorporate an agent to reduce or prevent agglomeration
during preservation, for example a small amount of a non-ionic surfactant,
especially an ethoxylated sorbitan ester, for example
polyoxyethylenesorbilan monolaurate, typically comprising about 20
ethyleneoxy units.
In another aspect, the present invention relates to a method for
preparing a stable lyophilized vaccine formulation, comprising the steps of:
(a) forming a composition of an anthrax antigen containing rPA and a
Iyoprotective amount of a reducing sugar; and
(b) freeze-drying said composition to form a stable lyophilized vaccine
formulation.
In preferred embodiments, the mixture or composition to be lyophilized
also contains a buffer, such as phosphate, a salt, such as NaCl, a detergent,
such as Tween, and an adjuvant, such as alhydrogel (alum), chitosan, mpl or
CpG, with CpG1018 and alhydrogel being especially preferred.
In one embodiment, the lyophilized composition also comprises a
bulking agent added in an amount sufficient to provide mass to the lyophilized
composition and contribute to the physical structure of the lyophilized bulk
material.
In a further aspect, the present invention relates to a kit comprising a
container that holds a lyophilized mixture or composition of an anthrax, or
other, vaccine containing rPA, trehalose and a pharmaceutically acceptable
carrier; and a set of instructions for reconstituting the lyophilized
composition
with a diluent to an rPA concentration of about 200 g/mL. In one
embodiment, a diluent is provided with the kit. Preferably, the diluent
contains
an adjuvant, such as alhydrogel.
4
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
The present invention also relates to a method of protecting against, or
treating, a microbial, especially a bacterial, infection in a mammal,
comprising
administering to a mammal either at risk of developing or afflicted with such
infection a therapeutically-effective amount of the vaccine composition of the
invention. In further embodiments thereof, the bacterial infection is an
anthrax
(Bacillus anthracis) infection and/or the mammal is a human being.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the amino acid sequence of a recombinant Protective
Antigen (rPA) from Bacillus anthracis (anthrax).
DEFINITIONS
As used herein, a "stable" formulation is one wherein the antigenic
protein, such as rPA, retains its physical and chemical stability and
integrity
upon storage, such as in the form of a lyophilized powder, solid or cake.
Analytical techniques for measuring protein stability are known in the art and
will not be described in detail herein. See, for example, Peptide and Protein
Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York,
N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).
For example, a "stable" formulation may be one wherein less than about 10%
and preferably less than about 5% of the protein is present as an aggregate in
the formulation. In other embodiments, any increase in aggregate formation
following lyophilization and storage of the lyophilized formulation can be
determined. For example, a "stable" lyophilized formulation may be one
wherein the increase in aggregate in the lyophilized formulation is less than
about 5% and preferably less than about 3%, when the lyophilized formulation
is stored at 2-8 C for at least 6 months, preferably 9 months, more
preferably
up to one year or longer, such as at least about 16 months.
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
As used herein, the term "about" means an amount that does not
substantially reduce the effectiveness of the component whose range or
amount is being recited by more than 10%. Wherever an amount or range is
recited using the term "about" it is to be understood that the invention
specifically contemplates the same range or amount as if the term "about" had
not been used. Thus, "in about the range of' and "in the range of" are
alternative embodiments of the methods and formulations of the invention.
As used herein, a "reconstituted" formulation is one that has been
prepared by dissolving a lyophilized protein formulation in a diluent such
that
the protein is dispersed in the reconstituted formulation. The reconstituted
formulation in suitable for administration to a patient.
As used herein "isotonic" refers to a formulation or composition of the
invention having substantially the same osmotic pressure as human blood.
Isotonic formulations will generally have an osmotic pressure from about 250
to 350mOsm/Kg. Isotonicity can be measured using a vapor pressure or ice-
freezing type osmometer, for example.
As used herein, the term "Iyoprotectant" means a molecule that, when
combined with an immunogenic protein, such as rPA, of the invention,
substantially reduces, if not prevents, chemical and/or physical instability
of
said protein upon Iyophilization and subsequent storage, sufficient to
maintain
the immunogenic properties of the protein and thereby insure continued utility
as a vaccine. Examples of such lyoprotectants include non-reducing sugars,
such as sucrose or trehalose, amino acids, such as monosodium glutamate or
histidine, methylamines like betaine, Iyotropic salts, like magnesium sulfate,
polyols, like trihydric or higher sugar alcohols, including glycerin,
erythritol,
glycerol, arabitol, xylitol, sorbitol, and mannitol, glycols, like propylene
glycol
and polyethylene glycol, and combinations of any or all of these.
