Description
Title of Invention: STABLE LIQUID FORMULATION OF
ETANERCEPT
Technical Field
[1] The present invention relates to a liquid formulation of etanercept (recombinant p75
sTNFR:Fc fusion protein), and more particularly, to a liquid formulation comprising
one or more stabilizers selected from the group consisting of methionine, lysine,
histidine, and pharmaceutically acceptable salts thereof in an amount sufficient to
reduce by-products of etanercept during storage.
Background Art
[2] Etanercept is a biological inflammation modulator that functions as a competitive
inhibitor of TNF-a, binding to cell surface TNF-a receptor, to inhibit TNF-a mediated
immune responses. Etanercept is a macromolecule with a molecular weight of ap
proximately 150 kDa, and is a homodimer of two Fc fusion proteins linked by a
disulfide bond, each Fc fusion protein consisting of a human soluble p75 TNF receptor
coupled to the Fc portion of human immunoglobulin G subclass 1 (Goldenberg,
Clinical Therapeutics, 21(1): 75-87, 1999; Moreland et al., Ann. Intern. Med., 130(6):
478-486, 1999).
[3] This prototypic fusion protein was first synthesized in the early 1990s by Bruce A.
Beutler at the University of Texas Southwestern Medical Center, and marketed by
Amgen under the trade name of Enbrel in 2002. Etanercept is a TNF-a inhibitor used
to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis, and under clinical
trials for the treatment of vasculitis, Alzheimer's disease, and Crohn's disease.
[4] Like protein drugs, antibody drugs have a very short half life, and chemical and
physical denaturation can be easily caused by unfavorable temperature, shear stress,
vibration, freeze-thawing, UV exposure, excessive pH change, organic solvents, and
microbial contamination. Chemical denaturation includes dimer dissociation,
oxidation, deamidation, isomerization, and polymerization, which are influenced by
the amino acids constituting the antibody and conditions of the solvent containing the
antibody (salt, pH and temperature). Physical denaturation includes loss of tertiary
structure, covalent/non-covalent aggregation and adhesion of monomers, which are in
fluenced by hydrophobic patches on the protein surface changed by antibodycontaining
surrounding environments such as solvents, complex protein structures such
as charge distribution, and thermal stability. The physical or chemical denaturation of
an antibody causes loss of its physiological activities. Since the denaturation is an irre
versible process, antibodies, once denatured, may not recover their native properties to
the initial state, leading to a reduction in their therapeutic efficacies. It has been also
suggested that aggregation of monomers causes immune responses. Therefore, many
studies have been conducted on antibody formulations containing a physiologically
effective amount without aggregates (Ishikawa et al., Biol. Pharm. Bull., 33(8):
1413-1417, 2010; Levin et al., The Development of Therapeutic Monoclonal Antibody
Products, editors. Boston (MA): BioProcess Technology Consultants, Inc, Chapter 9).
[5] There are many methods available for preventing protein denaturation in liquid for
mulations. In some protein drugs, the stability problems are addressed via
lyophilization. For instance, U.S. Patent No. 7,592,004 discloses a lyophilized for
mulation of daclizumab that is stabilized using 5 to 25 mM of a histidine buffer
solution (pH 5.5 to 6.5), 0.005 to 0.03% of a non-ionic surfactant, polysorbate and 100
to 300 mM of a non-reducing sugar, sucrose. U.S. Patent Publication No.
2010-0158925 discloses a lyophilized formulation of cetuximab that is stabilized using
a histidine buffer solution and lactobionic acid. However, the lyophilization process
generates freezing and drying stresses such as formation of ice crystals, pH change,
and high concentration of solute, and these stresses may cause antibody denaturation.
In addition, since a large-capacity lyophilizer is needed for the lyophilization process
during the production, high production costs arise during a large scale of production.
Dissolving the lyophilized product in sterile aqueous media for reconstitution before
use also poses an inconvenience.
[6] As an alternative to solve these limitations, a stabilizer is added in liquid for
mulations for the improvement of antibody stability. Surfactants, serum albumins,
polysaccharides, amino acids, polymers, salts or the like are known as stabilizers for
proteins including antibodies (Wang, Int. J. Pharm., 185: 129-188, 1999; Wang et al.,
J . Pharm. Sci., 96(1): 1-26, 2007).
[7] US Patent Publication No. 201 1-0070231 discloses a stable liquid composition of
IgG antibody, in which the stable pharmaceutical liquid formulation includes 50 mg/
mL or more of daclizumab in 20 to 60 mM of a succinate buffer solution (pH 5.5 to
6.5), 0.02 to 0.04% of polysorbate, and 75 to 150 mM of sodium chloride.
[8] US Patent Publication No. 201 1-0020328 discloses a therapeutically effective
amount of anti-CD20 antibody formulation, in which the pharmaceutically stable com
position includes 10 to 100 mM of sodium acetate, 25 to 100 mM of sodium chloride,
0.5 to 5% of arginine free base, 0.02 to 0.2 mM of EDTA and 0.01 to 0.2% of
polysorbate 80, and its pH is 5.0 to 7.0.
