U.S. patent application number 12/941917 was filed with the patent office on 2011-04-21 for hydroxyethyl starch-containing polypeptide compositions.
This patent application is currently assigned to Wyeth. Invention is credited to John Carpenter, Suchart Chongpraset, Rebecca Koval, Theodore W. Randolph, Nicholas W. Warne.
Application Number | 20110091415 12/941917 |
Document ID | / |
Family ID | 31498292 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091415 |
Kind Code |
A1 |
Warne; Nicholas W. ; et
al. |
April 21, 2011 |
Hydroxyethyl Starch-Containing Polypeptide Compositions
Abstract
The invention provides compositions containing hydroxyethyl
starch and polypeptides, including therapeutic polypeptides such as
interleukin-11, that provide for enhanced stability of the
polypeptide following storage at room temperature or elevated
temperatures.
Inventors: |
Warne; Nicholas W.;
(Andover, MA) ; Koval; Rebecca; (Billerica,
MA) ; Carpenter; John; (Littleton, CO) ;
Randolph; Theodore W.; (Longmont, CO) ; Chongpraset;
Suchart; (Bangkok, TH) |
Assignee: |
Wyeth
Madison
NJ
The Regent of the University of Colorado
Boulder
CO
|
Family ID: |
31498292 |
Appl. No.: |
12/941917 |
Filed: |
November 8, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12193987 |
Aug 19, 2008 |
|
|
|
12941917 |
|
|
|
|
11236213 |
Sep 27, 2005 |
7449444 |
|
|
12193987 |
|
|
|
|
10390053 |
Mar 17, 2003 |
6982080 |
|
|
11236213 |
|
|
|
|
60365044 |
Mar 15, 2002 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
514/1.1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 38/2073 20130101; A61K 47/36 20130101; A61P 1/00 20180101;
A61K 47/26 20130101; A61K 31/717 20130101; A61K 9/19 20130101; A61P
37/04 20180101; A61K 9/0019 20130101; A61P 35/00 20180101; A61P
7/00 20180101; A61K 31/7012 20130101; A61K 31/7012 20130101; A61K
2300/00 20130101; A61K 31/717 20130101; A61K 2300/00 20130101; A61K
38/2073 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
514/1.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 38/02 20060101 A61K038/02; A61P 29/00 20060101
A61P029/00; A61P 1/00 20060101 A61P001/00; A61P 37/04 20060101
A61P037/04; A61P 7/00 20060101 A61P007/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This work was supported by grants by the National Institutes
of Health Grant Nos. PHS-T32 CA 79446-02. The government has
certain rights in this invention.
Claims
1. A composition comprising a polypeptide, a disaccharide and a
hydroxyethyl starch wherein said composition has a glass transition
temperature of at least 60.degree. C.
2. The composition of claim 1, wherein said polypeptide does not
include an N-linked glycosylation site.
3. The composition of claim 1, wherein said polypeptide does not
include a cysteine amino acid.
4. The composition of claim 1, wherein said polypeptide has a basic
pI.
5. The composition of claim 1, wherein said composition has a glass
transition temperature of at least 75.degree. C.
6. The composition of claim 1, wherein said disaccharide is
selected from the group consisting of trehalose and sucrose.
7. The composition of claim 6, wherein said disaccharide is present
at a concentration of 0.5% to 6.0%.
8. The composition of claim 6, wherein said disaccharide is present
at a concentration of 1.5% to 6.0%.
9. The composition of claim 6, wherein said disaccharide is present
at a concentration of 2.5% to 5.0%.
10. The composition of claim 1, wherein said hydroxyethyl starch is
present at a concentration of 0.5% to 3.5%.
11. The composition of claim 1, wherein said hydroxyethyl starch is
present at a concentration of 2.5%.
12. The composition of claim 1, wherein said composition is stable
for at least 9 months when said composition is stored at 60.degree.
C.
13. A composition comprising an IL-11 polypeptide, a disaccharide,
and hydroxyethyl starch.
14. The composition of claim 13, wherein said IL-11 polypeptide is
stable for at least 9 months when said composition is stored at
60.degree. C.
15. The composition of claim 13, wherein said disaccharide is
selected from the group consisting of trehalose and sucrose.
16. The composition of claim 13, wherein said disaccharide is
present in said composition is present at a concentration of 0.5%
to 6.0%.
