Note: Descriptions are shown in the official language in which they were submitted.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
Compositions of A-Beta Peptide and Processes for Producing Same
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates generally to pharmaceutical compositions comprising
proteins, which are useful in raising a mammalian antibody response. More
specifically,
this invention relates to pharmaceutically acceptable compositions comprising
an amount
of amyloid beta peptide effective to elicit an immunogenic response in a
mammal and a
pharmaceutically acceptable diluent. Preferably, the diluent is a sterile
parenterally
acceptable aqueous phase.
State of the Art
Amyloid-beta peptide, also known as A-beta or A(3 peptide, is a cleavage
product
of the amyloid precursor protein (APP). It is the principal component of
amyloid
plaques in the mammalian brain that are fundamentally involved in and
characteristic of
2o Alzheimer's disease. A[3 peptide is a 39-43 amino acid chain varying in
length owing to
the variability of its processing from APP by several proteases.
Several mutations within the APP protein have been correlated with the
presence
of Alzheimer's disease. See, e.g., Goate et al, Nature 349, 704)(1991)
(valine~~~ to
isoleucine); Chartier Harlan et al. Nature 353, 844 (1991) (valine~l~ to
glycine); Murrell
et al., Science 254, 97 ( 1991 ) (valine~ 1' to phenylalanine); Mullan et al.,
Nature Genet. 1,
345 (1992) (a double mutation changing lysisne595-methionine596 to
asparagine59s-
leucine596). Such mutations are thought to cause Alzheimer's disease by
increased or
altered processing of APP to A[3 peptide, particularly processing of APP to
increased
3o amounts of A~342 and A~343. Mutations in other genes, such as the
presenilin genes, PS 1
and PS2, are thought indirectly to affect processing of APP to generate
increased
amounts of A~342 and A(343 (see Hardy, TINS 20, 154 (1997)). The observations
indicate that A(3 peptide, and particularly A(342, is a causative element in
Alzheimer's
disease. In the brain, the A(3 peptide aggregates and forms the amyloid
deposits
comprising the peptide organized into fibrils of (3-pleated sheet structures.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
2
The impetus of recent therapeutic research on the treatment or prevention of
Alzheimer's disease has focused on halting or slowing the production of A(3
peptide in
the brain or in blocking its post-release processing or deposition into
amyloid plaques.
One therapeutic approach of particular importance to the application of this
invention is
the use of A(3 peptide in inducing an immune response of the body against it.
See, for
example, PCT Publication No. W099/27944, which is incorporated by reference in
its
entirety for all purposes.
The present invention is directed to novel and unexpected methods for
practicing
the invention described in PCT Publication No. W099/27944. In particular, it
involves
administering certain formulations of the long forms of A(3 peptide to the
patient to
induce an immune response. However, as has been noted in the art, the longer
forms of
A(3 peptide are difficult to solubilize in conventional formulation systems.
Specifically, Hilbich, et al., J. Mol. Biol., 218 ( 1 ), pp. 149-64, ( 1991 )
report that
while A(31-43 peptide is soluble to a degree in pure water, the addition of
ionic
components, such as buffers or salts, or organic solvents cause the peptide to
precipitate
out of solution in the form of an amorphous aggregate. For example, Hilbich
found
phosphate-buffered saline ("PBS," which in this instance contained 137mM NaCI,
3 mM
KCI, 8 mM Na2HP04~2H20 and 2 mM KHZP04 at pH 7.5) rendered 90-94% of the
peptide in the composition insoluble. PBS is a conventional carrier for
parenteral
compositions, approximating the tonicity and pH level of the living system.
Five (5)
mM NaCI caused 42-50% of the peptide to precipitate. (Ibid., p.153, Table 2).
The
solution of peptide in pure water would be hypotonic and the pH of such a
solution was
determined to be 5.5 by Hilbich (idem.). Typically, the pH of the blood supply
in man is
about 7.4. Dyrks, et al. have also reported A(342 is insoluble at
physiological conditions.
Dyrks, T., Weidemann, A., Multhaup, G., et al. EMBO J.7, p. 949-57 (1988).
The conformation of A~3 peptide in the solution can be measured using circular
dichroism (C.D.) spectroscopy. Conformational studies of A~3 peptide and
fragments
using C.D. are reported by Hilbich, et al, idem. See also, monograph by M.
Manning
entitled: Protein Structure and Stability Assessment by Circular Dichrosim
Spectroscopy, from Biocatalyst design for Stability and Specificity; Himmel,
M. E. and
Georgiou, G., eds., ACS Symposium Series 516 (1993) at p. 36. This reference,
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
hereinafter referred to as the Manning reference, is hereby incorporated by
reference in
its entirety for all purposes.
Kline, et al., U.S. Patent Nos. 5,851,996 ('996 patent) and 5,753,624 ('624
patent) describe administration of very minute amounts (10-2mg or less) of A(3
peptide or
a fragment thereof administered sublingually in a liquid or solid carrier,
such as a
phenylated saline solution. The '996 patent states that the amyloid beta
protein "exists in
various structural forms" (col. 2, line 31 ) that can be used to treat
Alzheimer's disease. It
is not elsewhere defined what is meant by various structural forms, neither
are any
characterized beyond the 28 amino acid fragment used in the examples. The
doses of A~3
peptide are stated in the '996 patent to be from 10-x° to 10-2 mg (col.
8, lines 442-43).
In view of the above, the prior art has demonstrated the difficulty in
dissolving
and maintaining dissolution of A(3 peptide. Additionally, the lack of
solubility of the
long forms of A(3 peptide has presented difficulties from a sterilization and
standardization standpoint. Most standard methods of sterilization are
incompatible with
peptide formulations, including irradiation, autoclaving and chemical
sterilization
techniques, such as ethylene oxide gas and glutaraldehyde, all of which cause
peptide
degradation. Therefore, filtration of the peptide would be the method of
choice for
2o sterilization of a formulation of A(3 peptide. Unfortunately, the
insolubility of A(3
peptide causes clogging of the filtration membranes and prevents recovery of
sufficient
quantities of A(3 peptide for a commercial scale process.
SUMMARY OF THE INVENTION
This invention is directed to the surprising and unexpected discovery that
aqueous solutions comprising high concentrations of A(3 peptide can be
prepared by
adjusting the acidity/basicity of such an aqueous solution to a pH effective
to solubilize
A(3 peptide. Preferably, the pH is adjusted to a pH range of from about 8.5 to
about 12,
and more prefereably from about pH 9 to 10.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
This invention is further directed to the discovery that the solubilized
solutions of
A(3 peptide lend themselves to sterile filtration through a suitable micropore
filter with at
least 50% recovery of the A(3 peptide after the sterile filtration.
Preferably, at least about
70% of A(3 peptide is recovered after sterile filtration and, more preferably
at least 90%.
Such sterile solutions can be formulated as pharmaceutical compositions
comprising a
sufficient amount of A(3 peptide to effect an immunogenic response when
administered
to a mammal. Preferably, such administration is by parenteral administration,
in the
form of a suspension composition.
to Thus, in one aspect of the composition invention, after sterile filtration
the pH of
the composition is adjusted to a physiologically acceptable pH to form a
peptide
suspension containing at least 0.1 mg/ml of A(3 peptide. The composition being
useful
for parenteral administration. The pH of a suspension composition is between
about pH 5
and about 7, preferably between 5.5 and 6.5. A more preferred composition
comprises a
15 sufficient amount of QS-21 in conjunction with A(3 peptide to form a
visually clear,
sterile suspension.
This invention is still further directed to the discovery that the solubilized
and
sterile solutions of A[3 peptide can be lyophilized to provide for lyophilized
formulations
2o comprising A(3 peptide. These compositions can be reconstituted at the
appropriate time
to provide for an aqueous composition comprising A(3 peptide.
In another of its composition aspects, this invention is directed to an
aqueous
solution comprising at least 0.01 mg/ml of A(3 peptide wherein said aqueous
solution is
25 maintained at a pH sufficient to solubilize said A(3 peptide. Preferably,
the solution is
maintained at such a suitable pH by use of an effective amount of a
pharmaceutically
acceptable buffer.
In another of its composition aspects, this invention is directed to a sterile
3o aqueous solution comprising at least 0.01 mg/ml of A(3 peptide wherein said
aqueous
solution is maintained at a pH sufficient to solubilize said A(3 peptide.
Preferably, the
solution is maintained at such a pH by use of an effective amount of a
pharmaceutically
acceptable buffer.
CA 02374897 2001-11-22
WO 00/72870 PCT/iJS00/15302
In still another of its composition aspects, this invention is directed to
lyophilized
compositions comprising a lyophilized composition comprising A(3 peptide which
composition is prepared by the process of:
a) freezing a sterile aqueous solution comprising at least 0.01 mg/ml
of A(3 peptide wherein said aqueous solution is maintained at a pH sufficient
to
solubilize said A(3 peptide; and
b) lyophilizing the frozen composition prepared in a) above.
Preferably, the compositions of this invention comprise a long form (as
defined
below) of A(3 peptide. More preferably the composition comprises a
pharmaceutically
acceptable buffer which is preferably selected from the group consisting of
amino acids,
salts and derivatives thereof; pharmaceutically acceptable alkalizers, alkali
metal
hydroxides and ammonium hydroxides, organic and inorganic acids and salts
thereof;
and mixtures thereof.
In yet another of its composition aspects, this invention is directed to a
composition comprising an aqueous solution comprising at least 0.01 mg/ml of
A(3
peptide wherein said aqueous solution is maintained at a pH sufficient to
solubilize said
2o A(3 peptide and further wherein said A(3 peptide is substantially in a
random coil
conformation.
The compositions of this invention can be formulated into pharmaceutical
compositions suitable for delivery to a mammal having Alzheimer's disease or
at risk of
developing Alzheimer's disease. The composition aspects of the invention are
directed
to pharmaceutical compositions which are in a soluble random coil conformation
of A(3
peptide or a stable, aqueous suspension of at least 0.1 mg/ml of A(3 peptide
suspended in
said composition, or a lyophilized composition, any or all of which may be
sterile and
pareterally administerable.
