U.S. patent application number 10/504025 was filed with the patent office on 2005-06-02 for antibody-containing solution pharmaceuticals.
Invention is credited to Miyauchi, Eiichi, Mizushima, Hidefumi.
Application Number | 20050118163 10/504025 |
Document ID | / |
Family ID | 27678080 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118163 |
Kind Code |
A1 |
Mizushima, Hidefumi ; et
al. |
June 2, 2005 |
Antibody-containing solution pharmaceuticals
Abstract
An antibody-containing solution formulation comprising an
organic acid and a surfactant as stabilizers; a method for
suppressing the formation of visible insoluble matter and/or
insoluble particles due to the presence of metal ions in an
antibody-containing solution formulation, which comprises adding an
organic acid to the solution; a method for suppressing the
formation of visible insoluble matter and/or insoluble particles
during shaking and freezing-thawing of an antibody-containing
solution, which comprises adding a surfactant to the solution; and
a method for stabilizing an antibody-containing solution, which
comprises adding an organic acid and a surfactant.
Inventors: |
Mizushima, Hidefumi; (Tokyo,
JP) ; Miyauchi, Eiichi; (Tokyo, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Family ID: |
27678080 |
Appl. No.: |
10/504025 |
Filed: |
August 9, 2004 |
PCT Filed: |
February 14, 2003 |
PCT NO: |
PCT/JP03/01562 |
Current U.S.
Class: |
424/130.1 ;
514/557; 514/574 |
Current CPC
Class: |
C07K 16/3061 20130101;
A61K 39/39591 20130101; A61K 9/0019 20130101; A61P 37/04 20180101;
C07K 16/00 20130101; C07K 16/40 20130101; A61K 47/12 20130101; C07K
16/44 20130101; A61K 47/10 20130101; A61K 47/26 20130101; A61K
2039/505 20130101 |
Class at
Publication: |
424/130.1 ;
514/557; 514/574 |
International
Class: |
A61K 039/395; A61K
031/19 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2002 |
JP |
2002-036244 |
Claims
1. An antibody-containing solution formulation comprising an
organic acid and a surfactant as stabilizers.
2. The solution formulation according to claim 1, wherein the
organic acid is acetic acid or citric acid.
3. The solution formulation according to claim 2, wherein the
organic acid is acetic acid.
4. The solution formulation according to claim 3, wherein the
concentration of acetic acid is in the range of 10-50 mM.
5. The solution formulation according to claim 1, wherein the
surfactant is Polysorbate 80 or 20, or a poloxamer.
6. The solution formulation according to claim 5, wherein the
concentration of the surfactant is in the range of 0.01-10
mg/mL.
7. The solution formulation according to claim 1, which further
comprises sodium chloride.
8. The solution formulation according to any one of claims 1 to 7,
wherein the antibody is a recombinant antibody.
9. The solution formulation according to claim 8, wherein the
antibody is a chimeric antibody, a humanized antibody or a human
antibody.
10. The solution formulation according to claim 1, wherein the
antibody is an antibody of IgG class.
11. The solution formulation according to claim 10, wherein the
antibody of IgG class is an antibody of IgG1 class.
12. The solution formulation according to any one of claim 1,
wherein the antibody is an anti-interleukin-6 receptor antibody or
an anti-HM1.24 antibody.
13. The solution formulation according to claim 1, wherein the
antibody is an anti-HM1.24 antibody.
14. An anti-HM1.24 antibody-containing solution formulation
comprising 10-50 mM of acetic acid and 0.01-10 mg/mL of Polysorbate
80 as stabilizers.
15. A method for suppressing formation of visible insoluble matter
and/or insoluble particles caused by metal ions present in an
antibody-containing solution formulation, which comprises adding an
organic acid to the solution.
16. A method for suppressing formation of visible insoluble matter
and/or insoluble particles during shaking and freezing-thawing of
an antibody-containing solution, which comprises adding a
surfactant to the solution.
17. A method for stabilizing an antibody-containing solution, which
comprises adding an organic acid and a surfactant.
Description
TECHNICAL FIELD
[0001] The present invention relates to stable solution
formulations containing antibodies.
BACKGROUND ART
[0002] Advances in gene recombination technology have enabled the
pharmaceutical use of antibodies such as immunoglobulins,
monoclonal antibodies and humanized antibodies. To ensure a stable
supply of these antibodies, it is necessary to establish production
and storage conditions where the structure and activity of the
antibodies can be maintained.
[0003] When proteins are stored in a highly concentrated solution
form, they are usually associated with a problem of deterioration,
including the formation of insoluble aggregates, which is required
to be prevented. It is necessary to prevent such deterioration. For
example, the applicant has found that an anti-HM1.24 antibody has a
therapeutic effect on myeloma cells (JP KOKAI 11-092399), and also
has studied formulation of this antibody. However, the anti-HM1.24
antibody is an unstable protein and is more likely to undergo
physical and chemical changes (e.g., association, aggregation) as a
result of stresses in the purification process. Such stresses
include filtration stress during removal of virus and bacteria,
concentration stress, heat stress and light stress.
[0004] Also, in the case of using genetic engineering techniques to
obtain antibodies, antibody-producing cells are cultured in bulk,
the antibody-containing solutions are purified, frozen and stored
until thawing for use in drug formulation. However, repeating such
shaking and freezing-thawing steps causes the formation of antibody
aggregates and/or insoluble particles. Furthermore, long-term
storage causes decomposition of antibodies, resulting in formation
of decomposition products. These phenomena could eventually lead to
a reduced level of antibodies remaining in the solution.
[0005] There is also a problem that visible insoluble matter and
insoluble particles are formed in the presence of metal ions (Fe
ions) introduced during the production process. Since metal ions
(Fe ions), even when present in very small amounts in the solution,
contribute to the formation of visible insoluble matter and
insoluble particles, such ions should be removed completely.
However, there is a limit to removal methods such as precipitation,
complex formation, and so on. Thus, there has been a need to
develop a strategy to avoid the formation of visible insoluble
matter and insoluble particles even in the presence of metal
ions.
[0006] Many attempts have been made to store proteins in a solution
form, with the finding that stabilization effects are obtained by
addition of a stabilizer for preventing chemical and physical
changes. Examples of a stabilizer include high-molecular weight
materials such as proteins (e.g., human serum albumin, purified
gelatin) or low-molecular weight materials such as polyols, amino
acids and surfactants. However, when added as stabilizers,
organism-derived high-molecular weight materials like proteins are
disadvantageous in that very complicated processes are required to
remove contaminants, such as viruses and prions. With respect to
low-molecular weight materials, it is also preferable to use them
in as small amounts as possible.
