U.S. patent application number 14/613269 was filed with the patent office on 2015-06-04 for compositions of pegylated soluble tumor necrosis factor receptors and methods of preparing.
The applicant listed for this patent is Amgen Inc.. Invention is credited to Byeong S. CHANG, Bruce Authur KERWIN, Lei SHI.
Application Number | 20150150984 14/613269 |
Document ID | / |
Family ID | 29734431 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150150984 |
Kind Code |
A1 |
KERWIN; Bruce Authur ; et
al. |
June 4, 2015 |
COMPOSITIONS OF PEGYLATED SOLUBLE TUMOR NECROSIS FACTOR RECEPTORS
AND METHODS OF PREPARING
Abstract
The present invention provides for improved compositions
comprising a PEGsTNF-R1 which, in addition to having useful higher
concentrations, demonstrate decreased viscosity (<400 cP) and
improved stability.
Inventors: |
KERWIN; Bruce Authur;
(Bainbridge Island, WA) ; CHANG; Byeong S.;
(Thousand Oaks, CA) ; SHI; Lei; (Thousand Oaks,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amgen Inc. |
Thousand Oaks |
CA |
US |
|
|
Family ID: |
29734431 |
Appl. No.: |
14/613269 |
Filed: |
February 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12432452 |
Apr 29, 2009 |
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14613269 |
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10461839 |
Jun 12, 2003 |
7700722 |
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12432452 |
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10177566 |
Jun 20, 2002 |
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10461839 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 25/08 20180101;
A61P 27/02 20180101; A61P 43/00 20180101; A61P 25/04 20180101; A61P
1/18 20180101; A61P 37/00 20180101; A61P 39/00 20180101; A61P 19/00
20180101; A61K 31/198 20130101; A61K 47/60 20170801; A61K 47/12
20130101; A61P 17/06 20180101; A61P 19/02 20180101; A61P 29/00
20180101; A61P 17/00 20180101; A61P 11/00 20180101; A61K 9/0019
20130101; A61P 25/02 20180101; A61K 47/02 20130101; A61P 17/02
20180101; Y02A 50/401 20180101; A61K 9/19 20130101; A61K 47/26
20130101; A61P 1/04 20180101; A61K 47/183 20130101; A61P 31/04
20180101; A61P 41/00 20180101; A61P 35/02 20180101; A61P 9/10
20180101; A61P 7/06 20180101; A61P 9/04 20180101; A61P 9/00
20180101; A61K 47/10 20130101; A61P 25/00 20180101; A61P 21/00
20180101; A61P 3/10 20180101; A61K 38/1816 20130101; A61P 35/00
20180101; A61K 38/1793 20130101 |
International
Class: |
A61K 47/26 20060101
A61K047/26; A61K 9/00 20060101 A61K009/00; A61K 38/17 20060101
A61K038/17; A61K 47/48 20060101 A61K047/48; A61K 47/12 20060101
A61K047/12; A61K 47/02 20060101 A61K047/02 |
Claims
1. A pharmaceutical formulation comprising (a) a tonicity modifier
selected from the group consisting of mannitol, sorbitol, and
sucrose, (b) a polysorbate, and (c) a tumor necrosis factor (TNF)
inhibitor at a concentration of at least 45 mg/mL, wherein the TNF
inhibitor comprises a polypeptide, and wherein the viscosity of the
pharmaceutical formulation including the tonicity modifier is less
than 400 cP.
2. The pharmaceutical formulation of claim 1, comprising
mannitol.
3. The pharmaceutical formulation of claim 1, comprising
sucrose.
4. The pharmaceutical formulation of claim 1, wherein the
polysorbate is polysorbate 80.
5. The pharmaceutical formulation of claim 1, comprising mannitol
and polysorbate 80.
6. The pharmaceutical formulation of claim 1, comprising sucrose
and polysorbate 80.
7. The pharmaceutical formulation of claim 1, further comprising
acetate.
8. The pharmaceutical formulation of claim 7, comprising 10 mM
sodium acetate.
9. The pharmaceutical formulation of claim 1, further comprising
sodium citrate.
10. The pharmaceutical formulation of claim 1, further comprising
citric acid.
11. The pharmaceutical formulation of claim 1, wherein the pH of
the pharmaceutical formulation is between pH 5 and 6.
12. The pharmaceutical formulation of claim 1, wherein the
concentration of the TNF inhibitor is less than or equal to 55
mg/mL.
13. The pharmaceutical formulation of claim 1, further comprising
sodium chloride.
14. The pharmaceutical formulation of claim 1, further comprising
sodium phosphate.
15. The pharmaceutical formulation of claim 1, wherein the TNF
inhibitor comprises sTNF-RI, truncated sTNF-RI, sTNF-RII, or
truncated sTNF-RII.
16. The pharmaceutical formulation of claim 15, wherein the TNF
inhibitor comprises PEGsTNF-RI.
17. The pharmaceutical formulation of claim 1, wherein the TNF
inhibitor comprises 40 kDa TNF inhibitor .DELTA.51 or 40 kDa TNF
inhibitor .DELTA.53.
18. A pre-filled syringe containing the pharmaceutical formulation
of claim 1.
Description
[0001] This application is a continuation of U.S. nonprovisional
application Ser. No. 12/432,452, filed Apr. 29, 2009, pending,
which is a continuation of Ser. No. 10/461,839, filed Jun. 12,
2003, now U.S. Pat. No. 7,700,722, which is a continuation in part
of U.S. nonprovisional application Ser. No. 10/177,566, filed Jun.
20, 2002, abandoned, all of which are hereby incorporated by
reference.
[0002] The present application incorporates by reference in its
entirety all subject matter contained in the attached sequence
listing which is in txt format and is identified by the name of the
file, A-810-US-CNT2-SeqListFromA-810A.sub.--020315.ST25.txt,
created on Feb. 3, 2015, the size of which file is 2 KB.
BACKGROUND OF THE INVENTION
[0003] Inflammation is the body's defense reaction to injuries such
as those caused by mechanical damage, infection or antigenic
stimulation. An inflammatory reaction may be expressed
pathologically when inflammation is induced by an inappropriate
stimulus such as an autoantigen, is expressed in an exaggerated
manner, or persists well after the removal of the injurious agents.
Such inflammatory reaction may include the production of certain
cytokines.
[0004] While the etiology of inflammation is poorly understood,
considerable information has recently been gained regarding the
molecular aspects of inflammation. This research has led to
identification of certain cytokines which are believed to figure
prominently in the mediation of inflammation. Cytokines are
extracellular proteins that modify the behavior of cells,
particularly those cells that are in the immediate area of cytokine
synthesis and release. Tumor necrosis factors (TNFs) are a class of
cytokines produced by numerous cell types, including monocytes and
macrophages.
[0005] At least two TNFs have been previously described,
specifically TNF alpha (TNF-.alpha.) and TNF beta (TNF-.beta. or
lymphotoxin), and each is active as a trimeric molecule and is
believed to initiate cellular signaling by crosslinking receptors;
Engelmann et al., J. Biol. Chem., 265:14497-14504 (1990).
[0006] Several lines of evidence implicate TNF-.alpha. and
TNF-.beta. as major inflammatory cytokines. These known TNFs have
important physiological effects on a number of different target
cells which are involved in inflammatory responses to a variety of
stimuli such as infection and injury. The proteins cause both
fibroblasts and synovial cells to secrete latent collagenase and
prostaglandin E.sub.2 and cause osteocyte cells to stimulate bone
resorption. These proteins increase the surface adhesive properties
of endothelial cells for neutrophils. They also cause endothelial
cells to secrete coagulant activity and reduce their ability to
lyse clots. In addition they redirect the activity of adipocytes
away from the storage of lipids by inhibiting expression of the
enzyme lipoprotein lipase. TNFs also cause hepatocytes to
synthesize a class of proteins known as "acute phase reactants,"
which act on the hypothalamus as pyrogens; Selby et al., Lancet,
1(8583):483 (1988); Starnes, Jr. et al., J. Clin. Invest., 82:1321
(1988); Oliff et al., Cell, 50:555 (1987); and Waage et al.,
Lancet, 1(8529):355 (1987). Additionally, preclinical results with
various predictive animal models of inflammation, including
rheumatoid arthritis, have suggested that inhibition of TNF can
have a major impact on disease progression and severity; Dayer et
al., European Cytokine Network, 5(6):563-571 (1994) and Feldmann et
al., Annals Of The New York Academy Of Sciences, 66:272-278 (1995).