In one of its process aspects, this invention is directed to a process for
preparing a
sterile composition of a long form of A(3 peptide comprising:
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
adjusting the pH of an aqueous solution sufficient to solubilize the A(3
peptide therein;
dissolving into the solution an amount of the A(3 peptide sufficient to
achieve an immunogenic concentration for a mammal;
filtering the resulting solution through a uniform pore size membrane,
said pore size being in a range capable of excluding bacteria and passing
substantially all of the A(3 peptide through the membrane; and
optionally, for solutions containing 0.1 mg/mL or more of A(3 peptide,
adjusting the pH of the resulting solution to between about pH 5 to about pH 7
to
obtain a peptide suspension.
In another of its process aspects, this invention is directed to a process for
preventing or treating Alzheimer's disease in a mammal which method comprises
administering to said mammal a sufficient amount of a sterile aqueous
composition
comprising at least 0.05 mg/ml of A(3 peptide to induce an immunogenic
response in said
mammal.
Most preferably, the filtration processes of this invention employ A(3 peptide
which is substantially in the random coil conformation.
Brief Description of the Drawings
Figure 1 is a C.D. spectrum showing the mean residue ellipticity measurement
plotted as a function of wavelength for two different solutions of A~342. The
dotted line
shows the absorbance at pH 6 and is attributable to the (3-pleated sheet form
of the
molecule. The solid line is a plot of the absorbance of a solution of A(342 at
pH 9 and is
indicative of the random coil conformation of the peptide.
Figure 2 is a plot of A(342 placed into a solution against the peak area
calculation
3o for the amount of dissolved peptide as determined by reverse phase high
performance
liquid chromatography, demonstrating the solubility of A~342.
Detailed Description of the Invention
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
7
This invention is directed to compositions and methods employing
aqueous compositions comprising therapeutically effective concentrations of
A(3 peptide.
However, prior to discussing this invention in further detail, the following
terms will first
be defined.
Definitions:
The term "substantial identity" means that two peptide sequences, when
optimally aligned, such as by the programs GAP or BESTFIT using default gap
weights,
1 o share at least 65 percent sequence identity, preferably at least 80 or 90
percent sequence
identity, more preferably at least 95 percent sequence identity or more (e.g.,
99 percent
sequence identity or higher). Preferably, residue positions which are not
identical differ
by conservative amino acid substitutions.
For sequence comparison, typically one sequence acts as a reference sequence,
to
which test sequences are compared. A suitable reference sequence would be the
human
A(3 peptide sequence, specifically the 42 amino acid sequence as reported
below. Other
suitable forms would be the truncated forms: such as A(339, or the extended
form, A(343
(with an additional threonine group at the C-terminal end). When using a
sequence
comparison algorithm, test and reference sequences are input into a computer,
subsequence coordinates are designated, if necessary, and sequence algorithm
program
parameters are designated. The sequence comparison algorithm then calculates
the
percent sequence identity for the test sequences) relative to the reference
sequence,
based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the
local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),
by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by
the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
USA
85:2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer
Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally
Ausubel et
al., supra). One example of an algorithm that is suitable for determining
percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
8
analyses is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov~. Typically, default program parameters can be
used to
perform the sequence comparison, although customized parameters can also be
used.
For amino acid sequences, the BLASTP program uses as defaults a wordlength (W)
of 3,
an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff,
Proc. Natl. Acad. Sci. USA 89, 10915 (1989)).
For purposes of classifying amino acid substitutions as conservative or
nonconservative, amino acids are grouped as follows: Group I (hydrophobic side
chains):
1 o norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side
chains): cys, ser, thr;
Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn,
gln, his, lys,
arg; Group V (residues influencing chain orientation): gly, pro; and Group VI
(aromatic
side chains): trp, tyr, phe. Conservative substitutions involve substitutions
between
amino acids in the same class. Non-conservative substitutions constitute
exchanging a
member of one of these classes for a member of another.
APp69s, App7sy and APP~~~ refer, respectively, to the 695, 751, and 770 amino
acid residue long polypeptides encoded by the human APP gene. See Kang et al.,
Nature
325, 773 (1987); Ponte et al., Nature 331, 525 (1988); and Kitaguchi et al.,
Nature 331,
530 (1988). Amino acids within the human amyloid precursor protein (APP) are
assigned numbers according to the sequence of the APP770 isoform.
In the present invention and in the literature, the weight of A(3 peptide
represents
about 70% to about 85% A~3 peptide and about 15% to about 30% salt and water.
This is
determined by amino acid analysis and/or elemental nitrogen analysis. For
example,
when 0.1 mg A(342 peptide is corrected for the peptide content, this
represents 0.075 mg
of A~342 peptide and 0.025 mg water and salt; 0.6 mg A(340 peptide represents
0.45 mg
of A(340 peptide and 0.15 mg of water and salt; and 2.0 mg A(342 peptide
represents 1.5
mg of A(342 peptide and 0.5 mg of water and salt.
A(3 peptide, as used in this invention, refers to those segments of the A(3
peptide
capable of forming a (3-pleated sheet conformation and raising an immunogenic
response
when administered either alone or in conjunction with an adjuvant to a mammal.
It is
within the ordinary skill of the art to determine (3-pleated sheet
conformation as
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
9
described herein by use of, for example, circular dichroism measurements.
Immuongenicity may be determined as described in the Biological Activity
sections of
the Examples given below.
The term "long forms of A(3" peptide includes any of the naturally occurring
forms of A(338, A(339, A(340, A(341, A(342, and A(343 and peptide sequences
substantially identical thereto, and preferably the human forms. A(339, A(340,
A(341,
A~342 and A(343 refer to A~3 peptide containing amino acid residues 1-39, 1-
40, 1-41, 1-
42 and 1-43, respectively, amino acids being truncated from the C-terminal end
of the
to peptide. Thus, A(341, A(340 and A(339 differ from A(342 by the omission of
Ala, Ala-Ile,
and Ala-Ile-Val respectively from the C-terminus, as can be seen by reference
to the A(3
peptide sequence below. A~343 differs from A(342 by the presence of a
threonine residue
at the C-terminus. The sequences of these peptides and their relationship to
APP are
illustrated in Figure 1 of Hardy et al., TINS 20, 155 (1997).
A(342 has the sequence: H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-
Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-
Ile-Ile-Gly-Leu-Met-V al-Gly-Gly-V al-V al-IIe-Ala-OH.
2o The term "long forms of A[3" peptide also includes analogs thereof. Analogs
include allelic, species and induced variants. Analogs typically differ from
naturally
occurring peptides at one or a few positions, often by virtue of conservative
substitutions.
Analogs typically exhibit at least 80 or 90% sequence identity with natural
peptides.
Some analogs also include unnatural amino acids or modifications of N or C
terminal
amino acids. Examples of unnatural amino acids are a, a.-disubstituted amino
acids, N-
alkyl amino acids, lactic acid, 4-hydroxyproline, y-carboxyglutamate, e-N,N,N-
trimethyllysine, a -N-acetyllysine, O-phosphoserine, N-acetylserine, N-
formylmethionine, 3-methyl-histidine, 5-hydroxylysine, w-N-methylarginine.
3o A(3 may be utilized in the compositions and processes of the invention from
about
0.05 mg/ml to its upper solubility limit of about 2.0 mg/ml (referring to
Fig.2). Preferred
ranges of the peptide are from about 0.1 to about 0.8 mg/ml and more preferred
is a
range of from about 0.3 to about 0.6 mg/ml.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
It has been reported that the insoluble form of A~3 peptide found in amyloid
plaques is in the (3-pleated sheet conformation. Soto, C., et al.,
Neuroscience Letters,
186 (2-3), pp. 115-118, (1995). Also, Simmons, L.K., et al. Molec. Pharmacol.,
45 (3)
5 pp. 373-379 (1994) have reported that the (3-sheet conformation is
associated with the
neurotoxic effects of the peptide, while the random coil form is only weakly
toxic or
inactive. As noted above, the methods and compositions of this invention may
employ a
random coil conformation of A(3 peptide. Applicants demonstrate herein that
the random
coil conformation is capable of raising an immunogenic response in test
mammals. The
10 random coil conformation is most preferred in the microfiltration of the
processes.
The term "random coil" refers to an open chain conformation of the A(3
peptide.
The random coil is a secondary conformation of the peptide backbone and is not
ordered
into a regular conformation such as the a-helix or (3-pleated sheet forms.
Rather the
random coil is disordered from the hydrophobic folding and hydrogen bonding
interactions that characterize the other, more regularly arranged forms. A
random coil
may still possess some turns of the peptide backbone or partial ordering,
however, such
features are random and dynamic, and thus are not typical of all the random
coil
population. In the random coil conformation, the peptide exhibits good
solubility and
2o filterability. The a-helix and (3-pleated sheet conformations are well-
known peptide
conformations in the art. They are described, for example, in Lehninger's
Biochemistry
(2"d Ed.,Worth Publishers, 1975) at pp. 128-9 for the a-helix and for the (3-
pleated sheet
at pp. 133-4, which is incorporated by reference herein.
The random coil conformation of A~3 peptide can be characterized by its
circular
dichroism ("CD") spectrum and is easily distinguished from the (3-pleated
conformation.
As represented by a CD spectrum, a random coil is typified by a strong
negative band
between 190 and 200 nm, and little CD signal is seen at wavelengths longer
than 215
nm. If there is a CD signal at longer wavelengths is very weakly positive,
centered at
220-225 nm. This is shown in Figure 1 of the Drawings. The (3-pleated sheet
conformation on the other hand displays a sharp and strong positive band
centered at
about 200 nm. For a review of peptide secondary structure elucidation by C.D.
spectra,
please refer to the Manning reference cited supra.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
11
A(3 peptide is substantially in a random coil conformation when greater than
50%
of the A(3 peptide is the random coil conformation. Preferably, greater than
70% or 80%,
and most preferably, greater than 85% or 90% of the A~3 peptide is in the
random coil
conformation.
"Parenteral compositions" are those compositions which are sterilized and
suitable for administration directly into the body by, for example, injection
or infusion,
i.e. by routes which have immediate contact with the blood and without the
barrier or
immune system protections afforded by administration forms that enter the body
via the
dermal, mucosal, digestive or respiratory systems. For these
reasons,.sterility is a
necessity for a parenteral composition.
"Infusion", as a means of drug administration, involves a substantially
continuous
and slow flow of a drug solution into the blood stream over a relatively long
period.