17. The composition of claim 13, wherein said disaccharide is
present in said composition at a concentration of 1.5 to 6.0%.
18. The composition of claim 13, wherein said disaccharide is
present in said composition at a concentration of about 2.5% to
5.0%.
19. The composition of claim 15, wherein said disaccharide is
trehalose.
20. The composition of claim 15, wherein said disaccharide is
sucrose.
21. The composition of claim 15, wherein said hydroxyethyl starch
is present in said composition at a concentration of 0.5% to
3.5%.
22. The composition of claim 15, wherein said hydroxyethyl starch
is present in said composition at a concentration of 1.5% to
3.5%.
23. The composition of claim 15, wherein said hydroxyethyl starch
is present in said composition at a concentration of 2.5%.
24. The composition of claim 13 wherein said sucrose is present in
said composition at a concentration of 2.5% to 5.0%, and said
hydroxyethyl starch is present in said composition at a
concentration of 2.5%.
25. The composition of claim 13 wherein said trehalose is present
in said composition at a concentration of 2.5% to 5.0%, and said
hydroxyethyl starch is present in said composition at a
concentration of 2.5%.
26. A method of treating or preventing inflammation in a subject,
the method comprising administering to said subject a composition
comprising IL-11, a disaccharide, and hydroxyethyl starch.
27. The method of claim 26, wherein said subject is a human.
28. The method of claim 26, wherein said inflammation is associated
with inflammatory bowel disease.
29. The method of claim 28, wherein said inflammatory bowel disease
is Crohn's disease.
30. The method of claim 26, wherein said disaccharide is sucrose
present at a concentration of 2.5% to 5.0%, and said hydroxyethyl
starch is present at a concentration of 2.5%.
31. The method of claim 26, wherein said disaccharide is trehalose
present at a concentration of 2.5% to 5.0%, and said hydroxyethyl
starch is present at a concentration of 2.5%.
32. A method of enhancing an immune response in a subject, the
method comprising administering to said subject a composition
comprising IL-11, a disaccharide, and hydroxyethyl starch.
33. The method of claim 32, wherein said subject is a human.
34. The method of claim 33, wherein said composition is
administered in a route selected from the group consisting of
intramuscular, intravenous, intraarterial, intradermal,
intraperitoneal and subcutaneous delivery.
35. The method of claim 32 wherein said disaccharide is sucrose
present at a concentration of 2.5% to 5.0%, and said hydroxyethyl
starch is present at a concentration of 2.5%.
36. The method of claim 32 wherein said disaccharide is trehalose
present at a concentration of 2.5% to 5.0%, and said hydroxyethyl
starch is present at a concentration of 2.5%.
37. A method of increasing platelet production in a subject, the
method comprising administering a subject in need thereof a
composition comprising a polypeptide, a disaccharide, and
hydroxyethyl starch.
38. The method of claim 37, wherein said subject is a human.
39. The method of claim 38, wherein said subject has or is at risk
for developing a tumor.
40. The method of claim 39, wherein said tumor is a solid tumor or
a lymphoma.
41. The method of claim 39, wherein said subject is undergoing
treatment for said tumor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/193,987, filed Aug. 19, 2008, which is a continuation of
U.S. application Ser. No. 11/236,213, filed Sep. 27, 2005, which is
a continuation of U.S. application Ser. No. 10/390,053, filed Mar.
17, 2003 which claims priority to U.S. Application 60/365,044,
filed Mar. 15, 2002. The contents of these applications are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates to compositions containing
polypeptides, including therapeutic polypeptides such as
interleukin-11, that provide for enhanced stability of the
polypeptide following storage at room temperature or at elevated
temperatures.
BACKGROUND OF THE INVENTION
[0004] Recombinant human interleukin-11 (rhIL-11) is a
non-glycosylated polypeptide of 177 amino acids. The polypeptide
lacks cysteine residues and is highly basic (pI>10.5). rhIL-11
is used as a chemotherapeutic support agent, and is administered in
conjunction with other cancer treatments to increase platelet
levels. rhIL-11 has also been demonstrated to have anti
inflammatory effects and to be useful in treating conditions such
as Crohn's disease. rhIL-11 is a member of a family of human growth
factors that includes human growth hormone (hGH) and granulocyte
colony-stimulating factor (G-CSF).