Moreover, recent preliminary human clinical trials in rheumatoid
arthritis with inhibitors of TNF have shown promising results;
Rankin et al., British Journal Of Rheumatology, 3(4):4334-4342
(1995); Elliott et al., Lancet, 344:1105-1110 (1995); Tak et al.,
Arthritis and Rheumatism, 39:1077-1081 (1996); and Paleolog et al.,
Arthritis and Rheumatism, 39:1082-1091 (1996).
[0007] Protein inhibitors of TNF are disclosed in the art. EP
308378 reports that a protein derived from the urine of fever
patients has a TNF inhibiting activity. The effect of this protein
is presumably due to a competitive mechanism at the level of the
receptors. EP 308378 discloses a protein sufficiently pure to be
characterized by its N-terminus. The reference, however, does not
teach any DNA sequence or a recombinantly-produced TNF
inhibitor.
[0008] Recombinantly-produced TNF inhibitors have also been taught
in the art. For example, EP 393438 and EP 422339 teach the amino
acid and nucleic acid sequences of a mature, recombinant human "30
kDa TNF inhibitor" (also known as a p55 receptor and as sTNFR-I)
and a mature, recombinant human "40 kDa inhibitor" (also known as a
p75 receptor and as sTNFR-II) as well as modified forms thereof,
e.g., fragments, functional derivatives and variants. EP 393438 and
EP 422339 also disclose methods for isolating the genes responsible
for coding the inhibitors, cloning the gene in suitable vectors and
cell types, and expressing the gene to produce the inhibitors.
Mature recombinant human 30 kDa TNF inhibitor and mature
recombinant human 40 kDa TNF inhibitor have been demonstrated to be
capable of inhibiting TNF (EP 393438, EP 422339, PCT WO 92/16221
and PCT WO 95/34326).
[0009] sTNFR-I and sTNFR-II are members of the nerve growth
factor/TNF receptor superfamily of receptors which includes the
nerve growth factor receptor (NGF), the B cell antigen CD40, 4-1BB,
the rat T-cell antigen MRC OX40, the Fas antigen, and the CD27 and
CD30 antigens; Smith et al., Science, 248:1019-1023 (1990). The
most conserved feature amongst this group of cell surface receptors
is the cysteine-rich extracellular ligand binding domain, which can
be divided into four repeating motifs of about forty amino acids
and which contains 4-6 cysteine residues at positions which are
well conserved; Smith et al., supra.
[0010] EP 393438 further teaches a 40 kDa TNF inhibitor .DELTA.51
and a 40 kDa TNF inhibitor .DELTA.53, which are truncated versions
of the full-length recombinant 40 kDa TNF inhibitor protein wherein
51 or 53 amino acid residues, respectively, at the carboxyl
terminus of the mature protein are removed. Accordingly, a skilled
artisan would appreciate that the fourth domain of each of the 30
kDa TNF inhibitor and the 40 kDa inhibitor is not necessary for TNF
inhibition. In fact, various groups have confirmed this
understanding. Domain-deletion derivatives of the 30 kDa and 40 kDa
TNF inhibitors have been generated, and those derivatives without
the fourth domain retain full TNF binding activity while those
derivatives without the first, second or third domain,
respectively, do not retain TNF binding activity; Corcoran et al.,
Eur. J. Biochem., 223:831-840 (1994); Chih-Hsueh et al., The
Journal of Biological Chemistry, 270(6):2874-2878 (1995); and
Scallon et al., Cytokine, 7(8):759-770 (1995).
[0011] PCT WO US97/12244 describes functionally active truncated
forms of sTNFR-I and sTNFR-II (referred to as "truncated sTNFR(s)).
The truncated sTNFRs are modified forms of sTNFR-I and sTNFR-II
which do not contain the fourth domain (amino acid residues
Thr.sup.127-Asn.sup.161 of sTNFR-I and amino acid residues
Pro.sup.141-Thr.sup.179 of sTNFR-II); a portion of the third domain
(amino acid residues Asn.sup.111-Cys.sup.126 of sTNFR-I and amino
acid residues Pro.sup.123-Lys.sup.140 of sTNFR-II); and,
optionally, which do not contain a portion of the first domain
(amino acid residues Asp.sup.1-Cys.sup.19 of sTNFR-I and amino acid
residues Leu.sup.1-Cys.sup.32 of sTNFR-II).
[0012] PEG-rmet-Hu-sTNF-R1 (PEGsTNF-R1) as described herein is a
recombinant form of a functionally active truncated form of sTNFR-I
and sTNFR-II which has been PEGylated at the N-terminus with, e.g.,
a 30 kDa polyethylene glycol molecule. In our preliminary studies
with PEGsTNF-R1 it was found that as the PEGsTNF-R1 is
concentrated, the viscosity of the solution increases
exponentially. Large scale methods traditionally used for
concentrating proteins are known to be unsatisfactory when working
with such viscous solutions, and the increased viscosity may
prevent concentrating the protein to high concentrations without
damaging the final product. Because there may be instances in a
commercial setting where it will be necessary to have the protein
at a higher concentration (e.g., >45 mg/ml) in order to deliver
to required therapeutic dose, there is a need to develop
formulations which obtain such concentrations and with acceptable
low viscosities (e.g., <400 cP) to allow for the use of the
various delivery devices necessary for delivery of the therapeutic
dose. For example, in order to deliver the required therapeutic
dose of a PEGsTNF-R1 formulation wherein the PEGsTNF-R1
concentration is >45 mg/ml, and using a commercially available
autoinjector and pre-filled syringe as the delivery device, the
formulation should have a viscosity of <400 cP. Above this
viscosity, the strong possibility exists for the device or
container to fail. The present invention provides for PEGsTNF-R1
formulations having such concentrations and low viscosities,
thereby allowing for use of delivery devices which are more
convenient and patient-friendly.
SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to provide a new
and improved formulation of PEGsTNF-R1, wherein said formulation
has a concentration of at least 45 mg/ml without apparent damage to
the protein, and with decreased viscosity (<400 cP) and improved
stability.
[0014] Also provided are methods of preparing such formulations,
said methods being capable of being scaled up to accommodate a
commercial setting.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Polypeptide is defined herein as natural, synthetic, and
recombinant proteins or peptides having more than about 10 amino
acids, and having a desired biological activity. Proteins
contemplated for use herein would include but are not limited to
interferon consensus (see, U.S. Pat. Nos. 5,372,808, 5,541,293
4,897,471, and 4,695,623 hereby incorporated by reference including
drawings), granulocyte-colony stimulating factors (see, U.S. Pat.
Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT
Publication No. 94/17185, hereby incorporated by reference
including drawings), interleukins (see, U.S. Pat. No. 5,075,222,
hereby incorporated by reference including drawings),
erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,
5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference
including drawings), stem cell factor (PCT Publication Nos.
91/05795, 92/17505 and 95/17206, hereby incorporated by reference
including drawings), osteoprotegerin (PCT Publication No. 97/23614,
hereby incorporated by reference including drawings), novel
erythropoiesis stimulating protein (NESP) (PCT Publication No.