"Injection", on the other hand, is a rapid administration of a unit dose of a
solution or
suspension.
Intravascular (or intravenous or IV); intramuscular (IM); intraperitoneal
(IP);
subcutaneous (SC) ; and intrasternal all refer to different modes of
adminstration of
parenteral compositions. They describe in anatomical terms the area of the
body in which
the parenteral composition is introduced by injection or infusion. It is
contemplated that
the compositions of the present invention having a physiologically acceptable
pH can be
administered by any of the above modes, depending on the individual patient
and the
judgment of the administering physician. The higher pH solutions (pH > 8) may
most
advantageously be administered by a slow IV infusion or IV drip.
In understanding the terms "buffer" and "buffering agent" as used in the
invention, it should be recalled that the titration curve of an acid or base
has a relatively
3o flat zone extending to about 1.0 pH unit on either side of the titration
midpoint. At the
midpoint, an equivalent amount of the proton-donor and proton-acceptor species
of the
acid or base are present. In this zone, the pH of the system changes
relatively little when
small increments of H+ or OH- are added. This is the zone in which a conjugate
acid-
base pair acts as a buffer, a system which tends to resist change in pH when a
given
increment of H+ or OH- is added. At pH levels outside this zone there is less
capacity of
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
12
the buffer to resist changes to the pH. A buffer's power is maximum at the pH
of the
exact midpoint of its titration curve, i.e., where there are equal
concentrations of the
proton acceptor and proton donor and the pH is equal to the pK' (the acid
dissociation
constant). Buffer preparations are described in detail in Data for Biochemical
Research,
Rex, M.C., Oxford Science Publications, 1995.
Many physiological mechanisms operate within the body to maintain blood pH
within the narrow limits of 7.35 to 7.45. While some of the major buffering
mechanisms
are based on carbonic acid or phosphoric acid equilibria, many other
mechanisms
1 o involve amino acids and proteins. For example, the pH of tears is
maintained at 7.4 by
protein buffers. Single amino acids are also useful as buffers, although they
show a more
complex titration curve on account of having proton donor and proton acceptor
atoms
within the same molecule. A molecule of this type is referred to as
zwitterionic, i.e. it
exists in a form having both positively and negatively charged sites within
the same
15 molecule. Amino acids are capable of buffering both the addition of H+ ions
or OH-
ions, as shown below.
R R
OH H + O
H2N HsN
O O
neutral zwitterion
(dipolar form)
R R R
+ OH H+ + O OH- O
HsN H HsN ' ' HzN
O O O
zwitterion
For the purposes of this invention a "pharmaceutically acceptable buffer" is
defined broadly to include conjugate acid - base pairs as well as acidic or
basic
2o compounds that have the ability to adjust or to maintain the pH of a
solution of A[3
peptide at a desired level. In the solubilization/filtration processes of this
invention, that
is pH 8.5 or above or in another aspect below pH 4.
Preferably, such buffers are selected from the group consisting of amino
acids,
25 salts and derivatives thereof; pharmaceutically acceptable alkalizers,
alkali metal
hydroxides and ammonium hydroxides, organic and inorganic acids and salts
thereof;
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
13
and mixtures thereof. Buffering agents are employed in concentrations
sufficient to reach
and maintain the desired pH and thus the concentrations are dependent on the
acidity/basicity of the individual buffer or combination chosen. Selection of
an effective
concentration is within the ordinary skill of the art, using for example, pH
meters and
titration techniques.
Examples of classes of compounds useful in the present invention are
hydroxides,
including alkali metal hydroxides and ammonium hydroxides, alkalizers known in
the
pharmaceutical arts, including but not limited to tris, sodium borate
(NaZB407) and
disodium citrate, amino acids, salts or esters or amides of amino acids and
simple
derivatives thereof, for example, N-acetyl derivatives of amino acids.
Particularly
preferred buffers are glycine (e.g., sodium glycinate), arginine and lysine,
sodium
hydroxide and ammonium hydroxide.
Examples of pharmaceutically acceptable acids for practice of the methods of
the
present invention are, without limitation, hydrochloric acid, phosphoric acid,
citric acid,
acetic acid, malefic acid, malic acid and succinic acid, and the like..
Additionally these
acids may be used to titrate the pH value of a basic solution to a lower, more
physiologically acceptable level, resulting in a peptide suspension
composition.
Conversely, basic compounds such as those listed above can be used to titrate
a
low pH value of a filtered solution to a more physiologically acceptable pH,
also
resulting in a peptide suspension.
Combinations of the pH adjusting agents are also contemplated. Conjugate acid
base pairs as discussed above and exemplified by salts such as ammonium
acetate are
also within the scope of buffering agents for the invention. Such a conjugate
pair could
be formed, for example, by the titration of a basic solution of ammonium
hydroxide with
acetic acid to a more nearly neutral solution of ammonium acetate.
As used herein, the term "tonicity modifier" includes agents that contribute
to the
osmolality of the solution. Examples of tonicity modifiers suitable in the
present
invention include, but are not limited to saccharides (sugars) such as
mannitol, sucrose
and glucose and salts such as sodium chloride, potassium chloride and the
like.
Preferably, the tonicity agent will be employed in an amount to provide a
final osmotic
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
14
value of less than about 350 mOsm/kg and more preferably between about 250 to
about
350 mOsm/kg, and most preferably between about 280 to about 320 mOsm/kg. It
will be
noted that the charged compounds that serve as buffers in the formulations can
also
affect tonicity. Thus the tonicity of a buffered solution of A(3 peptide is
first determined
before being adjusted further by addition of tonicity modifying agents.
A chelating agent may optionally be used in the processes and compositions of
the invention. Examples of preferred chelating agents include
ethylenediaminetetraacetic
acid (EDTA), and its salts (such as sodium) which are normally employed at a
1 o concentration of 0.05 to 50 mM, more preferred at concentrations of 0.05
to 10 mM, or
about 0.1 to 5 mM being most preferred. Other known chelating (or sequestering
agents)
such as certain polyvinyl alcohols can also be used.
Optionally, the compositions may contain a surfactant or detergent such as
15 polysorbate (e.g. Tween~) or 4-(1,1,4,4-tetramethylbutyl)
phenyoxypolyethoxyehthanols
(Triton~), or polymers of polyethylenepolypropylene glycols (Pluronics~). The
surfactant ranges from about 0.005 to 1%, with about 0.02 to 0.75% preferred.
A
preferred polysorbate is PS-80, which is commercially available as Tween~ 80.
2o Wetting agents are also contemplated as excipeints useful in the invention.
The
polyethylene glycols, e.g. PEG 3350, are useful in modifying the association
of A(3
particles and the solubility thereof by associating with the polymer suface
and ordering
its hydrophilic moieties in the aqueous phase. Wetting agents may be present
form 0.5 to
% (w/v).
Pharmaceutically acceptable sugars (for example sucrose, dextrose, maltose or
lactose) or pharmaceutically acceptable sugar alcohols (for example mannitol,
xylitol or
sorbitol) have no influence on the medical effects of an active ingredient. In
one aspect
of this invention, sugars or sugar alcohols having a molecular weight of less
than 500,
3o and capable of easily dispersing and dissolving in water, can be used.
Examples of
sugars and sugar alcohols usable in the present invention include xylitol,
mannitol,
solbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose,
lactose, and the
like. They can be used alone, or as a mixture of two or more of these
compounds. The
CA 02374897 2001-11-22
WO 00/72870 PCTNS00/15302
most preferred sugar is mannitol, especially in the lyophilized compositions;
sucrose is
also preferred in the solution compositions.
It has also been found that the use of QS-21, an adjuvant, in the compositions
of
5 the invention interacts with the suspended protein in such a way that a
visually clear
suspension can be formed. This is a desirable interaction in that the peptide
is in the (3-
pleated sheet conformation but is suspended in the phase in very small
particles and may
provide more advantageous properties to the composition, such as added
stability of the
suspension or improved immunogenic properties. DPPC (dipalmitoyl phosphatidyl
10 choline) is known in the art and is anticipated to interact with A(3
peptide in a similar
fashion to QS-21 to provide the same small particle suspension and allow for
use of other
of the adjuvants in the invention, while providing a visually clear
suspension.
Alternatively, other adjuvants may be used in admixture with QS-21.
15 Lyophilization is a technique well known in the pharmaceutical arts, as are
techniques for stablizing peptides during and after the lyophilization
process.
Stabilization of a protein or peptide lyophilisate in an amino acid,
saccharide matrix is
also known to those of skill in the art. See, for example: Lueckel B., et al.,
Formulations
of sugars with amino acids or mannitol - Influence of concentration ratio on
the
properties of the freeze-concentrate and the lyophilizate, PHARM. DEV.
TECHNOL.
3(3) pp. 325-336 (1998). The Royal Pharmaceutical Society of GB Symp: 'New
Analytical Approaches to the Characterization of Biotechnology Products', June
1996
Luckel B.; et al., presented: A strategy for optimizing the lyophilization of
biotechnological products, PHARM. SCI. (UK) 3(1) pp. 3-8 (1997). Both of these
references are hereby incorporated by reference in their entirety for all
purposes.
The term "pharmaceutically acceptable" modifies any composition to mean that
the composition does not impart any deleterious or untoward effect on the
subject to
which it is administered and in the context in which it is administered. The
terms
"pharmaceutically acceptable diluent" and "pharmaceutical acceptable
excipient" refer to
any compound which preserves or does not alter the activity of the active
compounds)
and does not impart any deleterious or untoward effect on the subject to which
it is
administered and in the context in which it is administered, such as a non-
toxic pH
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
16
adjusting or buffering agent or a tonicity modifying agent or chelating agent,
and the
like.
Compositions or processes "comprising" one or more recited elements may
include other elements not specifically recited. For example, a composition
that
comprises A(3 peptide encompasses both A[3 peptide in the composition as
recited and
A(3 peptide as a component of a composition having other non-recited
components.