As used herein, the term "lyoprotecting amount" refers to the amount of
a Iyoprotectant, for example, trehalose, that when added to an immunogen,
6
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
such as rPA, of the invention, substantially preserves the physical and
chemical stability and integrity of the immunogen upon Iyophilization and
storage so that, once reconstituted, said immunogen retains its immunogenic
properties sufficiently to permit its use in a vaccine.
As used herein, the term "diluent" means a carrier that is
pharmaceutically acceptable (safe and non-toxic for administration to a
human) and is useful for the preparation of a reconstituted formulation.
Exemplary diluents include sterile water, bacteriostatic water for injection
(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile
saline
solution, Ringer's solution or dextrose solution.
As used herein, the term "preservative" means a compound that can be
added to the diluent to essentially reduce bacterial action in the
reconstituted
formulation, thus facilitating the production of a multi-use reconstituted
formulation, for example. Examples include octadecyldimethylbenzyl
ammonium chloride, hexamethonium chloride, benzalkonium chloride (a
mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups
are long-chain compounds), and benzethonium chloride. Other types of
preservatives include aromatic alcohols such as phenol, 2-phenoxyethanol,
thimerosal, benzethonium chloride, formaldehyde, butyl and benzyl alcohol,
allyl parabens such as methyl or propyl paraben, catechol, resorcinol,
cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative
herein is 2-phenoxyethanol.
As used herein, the term "bulking agent" refers to a compound that
adds mass to the lyophilized mixture or composition and contributes to the
physical structure of the lyophilized cake (e.g. facilitates the production of
an
essentially uniform lyophilized cake which maintains an open pore structure).
Exemplary bulking agents include mannitol, glycine, dextran, polyethylene
glycol and xorbitol. "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment include
7
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
those already with the disorder as well as those in which the disorder is to
be
prevented.
As used herein the term "effective amount" or "therapeutically effective
amount" means a dosage sufficient to treat, inhibit, or alleviate one or more
symptoms of a bacterial infection, especially anthrax infection. The precise
dosage may vary with such factors as the age, immune system status,
general health, and environmental circumstances of the patient, the nature
and extent of the infection (or anticipated infection) and the availability of
subsequent treatment/vaccination.
DETAILED DESCRIPTION OF THE INVENTION
The antigen to be formulated is generally prepared using techniques
well known in the art, including isolation from the organism as well as
synthesis by recombinant technology or direct chemical synthesis of the
polypeptide used in the antigen. The Protective Antigen used in the present
invention was prepared by recombinant technology and is referred to herein
as recombinant Protective Antigen (rPA).
In accordance with the present invention, there is provided a stable
reconstituted formulation, comprising a sub-unit vaccine antigen, for example,
an anti-microbial antigen, such as an anthrax antigen containing recombinant
Protective Antigen (rPA), and a pharmaceutically acceptable carrier, which
reconstituted formulation has been prepared from a lyophilized composition of
said anthrax antigen and a lyoprotective amount of trehalose. In separate and
preferred embodiments of such formulation or composition, intended for use
as a vaccine, rPA is present in the reconstituted formulation in the range of
about 100 to 300 g/ml, preferably about 150 to 250 pg/ml, most preferably
about 200 g/ml.
8
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
The reconstituted anthrax vaccine formulation of the invention may also
contain trehalose. In one embodiment, the trehalose is present in the
reconstituted formulation in the range of about 3% to 7%, preferably in the
range of about 4% to 6%, more preferably in the range of about 4.5% to 5.5%,
and most preferably at about 5% (w/v).
The stable reconstituted vaccine formulation of the invention is one
reconstituted from a lyophilized composition that was maintained at a
temperature of at least 5 C for a period of at least 1 month prior to
reconstitution, or at least 25 C for a period of at least 1 month prior to
reconstitution, or wherein said temperature is at least 30 C, 35 C, 40 C, 45
C,
50 C, 55 C, 60 C, 65 C, 70 C, or even higher, and for periods of at least 1
month, 2 months, 3 months, 4 months, 5 months, 6 months, 7, months, 8
months, 9 months, 10 months, or even longer, such as a year, or 16 months
or at least 2 years.