94/09257, hereby incorporated by reference including drawings),
leptin (OB protein)(see PCT publication Nos. 96/40912, 96/05309,
97/00128, 97/01010 and 97/06816 hereby incorporated by reference
including figures), megakaryocyte growth differentiation factor
(see, PCT Publication No. 95/26746 hereby incorporated by reference
including figures), tumor necrosis factor inhibitors, e.g., sTNF-R1
(see, PCT WO US97/12244 hereby incorporated by reference including
figures), interleukin-1 receptor antagonist (IL-1ra), brain derived
neurotrophic factor (BDNF), glial derived neurotrophic factor
(GDNF), keratinocyte growth factor (KGF) and thrombopoietin. The
term proteins, as used herein, includes peptides, polypeptides,
consensus molecules, analogs, derivatives or combinations
thereof.
[0016] In general, the sTNFRs contemplated for use in the present
invention are those described in PCT WO US97/12244, and references
cited therein. Specifically, the sTNFRs will be the truncated
sTNFRs described therein. The truncated sTNFRs may advantageously
be produced via recombinant techniques in bacterial, mammalian or
insect cell systems and may be either a glycosylated or
non-glycosylated forms of the protein. Alternatively, truncated
sTNFRs may be chemically synthesized. Currently preferred
production methods are described in PCT WO US97/12244.
[0017] Truncated sTNFRs each may typically be isolated and purified
to be substantially free from the presence of other proteinaceous
materials (i.e., non-truncated sTNFRs). Preferably, a truncated
sTNFR is about 80% free of other proteins which may be present due
to the production technique used in the manufacture of the
truncated sTNFR. More preferably a truncated sTNFR is about 90%
free of other proteins, particularly preferably about 95% free of
other proteins, and most preferably about >98% free of other
proteins. Currently preferred isolation and purification methods
are described in PCT WO US97/12244. It will be appreciated,
however, that the desired protein may be combined with other active
ingredients, chemical compositions and/or suitable pharmaceutical
formulation materials prior to administration.
[0018] In one aspect of the present invention, the truncated sTNFRs
will be derivatized by attaching the truncated sTNFRs to a water
soluble polymer. For example, the truncated sTNFRs will be
conjugated to one or more polyethylene glycol molecules in order to
improve pharmacokinetic performance by increasing the molecule's
apparent molecular weight.
[0019] Water soluble polymers are desirable because the protein to
which each is attached will not precipitate in an aqueous
environment, such as a physiological environment. Preferably, the
polymer will be pharmaceutically acceptable for the preparation of
a therapeutic product or composition. One skilled in the art will
be able to select the desired polymer based on such considerations
as whether the polymer/protein conjugate will be used
therapeutically and, if so, the desired dosage, circulation time
and resistance to proteolysis.
[0020] Suitable, clinically acceptable, water soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
polyethylene glycol propionaldehyde, copolymers of ethylene
glycol/propylene glycol, monomethoxy-polyethylene glycol,
carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA),
polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, poly (.beta.-amino acids)
(either homopolymers or random copolymers), poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers
(PPG) and other polyakylene oxides, polypropylene oxide/ethylene
oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol)
and other polyoxyethylated polyols, polyoxyethylated sorbitol, or
polyoxyethylated glucose, colonic acids or other carbohydrate
polymers, Ficoll or dextran and mixtures thereof.
[0021] As used herein, polyethylene glycol is meant to encompass
any of the forms that have been used to derivatize other proteins,
such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water.
[0022] The water soluble polymers each may be of any molecular
weight and may be branched or unbranched. The water soluble
polymers each typically have an average molecular weight of between
about 2 kDa to about 100 kDa (the term "about" indicating that in
preparations of a water soluble polymer, some molecules will weigh
more, some less, than the stated molecular weight). The average
molecular weight of each water soluble polymer preferably is
between about 5 kDa and about 50 kDa, more preferably between about
12 kDa and about 40 kDa and most preferably between about 20 kDa
and about 35 kDa.
[0023] Generally, the higher the molecular weight or the more
branches, the higher the polymer:protein ratio. Other sizes may be
used, depending on the desired therapeutic profile (e.g., the
duration of sustained release; the effects, if any, on biological
activity; the ease in handling; the degree or lack of antigenicity
and other known effects of a water soluble polymer on a therapeutic
protein).
[0024] The water soluble polymers each should be attached to the
protein with consideration of effects on functional or antigenic
domains of the protein. In general, chemical derivatization may be
performed under any suitable condition used to react a protein with
an activated polymer molecule. Activating groups which can be used
to link the water soluble polymer to one or more proteins include
the following: sulfone, maleimide, sulfhydryl, thiol, triflate,
tresylate, azidirine, oxirane and 5-pyridyl.
[0025] The water soluble polymers each are generally attached to
the protein at the .alpha.- or .epsilon.-amino groups of amino
acids or a reactive thiol group, but it is also contemplated that a
water soluble group could be attached to any reactive group of the
protein which is sufficiently reactive to become attached to a
water soluble group under suitable reaction conditions. Thus, a
water soluble polymer may be covalently bound to a protein via a
reactive group, such as a free amino or carboxyl group. The amino
acid residues having a free amino group may include lysine residues
and the N-terminal amino acid residue. Those having a free carboxyl
group may include aspartic acid residues, glutamic acid residues
and the C-terminal amino acid residue. Those having a reactive
thiol group include cysteine residues.
[0026] Methods for preparing proteins conjugated with water soluble
polymers will each generally comprise the steps of (a) reacting a
protein with a water soluble polymer under conditions whereby the
protein becomes attached to one or more water soluble polymers and
(b) obtaining the reaction product. Reaction conditions for each
conjugation may be selected from any of those known in the art or
those subsequently developed, but should be selected to avoid or
limit exposure to reaction conditions such as temperatures,
solvents and pH levels that would inactivate the protein to be
modified. In general, the optimal reaction conditions for the
reactions will be determined case-by-case based on known parameters
and the desired result. For example, the larger the ratio of water
soluble polymer:protein conjugate, the greater the percentage of
conjugated product. The optimum ratio (in terms of efficiency of
reaction in that there is no excess unreacted protein or polymer)
may be determined by factors such as the desired degree of
derivatization (e.g., mono-, di-, tri-, etc.), the molecular weight
of the polymer selected, whether the polymer is branched or
unbranched and the reaction conditions used. The ratio of water
soluble polymer (e.g., PEG) to protein will generally range from
1:1 to 100:1. One or more purified conjugates may be prepared from
each mixture by standard purification techniques, including among
others, dialysis, salting-out, ultrafiltration, ion-exchange
chromatography, gel filtration chromatography and
electrophoresis.
[0027] A variety of approaches have been used to attach the
polyethylene glycol molecules to the protein (PEGylation). For
example, Royer (U.S. Pat. No. 4,002,531) states that reductive
alkylation was used for attachment of polyethylene glycol molecules
to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337) disclose
PEG:protein conjugates involving, for example, enzymes and insulin.
Shaw (U.S. Pat. No. 4,904,584) disclose the modification of the
number of lysine residues in proteins for the attachment of
polyethylene glycol molecules via reactive amine groups. Hakimi et
al. (U.S. Pat. No. 5,834,594) disclose substantially
non-immunogenic water soluble PEG:protein conjugates, involving for
example, the proteins IL-2, interferon alpha, and IL-1ra. The
methods of Hakimi et al. involve the utilization of unique linkers
to connect the various free amino groups in the protein to PEG.
Kinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265) teach
methods allowing for selectively N-terminally chemically modified
proteins and analogs thereof, including G-CSF and consensus
interferon. Importantly, these modified proteins have advantages as
relates to protein stability, as well as providing for processing
advantages.