Further definitions are as follows:
to
Ahhreviati~n l~efiniti~n
C degrees Centigrade
cc cubic centimeter
C.D. circular dichroism
pH log[H], a measure of the hydryogen ion
content
and thus the acidity or basicity of
a solution
pK' acid dissociation constant
related to pH by: pH = pk' + log [H+ acceptor]
[H+ donor]
PS80 polysorbate 80 or Tween 80~ copolymer of
polysorbate and ethylene oxide; Merck Index
monograph no. 7559 (1 1t" Ed.)
urn micron
Min minute
ml milliliter
N normality -indication of molarity of a solution
M molarity, value stated in moles/liter
mM millimoles
DMSO dimethylsulfoxide
EDTA ethylenediamine tetraacetate, usually as
disodium salt
Tris tromethamine, tris(hydroxymethyl)aminomethane
Merck Index monograph no. 9684 (1 1t" Ed.)
RP HPLC reverse phase high performance liquid chromatography
RPM revolutions per minute
CFA / IFA complete Freund's adjuvant / incomplete Freund's
adjuvant (Chang et al., Advanced Drug Delivery
Reviews
32, 173-186 (1998)
MPL 3-O-deacylated monophosphoryl lipid A (MPLTM)
(see
for example GB 2220211).
QS 21 triterpene glycoside or saponin isolated
from the bark of
the Quillaja Saponaria Molina tree of South
America (see
Kensil et al., in Vaccine Design: The Subunit
and Adju-
vant Approach (eds. Powell & Newman, Plenum
Press,
NY, 1995); US Pat. No. 5,057,540). (StimulonTM
QS-21)
FIO for information only
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
17
Filtration is the process of removing contaminants by size exclusion from
fluids
passing through a membrane filter having a uniform pore size. Although micron-
sized
particles can be removed by use of non-membrane or depth materials such as
those found
s in fibrous media, only a membrane filter, having a precisely defined pore
size, can ensure
quantitative exclusion of particles having a smaller size. Thus with membrane
filtration
it is possible to quantitatively remove bacteria from a solution when passed
through a
microfilter and thus effect sterilization. Previously with A(3 peptide
purification the
peptide was so insoluble or remained aggregated to the point that the solution
could not
to be microfiltered at a commercial scale owing to blockage of the membrane by
particulates and/ or to poor recovery of the peptide in the resulting
microfiltered solution.
Preferred filters are generally defined as those filters having the capability
to
remove particles from 0.2 microns up.
The membrane filter is uniformly cast on a substrate, and generally performs
separations based on size exclusion at the membrane surface. However, a
surface type
membrane filter may trap particles both in the depth of its structure and at
the surface of
the membrane. In size exclusion separations, particles smaller than the pores
of the
2o membrane filter pass through while larger particles are retained at the
membrane surface.
Because these defined pore size filters do not "unload," (i.e. as the filter
begins to fill up
with trapped particles, increasing the flow pressure can allow passage of
small amounts
of trapped solids) they are the devices of choice for microbiological control.
"Sterilizing
grade" surface type membrane filters are well known in the pharmaceutical
industry.
In all filtration applications, the permeability of a filter medium can be
affected
by the chemical, molecular or electrostatic properties of the filtrate,
however it has been
found that the hydrophilic microfilters used in the present invention are
stable to the high
or, conversely, low pH environment depending on the method employed and
reliably
3o remove undesired particulate matter without clogging. It is within the
skill level of the
ordinary skilled artisan to be able to select and use hydrophilic
microfilters.
Commercially available product specifications or websites such as:
http://millispider.millipore.com/corporate/sitemap.nsf/catalogs (as of May
1999) enable
the selection of filters having the desired characteristics.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
18
Examples of preferred hydrophilic filters operative in the present invention
are
Millipore Durapore~, also called Millex GV, (Millipore Corporation,
headquartered in
Bedford, MA), a polyvinylidene fluoride hydrophilic polymer having good
stability and
low protein binding characteristics and a 0.22 ~m pore diameter; Millex GNTM,
a
hydrophilic nylon material having a 0.2 ~,m pore size; and Millex GPTM, a
hydrophilic
surface modified polyethersulfone polymer of pore size 0.22 Vim. More
preferred is the
Durapore filter owing to its stability at pH 9-9.5.
Preferably, substantially all of the A(3 peptide is recovered after
filtration.
Recovering substantially all the A(3 peptide is defined to mean greater than
about 50% of
the peptide is recovered after sterile filtration. Preferably, greater than
80% of the A[3
peptide is recovered after sterile filtration, and most preferably, greater
than 90% is
recovered after filtration.
Methods
The methods of this invention involve the preparation of aqueous
compositions comprising concentrations of at least O.Olmg/ml of the A~3
peptide. Such
compositions are prepared by adjusting the pH of the aqueous solution such
that the A~3
2o peptide will dissolve therein in requisite concentrations (e.g., at a
concentration of from
about 0.1 mg/ml to about 2.0 mg/ml).
Adjusting the pH of the aqueous solution is accomplished via conventional
methods typically exemplified by the addition of either an acid or a base to
arrive at the
desired pH. Preferably, the acid or base employed is pharmaceutically
acceptable at the
amounts employed. In order to maintain the pH of the solution over prolonged
storage, it
is preferable to employ a pharmaceutically acceptable buffer in the
composition. The
selection of a suitable buffer relative to the desired pH is within the skill
of the art.
3o Preferably, the pH of the aqueous composition is adjusted to between about
pH 2
to 4 or about pH 8.5 to 12. At such pHs, the A(3 peptide is surprisingly quite
soluble.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
19
When the process of the invention is practiced at low pH (about pH 2 to 4)
then
acids such as hydrohalide acids (e.g., HCI, HBr), phosphoric acid, citric acid
and acetic
acid and other pharmaceutically acceptable acids may be employed to lower the
pH of
the solution to the desired pH. Selection of suitable acids in within the
skill of the art.
When the process of the invention is practiced at high pH (about pH 8.5 to
12),
then pharmaceutically acceptable bases such as alkali metals, ammomium
hydroxides
(e.g., NaOH, NH40H) and the like may be employed to raise the pH of the
solution to
the desired pH. At high pH, the pH of the solution in a given buffer is
preferably
to adjusted to between 8.5 to 11. More preferred is a pH level of between pH 9
and 10.
Either prior to or subsequent to pH adjustment, requisite concentrations of
the A/3
peptide are added to the solution. It is preferred, of course, to add the A(3
peptide after
pH adjustment in order to immediately solubilize the peptide. Upon addition,
gentle
15 stirring and heating may be necessary to assist in the solubilization
process.
The addition of any optional additives such as pharmaceutically acceptable
buffers, tonicity agents, adjuvants, etc. to the composition can occur at any
convenient
time prior to or subsequent the addition of the A(3 peptide.
Once the aqueous solutions described above are prepared, sterile filtration
and/or
lyophilization can proceed according to procedures well known in the art which
procedures are exemplified by the examples below.
The following solutions are preferred buffer systems for solubilizing A~3
peptide
at target concentrations ranging from 0.6 to 2.0 mg/ml peptide:
Preferred Amino Acid Compositions:
10 mM Sodium Glycinate , pH 9.0, 9.5, or 10.0
and/or with 0.02 to 1.0 % (w/v) polysorbate 80 (PS-80)
optionally containing one or more tonicity modifiers sufficient for parenteral
administration;
10 mM L=Arginine-HCI, or 10 mM L-Lysinate, both at pH 9.0, 10.0
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
and/or with 0.02 to 1.0 % (w/v) polysorbate 80 (PS-80)
optionally containing one or more tonicity modifiers sufficient for parenteral
administration
5 The compositions of the present invention are preferably stored at low
temperature to discourage degradation or aggregation of the peptide chains.
The
preferred temperature range for storage of the A(3 peptide compositions is 2
to 8° C.
The following examples illustrate practice of the invention. These examples
are
l0 for illustrative purposes only and are not intended in any way to limit the
scope of the
invention claimed.
Examples
15 Example 1 - Peptide Solubility
Peptide and Reagents
A(342 as its trifluoroacetate salt was obtained from American Peptide Co., Lot
Numbers M05503T1, M10028T1 and used without further modification. Other salts
are
available and have been successfully employed in the processes of the
invention. For
2o examples of alternative counter-ion salts of the A~3 peptide see Table 2.
All acid, base and buffer solutions were prepared from laboratory grade
reagents,
and stored as sterile filtered stock solutions for convenience. Polysorbate 80
(Tween 80,
PS80): 4% stock solutions were prepared by w/v dilution of a qualified low
peroxide PS
80 solution (10% solutions of low peroxide polysorbitan mono-oleate are
available from
Aldrich-Sigma Chemicals, for example).