[0007] For stabilization of lyophilized antibody formulations,
there has been reported those comprising a sugar or amino sugar, an
amino acid, and a surfactant as stabilizers (JP TOKUHYO
2001-503781).
[0008] However, there has been a strong demand for easy-to-use
solution formulations that eliminates of dissolution and
reconstitution steps before use. Especially, there has been a need
for stable solution formulations containing antibodies.
DISCLOSURE OF THE INVENTION
[0009] The object of the present invention is to provide an
antibody-containing solution formulation that is stable for
long-term storage. This formulation is designed to suppress the
formation of visible insoluble matter and/or insoluble particles,
which are derived from the aggregation of antibodies caused by
physical stresses (e.g., freezing-thawing) during its production
process and by long-term storage; and to suppress the formation of
visible insoluble matter and/or insoluble particles in the presence
of metal ions (Fe ions) introduced during the production
process.
[0010] As a result of extensive and intensive efforts made to
achieve the above object, the inventors of the present invention
have found that the use of organic acids significantly suppresses
the formation of visible insoluble matter in the presence of iron,
and that the addition of surfactants very significantly suppresses
the formation of visible insoluble matter and/or insoluble
particles during shaking and freezing-thawing steps. These findings
have led to the completion of the present invention.
[0011] Namely, the present invention provides the following.
[0012] (1) An antibody-containing solution formulation comprising
an organic acid and a surfactant as stabilizers.
[0013] (2) The solution formulation according to (1) above, wherein
the organic acid is acetic acid or citric acid.
[0014] (3) The solution formulation according to (2) above, wherein
the organic acid is acetic acid.
[0015] (4) The solution formulation according to (3) above, wherein
the concentration of acetic acid is in the range of 10-50 mM.
[0016] (5) The solution formulation according to (1) above, wherein
the surfactant is Polysorbate 80 or 20.
[0017] (6) The solution formulation according to (5) above, wherein
the concentration of the surfactant is in the range of 0.01 to 10
mg/mL.
[0018] (7) The solution formulation according to (1) above, which
further comprises sodium chloride.
[0019] (8) The solution formulation according to any one of (1) to
(7) above, wherein the antibody is a recombinant antibody.
[0020] (9) The solution formulation according to (8) above, wherein
the antibody is a chimeric antibody, a humanized antibody or a
human antibody.
[0021] (10) The solution formulation according to (8) or (9) above,
wherein the antibody is an antibody of IgG class.
[0022] (11) The solution formulation according to (10) above,
wherein the antibody of IgG class is an antibody of IgG1 class.
[0023] (12) The solution formulation according to any one of (1) to
(11) above, wherein the antibody is an anti-interleukin-6 receptor
antibody or an anti-HM1.24 antibody.
[0024] (13) The solution formulation according to (12) above,
wherein the antibody is an anti-HM1.24 antibody.
[0025] (14) An anti-HM1.24 antibody-containing solution formulation
comprising 10-50 mM of acetic acid and 0.01-10 mg/mL of Polysorbate
80 as stabilizers.
[0026] (15) A method for suppressing formation of visible insoluble
matter and/or insoluble particles caused by metal ions presented in
an antibody-containing solution formulation, which comprises adding
an organic acid to the solution.
[0027] (16) A method for suppressing formation of visible insoluble
matter and/or insoluble particles during shaking and
freezing-thawing of an antibody-containing solution, which
comprises adding a surfactant to the solution.
[0028] (17) A method for stabilizing an antibody-containing
solution, which comprises adding an organic acid and a
surfactant.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] As used herein, the term "antibody-containing solution
formulation" is intended to mean a solution formulation containing
an antibody as an active ingredient and available for use in
administration to animals including human, preferably prepared
without using a lyophilization step.
[0030] As used herein, the term "antibody-containing solution" is
intended to mean a solution containing any antibody, whether the
antibody is native one or recombinant one. It is preferably a
culture medium of mammalian cells (e.g., CHO cells) containing
antibody molecules produced by culture, which may further be
subjected to partial purification or other certain treatment(s)
(bulk solution), or alternatively, the above-stated solution
formulation available for use in administration to animals
including human.
[0031] As used herein, the term "insoluble particles" is intended
to mean insoluble particulate matter of 10 .mu.m or larger, as
defined in Test for Insoluble Particles in Injections, Standard
Test Procedures, the Japanese Pharmacopoeia. The measurement of
insoluble particles may be accomplished by using a microscope, a
filter for collecting insoluble particles and a membrane filter for
measurement, but conveniently by using a light shielding type of an
automatic particle analyzer.
[0032] As used herein, the term "visible insoluble matter" is
intended to mean those easily detected by the unaided eye when a
solution formulation in a container is placed directly under a
white light at a brightness of about 3000 lux (2000 to 3750 lux)
against a black background, in accordance with the procedures
described in Section 2.9.20 of the European Pharmacopoeia, third
edition.
[0033] As used herein, the terms "aggregation products" and
"decomposition products" are intended to mean aggregates and
fragments, respectively, of antibody molecules used as an active
ingredient of the formulation. The content of these products may be
determined, for example, on the basis of peak ratio with gel
permeation chromatography as stated below.
[0034] There is no particular limitation on the antibodies used in
the solution formulation of the present invention, as long as they
can bind to a desired antigen. It is possible to use mouse
antibodies, rat antibodies, rabbit antibodies, sheep antibodies,
chimeric antibodies, humanized antibodies, human antibodies and the
like, as appropriate. Such antibodies may be polyclonal or
monoclonal, but are preferably monoclonal because uniform antibody
molecules can be produced stably. Polyclonal and monoclonal
antibodies can be prepared in a manner well known to those skilled
in the art.
[0035] In principle, monoclonal antibody-producing hybridomas can
be prepared using known techniques, as follows. Namely, a desired
antigen or a desired antigen-expressing cell is used as a
sensitizing antigen and immunized in accordance with conventional
procedures for immunization. The resulting immunocytes are then
fused with known parent cells using conventional procedures for
cell fusion, followed by selection of monoclonal antibody-producing
cells (hybridomas) through conventional screening procedures.