[0028] The preferred method of the present invention is the
selective N-terminal chemical modification as described by Kinstler
et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265). As taught by
Kinstler et al., one may selectively attach a water soluble polymer
to the N-terminus of the protein by performing the reaction at a pH
which allows one to take advantage of the pKa differences between
the .epsilon.-amino group of the lysine residues and that of the
.alpha.-amino group of the N-terminal residue of the protein. By
such selective derivatization, attachment of a water soluble
polymer to a protein is controlled: the conjugation with the
polymer takes place predominantly at the N-terminus of the protein
and no significant modification of other reactive groups, such as
the lysine side chain amino groups, occurs. Using reductive
alkylation, the water soluble polymer may be of the type described
above and should have a single reactive aldehyde for coupling to
the protein. Polyethylene glycol propionaldehyde, containing a
single reactive aldehyde, may be used.
[0029] By such selective derivatization, attachment of a water
soluble polymer (that contains a reactive group such as an
aldehyde) to a protein is controlled: the conjugation with the
polymer takes place predominantly at the N-terminus of the protein
and no significant modification of other reactive groups, such as
the lysine side chain amino groups, occurs. The preparation will
typically be greater than 90% monopolymer/protein conjugate, and
more typically greater than 95% monopolymer/protein conjugate, with
the remainder of observable molecules being unreacted (i.e.,
protein lacking the polymer moiety).
[0030] A specific embodiment of the present invention is an
unbranched monomethoxy-polyethylene glycol aldehyde molecule having
an average molecular weight of either about 20 kDa or about 33 kDa
(e.g., between 30 kDa and 35 kDa), or a tertiary-butyl polyethylene
glycol aldehyde having an average molecular weight of about 33 kDa
(e.g., between 30 kDa and 35 kDa) conjugated via reductive
alkylation to a truncated sTNFR, wherein the truncated sTNFR has
the amino acid sequence depicted in SEQ ID NO:1.
[0031] The pegylation also may specifically be carried out via
water soluble polymers having at least one reactive hydroxy group
(e.g. polyethylene glycol) reacted with a reagent having a reactive
carbonyl, nitrile or sulfone group to convert the hydroxyl group
into a reactive Michael acceptor, thereby forming an "activated
linker" useful in modifying various proteins to provide improved
biologically-active conjugates. "Reactive carbonyl, nitrile or
sulfone" means a carbonyl, nitrile or sulfone group to which a two
carbon group is bonded having a reactive site for thiol-specific
coupling on the second carbon from the carbonyl, nitrile or sulfone
group (WO 92/16221). The activated linkers can be monofunctional,
bifunctional, or multifunctional. Useful reagents having a reactive
sulfone group that can be used in the methods include, without
limitation, chlorosulfone, vinylsulfone and divinylsulfone.
[0032] PCT International Application No. US96/19459, the disclosure
of which is hereby incorporated by reference, teaches methods of
making sulfone-activated linkers by obtaining a compound having a
reactive hydroxyl group and converting the hydroxyl group to a
reactive Michael acceptor to form an activated linker, with the use
of tetrahydrofuran (THF) as the solvent for the conversion. Also
described is a process for purifying the activated linkers which
utilizes hydrophobic interaction chromatography to separate the
linkers based on size and end-group functionality.
[0033] Pharmaceutical compositions of the present invention will
generally include a therapeutically effective amount of a
chemically-modified derivative of truncated sTNFRs in admixture
with a vehicle. The primary solvent in a vehicle may be either
aqueous or non-aqueous in nature. In addition, the vehicle may
contain other pharmaceutically acceptable excipients. Excipient is
defined herein as a non-therapeutic agent added to a pharmaceutical
composition to provide a desired effect, e.g. stabilization,
isotonicity. Common attributes of desirable excipients are aqueous
solubility, non-toxicity, non-reactivity, rapid clearance from the
body, and the absence of immunogenicity. In addition, the
excipients should be capable of stabilizing the native conformation
of the protein so as to maintain the efficacy and safety of the
drug during processing, storage and administration to the
patient.
[0034] It is envisioned that the formulations of the present
invention will additionally contain a buffering agent, e.g., alkali
salts (sodium or potassium phosphate or their hydrogen or
dihydrogen salts), sodium citrate/citric acid, sodium
acetate/acetic acid, and any other pharmaceutically acceptable ph
buffering agent known in the art, to maintain the pH of the
solution within a desired range. Mixtures of these buffering agents
may also be used. The amount of buffering agent useful in the
composition depends largely on the particular buffer used and the
pH of the solution. For example, acetate is a more efficient buffer
at pH 5 than pH 6 so less acetate may be used in a solution at pH 5
than at pH 6. The preferred pH of the preferred formulations will
be in the range of 4.0 to 5.0, and pH-adjusting agents such as
hydrochloric acid, citric acid, sodium hydroxide, or a salt
thereof, may also be included in order to obtain the desired
pH.
[0035] The formulations of the present invention may further
include one or more tonicity modifiers to render the solution
isotonic with a patient's blood for injection. Typical tonicity
modifiers are well known in the art and include but are not limited
to various salts, amino acids or polysaccharides. Non-limiting
examples of suitable amino acids include glycine. Non-limiting
examples of suitable polysaccharides include sucrose, mannitol and
sorbitol. It is understood that more than one tonicity modifier may
be used at once, for example, sorbitol and glycine can be used in
combination to modify a formulation's tonicity.
[0036] It is also envisioned that other anti-oxidants may be
included in the formulations of the present invention.
Anti-oxidants contemplated for use in the preparation of the
formulations include amino acids such as glycine and lysine,
chelating agents such as EDTA and DTPA, and free-radical scavengers
such as sorbitol and mannitol.
[0037] Other effective administration forms such as parenteral
slow-release formulations, inhalant mists, orally-active
formulations, or suppositories are also envisioned. As such, the
formulations may also involve particulate preparations of polymeric
compounds such as bulk erosion polymers (e.g.,
poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer
blends, block copolymers of PEG, and lactic and glycolic acid,
poly(cyanoacrylates)); surface erosion polymers (e.g.,
poly(anhydrides) and poly(ortho esters)); hydrogel esters (e.g.,
pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone),
maleic anhydride-alkyl vinyl ether copolymers, cellulose,
hyaluronic acid derivatives, alginate, collagen, gelatin, albumin,
and starches and dextrans) and composition systems thereof; or
preparations of liposomes or microspheres. Such formulations may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the present proteins and
derivatives. The optimal pharmaceutical formulation for a desired
protein will be determined by one skilled in the art depending upon
the route of administration and desired dosage. Exemplary
pharmaceutical formulations are disclosed in Remington's
Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co.,
Easton, Pa. 18042, pages 1435-1712, the disclosure of which is
incorporated herein by reference.
[0038] Filtration is a pressure driven separation process that uses
membranes to separate components in a liquid or suspension based on
their size and charge differences. Membrane-based Tangential Flow
Filtration (TFF) unit operations are commonly used for clarifying,
concentrating, and purifying proteins. In TFF, the fluid is pumped
tangentially along the surface of the membrane. An applied pressure
serves to force a portion of the fluid through the membrane to the
filtrate side. The retained components do not build up at the
surface of the membrane, and instead they are swept along by the
tangential flow. TFF can be further categorized base on the size of
components being separated. A membrane pores size rating, typically
given as a micron value, indicates that particles larger than the
rating will be retained by the membrane. A nominal molecular weight
limits (NMWL) is an indication that most dissolved macromolecules
with molecular weights higher than the NMWL and some with molecular
weights lower than the NMWL will be retained by the membrane. A
components shape, its ability to deform, and its interaction with
other components in the solution all affect its retention.
[0039] Ultrafiltration (UF) is one of the most widely used forms of
TFF and is used to separate proteins from buffer components for
buffer exchange, desalting, and concentration. Depending on the
protein to be retained, membranes NMWLs in the range of 1 kD to
1000 kD are used. The typical sequences of steps in an
ultrafiltration process include cleaning the membranes and the
system, testing the integrity and permeability, equilibrating with
process buffer, concentrating the sample containing the product,
removing product from system, cleaning the membranes and the
system, testing integrity and permeability, and storing.