Peptide Solubilization:
Approx. 500 -700 ug A(3 peptide was weighed and dissolved with the appropriate
amount of buffer solution to give a theoretical 0.6, 0.8 or 1.0 mg/ml A(342
final
concentration. The peptide was directly weighed into 4 ml Wheaton glass vials
with
screw top caps. The appropriate amount of the buffer solution was added and
the peptide
was gently mixed for 15 to 30 minutes. Solutions were visually scored for
solubility as
follows: (+) = poor solubility, cloudy suspension; (++) = clear suspension
with some
insoluble particulates; (+++) = clear solution.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
21
Table 1. Visual Solubility of Solutions
Buffer Description Target Visual
conc. Solubility
A(342
(mg/ml)
0.01 N NaOH 1.0, 0.8 +++
0.01 N NH40H 1.0, 0.8 +++
50 mM Sodium Glycinate, pH 9.0 1.0 +++
mM Sodium Glycinate, pH 9.0 1.0, 0.8, +++
0.6
10 mM Sodium Glycinate, pH 9.5 0.6 +++
10 mM Sodium Glycinate, pH 10.0 1.0, 0.8 +++
10 mM Sodium Glycinate, pH 9.0, 0.02% 1.0 +++
PS80
10 mM Sodium Glycinate, pH 9.5, 0.1 0.6 +++
% PS 80, 5 % Sucrose
10 mM Sodium Glycinate, pH 9.5, 4 % 0.6 +++
Mannitol, 1 % Sucrose
10 mM Sodium Glycinate, pH 9.5, 4 % 0.6 +++
Mannitol
10 mM Sodium Glycinate, pH 10.0, 0.02% 1.0 +++
PS80
10 mM L-Arginine-HCI, pH 9.0 0.6 +++
10 mM L-Arginine-HCI, pH 10.0 0.8 +++
10 mM Sodium L-Lysinate, pH 9.0 0.8, 0.6 +++
10 mM Sodium L-Lysinate, pH 10.0 0.8 +++
10 mM Sodium Lysinate, pH 9.5, 4 % Mannitol0.6 +++
10 mM Ammonium Acetate, pH 9.0 1.0 +++
10 mM Ammonium Acetate, pH 9.0, 0.02 1.0 +++
% PS80
50 mM Tris-HCI, pH 10.0, EDTA (0.5 mM),1.0 + +
0.02 % PS-80
10 mM Sodium Borate, pH 9.0 1.0 +++
10 mM Sodium N-Acetyl-D-Glutamine, pH 0.8 +++
9.0
10 mM Glycine-HCI, pH 3.0 1.0 ++
0.01 N HCI 1.0 +++
0.01 M Phosphoric Acid 1.0 +++
DMSO (neat) 0.6 +++
10 mM Sodium Glycinate, pH 8.0 1.0 + +
10 mM L-Arginine-HCI, pH 9.0 0.8, 1.0 ++
10 mM Sodium Ammonium Bicarbonate, pH 0.8 ++
9.0
10 mM Sodium Ammonium Carbonate, pH 0.8 ++
9.0
50 mM Tris-HCI, pH 10.0, EDTA (0.5 mM) 1.0 + +
10 mM Sodium Borate, pH 10.0 1.0 ++
10 mM L-Arginine-HCI, pH 8.0 1.0 +
10 mM Ammonium Acetate, pH 8.0 1.0 +
10 mM Ammonium Acetate, pH 8.0, 0.02 1.0 +
% PS-80
50 mM Tris-HCI, pH 8.0, pH 9.0, or pH 1.0 +
10:0
50 mM Tris-HCI, pH 8.0 or pH 9.0, with 1.0 +
0.5mM EDTA
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
22
Buffer Description Target Visual
conc. Solubility
A(342
(mg/ml)
50 mM Tris-HCI pH 8.0 or pH 9.0, with 1.0 +
0.5 mM EDTA and
0.02 % PS-80
50 mM or 100 mM NaCI, pH 9.0 1.0 +
50 mM Sodium Phosphate, pH 8.0, pH 9.0 1.0 +
or pH 10.0
50 mM Sodium Glycinate, pH 8.0 or pH 1.0 +
10.0
mM Sodium N-Acetyl-D-Glucosamine, 0.8 +
pH 9.0 or pH 10.0
10 mM Sodium N-Acetyl-D-Glutamine, pH 0.8 +
10.0
10 mM Sodium Citrate, pH 3.0 or pH 4.0 1.0 +
Sodium Acetate, pH 4.0 __ ~ _1 0
10 mM .
The solutions ranked as +++ were visually clear at the target concentration.
The
pH range was from pH 9 to pH 10 for the buffered solutions. The pH of the
inorganic
5 solutions such as NaOH and NH40H were alkaline while HCl and phosphoric acid
were
strongly acidic. Solubility of the peptide was achieved from 0.6 to 1 mg/ml
peptide at
pH values approximately > pH 9. Additives such as polysorbate 80, sucrose,
mannitol
and EDTA did not affect the solubility of the peptide and assist in increasing
tonicity and
recovery after filtration, or may act as chelating agents. Acidic solutions
(approximately
l0 pH 4 or below) also solubilize the A(342 peptide at 0.6 to 1 mg/ml.
Solutions ranked as
++ visually appeared partially soluble when the peptide was dissolved at the
target
concentration. The peptide may be soluble at a lower concentration than tested
herein.
Those solutions ranked as + visually did not achieve complete solubility at
the target
concentration.
Example 2 - Solubility of A(3 peptide in buffered solutions
A~342 (0.6, 1.0, 1.5, 2.0, 3.0 and 3.5 mg/ml) was solubilized in 10 mM sodium
glycinate buffer, pH 9Ø Each solution was centrifuged in a bench top
centrifuge
(>10,000 RPM, -~- 10 mins) as cloudy suspensions were observed for
concentrations 2.0 -
3.5 mg/ml (solutions of 0.6 - 1.5 mg/ml were visually clear).
An aliquot of the supernatant from each solution was then analyzed by RP HPLC.
Table 2 contains the tabulated peptide peak areas and a graph demonstrating
the
solubility of A~342 in sodium glycinate buffer at pH 9.
CA 02374897 2001-11-22
WO 00/72870 PCT/LJS00/15302
23
Table 2. Solubility Limits, A(342 peptide, TFA salt
A/342 HPLC Peak Area
mg/mL
( 1 OmM glycine,
pH 9)
0.6 3613553
1 5921792
1.5 8850393
2 9213446
Addionally, the A~342 peptide was purified as the trifluoroacetate, ammonium,
chloride and sodium salts. These salts were dissolved in several buffers, at a
0.45
mg/mL A(342 peptide concentration, correcting for the counterion contribution
of the
various salts. Solutions of the peptide were filtered and the recoveries of
the peptides
were determined by comparing the RP-HPLC peak areas of the peptide before and
after
filtration. All salts were soluble and readily filtered at 0.45 mg/mL, as
shown in Table 3.
Table 3. Solubility of Alternate Salts of the A(342 peptide
0.45 mg/mL A(342 Recovery (%)
Ammonium salt in 1 mM NH40H 70
Ammonium salt in 2 mM NH40H 106
Ammonium salt in 10 mM Sodium Glycinate,115
pH 9.0
Ammonium salt in 10 mM Sodium Glycinate,96
5%
Sucrose, pH 9.0
Ammonium salt in 10 mM Sodium Glycinate,101
pH 9.5
Ammonium salt in 10 mM Sodium Glycinate,106
pH 10.0
Triflouroacetate salt in 10 mM Sodium 101
Glycinate, pH 9.0
Chloride salt inl0 mM Sodium Glycinate, 101
pH 9.0
Sodium salt in 10 mM Sodium Glycinate, 100
pH 9.0
Example 3 - Recovery of Solubilized Peptide from Representative Hydrophilic
Filters
Syringe filters tested (25mm filter diameters, 3.9 cm2 filter area): Millex
GV,
0.22 uM: a hydrophilic polyvinylidene difluoride (PVDF, Durapore~) membrane
with
low protein binding properties, Millex GN, 0.20 uM: a hydrophilic nylon
membrane
with low protein binding properties, and Millex GP, 0.22 uM: a hydrophilic
surface-
modified polyethenesulfone (PES) membrane with low protein binding properties.
The above listed filters were employed as representatives of the types of
2o commercially available hydrophilic microfilters. The filtration studies
were performed
using the following solubiliztion systems:
1. 0.6 mg/ml A(342 in 0.01 N NH40H
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
24
2. 0.6 mg/ml A(342 in 10 mM sodium glycinate, pH 9.0
3. 0.6 mg/ml A(342 in 10 mM sodium lysinate, pH 9.0
4. 0.6 mg/ml A(342 in 10 mM Arginine -HC1, pH 9.0
Approximately a 2 ml volume of each A[342 solution was filtered over each
filter.
Peptide concentrations were quantitated by reverse phase chromatography (RP
HPLC).
Filter recoveries were determined by comparing the peak areas of the peptide
before and
after microfiltration and the results are shown in Tables 3 and 4 below.
1o Table 3 - Filtration Recoveries
Filter Type
Formulation (% Recoveries)
0.6 mg/ml A(3 peptide Millex GV Millex GN Millex GP
in:
mM Sodium Glycinate99.1 97.2 98.0
10 mM Arginine-HCl 91.9 86.6 92.5
10 mM Sodium Lysinate 95.0 94.7 97.4
0.01 N NH40H 102.2 104.8 105.1
Example 4 - Filtration of A(342 in Sodium Glycine and Sodium Lysine Buffers
With and
Without Additional Excipients.
In these studies 0.6 mg/ml A~i42 was solubilized in 10 mM sodium glycinate
containing either 0.1 % polysorbate 80 (PS80), 0.9 % sodium chloride or
combinations
of 0.1 % PS80, 0.9 % NaCI, 5 % sucrose; 1% sucrose and/or 4 % mannitol.
2o Approximately 2 -5 mls of each formulation was filtered through a Millex GV
filter. An
aliquot of the filtrate was centrifuged on a bench top micro-centrifuge set at
>- 10,000
RPM for ~ 3 minutes. Filter recoveries were determined by comparing the RP-
HPLC
peak areas of the peptide before and after filtration.
Table 4 - Filtration Recoveries
For Various Buffered Formulations
Formulation ID % Recovery
0.6 mg/ml A(342 in:
10 mM Sodium Glycinate, pH 9.0 104.5
10 mM Sodium Glycinate, pH 9.0, 0.1 106.2
% PS-80
10 mM Sodium Glycinate, pH 9.0, 5 % 115.4
Sucrose
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
Formulation ID % Recovery
0.6 mg/ml A~342 in:
10 mM Sodium Glycinate, 4 % Mannitol, 103.0
pH 9.5
10 mM Sodium Glycinate, pH 9.5, 4 % Mannitol,98.0
1 %
Sucrose
10 mM Sodium Glycinate, pH 9.0, 0.9 % 76.7
NaCI
10 mM Sodium Glycinate, pH 9.0, 0.1 % 65.0
PS-80, 0.9 %
NaCI
10 mM L-Lysine/Citrate, pH 9.5 93.2
10 mM L-Lysine/Citrate, 4 % Mannitol, 80.0
pH 9.5
The Millex GV filter was established as a preferred filter for. A(3
formulations
owing to good recoveries of peptide and acceptability for use in commercial
processes.
Peptide formulations containing 0.9% NaCI were visually less soluble, leading
to lower
filter recoveries by RP HPLC. To increase the solubility of the peptide in the
presence of
inorganic salts, such as sodium chloride, it may be preferably to add a
sterilized solution
of the tonicity modifying agent after the microfiltration of the peptide in
the buffer
solution.
to On the other hand the tonicity modifying agents which are saccharides
(sugars)
display a different type of solution activity, and may favor the maintenance
of peptide
suspensions. This property, known as water-ordering, tends to "hydrate" the
peptide
chain in solution so that it adopts its thermodynamically more stable
conformation, a (3-
pleated sheet.