Preparation of hybridomas may be accomplished according to, for
example, the method of Milstein et al. (Kohler, G. and Milstein,
C., Methods Enzymol. (1981) 73:3-46). If an antigen used is less
immunogenic, such an antigen may be conjugated with an immunogenic
macromolecule (e.g., albumin) before use in immunization.
[0036] In addition, antibody genes are cloned from hybridomas,
integrated into appropriate vectors, and then transformed into
hosts to produce antibody molecules using gene recombination
technology. The genetically recombinant antibodies thus produced
may also be used in the present invention (see, e.g., Carl, A. K.
Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,
Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
More specifically, cDNA of antibody variable domains (V domains) is
synthesized from hybridoma mRNA using reverse transcriptase. Upon
obtaining DNA encoding the target antibody V domains, the DNA is
ligated to DNA encoding desired antibody constant domains (C
domains) and integrated into an expression vector. Alternatively,
the DNA encoding the antibody V domains may be integrated into an
expression vector carrying the DNA of the antibody C domains. The
DNA construct is integrated into an expression vector such that it
is expressed under control of an expression regulatory region,
e.g., an enhancer or a promoter. Host cells are then transformed
with this expression vector for antibody expression.
[0037] In the present invention, it is possible to use genetically
recombinant antibodies (e.g., chimeric antibodies, humanized
antibodies) that are artificially modified with a view to
attenuating the characteristics as heteroantigen to human. These
modified antibodies may be prepared in a known manner. A chimeric
antibody is composed of variable domains of heavy and light chains
from a non-human mammalian (e.g., mouse) antibody and constant
domains of heavy and light chains from a human antibody. To obtain
chimeric antibodies, DNAs encoding such mouse antibody variable
domains may be ligated to DNAs encoding the human antibody constant
domains, and then integrated into an expression vector, followed by
transformation into a host for antibody production.
[0038] Humanized antibodies are also called reshaped human
antibodies and are obtained by grafting complementarity determining
regions (CDRs) of non-human mammalian (e.g., mouse) antibodies to
replace those of human antibodies. Standard gene recombination
procedures for this purpose are also known. More specifically, a
DNA sequence designed to allow ligation between CDRs of mouse
antibody and framework regions (FRs) of human antibody is
synthesized by PCR from several oligonucleotides which are prepared
to have sections overlapping with one another at the ends. The DNA
thus obtained is ligated to DNA encoding human antibody constant
domains, and integrated into an expression vector, followed by
transformation into a host for antibody production (see European
Patent Publication No. EP 239400 and International Patent
Publication No. WO 96/02576). The FRs of human antibody, which is
ligated to CDRs, are selected such that the complementarity
determining regions form a favorable antigen-binding site. If
necessary, amino acid substitutions may be made in the framework
regions of antibody variable domains such that the complementarity
determining regions of reshaped humanized antibody may form an
appropriate antigen-binding site (Sato, K. et al., Cancer Res.
(1993) 53, 851-856).
[0039] Procedures for obtaining human antibodies are also known.
For example, human lymphocytes are sensitized in vitro with a
desired antigen or a desired antigen-expressing cell, and the
sensitized lymphocytes are then fused with human myeloma cells
(e.g., U266) to give desired human antibodies having binding
activity to the antigen (see JP KOKOKU 01-59878). Alternatively,
transgenic animals having the entire repertories of human antibody
genes may be immunized with an antigen to obtain desired human
antibodies (see International Publication Nos. WO 93/12227, WO
92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).
There are additional techniques using human antibody libraries to
give human antibodies by panning. For example, human antibody
variable domains may be expressed by phage display technology as a
single-chain antibody (scFv) on the surface of phages, followed by
selection of phages binding to the antigen. When genes of the
selected phages are analyzed, it is possible to determine DNA
sequences encoding human antibody variable domains binding to the
antigen. Once the DNA sequences of scFv binding to the antigen have
been identified, the sequences may be used to construct appropriate
expression vectors to obtain human antibodies. These techniques are
already well known and can be found in WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO
95/15388.
[0040] In a case where antibody genes are isolated and then
transformed into appropriate hosts to produce antibodies, any
suitable combination of host and expression vector can be used for
this purpose. When eukaryotic cells are used as hosts, animal
cells, plant cells and fungal cells may be used. Animal cells known
for this purpose include (1) mammalian cells such as CHO, COS,
myeloma, BHK (baby hamster kidney), HeLa and Vero, (2) amphibian
cells such as Xenopus oocytes, and (3) insect cells such as sf9,
sf21 and Tn5. Plant cells include those derived from Nicotiana
plants (e.g., Nicotiana tabacum), which may be subjected to callus
culture. Fungal cells include yeasts such as Saccharomyces (e.g.,
Saccharomyces serevisiae) and filamentous fungi such as Aspergillus
(e.g., Aspergillus niger). When prokaryotic cells are used, there
are production systems employing bacterial cells. Bacterial cells
known for this purpose are E. coli and Bacillus subtilis.
Antibodies can be obtained by introducing target antibody genes
into these cells via transformation and then culturing the
transformed cells in vitro.
[0041] Examples of antibodies to be contained in the stabilized
formulation of the present invention include, but are not limited
to, an anti-IL-6 receptor antibody, an anti-HM1.24 antigen
monoclonal antibody and an anti-parathyroid hormone-related peptide
antibody (anti-PTHrP antibody).
[0042] Examples of reshaped humanized antibodies preferred for use
in the present invention include a humanized anti-IL-6 receptor
antibody (hPM-1) (see International Publication No. WO92-19759), a
humanized anti-HM1.24 antigen monoclonal antibody (see
International Publication No. WO98-14580) and a humanized
anti-parathyroid hormone-related peptide antibody (anti-PTHrP
antibody; see International Publication No. WO98-13388).
[0043] Antibodies to be contained in the solution formulation of
the present invention may be of any immunoglobulin class,
preferably of IgG class including IgG1, IgG2, IgG3 and IgG4, and
more preferably of IgG1 class.
[0044] In the antibody-containing solution or solution formulation
of the present invention, the formation of visible insoluble matter
and/or insoluble particles, which is caused by metal ions (Fe ions)
introduced during production procedures, can be significantly
suppressed by addition of an organic acid. Preferred organic acids
are acetic acid and citric acid, with acetic acid being more
preferred. Such acids may be used in combination.