[0005] To minimize degradation, and to maintain the bioactivity of
the polypeptide, preparations containing rhIL-11 and related
polypeptides are typically provided as chilled preparations in
either a liquid or lyophilized state. Preparing, storing, and
transporting the chilled preparations can be labor-intensive,
costly and inconvenient for patients and health-care providers.
SUMMARY OF THE INVENTION
[0006] The invention is based in part on the discovery of
compositions that provide for enhanced stability of polypeptides
such as rhIL-11. Combining rhIL-11 with hydroxyethyl starch and
either of the disaccharides sucrose or trehalose has been found to
result in a composition in which the rhIL-11 is stable following
prolonged storage at room temperature. Unless otherwise noted,
"room temperature" means 25.+-.5.degree. C.
[0007] Any polypeptide for which prolonged storage at or near room
temperature is desired can be used in the compositions and methods
described herein. In some embodiments, the polypeptide is
non-glycosylated, lacks a cysteine residue, and/or has a basic
pI.
[0008] In one aspect, the invention provides a composition that
includes a polypeptide, a disaccharide, and an amylopectin
derivative. A preferred amylopectin derivative is a branched
amylopectin such as hydroxyethyl starch (HES).
[0009] Stability of the polypeptide in the composition can be
assessed by storing the composition at elevated temperatures for
extended periods of time. For example, the composition is
preferably formulated so that the polypeptide is stable for one or
more months (e.g., 1, 2, 3, 5, 6, 7, 8, or 9 or more months) when
the composition is stored at 60.degree. C.
[0010] Preferred disaccharides are trehalose or sucrose. In
preferred embodiments, the disaccharide is present in the
composition at a concentration of 1.5 to 6.0%. For example, the
disaccharide can be present at a concentration of 2.5-5.0%.
[0011] The hydroxyethyl starch is preferably present in the
composition at a concentration of 0.5 to 3.5%, e.g., the
hydroxyethyl starch may be present at a concentration of 2.5%.
[0012] In one embodiment, the invention provides a composition that
includes rhIL-11, sucrose, and hydroxyethyl starch. The sucrose is
present in the composition at a concentration of 2.5% to 5.0%, and
the hydroxyethyl starch is present in the composition at a
concentration of about 2.5%.
[0013] Also provided by the invention is a composition that
includes rhIL-11, trehalose, and hydroxyethyl starch. The trehalose
is present in the composition at a concentration of 2.5% to 5.0%,
and the hydroxyethyl starch is present in the composition at a
concentration of about 2.5%.
[0014] In a further aspect, the method provides a method of
treating or preventing inflammation in a subject by administering
to the subject a composition that includes rhIL-11, a disaccharide,
and hydroxyethyl starch. In some embodiments, the inflammation is
associated with inflammatory bowel disease, such as Crohn's
disease.
[0015] In one embodiment, the invention includes a method of
treating or preventing inflammation in a subject by administering
to a subject a composition that includes rhIL-11, sucrose, and
hydroxyethyl starch. The sucrose is present in the composition at a
concentration of 2.5% to 5.0%, and the hydroxyethyl starch is
present in the composition at a concentration of about 2.5%.
[0016] Also within the invention is a method of treating or
preventing inflammation in a subject by administering to a subject
a composition that includes rhIL-11, trehalose, and hydroxyethyl
starch, wherein the trehalose is present at a concentration of 2.5%
to 5.0%, and the hydroxyethyl starch is present at a concentration
of about 2.5%.
[0017] The invention further provides a method of enhancing an
immune response in, or increasing platelet levels of, a subject by
administering to the subject a composition that includes rhIL-11, a
disaccharide, and hydroxyethyl starch.
[0018] In a further embodiment, the method includes a method of
enhancing an immune response in a subject by administering to a
subject a composition that includes rhIL-11, sucrose, and
hydroxyethyl starch. The sucrose is present in the composition at a
concentration of 2.5% to 5.0%, and the hydroxyethyl starch is
present in the composition at a concentration of about 2.5%.
[0019] In a further aspect the invention includes a method of
increasing platelet levels in a subject by administering to the
subject a composition that includes rhIL-11, a disaccharide (e.g.,
sucrose or trehalose), and hydroxyethyl starch. The disaccharide is
present in the composition at a concentration of 2.5% to 5.0%, and
the hydroxyethyl starch is present in the composition at a
concentration of about 2.5%.
[0020] The subject used in the herein described methods can be,
e.g., a human, a non-human primate, a dog, a cat, horse, cow, pig,
sheep, rabbit, rat, or mouse.