The reconstituted anthrax vaccine formulation of the invention may also
contain a buffer. In specific embodiments thereof, the buffer (or salt) is
phosphate at a concentration of in the range of 3 mM to 7 mM, preferably in
the range of 4 mM to 6 mM, more preferably in the range of 4.5 mM to 5.5
mM, with concentrations of about 4 mM and about 5.25 mM being especially
preferred. In other preferred embodiments, the reconstituted formulation
comprises NaCl, for example, at about 0.39%. The reconstituted formulation
of the invention may also contain a detergent, such as Tween 20. In one
specific but non-limiting example thereof Tween 20 is present at about 0.02%.
The reconstituted anthrax vaccine formulation of the invention may also
contain an adjuvant, especially one selected from the group alhydrogel
(alum), chitosan, mpl and CpG, with CpG and alhydrogel preferred. In one
embodiment, the adjuvant is CpG1018, for example, present at about 2
mg/ml. In another embodiment, the adjuvant is alhydrogel, for example,
present at about 0.26% (w/v).
9
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
A complete but non-limiting example of such a formulation is one that
contains 200 g/ml rPA, 2 mg/ml CpG1018, 5% trehalose, 5.25 mM
phosphate, 0.39% NaCl, 0.02% Tween 20, 0.26% w/v alhydrogel (see
Example 1). In one embodiment of a vaccine formulation of the invention, the
reconstituted formulation is isotonic.
The present invention also provides a method for preparing a stable
reconstituted formulation, comprising the steps of:
(a) lyophilizing a mixture or composition of a sub-unit vaccine antigen,
for example, an anti-microbial antigen, such as an anthrax antigen containing
rPA, and a lyoprotective amount of trehalose; and
(b) reconstituting the lyophilized mixture or composition of step (a) in a
diluent such that the reconstituted formulation is stable and has an rPA
concentration of between about 0.1 and 10 times the rPA concentration in the
mixture or composition before lyophilization.
In other examples of the methods of the invention, the diluent used in
step (b) for reconstituting the mixture of step (a) comprises an adjuvant,
preferably selected from alhydrogel (alum), chitosan, MPL or CpG, most
preferably alhydrogel. In non-limiting examples, alhydrogel is present in the
range of about 0.2% to about 0.3%, or is present in the range of about 0.25%
to about 0.3%, or preferably is present at about 0.26%.
In other such examples, the diluent comprises a salt, such as
phosphate and/or NaCl. In selected examples, the phosphate is present in the
range of about 3 to 7 mM, or is present in the range of about 4 to 6 mM, or is
present in the range of about 4.5 to 5.5 mM, or the phosphate is preferably
present at about 5 mM. In other such examples, the NaCl is present in the
range of about 0.3% to about 0.5%, or is present in the range of about 0.35%
to about 0.45%, or is preferably present at about 0.39%. In a preferred
embodiment, the diluent has a pH about 7.4.
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
In a preferred embodiment, the diluent contains 0.26% alhydrogel in 5
mM phosphate and 0.39% NaCl, pH 7.4.
Sub-unit vaccines containing anti-microbial antigens and which can be
employed are usually recombinant protein-based antigens. Examples of such
anti-microbial antigens include Hepatitis B protective antigens, Herpes
Simplex Virus antigens, Influenza antigens, Congenital cytomegalovirus
(CMV) antigens, Tuberculosis antigens, HIV antigens, Diphtheria antigens,
Tetanus antigens, Pertussis antigens and Yersinia pestis protective antigens,
such as antigens comprising one, two or more antigenic proteins, for example
those disclosed in patent application W096/28551, incorporated herein by
reference. Most preferably, the antigen is an anthrax protective antigen, such
as recombinant protective antigen (rPA), especially that having the amino acid
sequence of SEQ ID NO: 1.
The present invention further provides a method for preparing a stable
lyophilized vaccine formulation, comprising the steps of:
(a) forming a mixture or composition of a sub-unit vaccine antigen, for
example, an anti-microbial antigen, such as anthrax antigen containing rPA,
and a lyoprotective amount of a reducing sugar; and
(b) freeze-drying said mixture or composition to form a stable
lyophilized vaccine formulation.
In specific examples of any of the methods of the invention, the
composition of step (a) contains rPA in the range of about 200 to 600 g/ml,
or about 300 to 500 g/ml, or about 350 to 450 g/ml, or preferably at about
400 g/ml. In other examples of the methods of the invention, the composition
of step (a) contains trehalose is present in the range of about 2 to 20%, or
in
the range of about 4 to 18%, or about 6 to 16%, or about 8 to 14%, or about 8
to 12%, or about 9 to 12%, or preferably at about 10% (w/v).