[0040] The most important factors in design of UF processes include
product yield, product quality, process time, and process
economics. Yield losses in a UF process can be generally be
attributed to sieving, solubility limitations, adsorption to the
membrane, and unrecoverable volumetric losses. While several key
process parameters such as transmembrane pressure, crossflow rate,
and membrane area need to be optimized, protein concentration is
one of the limiting factors in developing a TFF step. Since there
is the potential for highly concentrated areas to exist within the
TFF unit as the result of the polarization, high protein
concentration can exceed a solubility limitation and increase
fouling behavior at membrane surface. The significant increased
viscosity (e.g., >500 cP) associated with the concentration of
certain pegylated protein causes process difficulties in term of
maintaining crossflow rate and minimizing heat introduction. The
present invention provides an improved ultrafiltration process to
concentrate PEGsTNF-R1 to greater than 45 mg/ml by utilizing a
improved formulation and temperature effect.
[0041] The process of lyophilization is very well documented in
literature. Lyophilization is the process by which the moisture
content of the product is reduced by freezing and subsequent
sublimation under vacuum. The lyophilization process primarily
consists of three stages. The first stage involves freezing the
product and creating a frozen matrix suitable for drying. This step
impacts the drying characteristics in the next two stages. The
second stage is primary drying. Primary drying involves the removal
of the ice by sublimation by reducing the pressure (to typically
around 50-500 .mu.m Hg) of the product's environment while
maintaining the product temperature at a low, desirable level. The
third stage in the process is called secondary drying where the
bound water is removed until the residual moisture content reaches
below the target level.
[0042] Lyophilization improves product stability by (a) maintaining
the protein in an amorphous phase with its stabilizers, (b)
immobilizing the protein in a glassy phase below the glass
transition temperature (Tg') of the formulation, and (c) reducing
the residual moisture content to a low, desirable value.
Maintaining the protein in an amorphous phase with its stabilizers
helps in protecting the protein. Keeping the dried protein below
its glass transition temperature minimizes protein immobility on
all practical time-scales and therefore prevents degradation.
Reducing the amount of residual water minimizes all water-catalyzed
degradations.
[0043] A freeze dryer consists of a chamber with shelves on to
which the filled vials are loaded for lyophilization, a condenser
for capturing the product's sublimed water vapor as ice, a
refrigeration system that facilitates temperature control, and a
vacuum pump which can reduce the chamber pressure to
sub-atmospheric values. The chamber pressure is maintained at its
set-point by introducing, in a controlled manner, an inert, dry
bleed gas (such as nitrogen) at the front of the chamber. In most
cases, the chamber is separated from the condenser via a main
valve. The product is loaded onto the stainless steel shelves,
whose temperature is controlled via a heat-transfer fluid (silicone
oil) circulating through the shelves. Temperature of the
heat-transfer fluid is controlled via the refrigeration system.
[0044] The freezing stage is initiated by cooling the shelves to
the desired freezing temperature and holding the temperature
constant for equilibration. The cooled shelves help freeze the
product to the desired temperature. Following freezing, the chamber
pressure (measured by a capacitance manometer) is reduced to below
the saturated vapor pressure of ice at the frozen temperature. This
initiates primary drying. Since ambient pressure is below the
saturated vapor pressure at that temperature, part of the frozen
product instantaneously sublimes (the difference between the vapor
pressure of ice and the chamber pressure provides the driving force
for sublimation). Sublimation leads to pressure equilibration.
However, since the chamber pressure is constantly maintained below
the saturated vapor pressure of ice (at that temperature),
sublimation continues. The sublimed vapors are trapped at the
condenser as ice. Typically, the condenser coil or plates remain at
about -50.degree. C. to -70.degree. C. during the drying process.
When all the bulk water is removed via sublimation, primary drying
is complete. At this point, there is still some bound water
remaining in the product which can be removed by desorption at
higher temperatures experienced during secondary drying. So,
typically the shelf-temperature is raised at this stage and held,
until the desired residual moisture is achieved. At that point,
secondary drying is also complete, and the vials are stoppered in
the chamber. The chamber is aerated prior to the unloading of the
vials. Note that the above description is generic, and some
equipment design variations are available.
[0045] The objective of a lyophilization process is to achieve a
freeze-dried protein cake with acceptable appearance, biological
potency, ease of reconstitution, and long-term storage stability. A
prudently designed lyophilization cycle is one that is robust,
consumes less time and energy, and maintains product quality. Both
formulation-related and cycle-related factors contribute to
achieving this goal.
[0046] For freeze-dried products, the formulation and the
lyophilization process are intricately interrelated. As mentioned
earlier, to maintain product stability, the product temperature
needs to be below its glass transition temperature (Tg') both
during drying and storage. Therefore, a formulation with a higher
Tg' allows drying at a higher temperature compared with a
lower-Tg'-formulation and subsequently expedites the freeze-drying
time. Since Tg' of the formulation is approximately the
mass-average of Tg' values of all the amorphous components in the
formulation, the Tg' of the formulation can be raised by increasing
the weight fraction of high-Tg' components of the formulation
and/or by decreasing the weight fraction of low-Tg' components. Of
course, it is necessary that the chosen excipients regardless of
their Tg' values, protect the protein from possible
degradations.
[0047] The addition of a lyophilization excipient in the processes
described herein may be necessary. One or more excipients may be
added. The lyophilization excipient(s) contemplated for use in the
present processes include sucrose, lactose, mannitol, dextran,
sucrose, heparin, glycine, glucose, glutamic acid, gelatin,
sorbitol, histidine, dextrose, trehalose, methocel, hydroxy ethyl
cellulose, hydroxy ethyl starch, poly(ethylene glycol), poly(vinyl
pyrolidone) and polyvinyl alcohol, or various combinations thereof,
as well as other buffers, protein stabilizers, cryoprotectants, and
cyropreservatives commonly used by those skilled in the art. The
present invention provides an improved lyophilization and
reconstitution method for concentrating PEGsTNF-R1 to greater than
45 mg/ml.
[0048] Therapeutic uses of the compositions of the present
invention depend on the biologically active agent used. One skilled
in the art will readily be able to adapt a desired biologically
active agent to the present invention for its intended therapeutic
uses. Therapeutic uses for such agents are set forth in greater
detail in the following publications hereby incorporated by
reference including drawings. Therapeutic uses include but are not
limited to uses for proteins like consensus interferon (see, U.S.
Pat. Nos. 5,372,808, 5,541,293, hereby incorporated by reference
including drawings), interleukins (see, U.S. Pat. No. 5,075,222,
hereby incorporated by reference including drawings),
erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,
5,618,698 5,547,933, 5,621,080, 5,756,349, and 5,955,422, hereby
incorporated by reference including drawings), granulocyte-colony
stimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476,
5,582,823, 4,810,643 and PCT Publication No. 94/17185, hereby
incorporated by reference including drawings), megakaryocyte growth
differentiation factor (see, PCT Publication No. 95/26746), stem
cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206,
hereby incorporated by reference including drawings), OB protein
(see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010
and 97/06816 hereby incorporated by reference including figures),
and novel erythropoiesis stimulating protein (PCT Publication No.
94/09257, hereby incorporated by reference including drawings). In
addition, the present compositions may also be used for manufacture
of one or more medicaments for treatment or amelioration of the
conditions the biologically active agent is intended to treat.