[9] US Patent No. 7,785,592 discloses a stable formulation, in which 75 mg/mL or more
of palivizumab is stabilized by using a histidine buffer solution and glycine without
ionic salts and a surfactant, and its stability is maintained at 2 to 8°C for at least 15
months.
US Patent No. 6,991,790 discloses a stable aqueous pharmaceutical formulation,
including a therapeutically effective amount of an antibody, an acetate buffer solution
of approximately pH 4.8 to 5.5, a surfactant, and a polyol without an isotonic agent
such as sodium chloride.
US Patent No. 7,648,702 discloses a liquid formulation, in which a fusion protein of
the human p75 tumor necrosis factor receptor linked to the Fc portion of the human
immunoglobulin Gl (IgGl) is stabilized by using approximately 10 to 200 mM of Larginine
as an aggregation preventing agent. This patent is the only technique of using
etanercept as an active ingredient.
In order to prepare stable formulations, however, appropriate stabilizers should be
used considering the physicochemical properties of each active ingredient. When the
stabilizers are used in combination, competition therebetween and adverse effects may
lead to undesirable effects. In addition, the concentrations of antibodies should be
within the range suitable for the stabilization, and their concentrations are relatively
higher than those of protein drugs. Thus, much effort and caution are required to
stabilize antibodies in solutions (Shire et al., J. Pharm. Sci., 93(6): 1390-1402, 2004).
There are few studies on the stable liquid formulations of etanercept, and the only
method for stabilizing etanercept that the inventors are aware of is to use L-arginine in
the liquid formulation. Therefore, there is an urgent need to develop a new liquid for
mulation which is able to stably maintain the activity of etanercept for a long period
and which is more effective in etancercept stabilization than the known formulation
comprising L-arginine.
Disclosure of Invention
Technical Problem
The present inventors have made many efforts to develop a method for preparing a
liquid formulation capable of stably maintaining the activity of etanercept. As a result,
they found that one or more stabilizers selected from the group consisting of me
thionine, lysine, and histidine show remarkable effects on the stabilization of
etanercept in a solution, thereby completing the present invention.
Solution to Problem
An object of the present invention is to provide an aqueous formulation, comprising
stabilizers for reducing formation of etanercept by-products during storage in a
solution and maintaining its activity for a long period of time.
Advantageous Effects of Invention
The liquid formulation according to the present invention can effectively prevent
formation of etanercept by-products and stably maintain its pharmaceutical efficacies
for long-term storage. Therefore, the reconstitution procedure is not required before
administration, and the sterile formulation can be administered to patients to ensure
patient safety. Thus, it is expected to be applied to the fields in need of etanercept
treatment.
Best Mode for Carrying out the Invention
[18] In one aspect to achieve the above object, the present invention provides a liquid for
mulation, comprising one or more stabilizers selected from the group consisting of me
thionine, lysine, histidine, and pharmaceutically acceptable salts thereof.
[19] The formulation of the present invention may be a liquid formulation of etanercept
comprising, etanercept; and one or more stabilizers selected from the group consisting
of methionine, lysine, histidine, and pharmaceutically acceptable salts thereof.
[20] The formulation shows increased storage stability by reducing etanercept by-products
that are produced due to denaturation during storage.
[21] As a stabilizer, methionine, lysine, histidine, or pharmaceutically acceptable salts
thereof may be present in the formulation in an amount of 0.1 to 250 mM.
[22] Etanercept in the formulation may be present in an amount of 1 to 100 mg/mL.
[23] The formulation may further include one or more materials seleted from the group
consisting of a buffer, an isotonic agent, an excipient, and a preservative.
[24] The buffer may be selected from the group consisting of citrate, phosphate, succinate,
tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris, and may be
present in an amount of 0.1 to 100 mM.
[25] The isotonic agent may be selected from the group consisting of sodium chloride,
potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol,
polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose,
and may be present in an amount of 1 to 1000 mM.
[26] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or
pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
[27] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of lysine or phar
maceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
[28] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
[29] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically
acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of
sodium chloride.
[30] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of methionine or pharma
ceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000
mM of sodium chloride.
[31] The formulation of etanercept according to the present invention may be a liquid for
mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically
acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of
sodium chloride.
[32]
[33] Hereinafter, the present invention will be described in detail.
[34] The present invention has demonstrated for the first time that methionine, lysine,
histidine and pharmaceutically acceptable salts thereof have the effects of reducing
formation of etanercept by-products in solution and stably maintaining its activity for
long-term storage, and thus suggests a novel use thereof as stabilizers of a pharma
ceutically effective amount of etanercept.
[35] In order to prepare stable formulations, appropriate stabilizers should be used con
sidering the physicochemical properties of each active ingredient. According to the
difference in types, concentration, and combinations of materials included in a for
mulation, competition between the materials in the formulation can lead to undesirable
effects. Thus, it is difficult to prepare a stable drug specific formulation.