In other embodiments of the method, the composition or mixture of
step (a) contains a salt, preferably phosphate and/or NaCI (or saline). In
11
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
examples thereof, the phosphate is present in the range of about 0.3 to 0.7
mM, or in the range of about 0.35 to 0.65 mM, or about 0.4 to 0.6 mM, or
about 0.45 to 0.55 mM, or preferably is present at about 0.5 mM. In additional
such examples, the NaCl is present in the range of about 7 to 13 mM, or
about 8 to 12 mM, or about 9 to 11 mM, or about 9.5 to 10.5 mM, or
preferably is present at about 10 mM.
In other examples of the methods of the invention, the mixture or
composition of step (a) contains an adjuvant, preferably wherein the adjuvant
is selected from the group consisting of alhydrogel (alum), chitosan, mpl or
CpG. In preferred embodiments thereof, the adjuvant is CpG, such as
CpG1018. In examples thereof, CpG1018 is present in the range of about 2
mg/ml to about 6 mg/ml, or in the range of about 3 mg/ml to about 5 mg/ml, or
in the range of about 3.5 mg/ml to about 4.5 mg/ml, or CpG1018 is preferably
present at about 4 mg/ml.
Examples of suitable oligonucleotide compounds which may be
employed as additional adjuvants include those described in US6,207,648,
US6,589,940, US7,255,868 and US7,276,489 (the disclosures of which are
incorporated herein by reference).
In certain embodiments, the stabilized vaccine composition may also
incorporate an agent to reduce or prevent agglomeration during preservation,
for example a small amount of a non-ionic surfactant, especially an
ethoxylated sorbitan ester, for example polyoxyethylenesorbilan
monolaurate, typically comprising about 20 ethyleneoxy units.
In additional examples of the method, the mixture or composition of
step (a) further comprises a detergent, for example, Tween 20. In non-limiting
examples, Tween 20 is present in the range of about 0.3% to about 0.5%, or
is present in the range of about 0.35% to about 0.45%, or preferably is
present at about 0.04%.
12
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
In an especially preferred embodiment of the methods of the invention,
the mixture or composition in step (a) comprises 400 g/ml rPA, 4 mg/ml
CpG1018, 10% trehalose, 0.5 mM phosphate, 10 mM NaCl, and 0.04%
Tween 20.
The present invention, also provides for a composition suitable for
lyophilization, comprising an anthrax antigen containing recombinant
Protective Antigen (rPA) and a lyoprotective amount of trehalose. In non-
limiting embodiments of this composition, rPA is present in the range of about
100 fLg/mL to about 700 FLg/mL, or is present in the range of about 200 to 600
g/ml, or is present in the range of about 250 to 550 g/ml, or is present in
the
range of about 300 to 500 g/ml, or is present in the range of about 350 to
450 g/ml, is preferably present at about 400 pg/ml. In other examples of the
methods of the invention, the mixture or composition of step (a) contains
trehalose is present in the range of about 2 to 20%, or in the range of about
4
to 18%, or about 6 to 16%, or about 8 to 14%, or about 9 to 12%, or
preferably at about 10% (w/v).
In keeping with the invention, the lyoprotectant is added to the pre-
Iyophilized formulation or composition to be lyophilized. In preferred
embodiments, this additive is a non-reducing sugar, for example, sucrose or
trehalose, the latter being especially preferred. In one embodiment, the
amount of lyoprotectant added is such that, upon reconstitution, the resulting
formulation is isotonic. In addition, the amount of sugar added must be
sufficient to prevent the undesirable degradation or aggregation, for example,
of the antigenic protein, such as rPA, often occurring on lyophilization of
such
proteins.
In preferred embodiments of the invention, it may be desirable to add a
surfactant, detergent, to the pre-lyophilized composition and/or the
reconstituted formulation. Examples of useful surfactants include nonionic
surfactants such as polysorbates (e.g. polysorbates 20 (or Tween 20) or 80);
poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS);
13
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,
or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine;
linoleyl-,
myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palnidopropyl-, or
isostearamidopropyl-betaine (e.g lauroamidopropyl); myristamidopropyl-,
palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-
, or disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and
copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 and the
like). Tween 20 is a preferred surfactant or detergent. The amount added is
sufficient to reduce aggregation of the reconstituted protein and minimizes
the
formation of particulates after reconstitution. For example, the surfactant or
detergent may be present in the pre-lyophilized composition in an amount
from about 0.001-0.5%, and preferably from about 0.005-0.05%.