[0049] As relates specifically to PEGsTNFR1, the present invention
provides for methods for the treatment of certain diseases and
medical conditions (many of which can be characterized as
inflammatory diseases) that are mediated by TNF. A disease or
medical condition is considered to be a "TNF-mediated disease" if
the spontaneous or experimental disease is associated with elevated
levels of TNF in bodily fluids or in tissues adjacent to the focus
of the disease or indication within the body. TNF-mediated diseases
may also be recognized by the following two conditions: (1)
pathological findings associated with a disease can be mimicked
experimentally in animals by the administration of TNF and (2) the
pathology induced in experimental animal models of the disease can
be inhibited or abolished by treatment with agents which inhibit
the action of TNF. Many TNF-mediated diseases satisfy two of these
three conditions, and others will satisfy all three conditions. A
non-exclusive list of TNF-mediated diseases, as well as the related
sequela and symptoms associated therewith, is adult respiratory
distress syndrome; cachexia/anorexia; cancer (e.g., leukemias);
chronic fatigue syndrome; congestive heart failure; graft versus
host rejection; hyperalgesia; inflammatory bowel disease;
neuroinflammatory diseases; ischemic/reperfusion injury, including
cerebral ischemia (brain injury as a result of trauma, epilepsy,
hemorrhage or stroke, each of which may lead to neurodegeneration);
diabetes (e.g., juvenile onset Type 1 diabetes mellitus); multiple
sclerosis; ocular diseases; pain; pancreatitis; pulmonary fibrosis;
rheumatic diseases (e.g., rheumatoid arthritis, osteoarthritis,
juvenile (rheumatoid) arthritis, seronegative polyarthritis,
ankylosing spondylitis, Reiter's syndrome and reactive arthritis,
psoriatic arthritis, enteropathic arthritis, polymyositis,
dermatomyositis, scleroderma, systemic sclerosis, vasculitis,
cerebral vasculitis, Sjogren's syndrome, rheumatic fever,
polychondritis and polymyalgia rheumatica and giant cell
arteritis); septic shock; side effects from radiation therapy;
systemic lupus erythematous; temporal mandibular joint disease;
thyroiditis and tissue transplantation. Specifically, TNF-mediated
diseases (e.g., diseases mediated by TNF-.alpha. and/or TNF-.beta.)
may be treated by administering to a patient therapeutically
effective amounts of truncated sTNFRs or derivatives thereof.
[0050] The PEGsTNF-R1 products each may be administered to a
patient in therapeutically effective amounts for the treatment of
TNF-mediated diseases, as defined above, including such as
rheumatic diseases (e.g., lyme disease, juvenile (rheumatoid)
arthritis, osteoarthritis, psoriatic arthritis, rheumatoid
arthritis and staphylococcal-induced ("septic") arthritis). The
term "patient" is intended to encompass animals (e.g., cats, dogs
and horses) as well as humans.
[0051] A PEGsTNF-R1 product may be administered via topical,
enteral or parenteral administration including, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intra-articular, subcapsular, subarachnoid, intraspinal,
intraventricular and intrasternal injection and infusion. A
truncated sTNFR product may also be administered via oral
administration or be administered through mucus membranes, that is,
intranasally, sublingually, buccally or rectally for systemic
delivery.
[0052] It is preferred that PEGsTNF-R1 products be administered via
intra-articular, subcutaneous, intramuscular or intravenous
injection. Additionally, PEGsTNFR1 product may be administered by a
continuous infusion (e.g., constant or intermittent implanted or
external infusion flow-modulating devices) so as to continuously
provide the desired level of PEGsTNFR1 product in the blood for the
duration of the administration. This is preferably accomplished by
means of continuous infusion via, e.g., mini-pump such as osmotic
mini-pump. In these ways, one can be assured that the amount of
drug is maintained at the desired level and one can take blood
samples and monitor the amount of drug in the bloodstream. Various
pumps are commercially available, from suppliers such as MiniMed
Inc, Sylmar, Calif. (e.g., MT507) and Alza Corp., Palo Alto, Calif.
(e.g., Alzet osmotic pump, model 2MLI).
[0053] PEGsTNF-RI may be administered using an autoinjector type
device. These devices typically use a pre-filled syringe or
pre-filled cartridge with the device. The device is held against
the injection site, a needle inserts through the skin and injects
the drug in approximately 5-30 seconds depending on the device and
syringe configuration. The use of commercially available devices
and syringes requires viscosities of <400 cP for an injection to
occur in a reasonable time, i.e. <30 seconds and more preferably
<15 seconds. Commercial suppliers are Scandinavian Health Ltd.
Far Eastern World Center, 11th Floor-8, #77, Hsin Tai Wu Rood, Sec.
1, Hsih Chih, Taipei, Taiwan, R.O.C., and Owen Mumford Ltd. Brook
Hill, Woodstock, Oxford OX20 1TU, England.
[0054] It is also contemplated that other modes of continuous or
near-continuous dosing may be practiced. For example, chemical
derivatization may result in sustained release forms of the protein
which have the effect of continuous presence in the blood stream,
in predictable amounts based on a determined dosage regimen.
[0055] Modes of using PEGsTNF-R1 products for the treatment of
TNF-mediated diseases, including inflammatory conditions of a joint
(e.g., osteoarthritis, psoriatic arthritis and rheumatoid
arthritis), are set forth in European Patent Application 567566,
the teachings of which are hereby incorporated by reference. By way
of example but not limitation, in one specific embodiment PEGsTNFR1
products may be administered intra-articularly for the treatment of
rheumatoid arthritis and osteoarthritis. By way of example but not
limitation in another specific embodiment, PEGsTNF-R1 products may
be administered subcutaneously or intramuscularly for the treatment
of rheumatoid arthritis, inflammatory bowel disease,
cachexia/anorexia or multiple sclerosis. By way of example but not
limitation, in a still further specific embodiment PEGsTNF-R1
products may be administered intravenously for the treatment of
brain injury as a result of trauma, epilepsy, hemorrhage or stroke;
or administered intraventricularly for the treatment of brain
injury as a result of trauma. A preferred mode for the treatment of
arthritis includes: (1) a single intra-articular injection of a
PEGsTNF-R1 product given periodically as needed to prevent or
remedy the flare-up of arthritis and (2) periodic subcutaneous
injections of a PEGsTNFR1 product. The initiation of treatment for
septic shock should begin as soon as possible after septicemia or
the chance of septicemia is diagnosed. For example, treatment may
be begun immediately following surgery or an accident or any other
event that may carry the risk of initiating septic shock. Preferred
modes for the treatment of adult respiratory distress syndrome
include: (1) single or multiple intratracheal administrations of a
PEGsTNF-R1 product and (2) bolus or continuous intravenous infusion
of a PEGsTNF-R1 product. Regardless of the manner of
administration, the treatment of a TNF-mediated disease requires a
dose or total dose regimen of a PEGsTNF-R1 effective to reduce or
alleviate symptoms of the disease. Other factors in determining the
appropriate dosage can include the disease or condition to be
treated or prevented, the severity of the disease, the route of
administration, and the age, sex and medical condition of the
patient. Further refinement of the calculations necessary to
determine the appropriate dosage for treatment is routinely made by
those skilled in the art, especially in light of the dosage
information ad assays disclosed herein. The dosage can also be
determined through the use of known assays for determining dosages
used in conjunction with appropriate dose-response data. The
specific dose is calculated according to the approximate body
weight or body surface area of the patient.
[0056] The frequency of dosing depends on the pharmacokinetic
parameters of the PEGsTNF-R1 in the formulation used. The
PEGsTNF-R1 may be administered once, or in cases of severe and
prolonged disorders, administered daily in less frequent doses or
administered with an initial bolus dose followed by a continuous
dose or sustained delivery. When administered parenterally,
parenteral unit doses, for example, may each be up to 10 mg,
generally up to 15 mg and more generally up to 20 mg. When
administered into an articular cavity, the pharmaceutical
composition is preferably administered as a single injection from,
for example, a 3 to 10 ml syringe containing a dose, for example,
of between about 5 mg/ml to 10 mg/ml truncated sTNFR dissolved in
isotonic phosphate buffered saline. The preparation may be
administered into an articular cavity at a frequency, for example,
of once every 7 to 10 days. In such a manner, the administration is
continuously conducted, for example, 4 to 5 times while varying the
dose if necessary.