[0045] Addition of an organic acid(s) may be accomplished by
dissolving an antibody and other ingredients in an organic acid
buffer. To prepare a solution formulation, an antibody and other
ingredients may be dissolved in an aqueous buffer known in the art
of solution formulations, including acetate buffer and/or citrate
buffer (preferably sodium citrate buffer). The concentration of the
buffer used is usually 1 to 500 mM, preferably 5 to 100 mM, and
more preferably 10 to 50 mM.
[0046] Due to inclusion of an organic acid, the antibody-containing
solution formulation of the present invention achieved significant
suppression of insoluble contaminant formation, even in the
presence of Fe ions during storage at room temperature (25.degree.
C.), as compared to an antibody-containing solution formulation
supplemented with an inorganic acid.
[0047] In the present invention, addition of a surfactant allows
very significant suppression of visible insoluble matter and/or
insoluble particle formation during shaking and freezing-thawing of
the antibody-containing solution formulation. Typical examples of a
surfactant include:
[0048] nonionic surfactants (HLB 6 to 18) such as sorbitan fatty
acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate,
sorbitan monopalmitate), glycerine fatty acid esters (e.g.,
glycerine monocaprylate, glycerine monomyristate, glycerine
monostearate), polyglycerine fatty acid esters (e.g., decaglyceryl
monostearate, decaglyceryl distearate, decaglyceryl monolinoleate),
polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene
sorbitan monolaurate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan
monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene
sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters
(e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene
sorbitol tetraoleate), polyoxyethylene glycerine fatty acid esters
(e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol
fatty acid esters (e.g., polyethylene glycol distearate),
polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether),
polyoxyethylene polyoxypropylene alkyl ethers (e.g.,
polyoxyethylene polyoxypropylene glycol, polyoxyethylene
polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene
cetyl ether), polyoxyethylene alkylphenyl ethers (e.g.,
polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated
castor oils (e.g., polyoxyethylene castor oil, polyoxyethylene
hydrogenated castor oil), polyoxyethylene beeswax derivatives
(e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin
derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene
fatty acid amides (e.g., polyoxyethylene stearic acid amide);
[0049] anionic surfactants such as C.sub.10-C.sub.18 alkyl sulfate
(e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl
sulfate), polyoxyethylene C.sub.10-C.sub.18 alkyl ether sulfate
with an average of 2 to 4 moles of ethylene oxide units added
(e.g., sodium polyoxyethylene lauryl sulfate), and C.sub.8-C.sub.18
alkyl sulfosuccinate ester salts (e.g., sodium lauryl
sulfosuccinate ester); and
[0050] natural surfactants such as lecithin, glycerophospholipid,
sphingophospholipids (e.g., sphingomyelin), and sucrose esters of
C.sub.12-C.sub.18 fatty acids. The formulation of the present
invention may be supplemented with one or more of these
surfactants. Preferred surfactants for use in the solution
formulation of the present invention are polyoxyethylene sorbitan
fatty acid esters such as Polysorbate 20, 40, 60 or 80, with
Polysorbate 20 and 80 being particularly preferred. Also preferred
is polyoxyethylene polyoxypropylene glycol typified by poloxamers
(e.g., Pluronic F-68.RTM.), with Poloxamer 188 being particularly
preferred.
[0051] The amount of a surfactant to be added will vary depending
on the type of surfactant used. In the case of Polysorbate 20 or
Poloxamer 188, it is usually added in an amount of 0.001 to 100
mg/mL (0.0001% to 10%), preferably 0.005 to 50 mg/mL (0.0005% to
5%), and more preferably 0.01 to 10 mg/mL (0.001% to 1%). Even more
preferably, the amount is 0.025 to 0.25 mg/mL (0.0025% to 0.025%)
at which the maximum number of insoluble particles having a
diameter of no less than 25 .mu.m can be of no greater than 3 even
after shaking (200 strokes/min.times.30 minutes) and one cycle of
freezing-thawing (-80.degree. C./25.degree. C.). In the case of
Polysorbate 80, it is usually added in an amount of 0.001 to 100
mg/mL (0.0001% to 10%), preferably 0.005 to 50 mg/mL (0.0005% to
5%), and more preferably 0.01 to 10 mg/mL (0.001% to 1%). Even more
preferably, the amount is 0.025 to 1 mg/mL (0.0025% to 0.1%) at
which the maximum number of insoluble particles having a diameter
of no less than 25 .mu.m can be zero, and no formation of visible
insoluble matter is observed even after shaking (200
strokes/min.times.60 minutes) and 3 cycles of freezing-thawing
(-20.degree. C./5.degree. C.).
[0052] Preferably, the antibody-containing solution formulation of
the present invention is substantially free from protein
stabilizers such as human serum albumin and purified gelatin.
[0053] The antibody-containing solution formulation of the present
invention may further comprise sodium chloride in an amount of 10
to 300 mM, preferably 20 to 200 mM.
[0054] The antibody-containing solution formulation of the present
invention preferably has a pH of 4 to 8, more preferably 5 to 7.5.
However, the pH will vary depending on the type of antibody
contained and is not limited to this range. For example, in the
case of using an anti-HM1.24 antibody, a pH range of 5.5 to 6.5 is
most preferred for the purpose of avoiding aggregation induced by
heat stress, maintaining a high residue level, and suppressing the
formation of charged heteromolecules (including deamidated
products, etc.). The pH preferred for each antibody may be
determined in accordance with the examples shown below.
[0055] The formulation of the present invention may further
comprise, as a stabilizer, sugar alcohols such as mannitol and
sorbitol or saccharides such as nonreducing oligosaccharides
including nonreducing disaccharides (e.g., sucrose, trehalose) and
nonreducing trisaccharides (e.g., raffinose).
[0056] Likewise, the formulation of the present invention may
further comprise, as an isotonizing agent, polyethylene glycol or
saccharides such as dextran, mannitol, sorbitol, inositol, glucose,
fructose, lactose, xylose, mannose, maltose, sucrose, trehalose and
raffinose.