In specific embodiments of the invention, the lyoprotectant (such as
trehalose) may be used in combination with a bulking agent (e.g. mannitol or
glycine) to prepare the pre-lyophilization formulation. Use of such bulking
agents may facilitate production of a more uniform lyophilized cake (i.e.,
without excessive pockets and the like).
In other embodiments, the composition contains a salt, preferably
phosphate and/or NaCl (or saline). In examples thereof, the phosphate is
present in the range of about 0.3 to 0.7 mM, or in the range of about 0.35 to
0.65 mM, or about 0.4 to 0.6 mM, or about 0.45 to 0.55 mM, or preferably is
present at about 0.5 mM. In additional such examples, the NaCl is present in
the range of about 7 to 13 mM, or about 8 to 12 mM, or about 9 to 11 mM, or
about 9.5 to 10.5 mM, or preferably is present at about 10 mM.
In other examples, the composition contains an adjuvant, preferably
wherein the adjuvant is selected from the group consisting of alhydrogel
(alum), chitosan, mpl or CpG. In preferred embodiments thereof, the adjuvant
is CpG, such as CpG1018. In examples thereof, CpG1018 is present in the
14
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
range of about 2 mg/ml to about 6 mg/ml, or in the range of about 3 mg/ml to
about 5 mg/ml, or in the range of about 3.5 mg/ml to about 4.5 mg/ml, or
CpG1018 is preferably present at about 4 mg/ml.
In additional examples, the composition of further comprises a
detergent, for example, Tween 20. In non-limiting examples, Tween 20 is
present in the range of about 0.3% to about 0.5%, or is present in the range
of about 0.35% to about 0.45%, or preferably is present at about 0.04%.
In an especially preferred embodiment, the composition suitable for
lyophilization comprises 400 g/ml rPA, 4 mg/ml CpG1018, 10% trehalose,
0.5 mM phosphate, 10 mM NaCl, and 0.04% Tween 20.
The present invention also provides a method for preparing such a
composition, said method comprising essentially the procedure of step (a)
recited elsewhere herein for methods of the invention.
The phosphate present in said composition to be lyophilized can serve
the purposes of buffering the composition as well as the vaccine formulation
produced after reconstitution. However, phosphate is not intended as a
limiting buffer and other buffers may be used either in place or phosphate or
in combination with it. Examples of such buffers include histidine, phosphate,
Tris, citrate, succinate and other organic acids. The buffer concentration can
be from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM,
depending, for example, on the buffer and the desired isotonicity of the
vaccine formulation (e.g. of the reconstituted formulation). Such buffers may
also provide a lyoprotective effect in addition to that of trehalose.
The present invention also provides a kit comprising:
(a) a container that holds a lyophilized composition of an anthrax
vaccine containing rPA, trehalose and a pharmaceutically acceptable carrier;
and
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
(b) instructions for reconstituting the lyophilized composition with a
diluent to an rPA concentration of about 200 g/mL.
Such a kit may further comprise a container of a diluent suitable for
reconstituting the lyophilized composition. Specific embodiments of such
diluent are as described elsewhere herein for the diluent used in the methods
of the invention.
The kits of the invention commonly contain the lyophilized composition
in a vial or other suitable container, including, for example, bottles, vials
(e.g.
dual chamber vials), syringes (such as dual chamber syringes) and test tubes.
The container may be formed from a variety of materials such as glass or
plastic. The container holds the lyophilized formulation and the label on, or
associated with, the container may indicate directions for reconstitution
and/or
use. For example, the label may indicate that the lyophilized formulation is
reconstituted to protein concentrations as described above. The label may
further indicate that the formulation is useful or intended for subcutaneous
administration. The container holding the formulation may be a multi-use vial,
which allows for repeat administrations of the reconstituted formulation. The
kit, which is an article of manufacture, may further comprise a second
container comprising a suitable diluent of a type as disclosed herein. Upon
mixing of the diluent and the lyophilized formulation, the final rPA
concentration in the reconstituted formulation will generally be in a range as
disclosed herein, but generally at about 200 g/ml. The kit may further
include
other materials desirable from a commercial and user standpoint, including
other buffers, diluents, filters, needles, syringes, and package inserts with
instructions for use.