The A~3 peptide can be filtered at pH ranges from about 8.5 to about 12,
preferably from pH 9 to pH 10 with good recoveries. Filtration can be
accomplished
with a 0.2 um Millex GV (Millipore Durapore) membrane, which is acceptable for
a
manufacturing process.
2o For a stable, biologically active formulation of A(3 peptide in a
physiologically
acceptable presentation suitable for use in a clinical setting, the initial
step in the
formulation process will preferably include a pH 8.5 to 10 sterile filtration
of the peptide.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
26
Example 5 - Soluble Liquid Formulations
Assay Methods
Three lots of A~i42 peptide were manufactured by American Peptide Co (APC).
Confirmatory formulations, chemical and biological characterization of the APC
peptide
results were reproduced with A(342 manufactured by California Peptide
Research.
Formulation descriptions are as described in Parts 1, 2 and 3 below.
Stabilty Analyses.
Formulation-specific descriptions are provided in the individual sections
below.
1o Individual vials are analyzed periodically over several months storage at 2-
8°C.
Appearance, pH, peptide concentration and purity are routinely monitored over
the
course of the stability studies. A reverse phase HPLC (RP HPLC) system was
used to
quantitate the concentration and area percent purity of the A(3 peptide. The
RP HPLC
utilizes a polymeric reverse phase column, with an ammonium bicarbonate (or
15 tris)/acetonitrile gradient elution of the peptide and detection at 220 nm.
Peptide
concentrations are measured against a reference standard; purity is calculated
as the area
percent of A(3 peptide detected in the resulting integrated chromatogram.
Characterization.
2o Characterization of the solution structure of A(342 was obtained by
circular
dichroism studies, as shown in Figure 1.
Biological Activity.
Swiss Webster mice (4 -8 mice per group) were injected with A(342 in various
25 formulations and adjuvanted with CFA/IFA, MPL (Corixa Immunochemicals) or
QS 21
(Aquila Pharmaceuticals) at antigen/adjuvant concentrations noted in the
particular
study. Injections were routinely administered biweekly at 0, 2, and 4 weeks
unless
otherwise noted. Mice were bled following the 2-week and 4-week injections.
Sera
were analyzed in a standard sandwich ELISA assay, utilizing A(342 as the
antigen and
3o HRP-conjugated goat anti-mouse IgG as the reporter antibody. Antibody
titers were
reported in scatter plots or as geometric means of the data for each of the
animals in each
group, and generally compared to titers obtained using aggregated
A(342/CFA/IFA as the
control immunogen. See Table 8 and 10 titer results.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
27
1. Formulations.
A(3 peptide was solubilized at 0.6 mg/ml in 10 mM sodium glycinate, pH 9 - 9.5
buffer and filtered through a Millex GV 0.2 um filter. The formulation was
filled at a 0.5
ml volume in 2 cc glass vials (Gensia P/N X34-113-002) and capped with gray
butyl
stoppers (Gensia P/N X66-113-030) and seals. Vials were stored at 2-
8°C.
2. Stability Testing
1 o The concentration and purity of A~342 in the formulations were monitored
by RP
HPLC. Soluble samples were analyzed either neat or after microcentrifugation
of the
samples.
The following table (Table 5) presents the analytical data for the Soluble
Liquid
Formulation. No difference was seen between samples which were or were not
centrifuged prior to analysis, suggesting continuing solubility of the peptide
at the
intended concentration. The area % purity is comparable to the reference
standard: no
significant degradation of the peptide has been seen. The soluble formulation
has
remained stable over a period of 3 months when stored at 2°C to
8°C.
2o Table 5. Results after 3 months storage
A~3
0.6 mg/ml A(342 in: AppearancepH Area concentration
%
Purity (mg/ml)
Reference Standard n/a n/a 68 % n/a
10 mM Sodium Glycinate, Clear 8.6 69% 0.65
pH 9.0
solution
3. Characterization
Characterization of the A(3 formulation by circular dichroism demonstrates
that
the peptide assumes a random coil conformation in solution, with the
characteristic
negative ellipticity absorbance between 189-205 nm.
Biological Activity
The pH 9 soluble A(342 formulation, when injected with adjuvant, raised an
antibody titer in Swiss Webster mice. Injections at biweekly intervals, (0, 2,
4 weeks) of
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
28
33 ug A~342 with either 50 ug MPL or 25 ug QS21 adjuvants elicited an adequate
titer
response in comparison to controls.
Example 6 - Liquid Suspension Formulations
Glycine/Acetate Formulations
A(3 peptide was solubilized at 0.6 mg/ml in 10 mM sodium glycinate, pH 9 - 9.5
buffer alone or with various excipients. The peptide solutions were filtered
through a
1 o Millex GV filter and then titrated with 0.1 M acetic acid to~ pH ~ 6 to
create a suspension
of the peptide in the buffers. The formulations were filled at a 0.5 ml volume
in 2 cc
glass vials (Gensia P/N X34-113-002) and capped with gray butyl stoppers
(Gensia P/N
X66-113-030) and seals. Formulations were stored at 2-8°C.
Glycine/Citrate Formulations
0.6 mg/ml A(342 formulations were prepared in 10 mM sodium glycinate
containing 0.1% PS-80 alone or in combination with 5 % sucrose (25 mls). The
peptide
solutions were were filtered through a Millex GV filter and then titrated to
approximately
pH 6.0 with 0.1 M citric acid. Optionally, 0.9% sodium chloride was added to
the
glycine/citrate/PS 80 after titration to pH 6. The formulations were then
vialed at 0.5 ml
fills in 2 cc glass vials (Gensia P/N X34-113-002) and capped with gray butyl
stoppers
(Gensia P/N X66-113-030). Formulations were stored at 2-8°C.
The Glycine/Citrate formulations were further developed to include buffering
capacity at pH 6. Several 0.6 mg/ml A(3 peptide formulations at 100 ml were
prepared in
10 mM sodium glycinate pH 9, containing 5% sucrose with or without 0.1% PS 80.
Additional formulations at 0.1 mg/ml A(342 in 10 mM sodium glycinate
containing 5%
sucrose with or without 0.1% PS 80 were also prepared. The peptide solutions
were
3o filtered through Millex GV filters before pH adjustment. The pH was then
adjusted to
pH 6.0 with 10 mM and 20 mM sodium citrate buffer using a 1 M sodium citrate
pH 5.5
stock solution. The formulations were then vialed at 0.5 ml fills in 2 cc
glass vials
(Gensia P/N X34-113-002) and capped with gray butyl stoppers (Gensia P/N X66-
113-
030) and aluminium seals.
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
29
Stability Testing
The concentration and purity of A(342 in the formulations were monitored by RP
HPLC. Total peptide concentrations of the A(342 suspensions were measured by
resolubilization of the peptide with a v:v dilution with 2 % sodium dodecyl
sulfate (SDS)
in 0.01 N NaOH and 1 min heating at 100°C before analysis.
Alternatively, the
concentration of soluble A(342 in the suspension formulation was determined by
centrifuging the test sample in micro-centrifuge at >_ 10,000 rpm for 3-5
minutes.
Aliquots of either the resolubilized peptide or the supernatant were then
analyzed by RP
l0 HPLC. Note that during the course of these studies, chromatographic
improvements
were made to the RP HPLC method, resulting in better resolution and
quantitation.
Therefore the area % purity values are relative to the reference standard at
the time of
analysis and chromatograms are compared at each timepoint to assess
degradation. Both
pH and appearance were also monitored over the course of the stability
studies.
15 Table 6 presents the data for several liquid suspension formulations of
A(342. All
formulations are visually a suspension. These formulations are titrated to pH
6 with the
respective acid listed in the table. Depending upon the excipient, a peptide
suspension
may occur either immediately or over a period of up to 2 weeks or more. More
rapid
suspension formation can be achieved by addition of sodium chloride or
substitution of
2o citrate for acetate in the peptide formulation.
All formulations achieved the target concentration of 0.6 mg/ml peptide. The
area % purity for all formulations was comparable to the purity of the
reference standard.
No loss of peptide or degradation was seen over the 3 months storage at 2 to
8°C. The
pH and appearance remained comparable to the data presented in Table 1.
25 Both 0.9% NaCI and 5% sucrose provide physiologic tonicity for parenteral
administration. Likewise the suspension, at pH 6, is within the acceptable pH
range for
injection.
Table 6 Liquid Suspension Formulations
Formulation ID mg/ml
A(342
PercentTotal Supernatant
0.6 mg/ml A(3 in: AppearancepH PurityPeptide T=0 T
=2wks
mM Sodium suspension5.9 70% 0.54 0.23 .04
Glycinate/Acetate,
pH 6
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
10 mM Sodium suspension5.9 68% 0.65 0.21 .04
Glycinate/Acetate,
pH 6,
5 % Sucrose'
10 mM Sodium suspension6 67% 0.62 0.43 .08
Glycinate/Acetate,
pH 6,
0.1 % PS-80, 5 %
Sucrose
10 mM Sodium suspension5.9 68% 0.55 0.04 .04
Glycinate/Acetate,
pH 6,
0.1 % PS-80, 0.9
% NaCI
10 mM Sodium suspension6 70% 0.53 0.26 .OS
Glycinate/Acetate,
pH 6,
0.9 % NaCI'
10 mM Sodium suspensionnd 77% 0.63 <.O1 nd
Glycinate/Citrate,
pH 6,
0.1% PS 80, 5 %
Sucrose
10 mM Sodium suspensionnd 71 % 0.63 0.02 nd
Glycinate/Citrate,
pH 6,
0.1% PS 80, 0.9
% NaCI
nd: not done
Table 7 formulations were prepared with either l OmM or 20mM buffering
capacity provided with the citrate buffer to assure a pH of 6 is obtained
during the
manufacturing process. These formulations formed a suspension immediately. Due
to
the properties of the A(3 peptide, the change of conformation of the peptide
giving a
suspension occurs inside the sterile core. The use of a known buffer
concentration, as
opposed to titration, allows for ease of handling and reproducibility during
operations
1o within a sterile filling suite.