[0057] If desired, the antibody-containing solution formulation of
the present invention may further comprise a diluent, a
solubilizer, an excipient, a pH adjuster, a soothing agent, a
buffer, a sulfur-containing reducing agent, an antioxidant and the
like. For instance, examples of a sulfur-containing reducing agent
include those having a sulfhydryl group such as N-acetylcysteine,
N-acetylhomocysteine, thioctic acid, thiodiglycol,
thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and
a salt thereof, sodium thiosulfate, glutathione, and a
C.sub.1-C.sub.7 thioalkanoic acid. Examples of an antioxidant
include erythorbic acid, dibutylhydroxytoluene,
butylhydroxyanisole, .alpha.-tocopherol, tocopherol acetate,
L-ascorbic acid and a salt thereof, L-ascorbyl palmitate,
L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl
gallate and propyl gallate, as well as chelating agents such as
disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate
and sodium metaphosphate. Furthermore, the formulation of the
present invention may comprise other commonly used ingredients, for
example, inorganic salts such as sodium chloride, potassium
chloride, calcium chloride, sodium phosphate, potassium phosphate
and sodium bicarbonate, as well as organic salts such as sodium
citrate, potassium citrate and sodium acetate.
[0058] The antibody-containing solution formulation of the present
invention is usually administered by parenterally, for example, in
the dosage form of injections (e.g., subcutaneous, intravenous,
intramuscular or intraperitoneal injections) as well as
transdermal, transmucosal, transnasal and transpulmonary
preparations. However, it may also be administered orally.
[0059] The antibody-containing solution formulation of the present
invention can usually be provided in the form of containers having
a definite volume, including sealed and sterilized plastic or glass
vials, ampoules and syringes, as well as in the form of large
volume containers like bottles. In terms of convenience of
handling, pre-filled syringes are preferred.
[0060] The amount of an antibody in the formulation of the present
invention can be determined as appropriate for the type and
severity of disease to be treated, the age of a patient, etc. In
general, an antibody is incorporated in an amount of 0.1 to 200
mg/ml, preferably 1 to 120 mg/ml.
[0061] As shown in the examples below, the antibody-containing
solution formulation of the present invention was confirmed to
significantly suppress the formation of visible insoluble matter in
the presence of iron when an organic acid, particularly acetic acid
and/or citric acid, was added to an antibody solution. Thus,
addition of an organic acid significantly suppressed the formation
of visible insoluble matter and/or insoluble particles, which was
caused by metal ions (Fe ions) introduced during production
procedures. When further supplemented with a surfactant, the
antibody-containing solution formulation of the present invention
achieved very significant suppression of visible insoluble matter
and/or insoluble particle formation induced by shaking and
freezing-thawing.
[0062] The present invention will now be further described in the
following examples, which are not intended to limit the scope of
the invention. Based on the detailed description, various changes
and modifications will be apparent to those skilled in the art, and
such changes and modifications fall within the scope of the
invention.
EXAMPLES
[0063] Antibody Samples
[0064] A humanized anti-HM1.24 antigen monoclonal antibody
(hereinafter referred to as the "anti-HM1.24 antibody") was
produced in accordance with Reference Example 2 of International
Publication No. WO98-35698 and used in the examples.
[0065] The anti-HM1.24 antibody used in the examples was of IgG1
class.
[0066] Test Procedures
[0067] (1) Gel Permeation Chromatography (GPC)
[0068] Aggregation and decomposition products of the antibody are
separated by differences in molecular weight. In this test, dimers,
trimers and higher aggregates of the antibody are collectively
referred to as aggregation products, while two low-molecular-weight
fragments generated from the antibody are referred to as
decomposition product 1 and decomposition product 2, respectively.
The residue level of the main peak was also taken into
consideration.
[0069] The content was evaluated as the residue level based on the
initial level. Aggregation and decomposition products were both
expressed in percent (%).
[0070] GPC Conditions
[0071] Column: TSK gel G3000SWXL (TOSOH)
[0072] Guard column: TSK guard column SWXL (TOSOH)
[0073] Column temperature: kept constant around 25.degree. C.
[0074] Mobile phase: 50 mM phosphate buffer (pH 7.0)/300 mM sodium
chloride
[0075] Flow rate: about 0.5 mL/min
[0076] Detection wavelength: 280 nm
[0077] Calculation of Concentration
Concentration of anti-HM1.24 antibody (mg/mL)=(concentration of
standard sample.times.peak area of anti-HM1.24
antibody.times.amount of standard sample injected)/(total peak area
of standard sample.times.amount of test substance injected)
Residual level of anti-HM1.24 antibody (%)=[(anti-HM1.24 antibody
content after stress)/(initial anti-HM1.24 antibody
content)].times.100
Aggregation products (also for decomposition products) (%)=[(peak
area of aggregation (cleavage) products)/(total peak
area)].times.100
[0078] (2) Ion Exchange Chromatography (IEC)
[0079] Charged heteromolecules are separated, which are generated
by deterioration including deamidation of the antibody. Stability
was evaluated by changes in main peak area.
[0080] HPLC Conditions
[0081] Column: Poly CAT A (PolyLC Inc.)
[0082] Guard column: Poly CAT A Javelin guard (PolyLC Inc.)
[0083] Column temperature: kept constant around 25.degree. C.
[0084] Flow rate: 1 mL/min
[0085] Detection wavelength: 280 nm
[0086] Mobile phase: using a gradient of the following two eluents
A and B:
[0087] Eluent A: 25 mM MES buffer (0.05% sodium azide, pH 6.1)
[0088] Eluent B: 250 mM sodium chloride in 25 mM MES buffer (0.05%
sodium azide, pH 6.1)
[0089] Measurement
[0090] The column was equilibrated with 30% Eluent B and then
injected with a sample solution. A linear gradient was run to 70%
Eluent B over 32 minutes, followed by maintaining 70% Eluent B for
5 minutes. After washing with 100% Eluent B for 5 minutes or
longer, the column was equilibrated with 30% Eluent B for 15
minutes or longer and provided for the next measurement.
[0091] Analysis
[0092] Peak areas of the resulting HPLC chromatogram were analyzed
by automatic integration to determine the percentage of the main
peak area.
[0093] (3) Insoluble Particle Test
[0094] Measurement: according to the test for insoluble particles
in injections using a light shielding type of an automatic particle
analyzer, as described in Standard Test Procedures of the Japanese
Pharmacopoeia.
[0095] Instrument: a light shielding type of an automatic particle
analyzer (HIAC).
[0096] (4) Foreign Insoluble Matter Test
[0097] Measurement: Visible insoluble matter was detected by the
unaided eye against a black background according to the procedures
in Section 2.9.20 of the European Pharmacopoeia, third edition.