Generally, vaccines are prepared as injectables, in the form of aqueous
solutions or suspensions. Vaccines in an oil base are also well known such as
for inhaling. Solid forms which are dissolved or suspended prior to use may
also be formulated. Pharmaceutically acceptable carriers, diluents and
16
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
excipients are generally added that are compatible with the active ingredients
and acceptable for pharmaceutical use.
The pharmaceutical compositions and/or formulations useful herein
contain a pharmaceutically acceptable carrier, including any suitable diluent
or
excipient, which includes any pharmaceutical agent that does not itself induce
the production of antibodies harmful to the individual receiving the
composition, and which may be administered without undue toxicity.
Pharmaceutically acceptable carriers include, but are not limited to, liquids
such as water, saline, glycerol and ethanol, and the like, including carriers
useful in forming sprays for nasal and other respiratory tract delivery or for
delivery to the ophthalmic system. A thorough discussion of pharmaceutically
acceptable carriers, diluents, and other excipients is presented in
REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.
current edition). Those described elsewhere herein are especially preferred
for use in the formulations, composition s, methods and kits of the invention.
Vaccine compositions may further incorporate additional substances to
stabilize pH, or to function as adjuvants, wetting agents, or emulsifying
agents,
which can serve to improve the effectiveness of the vaccine.
Vaccines are generally formulated for parenteral administration and are
injected either subcutaneously or intramuscularly. Such vaccines can also be
formulated as suppositories or for oral administration, using methods known in
the art, or for administration through nasal or respiratory routes.
The amount of vaccine sufficient to confer immunity to pathogenic
bacteria, viruses, or other microbes is determined by methods well known to
those skilled in the art in view of the guidance provided herein. This
quantity will
be determined based upon the characteristics of the vaccine recipient and the
level of immunity required (as already noted above). Where vaccines are
administered by subcutaneous or intramuscular injection, a range of .5 to 500
g
purified protein may be given. As useful in the present invention, such
dosages
17
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
are commonly sufficient to provide about 1 g, possibly 10 g, even 50 g, and
as much as 100 g, up to 500 g of immunogenic protein, or immunogenic
polypeptide, or immunogenically active fragments thereof. In addition, more
than
one such active material may be present in the vaccine. Thus, more than one
antigenic structure may be used in formulating the vaccine, or vaccine
composition to use in the methods disclosed herein. This may include two or
more individually immunogenic proteins or polypeptides, proteins or
polypeptides showing immunogenic activity only when in combination, either
quantitatively equal in their respective concentrations or formulated to be
present in some ratio, either definite or indefinite.
A vaccine composition for use in the processes disclosed herein may
include one or more immunogenic proteins, one or more immunogenic
polypeptides, and/or one or more immunogenically active immunogens
comprising antigenic fragments of said immunogenic proteins and polypeptides,
the latter fragments being present in any proportions selected by the use of
the
present invention. The exact components, and their respective quantities,
making up the vaccines, and vaccine compositions, useful in the methods of the
present invention are determined, inter alia, by the nature of the disease to
be
treated or prevented, the severity of such condition where it already exists,
the
age, sex, and general health of the recipient, as well the personal and
professional experience and inclinations of the researcher and/or clinician
utilizing these methods.
For a vaccine of the present invention, such as a sub-unit vaccine for
use against anthrax, specific non-limiting examples of this vaccine
composition are those wherein said rPA is present at about 10 to 300 g/ml,
preferably 50 to 300 pg/ml, or wherein said rPA is present at about 100 to 300
pg/ml, more preferably about 150 to 250 g/ml, and most preferably wherein
said rPA is present at about 200 g/ml.
In other examples, the dose of antigen, preferably rPA, to be
administered is at least about 5 ,ug, or at least about 10 ,ug, or at least
about
18
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
25 pg, or at least about 50 ,ug, or at least about 75 ,ug, or at least about
100
pg, or at least about 150 pg, or at least about 200 pg, with a preferred dose
of
about 100 pg. A preferred dose volume is about 0.5 ml.
The present invention further relates to a vaccine comprising a purified
antigen, preferably an anthrax antigen, more preferably anthrax protective
antigen (PA) and most preferably a recombinant anthrax protective antigen
(rPA), such as that described in WO 2007/122373, pub. 1 November 2007,
(the disclosure of which is hereby incorporated by reference in its entirety)
bound to an adjuvant, preferably an alum-based adjuvant. In a preferred
embodiment, the present invention relates to a sub-unit vaccine comprising
alum-bound rPA, at least about 2 mM phosphate salt and at about pH 7.1.