Table 7. Buffered Suspensions
PercentTotal mg/ml
0.6 mg/ml A(342 in: AppearancepH Purity Aa Peptide
10 mM Sodium Citrate, 10 Suspension6.0 81 % 0.66
mM Glycine,
pH 6.0, 0.1 % PS 80, 5
% Sucrose
20 mM Sodium Citrate, 10 Suspension5.9 83% 0.69
mM Glycine,
pH 6.0, 0.1 % PS 80, 5
% Sucrose
20 mM Sodium Citrate, 10 Suspension6.0 81 % 0.67
mM Glycine,
pH 6.0, 5 % Sucrose
0.1 mg/ml A(342 in:
20 mM Sodium Citrate, 10 Suspension5.9 82% 0.2
mM Glycine,
pH 6.0, 0.1 % PS 80, 5
% Sucrose
20 mM Sodium Citrate, 10 Suspension6.0 81 % 0.09
mM Glycine,
pH 6.0, 5 % Sucrose
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
31
These formulations were further expanded. Either the triflouroacetate or the
chloride salts of the A(342 peptide were solubilized in 10 mM sodium
glycinate, pH 9 -
9.5. Concentrations were adjusted for salt content to yield a A(342
concentration of 0.45
mg/mL. Polysorbate 80, at concentrations ranging from 0.02% to 0.5% may be
added to
the peptide solutions prior to filtration. Likewise, sucrose and sodium
choride may be
added to the formulations, either before or after the addition of the acid.
The pH of the
peptide solutions was adjusted with either citrate or HCl acids, as noted
below. All
formulations formed suspensions, and resulted in the expected concentration of
A(342
peptide.
Table 7b
Suspension Formulations
0.45 mg/mL A(342 in:
10 mM Sodium Glycinate, 20 mM Citrate, 0.1 % PS-80, pH 6.0
10 mM Sodium Glycinate, 20 mM Citrate, 0.5% PS-80, pH 6.0
10 mM Sodium Glycinate, 20 mM Citrate, 154 mM NaCI, pH 6.0
10 mM Sodium Glycinate, 20 mM Citrate, 154 mM NaCI, 0.1% PS-80, pH 6.0
10 mM Sodium Glycinate, 154 mM NaCI, pH 6.0 (HCI)
10 mM Sodium Glycinate, 154 mM NaCI, 0.1 % PS-80, pH 6.0 (NCI)
Characterization
Characterization of the A~342, pH 6 suspensions by circular dichroism and FT-
IR
demonstrates that the peptide assumes a beta sheet conformation as the peptide
forms a
suspension. The beta sheet structures are comparable in the 0.6 mg/ml
suspensions,
regardless of the buffering acid (citrate, acetate, phosophate) or additional
excipients
(sucrose, NaCI) added to the formulations. The addition of PS-80 appears to
generate a
more uniformly dispersed suspension.
Biological Activity
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
32
The A(3 peptide suspension, when injected with adjuvant, raises an antibody
titer
in Swiss Webster mice. Injections at biweekly intervals, (0, 2, 4 weeks) as
shown in
Table 8 elicited an adequate titer response in comparison to the control.
Table 8. Antibody Response to Suspension Formulations
Peptide Formulation ug Peptideug Antibody Titers
0.6 mg/ml A(3 in: Adjuvant Geometric
means
2d bleed*
mM Sodium Glycinate/Acetate,33 ug 50 ug ~ 7000
pH 6,
5 % Sucrose MPL*
10 mM Sodium Glycinate/Acetate,33 ug 25 ug QS ~ 10 000
pH 6, .
5 % Sucrose 21
10 mM Sodium Glycinate/Acetate,33 ug 50 ug ~ 10 000
pH 6,
0.1 % PS-80, 5 % Sucrose MPL*
10 mM Sodium Glycinate/Acetate,33 ug 25 ug QS ~ 7000
pH 6,
0.1 % PS-80, 5 % Sucrose 21
10 mM Sodium Glycinate/Citrate,33 ug 50 ug ~ 10 000
pH 6,
0.1 % PS 80, 5 % Sucrose MPL*
10 mM Sodium Glycinate/Citrate,33 ug 25 ug QS ~ 8,000
pH 6,
0.1 % PS 80, 5 % Sucrose 21
10 mM Sodium Glycinate/Citrate,33 ug 50 ug ~ 12 000
pH 6,
0.9 % NaCI MPL*
10 mM Sodium Glycinate/Citrate,33 ug 25 ug QS ~ 14 000
pH 6,
0.9 % NaCI 21
Control: Calif. Peptide 33 ug CFA/IFA ~ 1,200
lot MF0639
MYL tormulahon contammg triethanolamme
** Titers calculated as units at 50% max. OD
1o Example 7 - Lyophilized Formulations
Formulations.
Four combinations of 0.6 mg/ml A(342 in 10 mM glycinate or lysine buffer with
mannitol or mannitol and sucrose were prepared and sterile filtered through a
Millex GV
filter. One formulation, 0.6 mg/ml A(342 in 10 mM sodium glycinate pH 9.5, 4%
mannitol, was vialed and stoppered without titration to a physiological pH.
The
remaining three solutions (lysine/citrate/4% mannitol; glycinate/citrate/4%
mannitol;
glycinate/HCl/4% mannitol/1% sucrose) were then titrated to pH 7.5 with either
citric
acid or HCl as noted. The formulations were then vialed at 0.5 ml fills in 2
cc glass vials
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
33
(Gensia P/N X34-113-002) and loosely capped with grey butyl lyophilization
stoppers
(Gensia P/N X66-113-030).
Lyophilization.
The formulations were lyophilized in a programmable Virtis lyophilizer
(provided by Gensia Sicor). Mannitol was chosen as the primary matrix
component. To
achieve crystallization of the mannitol, the formulations were frozen with
thermal
cycling (annealing), followed by primary and secondary drying of the cakes.
Stability Analyses.
The concentration and purity of the lyophilized formulations was monitored by
RP HPLC. Each formulation was reconstituted with 1.0 ml water for injection
(WFI).
Formulations were analyzed immediately after reconstitution (with or without
centrifugation in a bench top micro-centrifuge at >_ 10, 000 rpm for 3-5
minutes) or
resolubilized as described previously.
The following table (Table 9) tabulates the concentration of the four A(342
formulations following lyophilization and reconstitution. As can be seen from
the data,
these formulations were soluble. Samples were analyzed both as the
reconstituted
2o formulation, and as the reconstituted formulation "resolubilized" as
described for the
peptide suspensions. No significant difference was observed in the
reconstituted
formulation versus the total peptide concentration (resolubilized sample). No
significant
degradation or loss in peptide was seen over the course of 3 months storage at
2 to 8 °C
as determined by RP HPLC as in the stability testing for Example 5.
Table 9. Lyophilized formulations
Formulation mg/ml A(342
0.6 mg/ml A(342 AppearancepH % PurityReconstitutedResolubilized
in:
peptide peptide
10 mM Sodium Glycinate,Clear 8.2 82% 0.59 0.61
pH 9.5, 4 % Mannitolsolution
10 mM Sodium Clear 7.4 82% 0.57 0.60
Glycinate/citrate,solution
pH 7.5,
4 % Mannitol
10 mM Sodium Clear 7.6 81 % 0.57 0.55
Lysinate/citrate, solution
pH 7.5,
4 % Mannitol
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
34
mM Sodium Clear 7.4 81 % 0.56 0.59
Glycinate/HCL, solution
pH 7.5, 4
Mannitol, 1 % sucrose
Characterization
Characterization of the A(342 pH 7.5 lyophilized formulations by circular
5 dichroism and FTIR demonstrates that the peptide remains in a random coil
conformation throughout lyophilization and reconstitution. This observation is
consistent with the A~342 sodium glycinate pH 9 formulation which is also
soluble,
filterable and assumes a random coil conformation in solution.
Biological Activity
The A(342 lyophilized formulation, 0.6 mg/ml A(342 in l OmM sodium
glycinate/HCl/4% mannitol/1% sucrose, pH 7.5, was chosen as a representative
lyophilized formulation. This product, when mixed with either MPL or QS21
adjuvant,
raises an antibody titer in Swiss Webster mice. Geometric mean titers are
shown in
Table 10 in comparison to controls.
Table 10 Antibody Response to Lyophilized Preparations
Peptide Formulation ug ug Antibody Titers
0.6 mg/ml A(342 in: Peptide Adjuvant Geometric
means
2d bleed**
10 mM Sodium Glycinate/HCL,33 ug 50 ug ~ 14 000
pH
7.5, 4 % Mannitol, 1 % MPL*
sucrose
10 mM Sodium Glycinate/HCL,33 ug 25 ug ~ 3 000
pH QS
7.5, 4 % Mannitol, 1 % 21
sucrose
Control: Cali Peptide lot 33 ug CFA/IFA ~ 1,200
MF0639
MYL tormularion contammg trietrianolamme
**Titers calculated as units at 50% max. OD
Example 8 - Scale-up using GMP manufacturing standards
The peptide suspension was scaled up to a 1.5-liter scale, manufactured and
filled
at a contract GMP drug product manufacturer. Two concentrations were filled:
0.6
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
mg/ml and 0.1 mg/ml. Both concentrations were successfully scaled up, filled,
and
remain stable at 2 - 8°C and at 25°C after two months.
A~3 peptide was dissolved at either 0.6 mg/ml or 0.1 mg/ml concentrations in a
10
mM sodium glycinate pH 9 buffer containing 5% sucrose. The peptide solution
was
5 sterile filtered through a Millipak 20 Millipore Durapore 0.2 um sterilizing
filter, into the
aseptic core. RP HPLC analysis demonstrated no significant loss of peptide
throughout
the filtration process. A 1 M sodium citrate pH 5.5 buffer was compounded, and
likewise sterile filtered through a 0.2 um Durapore filter into the aseptic
core. The
peptide solution was weighed, and an appropriate amount of sodium citrate
buffer added
1o to yield a 20 mM citrate, pH 6 formulation.
At the 0.6 mg/ml concentration, the peptide immediately formed a suspension
upon addition of the citrate buffer; the in-process pH measurement was 6.4.
The peptide
suspension was constantly stirred, filled at 1.2 ml per vial into 2cc
borosilicate glass
vials, and sealed with West 4416 stoppers and seals. The 0.1 mg/ml
concentration
15 remained soluble (and was filled soluble) for several hours before forming
a suspension
in the vials by the next day.