[0021] Also provided by the invention is a method of making a
stable rhIL-11-containing composition. The method includes
providing an rhIL-11 polypeptide and contacting the rhIL-11 with a
disaccharide and hydroxyethyl starch, thereby making a stable
rhIL-11-containing composition. Preferably, the rhIL-11 polypeptide
is stable in the composition for at least 1 month when the
composition is stored at 60.degree. C. In preferred embodiments,
the rhIL-11 polypeptide is stable in the composition for at least
2, 3, 4, 5, 6, 7, 8 or 9 or more months when the composition is
stored at 60.degree. C. The disaccharide in the composition is
preferably sucrose or trehalose, and is preferably present in the
composition at a concentration of 2.5% to 5.0%. The hydroxyethyl
starch is preferably present in the composition at a concentration
of about 2.5%.
[0022] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present Specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0023] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 immediately after freeze-drying. FIG. 1A
shows the spectrum of native rhIL-11 (solid line), protein
lyophilized with no excipients (short dash) and 2.5% HES (dash and
dot). FIG. 1B shows the spectrum of native rhIL-11 (solid line),
protein lyophilized with no excipients (dash and dot), 2.5% sucrose
(short dash) and 2.5% trehalose (dotted). FIG. 1C shows the
spectrum of native rhIL-11 (solid line), protein lyophilized with
no excipients (dash and dot), mixture of 2.5% HES and 2.5% sucrose
(short dash), mixture of 2.5% HES and 2.5% trehalose (dotted).
[0025] FIG. 2 is a representative DSC thermogram. FIG. 2A shows
disaccharides (a) 2.5% sucrose, (b) 5.0% sucrose, (c) 5.0%
trehalose and (d) 2.5% trehalose formulations. FIG. 2B shows
Colyophilized formulations: (e) 5.0% sucrose+2.5% HES (f) 2.5%
sucrose+2.5% HES, (g) 5.0% trehalose+2.5% HES, (h) 2.5%
trehalose+2.5% HES.
[0026] FIG. 3 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 lyophilized with 2.5% (w/v) HES: (solid)
native rhIL-11, (dotted) spectra taken immediately after
freeze-drying, (dashed) spectra taken after 3 months, (dot-dashed)
after 6 months, (long dashed) after 9 months. FIG. 3A shows data at
40.degree. C., and FIG. 3B at 60.degree. C.
[0027] FIG. 4 is a plot showing the formation of soluble aggregates
of rhIL-11 as a function of time as determined by size exclusion
chromatography. Formulations stored at 40.degree. C. (FIGS. 4A and
C) or 60.degree. C. (FIGS. 4B and D) in the presence of 2.5%
sucrose (circle), 5.0% sucrose (triangle down), 2.5% trehalose
(square), 5.0% trehalose, (diamond) and 2.5% (w/v) HES (triangle
up). FIGS. 4A and B represent the disaccharides or HES formulations
and FIGS. 4C and D correspond to the combination of disaccharides
with HES.
[0028] FIG. 5 is a plot showing the formation of cleavage products
of rhIL-11 as a function of time as determined by reversed phase
chromatography. Formulations stored at 40.degree. C. (FIGS. 5A and
C) or 60.degree. C. (FIGS. 5B and D) in the presence of 2.5%
sucrose (circle), 5.0% sucrose (triangle down), 2.5% trehalose
(square), 5.0% trehalose, (diamond) and 2.5% (w/v) HES (triangle
up). FIGS. 5A and B represent the disaccharides or HES formulations
and FIGS. 5C and D correspond to the combination of disaccharides
with HES.
[0029] FIG. 6 is a plot showing methionine 58 oxidation of rhIL-11
as a function of time as determined by reversed phase
chromatography. Formulations stored at 40.degree. C. (FIGS. 6A and
C) or 60.degree. C. (FIGS. 6B and D) in the presence of 2.5%
sucrose (circle), 5.0% sucrose (triangle down), 2.5% trehalose
(square), 5.0% trehalose, (diamond) and 2.5% (w/v) HES (triangle
up). FIGS. 6A and B represent the disaccharides or HES formulations
and FIGS. 6C and D correspond to the combination of disaccharides
with HES.
[0030] FIG. 7 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 lyophilized with 2.5% trehalose (FIGS.