[0057] In some cases, PEGsTNF-R1 products may be administered as an
adjunct to other therapy and also with other pharmaceutical
formulations suitable for the indication being treated. A
PEGsTNF-R1 product and any of one or more traditional or new
anti-inflammatory drugs may be administered separately or in
combination.
[0058] Present treatment of TNF-mediated diseases, as defined
above, including acute and chronic inflammation such as rheumatic
diseases (e.g., lyme disease, juvenile (rheumatoid) arthritis,
osteoarthritis, psoriatic arthritis, rheumatoid arthritis and
staphylococcal-induced ("septic") arthritis) includes first line
drugs for control of pain and inflammation classified as
non-steroidal, anti-inflammatory drugs (NSAIDs). Secondary
treatments include corticosteroids, slow acting antirheumatic drugs
(SAARDs) or disease modifying (DM) drugs. Additional TNF-mediated
diseases contemplated are those described in PCT WO US97/12244.
[0059] Preferred PEGsTNF-R1 formulations contemplated for use in
the present invention will contain one or more buffering agents
such as, but not limited to acetate, histidine or phosphate; a
tonicity modifier such as, but not limited to sucrose, sorbitol,
mannitol, or glycine; an antioxidant such as, but not limited to
methionine, EDTA, or ascorbate; an antimicrobial agent such as, but
not limited to benzyl alcohol or phenol; a surfactant such as, but
not limited to polysorbate 20 or polysorbate 80.
[0060] It is contemplated that when sorbitol is the tonicity
modifier, it is between zero and 5.48%, more preferably 1% to
5.48%, more preferably 1.5% to 5.48%, more preferably 2% to 5.48%,
and even more preferably 2.56% to 5.48%. It is contemplated that
when glycine is the tonicity modifier, it is between zero and
2.19%, more preferably 1% to 2.19%, more preferably 1.25% to 2.19%,
and even more preferably 1.5% to 2.19%. In one particular
embodiment, the formulation comprises acetate buffer at between pH
4-5 and 2.56% Sorbitol. It is understood that the above percentages
are based on weight/volume.
[0061] The following examples are offered to more fully illustrate
the invention, but are not to be construed as limiting the scope
thereof. Additional methods for reducing the solution viscosity of
a PEGsTNFR1 formulation may include site-directed mutagenesis of
specific amino acids or removal of amino acids sequences contained
within the coding region of the sTNFR1 amino acid sequence.
Example 1
[0062] This example describes experiments wherein PEGsTNF-RI at 45
cP and 336 cP was loaded into 1 ml syringes and injected with
commercially available autoinjectors. The results of these studies
are shown in the following table (Table 1). The standard
autoinjector was modified to deliver the 336 cP solution.
TABLE-US-00001 TABLE 1 Viscosity Needle Injection (cP) Size time
(sec) 45 26 10.5 45 27 42 336 23 9 336 25 78
The data demonstrate that while the autoinjector can deliver the
low viscosity solution in less than 15 sec with the 26G syringe, a
significantly larger needle was necessary to make an equivalent
injection with the higher viscosity material. Smaller needles (26
or 27 G) are preferred over the larger needles (23 G) to reduce
injection pain. This shows the importance of developing
formulations of PEGsTNF-R1 having sufficiently high concentrations
and with sufficiently low viscosities.
Example 2
[0063] This example describes experiments wherein various
concentrated samples of PEGsTNF-R1 were prepared and then viscosity
measurements taken on concentrated samples.
[0064] The samples for this experiment were prepared by room
temperature diafiltration at the indicated pH in 10 mM sodium
acetate. The membranes used for the diafiltration were in the form
of cassettes, and the membrane types were regenerated cellulose
with nominal molecular weight cut-off value of 5 kD and 10 kD. The
starting material enters through the feed port and buffer-exchanged
product exits through the retentate port. Filtrate was removed from
filtrate ports. Transmembrane pressure, crossflow rate, and
filtrate flowrate were monitored and controlled during the process.
After concentration of the protein, 140 mM sodium chloride (NaCl),
5.48% sorbitol, or 2.19% glycine was added and viscosity
measurements taken.
[0065] Viscosity was measured using a Brookfield viscometer
(Brookfield Instruments, USA). The system was temperature stated at
16.degree. C. using a circulating water bath. Viscosity
measurements were recorded after equilibration of the system. The
results of this analysis are depicted in Table 2.
TABLE-US-00002 TABLE 2 pH of Final protein Viscosity Sample #
sample concentration Excipients (cP) c 5.0 59 mg/ml none 532 2 4.5
55 mg/ml NaCl (0.9%) 550 3 4.5 55 mg/ml Sorbitol (5.48%) 276 4 4.5
55 mg/ml Glycine (2.19%) 255 5 4.5 55 mg/ml NaCl (0.30%) 355
Glycine (0.723%) Sorbitol (1.81%) 6 4.5 55 mg/ml Glycine (1.10%)
255 Sorbitol (2.74%) 7 5.0 55 mg/ml NaCl (0.9%) 719 8 5.0 55 mg/ml
Sorbitol (5.48%) 335 9 5.0 55 mg/ml Glycine (2.19%) 327 10 5.0 55
mg/ml NaCl (0.30%) 544 Glycine (0.723%) Sorbitol (1.81%) 11 5.0 55
mg/ml Glycine (1.10%) Sorbitol (2.74%) 326 12 4.5 45 mg/ml NaCl
(0.9%) 245 13 4.5 45 mg/ml Sorbitol (5.48%) 134 14 4.5 45 mg/ml
Glycine (2.19%) 125 15 4.5 45 mg/ml NaCl (0.30%) 167 Glycine
(0.723%) Sorbitol (1.81%) 16 4.5 45 mg/ml Glycine (1.10%) 126
Sorbitol (2.74%) 17 5.0 45 mg/ml NaCl (0.9%) 309 18 5.0 45 mg/ml
Sorbitol (5.48%) 157 19 5.0 45 mg/ml Glycine (2.19%) 164 20 5.0 45
mg/ml NaCl (0.30%) 220 Glycine (0.723%) Sorbitol (1.81%) 21 5.0 45
mg/ml Glycine (1.10%) 154 Sorbitol (2.74%)
This example demonstrates that pH, protein concentration and
additional excipients all affect the final viscosity, and that
formulations having concentrations of up to 55 mg/ml and
viscosities<400 cP can be obtained using sorbitol and/or glycine
as a formulation excipient.
Example 3
[0066] This example describes experiments wherein samples of
PEGsTNF-R1 were lyophilized using varying pH's, protein
concentrations, and lyophilization methods. The lypohilized samples
were then reconstituted to concentrations>50 mg/ml and viscosity
measurements taken.
[0067] PEGsTNF-R1 at 25 mg/ml for lyophilization was prepared as
follows: PEGsTNF-R1 was buffer exchanged into water, concentrated
using an Amicon stirred cell device, and diluted with 10.times.
concentrated buffer to 25 mg/ml in 10 mM histidine, pH 4.0 or 5.5,
1% (w/v) sucrose, 2% (w/v) glycine and 0.01% polysorbate 20.
[0068] PEGsTNF-R1 at 60 mg/ml for lyophilization was prepared as
follows: PEGsTNFR1 was buffer exchanged into water, concentrated
using an Amicon stirred cell device, and diluted with 10.times.
concentrated buffer to 25 mg/ml in 10 mM histidine, pH 4.0 or 5.5,
1.0% (w/v) sucrose, 2% (w/v) glycine and 0.01% polysorbate 20.