Table 11 presents the data generated for this 0.6 mg/ml A(342 suspension:
Table 11
25
A(3 Peptide
0.6 mg/ml suspension
in 10 mM glycine, 20 mM citrate, 5% sucrose, pH 6
Test Specification Result
Appearance Hazy colorless suspension,Pass
substantially free
of
contaminant particles
PH 5.5-6.5 6.3
Concentration 0.5 - 0.7 mg/ml 0.64 mg/ml
Area Percent PurityFIO 84.4%
(HPLC)
Volume, ml FIO 1.13 ml
Bulk Sterility FIO No growth
Final Container No growth Pass
Sterility
Bacterial endotoxinsFIO < 2 EU/ml
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
36
Example 9
Buffered suspensions of A(31-42 with QS-1 adjuvant
Single vial formulations incorporating A(31-42 and an adjuvant were studied
using QS-21,
a triterpene glycoside having immune stimulating activity, and surprisingly
found to result
in the formation of a visually clear suspension of the peptide.
1. Single Vial Formulation of A(31-42 /QS-21
to Solubilization of lyophilized QS-21 with A(31-42 solution in 10 mM sodium
glycinate at pH 9Ø In each of the following examples, lyophilized QS-21 was
solubilized with A~ 1-42 (TFA salt, 0.45 mg/mL) solution in 10 mM Glycine
(Gly),
pH 9Ø The pH of the composition containing the solubilized QS-21 was then
rapidly
adjusted to 6.0 by the addition of citrate buffer (Cit) to form a visually
clear
suspension. Turbidity measurements were determined with a spectrophotometer
set at
a wavelength of 405 mn to measure the clarity of the resulting suspensions.
The
results indicate that the particle size of the suspended peptide is smaller
than the
wavelength of the reflected light. These formulations have demonstrated 3
months
stability with storage at 2-8°C with ongoing stability monitoring; all
formulations
were found to contain A(31-42 predominantly in the (3-sheet conformation; all
formulations showed no turbidity when analyzed in a spectrophotometer at 405
nm.
The 0.45 mg/mL A(31-42 , 0.2 mg/mL QS-21, 10 mM Gly, 20 mM Cit, 5 % Sucrose,
pH 6.0 formulation was tested in the mouse titer assay, resulting in a high
titer
response.
Table 12A
Formulation Appearance
0.45 mg/mL A~31-42 , 0.2 mg/mL QS-21, 10 mM Gly,Clear
20 mM Cit,
5 % Sucrose, pH 6.0'
0.45 mg/mL A(31-42 , 0.1 mg/mL QS-21, 10 mM Gly,Clear
20 mM Cit,
5 % Sucrose, pH 6.0
0.225 mg/mL A[31-42 , 0.3 mg/mL QS-21, 10 mM Clear
Gly, 20 mM Cit,
5 % Sucrose, pH 6.0
0.225
mg/mL
A(31-42
,
0.15
mg/mL
QS-21,
10
mM
Gly,
20
mM
Cit,
Clear
5
%
Sucrose,
pH
6.0
'Found
to
have
high
mouse
titer
response
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
37
2. A(342 Titrations with QS-21
A(342 (2 mg/mL) was solubilized in 10 mM Glycine, pH 9.5. The pH was
readjusted to
9.5 with 1 N NaOH. The A(342 solution was then filtered through a Millex GV
filter.
QS-21 (5 mg/mL) was solubilized in 10 mM Citrate, pH 6.0 and was then filtered
through Millex GV filter. Aliquots of A(342 were mixed with aliquots of QS-21
to give
the desired ratio, mixed, and diluted with 10 mM Glycine, pH 9.5 and 1 M
Citrate, pH
5.2, to yield the final concentrations noted in Table 12 below.
1o Table 12B. Initial AN1792/QS21 Co-Formulations
Formulations Appearance OD4os nm
In 10 mM Glycine, 20 mM Citrate, pH 6.0
1.0 mg/mL A(342, 1.0 mg/mL QS-21 clear 0.018
1.0 mg/mL A(342, 0.5 mg/mL QS-21 opaque (+) 0.013
1.0 mg/mL A(342, 0.25 mg/mL QS-21 opaque (++) 0.030
1.0 mg/mL A(342, 0.125 mg/mL QS-21 opaque (+++)0.175
1.0 mg/mL A(342, 0.063 mg/mL QS-21 opaque (+++)0.308
1.0 mg/mL A(342, 0.031 mg/mL QS-21 opaque (+++)0.315
1.0 mg/mL A(342, 0.016 mg/mL QS-21 opaque (+++)0.268
1.0 mg/mL A(342, 0.0 mg/mL QS-21 opaque (+++)0.213
0.5 mg/mL A(342, 1.0 mg/mL QS-21 clear 0.005
0.5 mg/mL A(342, 0.5 mg/mL QS-21 clear 0.006
0.5 mg/mL A(342, 0.25 mg/mL QS-21 clear 0.005
0.5 mg/mL A(342, 0.125 mg/mL QS-21 opaque (+) 0.021
0.5 mg/mL A(342, 0.063 mg/mL QS-21 opaque (++) 0.148
0.5 mg/mL A(342, 0.031 mg/mL QS-21 opaque (+++)0.172
0.5 mg/mL A(342, 0.016 mg/mL QS-21 opaque (+++)0.162
0.5 mg/mL A(342, 0.0 mg/mL QS-21 N/A N/A
0.3 mg/mL A(342, 1.0 mg/mL QS-21 clear 0.004
0.3 mg/mL A(342, 0.5 mg/mL QS-21 clear 0.004
0.3 mg/mL A(342, 0.25 mg/mL QS-21 clear 0.006
0.3 mg/mL A(342, 0.125 mg/mL QS-21 clear 0.005
0.3 mg/mL A(342, 0.063 mg/mL QS-21 opaque (+) 0.013
0.3 mg/mL A~342, 0.031 mg/mL QS-21 opaque (+) 0.077
0.3 mg/mL A(342, 0.016 mg/mL QS-21 opaque (+) 0.068
0.3 mg/mL A(342, 0.0 mg/mL QS-21 opaque (++) 0.046
0.1 mg/mL A~342, 1.0 mg/mL QS-21 clear 0.004
0.1 mg/mL A~i42, 0.5 mg/mL QS-21 clear 0.002
0.1 mg/mL A(342, 0.25 mg/mL QS-21 - - clear 0.002
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
38
0.1 mg/mL A(342, 0.125 mg/mL QS-21 clear 0.001
0.1 mg/mL A(342, 0.063 mg/mL QS-21 clear 0.002
0.1 mg/mL A/342, 0.031 mg/mL QS-21 clear 0.003
0.1 mg/mL A(342, 0.016 mg/mL QS-21 clear 0.004
0.1 mg/mL A(342, 0.0 mg/mL QS-21 clear 0.008
3. Additional formulations of A(342 and QS-21
A~342, chloride salt, was solubilized atl mg/mLin 10 sodium glycinate, , pH
9.0 -
9.5 with or without 5 % sucrose, 0.1 % PS-80, or 0.4 % PS-80. Peptide
solutions
were sterile filtered through Millex GV syringe filters. Lyophilized QS-21 was
solubilized at 5 mg/mL in 10 mM citrate, pH 6.0, and sterile filtered through
a
Millex GV syringe filter.
Appropriate volumes of the A(342 solutions and the QS-21 solutions were
combined to
yield the final concentrations of A/342 and QS-21 noted in the formulations in
Table XX
1 o below. Last, the pH was lowered with 1 M Citrate buffer, pH 5.4, to give a
final pH of 6
in 20 mM citrate buffer. The visual appearance of the formulations was rated
as clear to
cloudy (+ to +++). Varying the concentrations and ratios of A(342 and QS-21
can
moderate the visual appearance of the formulations. Furthermore, sugars and
surfactants
are acceptable additional excipients to the formulations.
Table 12C. Additional Formulations of A(342 and QS-21
Formulations Appearance
QS-21 A(342 A(342/ No 5 % 0.1 % 0.4
(mg/mL) (mg/mL) QS-21 ExcipientSucrose PS-80 PS-80
Ratio
0.05 0.05 1.0 Clear Clear Clear Clear
0.1 0.1 1.0 Clear Clear Clear Clear
0.2 0.2 1.0 Clear Clear Clear Cloudy
(+)
0.1 0.23 2.3 Cloudy Cloudy Cloudy Cloudy
(+) (+) (+) (+)
0.2 0.23 1.1 Clear Clear Clear Cloudy
(+)
0.3 0.23 0.8 Clear Clear Clear Cloudy
(+)
0.1 0.45 4.5 Cloudy Cloudy Clear Cloudy
(++) (++) (++)
0.2 0.45 2.3 Cloudy Cloudy Cloudy Cloudy
(++) (++) (+) (++)
0.3 0.45 1.5 Clear Clear Clear Cloudy
(++)
CA 02374897 2001-11-22
WO 00/72870 PCT/US00/15302
39
Example 10 - Measurement and Interpretation of C.D. Spectra of A(342
Formulations
The circular dichroism data were collected using an Aviv model 62-DS
spectropolarimeter (Lakewood, NJ). Samples were prepared appropiately for
collection
of near UV and far UV spectra, respectively, and loaded into a strain-free 1
mm
pathlength quartz cell. The sample holder was held at a steady temperature of
25° C.
Data were collected at 0.5 nm intervals using a 4 second averaging time. In
the far UV
region ( ~, ~ 180-250 nm), signals from the peptide backbone dominate the
spectrum and
estimates of the secondary structure composition can be obtained. Examination
of the
l0 near UV ( ~, ~ 1250-350 nm) region provides quite different information: in
this region
the primary signals arise from aromatic side chains (Phe, Tyr, and Trp). The
sign and
magnitude of the signal indicates the degree of flexibility at each site as
well as the
orientation of the side chain relative to the peptide backbone. As the number
of these
chromophores is less than the number of amide groups, and because the
chromophores
are distributed throughout the molecule, they provide some indication of local
structure.
Numerous modifications and variations in the invention as described in the
above
illustrative examples are expected to occur to those skilled in the art and,
consequently,
only such limitations as appear in the appended claims should be placed
thereon.
2o Accordingly, it is intended in the appended claims to cover all such
equivalent variations,
which come within the scope of the invention as claimed.