7A and B) and 5.0% trehalose (FIGS. 7C and D): (solid) native
rhIL-11, (dotted) spectra immediately after freeze-drying, (dashed)
spectra taken after 3 months, (dot-dashed) after 6 months, (long
dashed) after 9 months. FIGS. 7A and C show data at 40.degree. C.,
and FIGS. 7B and D at 60.degree. C.
[0031] FIG. 8 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 lyophilized with 2.5% sucrose (FIGS. 8A
and B) and 5.0% sucrose (FIGS. 8C and D): (solid) native rhIL-11,
(dotted) spectra immediately after freeze-drying, (dashed) spectra
taken after 3 months, (dot-dashed) after 6 months, (long dashed)
after 9 months. FIGS. 8A and C show data at 40.degree. C., and
FIGS. 8B and D at 60.degree. C.
[0032] FIG. 9 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 dried with 2.5% trehalose plus 2.5% HES
(FIGS. 9A and B) and 5.0% trehalose plus 2.5% HES (FIGS. 9C and D):
(solid) native rhIL-11, (dotted) spectra taken immediately after
freeze-drying, (dashed) spectra taken after 3 months, (dot-dashed)
after 6 months, (long dashed) after 9 months. FIGS. 9A and C show
data at 40.degree. C., and FIGS. 9B and D at 60.degree. C.
[0033] FIG. 10 is an IR spectrum plot showing the second-derivative
amide I spectra of rhIL-11 dried with 2.5% sucrose plus 2.5% HES
(FIGS. 10A and B) and 5.0% sucrose plus 2.5% HES (FIGS. 10C and D):
(solid) native rhIL-11, (dotted) spectra taken immediately after
freeze-drying, (dashed) spectra taken after 3 months, (dot-dashed)
after 6 months, (long dashed) after 9 months. FIGS. 10A and C show
data at 40.degree. C., and FIGS. 10B and D at 60.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The invention provides compositions that include a
polypeptide (e.g., a therapeutically useful polypeptide), a
disaccharide and an amylopectin derivative, including branched
amylopectins such as hydroxyethyl starch (HES).
[0035] Without being limited by any particular mechanism, it is
likely that the combination of a hydroxyethyl starch (HES) with a
disaccharide, such as sucrose or trehalose, increases the glass
transition temperature, T.sub.g, of the dried polypeptide
compositions of the invention, compared to protein dried with
disaccharides alone, and results in improved protein stability
during storage.
[0036] The invention also provides methods of treating or
preventing various disorders, such as inflammatory bowel diseases
(e.g., Crohn's disease, ulcerative colitis, indeterminate colitis,
and infectious colitis), mucositis (e.g., oral mucositis,
gastrointestinal mucositis, nasal mucositis, and proctitis),
necrotizing enterocolitis, aphthous ulcers, psoriasis, pharyngitis,
esophagitis, peptic ulcers, gingivitis, periodontitis, and ocular
diseases (e.g., conjunctivitis, retinitis, and uveitis) in a
subject by administering to the subject a composition that includes
rhIL-11, a disaccharide, and hydroxyethyl starch.
[0037] Interleukin 11 (IL-11) is a pleiotropic cytokine that
stimulates primitive lymphohematopoietic progenitor cells and acts
in synergy with other hematopoietic growth factors to stimulate the
proliferation and maturation of megakaryocytes. IL-11 is described
in detail in International Application PCT/US90/06803, published
May 30, 1991; as well as in U.S. Pat. No. 5,215,895; issued Jun. 1,
1993. A cloned human IL-11 was previously deposited with the ATCC,
10801 University Boulevard, Manassas, Va. 20110-2209, on Mar. 30,
1990 under ATCC No. 68284. Moreover, as described in U.S. Pat. No.
5,270,181; issued Dec. 14, 1993; and U.S. Pat. No. 5,292,646;
issued Mar. 8, 1994; IL-11 may also be produced recombinantly as a
fusion protein with another protein. IL-11 can be produced in a
variety of host cells by resort to now conventional genetic
engineering techniques. In addition, IL-11 can be obtained from
various cell lines, for example, the human lung fibroblast cell
line, MRC-5 (ATCC Accession No. CCL 171) and Paul et al., the human
trophoblastic cell line, TPA30-1 (ATCC Accession No. CRL 1583).