Example 1
Effects of Organic Acid Addition
[0098] Anti-HM1.24 antibody formulations prepared in different
buffers (phosphate, acetate, citrate) were supplemented with iron
(FeCl.sub.3), and observed for the formation of visible insoluble
matter by the unaided eye against a black background in accordance
with the foreign insoluble matter test. The samples were stored at
room temperature (about 25.degree. C.). Table 1 summarizes the
composition of tested formulations, and Table 2 shows the results
obtained.
1TABLE 1 Composition of tested formulations Sample No. 1 2 3 4 5 6
7 8 9 10 11 12 Anti-HM1.24 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
2.5 2.5 antibody (mg/mL) Polysorbate 80 0.025 0.025 0.025 0.025
0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 (%) Buffer type Ac
Ac Ac Ac Phos Phos Phos Phos Cit Cit Cit Cit Buffer conc. 20 20 20
20 20 20 20 20 20 20 20 20 (mM) Sodium 100 100 100 100 100 100 100
100 100 100 100 100 chloride (mM) pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 6.0 6.0 FeCl.sub.3 conc. 0 10 100 1000 0 10 100 1000 0
10 100 1000 (.mu.g/L) Ac: acetate buffer, Phos: phosphate buffer,
Cit: citrate buffer
[0099]
2TABLE 2 Results of foreign insoluble matter test on samples
supplemented with iron (FeCl.sub.3) Sample No. Buffer Conc. Initial
1 day 4 days 5 days 6 days 7 days 8 days 1 Acetate 0 .mu.g/L - - -
- - .+-. - 2 10 .mu.g/L - - .+-. .+-. + .+-. + 3 100 .mu.g/L - -
.+-. .+-. .+-. .+-. + 4 1000 .mu.g/L - - .+-. .+-. .+-. .+-. + 5
Phosphate 0 .mu.g/L - - - - - - - 6 10 .mu.g/L - .+-. .+-. .+-.
.+-. + .+-. 7 100 .mu.g/L - - .+-. + + + + 8 1000 .mu.g/L - - ++ ++
++ +++ +++ 9 Citrate 0 .mu.g/L - - - - - - .+-. 10 10 .mu.g/L - - -
- - .+-. + 11 100 .mu.g/L - - - - .+-. .+-. + 12 1000 .mu.g/L - - -
- - .+-. .+-. not observed .fwdarw. highly observed (Evaluation)
(-) < (.+-.) < (+) < (++) < (+++)
[0100] As is evident from the above results, the anti-HM1.24
antibody formulations prepared in acetate and citrate buffers
significantly suppressed the formation of visible insoluble matter
in the presence of iron, as compared to the formulation prepared in
phosphate buffer.
Example 2
Effects of Surfactant Addition
[0101] Polysorbate 80, Polysorbate 20 and Poloxamer 188 were used
in the shaking test, freezing-thawing test and storage stability
test of anti-HM1.24 antibody formulations (2.5 to 10 mg/mL) to
study surfactant-induced effects on the formulations.
[0102] (1) Physical Stress Test
[0103] Surfactant-induced effects on physical stresses (shaking and
freezing-thawing) were studied in terms of insoluble particle or
visible insoluble matter formation.
[0104] (1-1) Test on 2.5 mg/mL Solutions
[0105] The evaluation was conducted as follows. Table 3 shows the
composition of tested formulations along with the results
obtained.
[0106] Test sample: 2 mL per 5 mL vial (Samples 13-22)
[0107] Evaluation: insoluble particle test using a light shielding
type of an automatic particle analyzer (HIAC)
[0108] Evaluation conditions:
[0109] (i) Shaking Test
[0110] Shaking conditions: 200 strokes/min..times.30 min.
[0111] Shaker: RECIPRO SHAKER SR-I
[0112] (Taiyo Scientific Industrial Co., Ltd.)
[0113] (ii) Freezing-Thawing Test
[0114] Freezing-thawing conditions: -80.degree.
C..fwdarw.25.degree. C..times.1 cycle
3TABLE 3 Composition of tested formulations and results Sample No.
13 14 15 16 17 18 19 20 21 22 Anti-HM1.24 antibody (mg/mL) 2.5 2.5
2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Polysorbate 80 (%) 0.025 0.01
0.0025 -- -- -- -- -- -- -- Polysorbate 20 (%) -- -- -- 0.025 0.01
0.0025 -- -- -- -- Poloxamer 188 (%) -- -- -- -- -- -- 0.025 0.01
0.0025 -- Sodium chloride (mM) 100 100 100 100 100 100 100 100 100
--100 Acetate buffer (mM) 20 20 20 20 20 20 20 20 20 20 PH 6.0 6.0
6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 Initial counts of particles
.gtoreq.10 .mu.m 2 9 6 6 9 3 9 1 5 19 (Counts/mL) .gtoreq.25 .mu.m
0 1 0 0 0 0 0 0 0 0 After shaking .gtoreq.10 .mu.m 78 49 30 66 89
74 131 32 44 5922 (Counts/mL) .gtoreq.25 .mu.m 3 1 0 1 1 0 1 0 1
1485 After freezing-thawing .gtoreq.10 .mu.m 6 1 2 3 2 2 3 2 4 329
(Counts/mL) .gtoreq.25 .mu.m 1 0 0 2 0 0 0 2 1 47
[0115] The surfactants used (Polysorbate 80, Polysorbate 20,
Poloxamer 188) were found to produce a significant effect when
added in the range of 0.0025% to 0.025%, in view of suppression of
the formation of insoluble particles or visible insoluble
matter.
[0116] (1-2) Test on 10 mg/mL Solutions
[0117] The evaluation was conducted as follows. Table 4 shows the
composition of tested formulations along with the results
obtained.
[0118] Test sample: 10 mL per 20 mL vial (Samples 23-28)
[0119] Evaluation:
[0120] (i) Insoluble Particle Test Using a Light Shielding Type of
an Automatic Particle Analyzer (HIAC)
[0121] (ii) Insoluble Contamination Test According to the EP Method
(Against a Black Background)
[0122] Evaluation conditions:
[0123] (i) Shaking Test
[0124] Shaking conditions: 200 strokes/min..times.60 min.
[0125] Shaker: RECIPRO SHAKER SR-I
[0126] (Taiyo Scientific Industrial Co., Ltd.)
[0127] (ii) Freezing-Thawing Test
[0128] Freezing-thawing conditions: -20.degree. C. 5.degree.
C..times.3 cycles
4TABLE 4 Composition of tested formulations and results Sample No.