[36] In the light of the above background, the present inventors have found that the liquid
formulation comprising one or more stabilizers selected from the group consisting of
methionine, lysine, and histidine, and pharmaceutically acceptable salts thereof
increases storage stability of etanercept compared to the formulation having no sta
bilizers, by reducing etanercept by-products during storage in a solution.
[37] Specifically, in one embodiment of the present invention, in order to investigate the
optimal stabilizer for the preparation of the stable liquid formulation of etanercept, a
variety of amino acids, surfactants, and polymers are added to examine their stabi
lization effects. As a result, lysine, histidine, methionine, or the combination thereof
was found to show remarkable effects on stabilizing etanercept by preventing its denaturation
during storage in a solution at high temperature.
[38] Further, the formulation according to the present invention was found to show
etanercept stabilizing effects which were equivalent to or higher than that of the for
mulation comprising L-arginine which is the known stable formulation of etanercept
(US Patent No. 7,648,702). In particular, methionine was found to show the very
excellent effect of stabilizing etanercept better than L-arginine.
[39] Specifically, etanercept by-products formed during storage were examined by Size
Exclusion-HPLC (SE-HPLC). As a result, compared to the total amount of impurities
of the etanercept formulation that was prepared by using L-arginine as the stabilizer in
US Patent No. 7,648,702, the formulation prepared using lysine as the stabilizer
showed a similar amount of impurities, and the formulation prepared using histidine or
methionine as the stabilizer showed a lower amount of impurities (see Table 2). Hy
drophobic Interaction-HPLC (HI-HPLC) analysis also showed similar results. These
results indicate that histidine, lysine, and methionine prevent etanercept denaturation to
inhibit formation of by-products thereof (see Table 3).
[40] Therefore, the liquid formulation according to the present invention can stably
maintain pharmaceutical efficacies of etanercept for a long period of time by e f
fectively reducing formation of etanercept by-products.
[41]
[42] As used herein, the term "etanercept (recombinant p75 sTNFR:Fc fusion protein)"
refers to a protein which is a homodimer form of two Fc fusion proteins linked by
disulfide bonds, each Fc fusion protein consisting of a human soluble 75 kilodalton
(p75) TNF(tumor necrosis factor) receptor coupled to the Fc portion of human IgGl.
[43] Specifically, etanercept is a homodimer form of two Fc fusion proteins linked by 3
disulfide bonds, each Fc fusion protein consisting of the extracellular ligand-binding
portion of the human soluble p75 TNF receptor linked to the Fc portion of human
IgGl. The Fc component of etanercept contains the CH2 domain, the CH3 domain and
hinge region, but not the CHI domain of IgGl. The etanercept may have a molecular
weight of approximately 150 kilodaltons(kDa). This etanercept may be currently sold
under the trade name ENBREL® (Amgen Inc., Thousand Oaks, CA.), and have CAS
number 185243-69-0.
[44] Etanercept may be produced by recombinant DNA technology, but is not limited
thereto.
[45] The etanercept of the present invention is a biological inflammation modulator that
functions as a competitive inhibitor of TNF-a, binding to cell surface TNF-a receptor,
to inhibit TNF-a mediated immune responses, and is used to treat rheumatoid arthritis,
psoriasis, and ankylosing spondylitis, and is under clinical trials for the treatment of
vasculitis, Alzheimer's disease, and Crohn's disease.
[46] In the liquid formulation according to the present invention, the etanercept is
contained in a therapeutically effective amount. Preferably, etanercept is present in an
amount of 1 to 100 mg/ml.
[47] As used herein, the term "stabilized liquid formulation" or "stable liquid formulation"
refers to a formulation that retains the physical and chemical identity and integrity of
the therapeutically active ingredient, etanercept, during storage in a solution. An an
alytical measurement of the etanercep stability may be performed by protein stability
assay widely known in the art. The stability may be measured at a predetermined tem
perature for a predetermined time. For rapid assay, the formulation may be stored at a
higher or "elevated" temperature, for example, 40°C for 2 weeks to 1 month or longer,
and its time-dependent stability measured at this time.
[48] As used herein, the term "stabilizer" refers to a specific chemical that interacts with a
biological molecule and/or a general pharmaceutical excipient in a formulation to
improve its stability. The stabilizer generally protects proteins from air/solution
interface-induced stress and solution/surface-induced stress which cause protein ag
gregation. In the present invention, the stabilizer is a component that reduces formation
of etanercept by-products during storage in a solution to maintain its activity for a long
period of time, and is preferably one or more selected from the group consisting of me
thionine, lysine, histidine and pharmaceutically acceptable salts thereof.
[49] As for the amino acids, both L-forms and D-forms are included in the scope of the
present invention. Not only the amino acids themselves such as methionine, lysine, and
histidine, but also anlogues, solvates, hydrates and stereoisomers, and the pharma
ceutically acceptable salts thereof are within the scope of the present invention as long
as they show substantially the same effect.