Described in Proc Natl Acad Sci USA 87:7512 (1990) is a cDNA
encoding human IL-11 as well as the deduced amino acid sequence
(amino acids 1 to 199). U.S. Pat. No. 5,292,646, supra, describes a
des-Pro form of IL-11 in which the N-terminal proline of the mature
form of IL-11 (amino acids 22-199) has been removed (amino acids
23-199). As is appreciated by one skilled in the art, any form of
IL-11 that retains IL-11 activity is useful according to the
present invention.
[0038] In addition to recombinant techniques, IL-11 can also be
produced by known conventional chemical synthesis. Methods for
constructing the polypeptides useful in the present invention by
synthetic means are known to those of skill in the art. The
synthetically constructed cytokine polypeptide sequences, by virtue
of sharing primary, secondary, or tertiary structural and
conformational characteristics with the natural cytokine
polypeptides are anticipated to possess biological activities in
common therewith. Such synthetically constructed cytokine
polypeptide sequences or fragments thereof, which duplicate or
partially duplicate the functionality thereof may also be used in
the method of this invention. Thus, they may be employed as
biologically active or immunological substitutes for the natural,
purified cytokines useful in the present invention.
[0039] Modifications in the protein, peptide or DNA sequences of
these cytokines or active fragments thereof may also produce
proteins that may be employed in the compositions and methods of
this invention. Such modified cytokines can be made by one skilled
in the art using known techniques. Modifications of interest in the
cytokine sequences, e.g., the IL-11 sequence, may include the
replacement, insertion or deletion of one or more selected amino
acid residues in the coding sequences. Mutagenic techniques for
such replacement, insertion or deletion are well known to one
skilled in the art. (See, e.g., U.S. Pat. No. 4,518,584.)
[0040] Other specific mutations of the sequences of the cytokine
polypeptides which may be useful therapeutically as described
herein may involve, e.g., the insertion of one or more
glycosylation sites. An asparagine-linked glycosylation recognition
site can be inserted into the sequence by the deletion,
substitution or addition of amino acids into the peptide sequence
or nucleotides into the DNA sequence. Such changes may be made at
any site of the molecule that is modified by addition of O-linked
carbohydrate. Expression of such altered nucleotide or peptide
sequences produces variants which may be glycosylated at those
sites.
[0041] Additional analogs and derivatives of the sequence of the
selected cytokine which would be expected to retain or prolong its
activity in whole or in part, and which are expected to be useful
in the present method, may also be easily made by one of skill in
the art. One such modification may be the attachment of
polyethylene glycol (PEG) onto existing lysine residues in the
cytokine sequence or the insertion of one or more lysine residues
or other amino acid residues that can react with PEG or PEG
derivatives into the sequence by conventional techniques to enable
the attachment of PEG moieties.
[0042] Additional analogs of these selected cytokines may also be
characterized by allelic variations in the DNA sequences encoding
them, or induced variations in the DNA sequences encoding them. It
is anticipated that all analogs disclosed in the above-referenced
publications, including those characterized by DNA sequences
capable of hybridizing to the disclosed cytokine sequences under
stringent hybridization conditions or non-stringent conditions
(Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d edit.,
Cold Spring Harbor Laboratory, New York (1989)) will be similarly
useful in this invention.
[0043] Also considered useful in the compositions and methods
disclosed herein are fusion molecules, prepared by fusing the
sequence or a biologically active fragment of the sequence of one
cytokine to another cytokine or proteinaceous therapeutic agent,
e.g., IL-11 fused to IL-6 (see, e.g., methods for fusion described
in PCT/US91/06186 (WO92/04455), published Mar. 19, 1992).
Alternatively, combinations of the cytokines may be administered
together according to the method.
[0044] Thus, where in the description of the methods of this
invention IL-11 is mentioned by name, it is understood by those of
skill in the art that IL-11 encompasses the protein produced by the
sequences presently disclosed in the art, as well as proteins
characterized by the modifications described above yet which retain
substantially similar activity.
Hydroxyethyl Starch (HES)
[0045] While any branched polysaccharide (e.g., an amylopectin) can
be used in the compositions and methods described herein, HES is a
preferred stabilizer because it is non-toxic, is biodegradable, has
excellent glass-forming properties, and is clinically approved for
therapeutic uses.
[0046] Hydroxyethyl starch is a derivative of amylopectin, which is
a highly branched compound of about 250-300 K that is soluble in
water and virtually insoluble in organic solvents. HES includes
about 0.4 to about 0.5 hydroxyethyl units per component glucose
monomer. In humans and other animals, amylopectin is rapidly
hydrolyzed by alpha-amylase. The activity of alpha-amylase depends
on the position of the hydroxyethyl groups at positions C2, C3, and
C6 on the glucose molecule. HES can be obtained from a variety of
vendors.