The vaccine formulation used for in vivo administration are commonly
sterile, which can be accomplished by filtration through sterile filtration
membranes, prior to, or following, lyophilization and reconstitution.
Alternatively, sterility of the entire composition may be accomplished by
autoclaving the ingredients, except for protein, at about 120 C for up to
about
30 minutes, as one non-limiting example.
For the actual lyophilization procedure itself, different commercially
devices are available, including Hu1150.TM. (Hull, USA) or GT20.TM.
(Leybold-Heraeus, Germany) BOC Edwards, Christ GTS, FTS Lyostar freeze-
dryers. Freeze-drying is accomplished by freezing the formulation and
subsequently subliming ice from the frozen content at a temperature suitable
for primary drying. Under this condition, the product temperature is below the
eutectic point or the collapse temperature of the formulation. Typically, the
shelf temperature for the primary drying will range from about -30 to 25 C.
(provided the product remains frozen during primary drying) at a suitable
pressure, ranging typically from about 50 to 250 mTorr. The formulation, size
and type of the container holding the sample (e.g., glass vial) and the volume
of liquid will mainly dictate the time required for drying, which can range
from
a few hours to several days (e.g. 40-60 .hrs). A secondary drying stage may
19
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
be carried out at about 0-40 C, depending primarily on the type and size of
container and the type of protein employed. However, it was found herein that
a secondary drying step may not be necessary. For example, the shelf
temperature throughout the entire water removal phase of Iyophilization may
be from about 15-30 C (e.g., about 20 C). The time and pressure required
for secondary drying will be that which produces a suitable lyophilized cake,
dependent, e.g., on the temperature and other parameters. The secondary
drying time is dictated by the desired residual moisture level in the product
and typically takes at least about 5 hours (e.g. 10-15 hours). The pressure
may be the same as that employed during the primary drying step. Freeze-
drying conditions can be varied depending on the formulation and vial size.
The resulting lyophilized powder will commonly possess a moisture content
thereof that is less than about 5%, and preferably less than about 3%.
Examples of freeze-drying cycles useful in the methods of the invention
are shown in Tables 1 and 2:
Table 1. Freeze Drying Cycle
STEP TIME (h/m Temp ( C) Jac (pB) Run Time
Load 10m 10m
Ramp 4h -40 h 10m
Hold 4h -40 8h 10m
Hold 45m -40 200 8h 55m
Ramp 40m -30 200 9h 35m
Hold 18h -30 200 27h 35m
Ramp 8h 20m 20 200 35h 55m
Hold 4h 20 200 39h 55m
Ramp 1h 5 200 0h 55m
Hold 35m 5 200 1 h 55m
Stoppering 5 200
Freeze drying of the samples of Table 1 was accomplished using an LS40
freeze drier obtained from Severn Sciences Ltd.
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
Table 2. Freeze-dry cycle.
STEP Time Temp Vac
(Min) C) (mTorr
Load 0 20
Ramp 240 -40
Hold 180 -40
Hold 45 -40 113
Ramp 40 -30 113
Hold 2400 -30 113
Ramp 500 20 113
Hold 480 20 113
Ramp 60 5 113
Hold 35 5 113
Stoppering 5 113
This modified cycle of Table 2 was performed using a Christ freeze-dryer.
In each cycle, the product was stoppered under vacuum before
introducing air into the chamber. Aluminium seals were then applied to the
vials and crimped, on removal from the freeze dryer.
The reconstituted formulation is administered to a patient in need of
treatment with the protein, preferably a human, in accord with known
methods, such as intravenous administration as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal,
oral, topical, or inhalation routes.
The lyophilized vaccine compositions prepared by the methods of the
invention are typically maintained in the freeze-dried state until the time of
use. When ready for administration to a patient, the lyophilized formulation
is
reconstituted with a diluent as described herein.
Reconstitution commonly occurs at room temperature to ensure
complete hydration, although other temperatures may be employed as
desired. The time required for reconstitution will depend, e.g., on the type
of
diluent, amount of excipient(s) and protein, such as rPA. Examples of diluents
21
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
include sterile water, bacteriostatic water for injection (BWFI), a pH
buffered
solution (e.g. phosphate-buffered saline is especially preferred), sterile
saline
solution, Ringer's solution or dextrose solution. The diluent may optionally
contain a preservative. Aromatic alcohols such as benzyl or phenol alcohol
are preferred preservatives. The amount of preservative employed is
determined by assessing different preservative concentrations for
compatibility with the protein and preservative efficacy testing. For example,
if
the preservative is an aromatic alcohol (such as benzyl alcohol), it can be
present in an amount from about 0.1-2.0% and preferably from about 0.5-
1.5%, but most preferably about 1.0-1.2%.