[50] As used herein, the term "pharmaceutically acceptable salts" refers to compounds
that are prepared in the form of salts of the amino acids, such as methionine, lysine,
and histidine, within the range retaining their functions as the stabilizers of the present
invention. Specifically, it may form a salt by acid addition, and for example, it may
form a salt with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid,
phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acid (e.g., acetic
acid, halo acetic acid such as trifluoroacetic acid, propionic acid, maleic acid, succinic
acid, malic acid, citric acid, tartaric acid, salicylic acid), sugar acid (glucuronic acid,
galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharide (e.g., hyaluronic
acid, chondroitin sulfate, arginine acid), organic sulfonic acid including sulfonic acid
sugar ester such as chondroitin sulfate (e.g., methane sulfonic acid, p-toluene sulfonic
acid).
[51] As used herein, the term "analogues" refers to compounds that show the same
functions as the amino acids, such as methionine, lysine, and histidine, used as sta
bilizers of the present invention. In the present invention, the amino acids such as methionine,
lysine, and histidine include the analogues of thereof. In the present
invention, examples of the analogues may include methionine analogues, namely, se
lenomethionine, hydroxy methyl butanoic acid, ethionine, and trifluoromethionine, but
are not limited thereto.
[52] As used herein, the term "by-products" refers to the undesirable products that reduce
the ratio of the therapeutically active ingredient, etanercept, in the formulation. The
typical by-products include "low molecular weight products" resulting from etanercept
denaturation by deamination or hydrolysis, "high molecular weight products" such as
oligomers and aggregates, or mixtures thereof.
[53] As used herein, the term "high molecular weight products" includes etanercept
fragments that are subsequently aggregated by denaturation (e.g., produced by
polypeptide degradation due to deamination or hydrolysis) or mixtures thereof.
Typically, the high molecular weight products are complexes having higher molecular
weights than the therapeutic monomer etanercept, and may have a molecular weight of
more than approximately 150 kDa.
[54] As used herein, the term "low molecular weight products" includes, for example,
therapeutic polypeptides produced by deamination or hydrolysis, namely, etanercept
fragments. Typically, the low molecular weight products are complexes having lower
molecular weights than the therapeutic monomer etanercept, and may have a molecular
weight of less than approximately 150 kDa.
[55]
[56] The liquid formulation of the present invention includes one or more stabilizers
selected from the group consisting of methionine, lysine, histidine, and pharma
ceutically acceptable salts thereof. Specifically, the liquid formulation comprises me
thionine, lysine, or histidine as a stabilizer, or two stabilizers such as methionine and
lysine, methionine and histidine, or lysine and histidine, or three stabilizers such as me
thionine, lysine, and histidine. In the above formulation comprising combinations of
the amino acids, the pharmaceutically acceptable salts of methionine, lysine, histidine
respectively may be added further, or methionine, lysine, and histidine may be the
modified forms of the pharmaceutically acceptable salts respectively. The liquid for
mulation comprises preferably methionine, lysine, histidine, methionine and lysine,
methionine and histidine, or lysine and histidine, and more preferably methionine as a
stabilizer.
[57] Methionine is an essential amino acid, and has a chemical formula of C5H NO2S and
a molecular weight of 149.21. Methionine is a non-polar amino acid, and contains a
thioether group(-S-CH 3) in its side chain. It has a pKi value of 2.28, pK2value of 9.21,
and isoelectronic point (PI) value of 5.74. Methionine prevents oxidation of antibodies
in liquid formulations to stabilize antibodies by competing with the methionine
residues in antibodies for reaction with the free hydroxyl radicals (Lam et al., J . Pharm.
Sci., 86(11): 1250-1255, 1997; Wang, Int. J. Pharm., 185: 129-188, 1999).
[58] Lysine is an essential amino acid, and has a chemical formula of C H14N20 2 and a
molecular weight of 146. 19. Lysine is a basic amino acid, and has a pK^ value of 2.18,
pK2value of 8.95, pK3value of 10.53 and PI value of 9.74.
[59] Histidine is an essential amino acid with an imidazole functional group having
positive charge in its side chain, and has a chemical formula of C H N 3O2 and a
molecular weight of 155. 15. Histidine is a basic amino acid, and has a pK^ value of
1.82, pK2value of 9.17, pK3value of 6.0 and PI value of 7.59.
[60] In contrast, L-arginine contained in the known commercially available formulation of
etanercept is an essential amino acid, and has a chemical formula of C H14N40 2 and a
molecular weight of 174.2. Arginine is a basic amino acid, and has a pK^ value of 2.17,
pK2value of 9.04, pK3value of 12.48 and PI value of 10.76. That is, methionine,
lysine, and histidine used as a stabilizer in the present invention are totally different
from arginine as a known stabilizer for etanercept in construct, chemical formula,
physicochemical properties, and ionization tendency. The present invention has
demonstrated for the first time that amino acids such methionine, lysine, and histidine
are the appropriate stabilizers for etanercept.
[61] The content of the stabilizer in the liquid formulation of the present invention is 0.1
to 250 mM, preferably 1 to 100 mM, and more preferably 5 to 50 mM.
[62] The liquid formulation of the present invention may further include any material
which is generally contained in formulations of protein drugs or antibody drugs to
increase etanercept stability according to one or more stabilizers selected from the
group consisting of methionine, lysine, histidine, and pharmaceutically acceptable salts
thereof except for those deteriorating the function of the present invention. For
example, L-arginine known as a stabilizer for etanercept may be added to the liquid
formulation of the present invention.