Disaccharides
[0047] Preferred disaccharides are sucrose and trehalose.
Disaccharides can be obtained from a variety of vendors.
Preparing Compositions that Include a Polypeptide, Disaccharide,
and Hydroxyethyl Starch
[0048] Compositions containing a polypeptide, disaccharide and
hydroxyethyl starch can be prepared by buffer exchanging the
polypeptide into the appropriate solution. This process can be
accomplished, by one skilled in the art, by diafiltration,
dialysis, chromatography, crystallization followed by
reconstitution or some combination of freeze-drying or spray-drying
followed by reconstitution with water or a combination of water and
disaccharides and/or hydroxyethyl starch. Once the solution
containing the polypeptide, disaccharide and hydroxyethyl starch
has been prepared, it is dehydrated by means of freeze-drying,
spray-drying, vacuum drying or by the use of supercritical fluid
based dehydration. The resultant lyophilized powder represents the
stable composition of the polypeptide, disaccharide and
hydroxyethyl starch.
Methods of Treatment with IL-11
[0049] A suitable treatment regimen for patients undergoing
treatment, including for example prophylactic treatment, may be
determined by the attending physician based upon such factors as
the patient's age, sex, weight, and general health. Generally, a
suitable dose of IL-11 ranges broadly, preferably between 1 and 100
.mu.g/kg body weight, e.g., 5 to 90 .mu.g/kg body weight. 10 to 80
.mu.g/kg body weight, 20 to 70 .mu.g/kg body weight, or 30 to 65
.mu.g/kg body weight. Another suitable dose is in the range of
about 25 to 50 .mu.g/kg body weight. If desired, these doses can be
adjusted to units. A unit is conventionally described as the
concentration of polypeptide which leads to half-maximal
stimulation in a suitable assay, e.g., for IL-11, the T1165 assay
described in PCT/US90/06803. Doses may be administered daily for
between one day and six months, or for as long as is deemed
necessary and safe, as is readily ascertained by standard tests by
the attending physician, depending upon the nature of the disorder
being treated. Where appropriate, the dosages may be adjusted
upward or downward, for example, a dosing regimen requiring
administration of IL-11 at a dose of 25 .mu.g/kg, daily for one
week, or fewer days, or multiple weeks if indicated. The progress
of treatment is appropriately monitored by measurement of markers
associated with the disorder being treated to determine if such a
dose results in a decrease of, for example, TNF.alpha. levels (or
corresponding marker) and if not, increasing the dose two-fold for
an additional time period of treatment and measurement of marker
levels until an effective dosing regimen is reached.
[0050] Compositions of the invention may be administered rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
bucally, or as a nasal spray. The term "parenteral" as used herein
refers to modes of administration that include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
EXAMPLES
[0051] The invention will be further illustrated in the following
non-limiting examples. The examples describe the prediction of
protein stability in a dried solid after formulation and
lyophilization of composition that includes the polypeptide,
hydroxyethyl starch (HES), and sucrose or trehalose (Example 1).
Also shown are examples describing IL-11 properties in compositions
after short-term storage (Example 2), and long-term storage nine
months at three different temperatures (Example 3).
[0052] In some examples, protein stability was predicted by
measuring the T.sub.g of the lyophilized cake. The T.sub.g, or
glass transition temperature, is a temperature range over which a
material undergoes a transformation from a glass state to rubbery
texture state and undergoes a change in mobility. The T.sub.g-glass
transition is a useful indicator of the stability of a lyophilized
polypeptide-containing composition: a low T.sub.g-glass transition
temperature indicates a relatively unstable lyophilized
composition. The T.sub.g temperature was determined using
differential scanning calorimetry (DSC).
[0053] Protein structure was determined using infrared
spectroscopy, size exclusion high performance liquid chromatography
(SEC-HPLC) and reversed phase high performance liquid
chromatography (RP-HPLC).