The present invention further provides a method of protecting against,
or a method of treating, a microbial infection, preferably a bacterial
infection,
comprising administering to a mammal at risk of such infection a
therapeutically-effective amount of a stable vaccine formulation as described
herein. The infecting organism may be any one or more of those recited
herein and the formulation being one recited herein and/or a formulation
reconstituted from a vaccine composition or mixture freeze-dried according to
the methods of the invention. In a preferred embodiment, the bacterial
infection is an anthrax infection (i.e., caused by Bacillus anthracis). In one
example, the animal to be protected is a mammal, especially a human patient.
A preferred formulation for use in such treatment is a vaccine composition
containing 200 g/ml rPA, 2 mg/ml CpG1018, 5% trehalose, 5.25 mM
phosphate, 0.39% NaCl, 0.02% Tween 20, 0.26% w/v alhydrogel.
In carrying out the procedures of the present invention it is of course to
be understood that reference to particular buffers, media, reagents, and the
like are not intended to be limiting, but are to be read so as to include all
related materials that one of ordinary skill in the art would recognize as
being
of interest or value in the particular context in which that discussion is
presented. For example, it is often possible to substitute one buffer system
for
another and still achieve similar, if not identical, results. Those of skill
in the
art will have sufficient knowledge of such systems and methodologies so as to
22
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
be able, without undue experimentation, to make such substitutions as will
optimally serve their purposes in using the methods and procedures disclosed
herein, where said materials are not essential to the invention.
The invention is described in more detail in the following non-limiting
example. It is to be understood that these methods and examples in no way
limit
the invention to the embodiments described herein and that other embodiments
and uses will no doubt suggest themselves to those skilled in the art.
Example 1
Stability of Trehalose-containing Lyophilized Vaccine
400 g/ml rPA, 4 mg/ml CpG1018 in 10% trehalose, 0.5 mM
phosphate, 10 mM NaCl, 0.04% Tween 20 was lyophilized. This formulation
composition prior to lyophilization is twice the concentration of the
reconstituted material. This formulation was then reconstituted with 0.26%
alhydrogel in 5mM phosphate, 0.39% saline, pH 7.4. The reconstituted
formulation was 200 g/ml rPA, 2mg/mI CpG1018 in 5% trehalose, 5.25 mM
phosphate, 0.39% NaCl, 0.02% Tween 20, 0.26% alhydrogel. All % values
are w/v.
At 70 C, rPA retained potency after up to 8 months. Because 70 C is
above the Tg (glass transition temperature, where the trehalose sugar glass
starts to soften and is no longer brittle) it would have been expected to find
increased degradation but no such increased degradation was observed.
Thus, this lyophilized formulation was unexpectedly stable in the presence of
trehalose.
23
CA 02750321 2011-07-20
WO 2010/084298 PCT/GB2009/050051
Example 2
Stability of the Lyophilized Vaccine
Female adult A/J mice were immunized on day 0 intra-muscularly (i.m.)
with 0.1 ml of the respective samples indicated in Table 3, which were
formulated with diluent prior to administration. All the mice were challenged
with B. anthracis STI strain spores on day 21 and survival was determined on
day 11.
The results of Table 3 show a much greater stability for a lyophilized
vaccine than would have been expected based on previous experience with
such vaccines (compare, for example, the results for storage at 70 C with that
for 5 C. The diluent was comprised of: 0.26% (w/w) alhydrogel (0.4% AI(OH)3)
in 0.39% (w/v) sodium chloride buffered with 5 mM phosphate, pH 7.4. The
naive animals were negative controls that had not received any treatment but
were challenged at the day 21 with the same dose of spores as the test
animals.
Table 3. Results of immunization following storage.
Sample Storage Storage Time Challenge Survival
Temperature (months) (day 11)
C
Lyo rPA/trehalose 5 C 16 10/10
+ diluent
(lot#050207A)
Lyo rPA/trehalose 55 C 16 10/10
+ diluent
(lot#050207A)
Lyo rPA/trehalose 5 C 7 10/10
+ diluent
(lot#290807A)
Lyo rPA/trehalose 70 C 7 10/10
+ diluent
(lot#290807A)
Naive N/A N/A 0/5
24