[63] The liquid formulation according to the present invention may further include a
buffer in addition to the stabilizer. As used herein, the term "buffer" refers to the
component that improves isotonicity and chemical stability of the formulation, and
functions to maintain physiologically suitable pH. The buffer prevents a rapid pH
change of the liquid formulation to maintain pH of the solution for the stabilization of
etanercept. Preferred examples of the buffer include citrate, phosphate, succinate,
tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris, but are not
limited thereto. In the specific embodiment, the buffer is a phosphate buffer. The
buffer may be used either alone or in combinations of two or more thereof.
[64] In the various embodiments of the present invention, the formulation should have pH
of approximately 5 to 7.5 or pH of approximately 5.8 to 6.8. In the specific embodiment,
the formulation has pH of approximately 6.0 to 6.6. The ranges from the in
termediate to the above mentioned pH levels, for example, approximately pH 5.2 to ap
proximately pH 6.4 (e.g., pH 6.2) are also intended to be part of this invention. For
example, ranges of values using a combination of any of the above mentioned values
as upper and/or lower limits are intended to be included. If necessary, the pH may be
adjusted by techniques known in the art. For example, the pH may be adjusted to any
desirable range by addition of HC1 or by addition of histidine, if necessary.
[65] In another embodiment, the buffer is present at a concentration of 0.1 to 100 mM,
preferably 1 to 50 mM, and more preferably 5 to 25 mM. The ranges from the in
termediate to the above mentioned concentrations are also intended to be part of this
invention. For example, ranges of concentrations using a combination of any of the
above mentioned concentrations as upper and/or lower limits are intended to be
included. In the specific embodiment, the buffer should be present in an amount
sufficient to maintain the physiologically sufficient pH.
[66] The liquid formulation according to the present invention may further include an
isotonic agent in addition to the stabilizer. As used herein, the term "isotonic agent"
refers to a component that functions to partially maintain isotonicity of the formulation
and the protein level, and partially maintain the level, ratio, or proportion of the thera
peutically active polypeptide present in the formulation. The isotonic agent possesses
the same osmotic pressure as blood plasma, and so can be intravenously infused into a
subject without changing the osmotic pressure of the subject's blood plasma. Indeed, in
one embodiment according to the present invention, isotonic agent is present in an
amount sufficient to render the formulation suitable for intravenous infusion. Often,
the isotonic agent serves as a bulking agent as well. As such, the isotonic agent may
allow the protein to overcome various stresses such as freezing and shear.
[67] The isotonic agent serves to maintain the proper osmotic pressure in the body, when
etanercept in the solution is administered into the body. Examples of the isotonic agent
may include the commonly used sodium chloride, potassium chloride, boric acid,
sodium borate, mannitol, glycerin, propylene glycol, polyethylene glycol, maltose,
sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose, but is not limited thereto. In
the specific embodiment, the isotonic agent is NaCl. These isotonic agents may be
used either alone or in combinations of two or more thereof.
[68] In still another embodiment, the isotonic agent (e.g., NaCl) is present at a con
centration of 1 to 1000 mM, preferably 10 to 500 mM, and more preferably 50 to 250
mM. The ranges from the intermediate to the above mentioned concentrations are also
intended to be part of this invention. For example, ranges of concentrations using a
combination of any of the above mentioned concentrations as upper and/or lower limits
are intended to be included. The isotonic agent should be present in an amount
sufficient to maintain osmosis of the formulation.
[69] The liquid formulation according to the present invention may further include a phar
maceutically acceptable excipient, and examples of the excipient may include sugars
and polyols, surfactants, polymers or the like. Examples of the sugars and polyols may
include sucrose, trehalose, lactose, maltose, galactose, mannitol, sorbitol, glycerol,
examples of the surfactants may include non-ionic surfactants such as polysorbate 20,
polysorbate 80, and poloxamer, and examples of the polymers may include dextran,
polyethylene glycol, carboxyl methylcellulose, hyaluronic acid, and cyclodextrin.
[70] The liquid formulation according to the present invention may further include a
preservative. The preservative means a chemical that is added to pharmaceutical for
mulations as an antimicrobial agent. Examples of the preservative may include benzalkonium
chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol,
methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chloro-cresol,
phenylmercuric nitrate, thimerosal, and benzoic acid, but are not limited thereto. These
preservatives may be used either alone or in combinations of two or more thereof.
[71] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or
pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride. More specifically, the formulation of the present
invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1
to 100 mM of methionine or pharmaceutically acceptable salts thereof, 0.1 to 50 mM
of phosphate buffer, and 1 to 500 mM of sodium chloride, at pH 6.0 to 6.6.
[72] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride. More specifically, the formulation of the present
invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1
to 100 mM of histidine or pharmaceutically acceptable salts thereof, 0.1 to 50 mM of
phosphate buffer, and 1 to 500 mM of sodium chloride, at pH 6.0 to 6.6.