23 24 25 26 27 28 Anti-HM1.24 antibody (mg/mL) 10 10 10 10 10 10
Polysorbate 80 (%) 0 0.00025 0.0025 0.025 0.05 0.1 Acetic acid (mM)
10 10 10 10 10 10 Sodium chloride (mM) 100 100 100 100 100 100 PH
6.0 6.0 6.0 6.0 6.0 6.0 Initial counts of particles .gtoreq.10
.mu.m 2 0 0 0 1 0 (Counts/mL) .gtoreq.25 .mu.m 0 0 0 0 0 0 After
shaking .gtoreq.10 .mu.m 44079 0 0 0 0 4 (Counts/mL) .gtoreq.25
.mu.m 15699 0 0 0 0 0 After freezing-thawing .gtoreq.10 .mu.m 5054
1 0 0 0 0 (.times.3 cycles) (Counts/mL) .gtoreq.25 .mu.m 333 0 0 0
0 0 Insoluble Initial - - - - - - contamination Shaken + - - - - -
test Frozen-thawed + + - - - - (.times.3 times) +: observed, -: not
observed
[0129] Polysorbate 80 was found to produce an effect when added in
the range of 0.0025% to 0.1%, in view of suppression of the
formation of visible insoluble matter and insoluble particles.
[0130] (2) Storage Stability Test
[0131] Polysorbate 80 was tested for its inhibitory effect on the
time-dependent formation of visible insoluble matter in anti-HM1.24
antibody solutions during storage at 5.degree. C.
[0132] (2-1) Test on 2.5 and 5.0 mg/mL Solutions
[0133] The evaluation was conducted as follows. Table 5 shows the
composition of tested formulations along with the results
obtained.
[0134] Test sample: 5 mL per 10 mL vial (Samples 29-38)
[0135] Evaluation: insoluble contamination test according to the EP
method (against a black background)
[0136] Storage conditions: at 5.degree. C. for 3 months (5.degree.
C.-3M)
5TABLE 5 Composition of tested formulations and results Sample No.
29 30 31 32 33 34 35 36 37 38 Anti-HM1.24 antibody 2.5 2.5 2.5 2.5
2.5 5.0 5.0 5.0 5.0 5.0 (mg/mL) Polysorbate 80 (%) - 0.001 0.0025
0.01 0.025 - 0.002 0.005 0.02 0.05 Sodium chloride (mM) 100 100 100
100 100 100 100 100 100 100 Acetate buffer (mM) 20 20 20 20 20 20
20 20 20 20 PH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 Vial ID1
Initial - - - - - + - - - - 3 M + + - - - ++ - - - - Vial ID2
Initial - - - - - - - - - - 3 M + - - - - ++ - - - + Vial ID3
Initial ++ - - - - - - - - - 3 M + + + - - ++ - - + - Vial ID4
Initial - - - - - - - - - - 3 M + + - - - ++ + + - - Vial ID5
Initial - - - - - - - - 3 M + - - - ++ + - - ++: highly observed,
+: slightly observed, -: not observed
[0137] After storage at 5.degree. C. for 3 months, a remarkable
formation of visible insoluble matter was observed in the
formulations containing no Polysorbate 80 (Samples 29 and 34),
whereas a significant inhibitory effect on formation of
contamination was observed when Polysorbate 80 was added to the
formulations in the range of 0.001% to 0.05%.
[0138] (2-2) Test on 10 mg/mL Solutions
[0139] The evaluation was conducted as follows. Table 6 shows the
composition of tested formulations along with the results
obtained.
[0140] Test sample: 10 mL per 20 mL vial (Samples 39-42)
[0141] Evaluation: foreign insoluble matter test according to the
EP method (against a black background)
[0142] Storage conditions: at 5.degree. C. for 3, 6 and 12 months
(5.degree. C.-3, 6, 12M)
6TABLE 6 Composition of tested formulations and results Sample No.
39 40 41 42 Anti-HM1.24 antibody (mg/mL) 10 10 10 10 Polysorbate 80
(%) 0 0.005 0.025 0.05 Sodium chloride (mM) 100 100 100 100 Acetate
buffer (mM) 10 10 10 10 PH 6.0 6.0 6.0 6.0 Foreign Initial - - - -
insoluble 5.degree. C.-3 M + - - - matter test 5.degree. C.-6 M + -
- - 5.degree. C.-12 M + - - - +: observed, -: not observed
[0143] When added at 0.005% or more, Polysorbate 80 was found to
produce a significant effect on the formation of visible insoluble
matter.
Example 3
Dependency on pH
[0144] To confirm the optimum pH for the anti-HM1.24 antibody in
the concentration range of 2.5 to 10 mg/mL, the heat resistance
test and the storage stability test were conducted.
[0145] (1) Test on 2.5 mg/mL Formulations
[0146] The evaluation was conducted as follows. Tables 7 and 8 show
the composition of tested formulations and the results obtained,
respectively.
[0147] Test sample: 1 mL per 5 mL vial (Samples 43-47)
[0148] Evaluation: heat resistance test, storage stability test
[0149] Storage conditions:
[0150] at 50.degree. C. for 3 moths (50.degree. C.-3M)(GPC)
[0151] at 5.degree. C. for 6 months (5.degree. C.-6M)(IEC)
7TABLE 7 Composition of tested formulations Sample No. 43 44 45 46
47 Anti-HM1.24 antibody 2.5 2.5 2.5 2.5 2.5 (mg/mL) Acetate buffer
(mM) 20 20 20 20 20 Sodium chloride (mM) 100 100 100 100 100
Polysorbate 80 (%) 0.025 0.025 0.025 0.025 0.025 PH 5.0 5.5 6.0 6.5
7.0
[0152]
8TABLE 8 Evaluation results of heat resistance test (50.degree.
C.-3M) and storage stability test (5.degree. C.-6M) GPC (50.degree.
C.-3M) Sample Residue Aggregation Decomposition Decomposition IEC
(5.degree. C.-6M) No. pH (%) products (%) product 1 (%) product 2
(%) Main peak (%) 43 5.0 53.7 35.8 ND 12.6 93.1 44 5.5 68.1 29.6 ND
9.5 92.8 45 6.0 76.6 19.6 4.9 8.2 92.5 46 6.5 78.2 15.2 8.9 7.8
90.7 47 7.0 77.8 12.4 11.4 8.5 88.0
[0153] (2) Test on 10 mg/mL Formulations
[0154] The evaluation was conducted as follows. Tables 9 and 10
show the composition of tested formulations and the results
obtained, respectively.