[0069] Samples were then lyophilized using the low temperature
method or high temperature method as described in the Materials and
Methods section below. After lyophilization, the samples were
reconstituted with water to the desired protein concentration and
with the following excipient concentrations: 5 mM histidine, pH 4.0
or 5.5, 0.5% (w/v) sucrose, 1% (w/v) glycine and 0.005% polysorbate
20. Viscosity was measured using a Haake falling ball
microviscometer (Haake Instruments, Germany). The system was
temperature stated at 26.degree. C. using a circulating water bath.
Viscosity measurements were recorded after equilibration of the
system. The results of this analysis are depicted in Table 3.
TABLE-US-00003 TABLE 3 Final protein concen- Solution tration
Lyophili- for after Sample zation reconsti- reconsti- Viscosity #
method tution tution (cP) 1 Lyophilized Low Sterile 57 mg/ml 230 at
25 mg/ml Temperature water and pH 4.0 method 2 Lyophilized Low
Sterile 57 mg/ml 515 at 25 mg/ml Temperature water and pH 5.5
method 3 Lyophilized Low Sterile 72.2 mg/ml 410* at 25 mg/ml
Temperature water and pH 4 method 4 Lyophilized Low 10% 72.9 mg/ml
570* at 25 mg/ml Temperature sucrose and pH 4 method 5 Lyophilized
Low Sterile 60 mg/ml 246 at 60 mg/ml Temperature water and pH 4
method 6 Lyophilized High Sterile 60 mg/ml 266 at 60 mg/ml
Temperature water and pH 4 method *Due to the high viscosity,
samples were measured at 37.degree. C.
This example again demonstrates that pH, protein concentration and
additional excipients all affect the final viscosity, and that
formulations having concentrations of at least 57 mg/ml and
viscosities<400 cP can be obtained using various
ultrafiltration/lyophilization techniques.
Example 4
[0070] This example describes experiments wherein samples of
PEGsTNFR1, at various concentrations, and containing various
excipients, were tested for stability.
[0071] Samples for stability studies of PEGsTNFR1 at concentrations
of 15 mg/ml were prepared by buffer exchanging the protein into
deionized water using the tangential flow system described above.
Excipients (e.g., histidine, acetate) were then added from stock
solutions to their final concentrations and pH. The samples were
then sterile filtered and 1 ml aliquots filled in 3 cc glass vials
and incubated at the indicated temperature.
[0072] Samples for stability studies of PEGsTNFR1 at
concentrations.gtoreq.45 mg/ml were buffer exchanged into 10 mM
acetate. The pH after concentrating was 4.9. Excipients were added
from stock solutions to the indicated concentrations. The samples
were then sterile filtered and 1 ml aliquots filled in 3 cc glass
vials and incubated at the indicated temperature.
[0073] Stability of PEGsTNFR1 was determined by high performance
size exclusion chromatography. A TosoHaas TSKGSW3000xL
(7.8.times.300 mm) size exclusion column was equilibrated in buffer
containing 10 mM sodium acetate pH 5.0, 0.5M sodium chloride, 10%
ethanol (v/v). Protein was eluted using a flow rate of 0.5 ml/min.
The results of this analysis are depicted in Table 4.
TABLE-US-00004 TABLE 4 % Main Protein Incubation Peak by Buffer/
Tonicity Temp SEC at Excipients Modifier pH (mg/ml) (.degree. C.)
12 weeks 10 mM Acetate 140 mM NaCl 4 15 4 92.9 10 mM Acetate 140 mM
NaCl 5.5 15 4 93.4 10 mM Histidine 140 mM NaCl 5.5 15 4 93.3 10 mM
Acetate 140 mM NaCl 4 15 37 67.2 10 mM Acetate 140 mM NaCl 5.5 15
37 87.3 10 mM Histidine 140 mM NaCl 5.5 15 37 82.0 10 mM Acetate
NaCl (140 mM) 4.9 52 4 97.4 10 mM Acetate Sorbitol (5.48%) 4.9 45 4
97.0 10 mM Acetate Glycine (2.19%) 4.9 46 4 96.9 10 mM Acetate NaCl
(140 mM) 4.9 52 37 73.1 10 mM Acetate Sorbitol (5.48%) 4.9 45 37
86.1 10 mM Acetate Glycine (2.19%) 4.9 46 37 88.4
[0074] The data demonstrate that pH, temperature, protein
concentration and choice of excipients are all factors which affect
the stability and that stable formulations having concentrations of
>45 mg/ml can be prepared.
Materials and Methods
[0075] All chemicals were ACS grade or better.
[0076] The sTNFRs used in the present invention were prepared
according to the above incorporated-by-reference PCT WO
US97/12244.
[0077] The PEGsTNFR1 formulations used in the present invention
were prepared using the selective N-terminal chemical modification
as described by Kinstler et al. (U.S. Pat. Nos. 5,824,784 and
5,985,265).
[0078] The low temperature lyophilization of the PEGsTNF-R1 was
carried out as follows: Vials were loaded onto a shelf equilibrated
at 4.degree. C. The shelf temperature was decreased to -50.degree.
C. at a cooling rate of 36.degree. C./hr. After holding at
-50.degree. C. for two hours, the shelf temperature was increased
to -15.degree. C. at a heating rate of 35.degree. C./hr and held
there for two hours and 30 minutes. The samples were then brought
back to -50.degree. C. at a cooling rate of -23.degree. C./hr. The
primary drying was started by evacuating the chamber to 80 mTorr
and held at -50.degree. C. for an additional 30 minutes. The shelf
temperature was brought to -25.degree. C. at a heating rate of
12.5.degree. C./hr, then kept at -25.degree. C. for seventeen
hours. The secondary drying was initiated by bringing the shelf
temperature to 30.degree. C. by heating at a rate of 5.5.degree.
C./hr. After 12 hours at 30.degree. C., the secondary drying was
complete.
[0079] The high temperature lyophilization of the PEGsTNF-R1 was
carried out as follows: Vials were loaded onto a shelf equilibrated
at 4.degree. C. The shelf temperature was decreased to -40.degree.
C. at a cooling rate of 15.degree. C./hr. After holding at
-40.degree. C. for two hours, the shelf temperature was increased
to -15.degree. C. at a heating rate of 10.degree. C./hr and held
there for two hours. The primary drying was started by evacuating
the chamber to 80 mmHg. The shelf temperature was kept at
-15.degree. C. for one hour and then increased to 10.degree. C. at
a heating rate of 10.degree. C./hr. The primary drying was
continued for 30 hours at 10.degree. C. The secondary drying was
continued by increasing the shelf temperature to 30.degree. C. at a
heating rate of 10.degree. C./hr. After 14 hours at 30.degree. C.,
the secondary drying was complete.
[0080] The present invention has been described in terms of
particular embodiments found or proposed to comprise preferred
modes for the practice of the invention. It will be appreciated by
those of ordinary skill in the art that, in light of the present
disclosure, numerous modifications and changes can be made in the
particular embodiments exemplified without departing from the
intended scope of the invention.
Sequence CWU 1
1
11106PRTHomo sapiens 1Met Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile
His Pro Gln Asn Asn 1 5 10 15 Ser Ile Cys Cys Thr Lys Cys His Lys
Gly Thr Tyr Leu Tyr Asn Asp 20 25 30 Cys Pro Gly Pro Gly Gln Asp
Thr Asp Cys Arg Glu Cys Glu Ser Gly 35 40 45 Ser Phe Thr Ala Ser
Glu Asn His Leu Arg His Cys Leu Ser Cys Ser 50 55 60 Lys Cys Arg
Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val 65 70 75 80 Asp
Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr 85 90
95 Trp Ser Phe Asn Leu Phe Gln Cys Phe Asn 100 105
* * * * *