[73] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of lysine or
pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride. More specifically, the formulation of the present
invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1
to 100 mM of lysine or pharmaceutically acceptable salts thereof, 0.1 to 50 mM of
phosphate buffer, and 1 to 500 mM of sodium chloride, at pH 6.0 to 6.6.
[74] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically
acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of
sodium chloride. More specifically, the formulation of the present invention is the
stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 1 to 50 mM of
histidine or pharmaceutically acceptable salts thereof, 1 to 100 mM of lysine or phar
maceutically acceptable salts thereof, 0.1 to 50 mM of phosphate buffer, and 1 to 500
mM of sodium chloride, at pH 6.0 to 6.6.
[75] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of methionine or pharma
ceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000
mM of sodium chloride. More specifically, the formulation of the present invention is
the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 1 to 50 mM of
histidine or pharmaceutically acceptable salts thereof, 1 to 100 mM of methionine or
pharmaceutically acceptable salts thereof, 0.1 to 50 mM of phosphate buffer, and 1 to
500 mM of sodium chloride, at pH 6.0 to 6.6.
[76] In the specific embodiment, the formulation of the present invention is a stable liquid
formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or
pharmaceutically acceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically
acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of
sodium chloride. More specifically, the formulation of the present invention is the
stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 1 to 50 mM of me
thionine or pharmaceutically acceptable salts thereof, 1 to 50 mM of lysinee or phar
maceutically acceptable salts thereof, 0.1 to 50 mM of phosphate buffer, and 1 to 500
mM of sodium chloride, at pH 6.0 to 6.6.
[77] The formulation of the present invention can be used for treatment of a disease in
which etanercept is therapeutically effective. Etanercept is a biological inflammation
modulator to inhibit TNF-a mediated immune responses, and the formulation of the
present invention is used to treat rheumatoid arthritis, psoriasis, ankylosing spondylitis,
vasculitis, Alzheimer's disease, or Crohn's disease, not limited thereto. The formulation
of the present invention may be administered by oral or parenteral, i.e., subcutanelusly,
intramuscularly, ntraperitoneail, intra-abdominal, transdermal route, and/or intra
venously, but is not limited thereto.
[78]
[79] In another aspect, the present invention provides a method for incresing stability of
etanercept using a liquid formulation comprising one or more stabilizers selected from
the group consisting of methionine, lysine, histidine, and pharmaceutically acceptable
salts thereof.
[80] The liquid formulation can increase storage stability of etanercept by reducing
etanercept by-products that are produced due to denaturation during storage.
[81]
Mode for the Invention
[82] Hereinafter, the present invention will be described in more detail with reference to
Examples. However, these Examples are for illustrative purposes only, and the
invention is not intended to be limited by these Examples.
[83]
[84] Stability test of etanercept aqueous formulation according to
addition of stabilizer
[85] In order to investigate the optimal stabilizer for the preparation of the stable aqueous
formulation of etanercept, histidine as a stabilizer was added to a 5 mM phosphate
solution to which etanercept was added to a concentration of 50 mg/mL, and sodium
chloride was added thereto as an isotonic agentto prepare Formulation 1.
[86] In addition, etanercept was added to a 10 mM phosphate solution to a concentration
of 50 mg/mL, and sodium chloride was added thereto as an isotonic agent. Then, each
of lysine, arginine, methionine, glycine, polysorbate 20, polysorbate 80, poloxamer
188, propylene glycol, protamine sulfate, sucrose, and guanidine HC1 was added to
prepare Formulations 2 to 12.
[87] Finally, sodium chloride as the isotonic agent was added to a 10 mM phosphate
solution to which etanercept was added to a concentration of 50 mg/mL to prepare a
stabilizer-free formulation. The stabilizer compositions of the formulations are shown
in the following Table 1. Each 0.5 mL of the prepared formulation was put in 1.0 mL
glass syringes, sealed, and stored at 50°C.
[88] Table 1
[Table 1]
[89]
[90] At 7 days after storage at 50°C, Formulations 1 to 12 and the stabilizer-free for
mulation of Table 1 were analyzed by SE-HPLC to examine the types of low
molecular weight products and high molecular weight products such as oligomers and
aggregates resulting from etanercept denaturation, and the total amount of these
products was represented as Total Impurity. In addition, structural changes of
etanercept were examined by HI-HPLC. In HI-HPLC, etanercept-related substances
were separated into 4 peaks, including pre-Peak, Peak 1, Peak 2, and Peak 3. Pre-Peak
and Peak 1 represent low molecular weight products, Peak 2 represents etanercept, and
Peak 3 represents dimers with low aggregation or activity. Thus, the total amount of
pre-Peak, Peak 1, and Peak 3 was represented as Total Impurity. The results are shown
in Tables 2 and 3, respectively.