[0155] Test sample: 1 mL per 5 mL vial (Samples 48-54)
[0156] Evaluation: heat resistance test
[0157] Storage conditions:
[0158] at 50.degree. C. for 1 month (50.degree. C.-1M)(GPC)
[0159] at 40.degree. C. for 1 month (40.degree. C.-1M)(IEC)
9TABLE 9 Composition of tested formulations Sample No. 48 49 50 51
52 53 54 Anti-HM1.24 antibody 10 10 10 10 10 10 10 (mg/mL)
Polysorbate 80 (%) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Acetate
buffer (mM) 10 10 10 10 10 10 10 Sodium chloride (mM) 100 100 100
100 100 100 100 PH 5.50 5.75 6.00 6.25 6.50 6.75 7.00
[0160]
10TABLE 10 Evaluation results of heat resistance test (50.degree.
C.-1M, 40.degree. C.-1M) GPC (50.degree. C.-1M) Sample Residue
Aggregation Decomposition Decomposition IEC (40.degree. C.-1M) No.
pH (%) products (%) product 1 (%) product 2 (%) Main peak (%) 48
5.50 79.1 14.5 5.4 2.5 67.2 49 5.75 84.2 11.3 5.8 2.3 64.4 50 6.00
85.6 10.0 5.9 2.2 62.5 51 6.25 85.5 10.1 6.1 2.2 58.5 52 6.50 86.8
8.6 6.5 2.2 57.8 53 6.75 87.1 7.9 6.8 2.2 57.0 54 7.00 86.5 7.7 7.6
2.5 55.0
[0161] On the basis of these results, it is confirmed that, in the
range of pH 5.5 to pH 6.5, the formation of aggregation products
and the transition into charged heteromolecules were suppressed.
Further, the following were confirmed.
[0162] The formation of aggregation products was accelerated under
lower pH conditions.
[0163] The formation of decomposition product 2 was not
substantially dependent on pH.
[0164] The formation of decomposition product 1 was accelerated at
higher pH.
[0165] The residue level of the main peak in IEC was higher at
lower pH.
Example 4
Dependency on Sodium Chloride Concentration
[0166] The heat resistance test was conducted to study the effect
of sodium chloride concentration on the stability of the
anti-HM1.24 antibody.
[0167] (1) Test on 2.5 mg/mL Formulations
[0168] The evaluation was conducted as follows. Tables 11 and 12
show the composition of tested formulations and the results
obtained, respectively.
[0169] Test sample: 1 mL per 5 mL vial (Samples 55-58)
[0170] Evaluation: heat resistance test
[0171] Storage conditions: at 50.degree. C. for 3 months
(50.degree. C.-3M)(GPC)
11TABLE 11 Composition of tested formulations Sample No. 55 56 57
58 Anti-HM1.24 antibody (mg/mL) 2.5 2.5 2.5 2.5 Polysorbate 80 (%)
0.025 0.025 0.025 0.025 Acetate buffer (mM) 20 20 20 20 PH 6.0 6.0
6.0 6.0 Sodium chloride (mM) 23 100 200 500
[0172]
12TABLE 12 Evaluation results of heat resistance test (50.degree.
C.-3M) Aggregation Decomposition Decomposition Sample NaCl Residue
products product 1 product 2 No. (mM) (%) (%) (%) (%) 55 23 75.4
17.6 ND 7.1 56 100 76.8 15.5 ND 7.6 57 200 76.5 15.8 ND 7.8 58 500
68.0 14.5 8.7 8.6
[0173] When sodium chloride was added to the sample such that the
final concentration was 500 mM, a reduced residue level of
monomeric molecules was observed, whereas the other samples showed
no difference in stability.
[0174] (2) Test on 10 mg/mL Formulations
[0175] The evaluation was conducted as follows. Tables 13 and 14
show the composition of tested formulations and the results
obtained, respectively.
[0176] Test sample: 1 mL per 5 mL vial (Samples 59-61)
[0177] Evaluation: heat resistance test
[0178] Storage conditions: at 50.degree. C. for 1 month (50.degree.
C.-1M)(GPC)
13TABLE 13 Composition of tested formulations Sample No. 59 60 61
Anti-HM1.24 antibody (mg/mL) 10 10 10 Polysorbate 80 (%) 0.05 0.05
0.05 Acetate buffer (mM) 10 10 10 PH 6.0 6.0 6.0 Sodium chloride
(mM) 100 150 200
[0179]
14TABLE 14 Evaluation results of heat resistance test (50.degree.
C.-1M) Aggregation Decomposition Decomposition Sample NaCl Residue
products product 1 product 2 No. (mM) (%) (%) (%) (%) 59 100 90.6
7.0 4.5 1.9 60 150 89.2 7.6 4.5 2.0 61 200 88.9 7.8 4.7 2.1
[0180] Over the tested concentration range of sodium chloride (23
to 200 mM), there was no difference in the stability of the
anti-HM1.24 antibody formulations.
Example 5
Dependency on Acetic Acid Concentration
[0181] The heat resistance test was conducted to study the effect
of acetic acid concentration on the stability of the anti-HM1.24
antibody.
[0182] (1) Test on 10 mg/mL Formulations
[0183] The evaluation was conducted as follows. Tables 15 and 16
show the composition of tested formulations and the results
obtained, respectively.
[0184] Test sample: 1 mL per 5 mL vial (Samples 59, 62 and 63)
[0185] Evaluation: heat resistance test
[0186] Storage conditions: at 50.degree. C. for 1 month (50.degree.
C.-1M)(GPC)
15TABLE 15 Composition of tested formulations Sample No. 59 62 63
Anti-HM1.24 antibody (mg/mL) 10 10 10 Polysorbate 80 (%) 0.05 0.05
0.05 Acetate buffer (mM) 10 20 50 pH 6.0 6.0 6.0 Sodium chloride
(mM) 100 100 100
[0187]
16TABLE 16 Evaluation results from heat resistance test (50.degree.
C.-1M) Aggre- Acetic gation Decomposition Decomposition Sample acid
Residue products product 1 product 2 No. (mM) (%) (%) (%) (%) 59 10
90.6 7.0 4.5 1.9 62 20 89.7 7.9 4.8 2.1 63 50 89.0 8.3 4.9 2.1
[0188] On the basis of these results, it is confirmed that the
anti-HM1.24 antibody formulations were stable over the range of 10
to 50 mM acetic acid.
* * * * *