Table 2
[Table 2]
[92] Table 3
[Table 3]
[93]
[94] As shown in the SE-HPLC results of Table 2, when stored at 50°C for 7 days, For
mulation 1 containing histidine as a stabilizer showed total impurity of 22.2%, For
mulation 2 containing lysine as a stabilizer showed total impurity of 24.8%, For
mulation 3 containing arginine as a stabilizer showed total impurity of 24.2%, For
mulation 4 containing methionine as a stabilizer showed total impurity of 20.8%. All
of them showed remarkably low Total Impurity, compared to stabilizer-free for
mulation showing Total Impurity of 30.2%. When these formulations were compared
to Formulation 3 using L-arginine as a stabilizer (US Patent No. 7,648,702), For
mulation 2 containing lysine showed similar total impurity of 24.8%, and Formulation
1 containing histidine and Formulation 4 containing methionine showed reduced Total
Impurity of 22.2% and 20.8%, respectively.
The HI-HPLC results of Table 3 are similar to the SE-HPLC results. The stabilizerfree
formulation showed Total Impurity of 41.9%. On the contrary, each of For
mulations 1, 2, 3, and 4 showed Total Impurity of 33.7%, 34.9%, 34.3%, and 30.3%
respectively, indicating that histidine, lysine, L-arginine, and methionine added in each
formulation have the effects of preventing etanercept denaturation. In particular, For
mulation 4 containing methionine showed the lowest Total Impurity, indicating that
methionine is most effective in etanercept stabilization.
[96] Consequently, the present invention demonstrated that lysine, histidine, and me
thionine have effects of stabilizing etanercept by preventing its denaturation, which are
equivalent to or higher than that of L-arginine, and in particular, methionine is more
effective in etanercept stabilization than L-arginine.
[97]
[98] Stability test of etanercept aqueous formulation according to me¬
thionine concentration
[99] In order to investigate the optimal concentration of methionine for the stabilization of
etanercept, 120 mM sodium chloride was added to 10 mM phosphate solutions, and
each of 25 mM and 12.5 mM methionine was added thereto, after which etanercept in
a concentration of 50 mg/mL so was added as to prepare Formulations 13 and 14, r e
spectively. According to the preparation of a commercially available etanercept
aqueous formulation, 25 mM L-arginine was added to 100 mM sodium chloride and
1% sucrose in a 10 mM phosphate solution, and etanercept was added to a con
centration of 50 mg/mL so as to prepare a control. Each 0.5 mL of the formulation was
put in 1.0 mL glass syringes, sealed, and stored at 40°C, 25°C and 4°C. The com
positions of the prepared aqueous formulations are shown in the following Table 4.
[100] Table 4
[Table 4]
[101] After Formulation 13, Formulation 14, and the control were stored at 40°C for 1 and
3 weeks, and at 25°C and 4°C for 4 and 8 weeks each, their Total Impurity was
measured by SE-HPLC and HI-HPLC, as in Example 1. The results are shown in
Tables 5 and 6, respectively.
[102] Table 5
[Table 5]
[103] Table 6
[Table 6]
[104]
[105] As shown in the results of Tables 5 and 6, Formulation 13 and Formulation 14
containing methionine as the stabilizer showed low impurity production under all of
the storage conditions of 40°C, 25°C and 4°C, compared to the control group prepared
according to the preparation method of a commercially available etanercept aqueous
formulation. Specifically, the SE-HPLC and HI-HPLC results showed that For
mulation 13 containing 25 mM methionine and Formulation 14 containing 12.5 mM
methionine showed lower impurity production at 40°C and 3 weeks, and at 25°C and 8
weeks, and similar impurity production at 4°C and 8 weeks, compared to the control.
These results indicate that the methionine-containing formulation of the present
invention is a more stable formulation for maintaining the etanercept activity for a long
period of time than the commercially available formulation.
[106]
[107] In conclusion, the present invention demonstrated that the etanercept aqueous for
mulation containing methionine prevents denaturation of etanercept to maintain its
activity for a long period of time, and methionine is very effective as a stabilizer in the
etanercept aqueous formulation.
[108]
[109] Stability test of etanercept aqueous formulation comprising
histidine and lysine, or histidine and methionine
[110] Histidine 5mM and lysine 25mM as a stabilizer were added to a 10 mM phosphate
solution to which etanercept was added to a concentration of 50 mg/mL, and sodium
chloride was added thereto as an isotonic agent to prepare Formulation 15.
[Ill] In addition, Histidine 5mM and methionine 25mM as a stabilizer were added to a 10
mM phosphate solution to which etanercept was added to a concentration of 50 mg/
mL, and sodium chloride was added thereto as an isotonic agentto prepare Formulation
16.
[112] Finally, sodium chloride as the isotonic agent was added to a 10 mM phosphate
solution and etanerceptwas added thereto to a concentration of 50 mg/mL to prepare a
stabilizer-free formulation.
[113] Each 0.5 mL of the prepared formulation was put in 1.0 mL glass syringes, sealed,
and stored at 50°C.
[114] The stabilizer compositions of the formulations are shown in the following Table 7.
[115] Table 7
[Table 7]
[116]
[117] At 7 days after storage at 50°C, Formulations 15, 16 and stabilizer-free formulation
of Table 7 were analyzed by SE-HPLC as example 1 to measure their Total Impurity.
The results are shown in Tables 8.
[118] Table 8
[Table 8]
[119]
[120] As shown in the SE-HPLC results of Table 8, when stored at 50°C for 7 days, For
mulation 16 containing histidine as a stabilizer showed total impurity of 21.5%, and
showed remarkably low Total Impurity, compared to the stabilizer-free formulation
showing Total Impurity of 30.2%. And Formulation 16 showed remarkably low Total
Impurity, compared to Formulation 1 containing lOmM histidine showing Total
Impurity of 22.2%, and Formulation 2 containing lysine 25mM showing Total
Impurity of 24.8%.
[121] That is, the present invention demonstrated that the formulation comprising histidine
and lysine has the remarkably excellent effects of preventing etanercept denaturation
by reducing the amount of total impurity for storage, compared to the stabilizer-free
formulation and each formulation comprising histidine, lysine, and methionine r e
spectively.
[122] In addition, as shown in the SE-HPLC results of Table 8, when stored at 50°C for 7
days, Formulation 16 containing histidine as a stabilizer showed Total Impurity of
20.9%, which is remarkably low, compared to the stabilizer-free formulation showing
Total Impurity of 30.2%. In particular, Formulation 16 showed low Total Impurity
compared to Formulation 15 containing histidine and lysine as a stabilizer.
[123] That is, the present invention demonstrated that the formulation comprising histidine
and methionine has remarkably effects on the preventing of etanercept denaturation by
reducing the amount of Total Impurity for storage, compared to the stabilizer-free for
mulation.
[124]
[125] Stability test of etanercept aqueous formulation comprising me¬
thionine and lysine
[126] Methionine 12.5mM and lysine 12.5mM as a stabilizer were added to a 10 mM
phosphate solution to which etanercept was added to a concentration of 50 mg/mL, and
sodium chloride was added thereto as an isotonic agent to prepare Formulation 17.
[127] In addition, sodium chloride as the isotonic agent was added to a 10 mM phosphate
solution and etanercept was added thereto to a concentration of 50 mg/mL to prepare a
stabilizer-free formulation.
[128] Each 0.5 mL of the prepared formulation was put in 1.0 mL glass syringes, sealed,
and stored at 50°C. The stabilizer compositions of the formulations are shown in the
following Table 9.
[129] Table 9
[Table 9]
[130]
[131] At 7 days after storage at 50°C, Formulations 17, and stabilizer-free formulation of
Table 9 were analyzed by SE-HPLC as example 1 to measure their Total Impurity. The
results are shown in Table 10.
[132] Table 10
[Table 10]
[133]
[134] As shown in the SE-HPLC results of Table 10, when stored at 50°C for 7 days, For
mulation 17 containing methionine and lysine as a stabilizer showed Total Impurity of
27%, which is remarkably low, compared to the stabilizer- free formulation showing
Total Impurity of 32.8%.
[135] That is, the present invention demonstrated that the formulation comprising me
thionine and lysine has remarkably effects on the stabilizing of etanercept by reducing
the amount of Total Impurity, compared to the stabilizer- free formulation.
[136]
Claims
1.A liquid formulation of etanercept, comprising etanercept; and one or
more stabilizers selected from the group consisting of methionine,
lysine, histidine, and pharmaceutically acceptable salts thereof.
2.The liquid formulation according to claim 1, wherein the stabilizer is
methionine and/or pharmaceutically acceptable salts thereof.
3.The liquid formulation according to claim 1, wherein the stabilizer is
present in an amount of 0.1 to 250 mM.
4.The liquid formulation according to claim 1, wherein the etanercept is
present in an amount of 1 to 100 mg/mL.
5.The liquid formulation according to claim 1, which have increased
storage stability of etanercept compared to the formulation having no
stabilizers, by reducing etanercept by-products that are produced due to
denaturation during storage.
6.The liquid formulation according to claim 1, which further comprises
one or more materials selected from the group consisting of a buffer, an
isotonic agent, an excipient, or a preservative.
7.The liquid formulation according to claim 6, wherein the buffer is
selected from the group consisting of citrate, phosphate, succinate,
tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and
Tris.
8.The liquid formulation according to claim 6, wherein the buffer is
present in an amount of 0.1 to 100 mM.
9.The liquid formulation according to claim 6, wherein the isotonic agent
is selected from the group consisting of sodium chloride, potassium
chloride, boric acid, sodium borate, mannitol, glycerin, propylene
glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol,
xylitol, sorbitol, and glucose.
10.The liquid formulation according to claim 6, wherein the isotonic agent
is present in an amount of 1 to 1000 mM.
11.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically ac
ceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
12.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of lysine or pharmaceutically ac
ceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
13.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically ac
ceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
14.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically ac
ceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically ac
ceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.
15.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically ac
ceptable salts thereof, 0.1 to 250 mM of methionine or pharma
ceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer,
and 1 to 1000 mM of sodium chloride.
16.The liquid formulation according to claim 1, comprising 1 to 100 mg/
mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically ac
ceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically ac
ceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to
1000 mM of sodium chloride.