Example 1
Preparation and Screening of Compositions Including IL-11, a
Disaccharide and/or Hydroxyethyl Starch
Materials and Methods
Materials
[0054] Recombinant Human Interleukin-11 (rhIL-11) was produced at
Wyeth BioPharma (Andover, Mass.). The protein was expressed in
Escherichia coli as a part of a fusion protein with a thioredoxin,
from which it was cleaved and purified to homogeneity using
conventional chromatography as previously reported. (Czupryn et
al., J Biol. Chem. 270, 978-985 (1995)). Sucrose and trehalose were
purchased from Pfanstiehl laboratories (Waukegan, Ill.).
Hydroxyethyl starch HES (Viastarch) was purchase from Fresenius,
Austria, and had a mean molecular weight of 200 kD. (Searles et
al., J Pharm Sci. 90, 860-871 (2001)). All other reagents were
obtained from Sigma Chemicals Co. (St. Louis, Mo.).
Lyophilization
[0055] Samples were freeze-dried with a FTS Durastop lyophilizer
equipped with a Dura dry MP condenser unit (Stone Ridge, N.Y.).
(Allison et al., J. Pharm. Sci. 89, 199-214 (2000)). Formulations
of rhIL-11 were prepared in 10 mM Tris buffer pH 7.0 to a final
protein concentration of 3.9 mg/mL, with sucrose (1.0, 2.5, 5.0 or
10.0% wt/vol), trehalose (1.0, 2.5, 5.0 or 10.0%) or HES (2.5%).
Formulations containing 2.5% HES with 1.0, 2.5, 5.0 or 10.0%
disaccharide were also prepared. Samples aliquots (1 mL) were
transferred into 5 mL lyophilization vials (West Co.) and placed on
the lyophilizer shelf, which was at room temperature.
[0056] Samples were frozen by cooling the shelves to -40.degree. C.
at a rate of 2.5.degree. C./min. After the samples were at this
temperature for 4 hours, primary drying was started by reducing
chamber pressure to 100 .mu.mHg, and continued by maintaining the
shelf temperature at -40.degree. C. for 48 hours. Secondary drying
(100 .mu.mHg chamber pressure) was initiated by increasing shelf
temperature to 25.degree. C. at a rate of 2.degree. C./min. This
temperature was maintained for 12 hours. This lyophilization
conservative cycle was designed to assure that the formulations
containing disaccharides alone as excipients did not collapse
during processing. All formulations formed pharmaceutically elegant
cakes. The residual water content of all formulations were 0.5-0.9%
by mass as determined by Karl-Fisher titration. (Allison et al., J.
Pharm. Sci. 89, 199-214 (2000)).
Storage Stability
[0057] After lyophilization, the vials were sealed under vacuum and
placed in incubators at 40 and 60.degree. C. Samples of each
formulation were removed at the indicated time points and stored at
-20.degree. C. until analyzed.
Differential Scanning calorimetry
[0058] Thermal analysis of dried Interleukin-11 formulations was
performed using a Perkin-Elmer DSC-7 differential scanning
calorimeter (Norwalk, Conn.). Dried samples (5 mg) were sealed in
aluminum sample pans in a dry nitrogen-purged glove box, and
analyzed with an empty pan in the reference oven. Samples were
warmed at 5.degree. C./min, and thermal data were obtained. Samples
were scanned twice. (Hatley, Develop Biol Standard. 74, 105-122
(1991)). During the first scan, samples were heated to the onset of
the T.sub.g and cooled to 0.degree. C. During the second scan the
sample was heated to 190.degree. C., and the T.sub.g value was
determined. Glass transition temperatures were reported as the
midpoint in the second-order transition in the baseline. The
results are presented as approximate average values for triplicate
samples as shown in Table 1.
TABLE-US-00001 TABLE 1 Percent Hydroxyl Ethyl Starch Amorphous
Component None 2.5% None 140.degree. C..sup.a ND 1% sucrose
65.0.degree. C. ND 2.5% sucrose 60.8 .+-. 3.8.degree. C. 83.7 .+-.
3.2.degree. C. 5% sucrose 60.7 .+-. 4.5.degree. C. 80.1 .+-.
5.7.degree. C. 10% sucrose 55.degree. C. 65.degree. C. 1% trehalose
75.degree. C. ND 2.5% trehalose 103.7 .+-. 1.9.degree. C. 124.7
.+-. 4.5.degree. C. 5% trehalose 104.3 .+-. 4.6.degree. C. 119.7
.+-. 2.8.degree. C. 10% trehalose 103.degree. C. 115.degree. C.
.sup.atemperature calculated was a melting temperature, not a
T.sub.g. ND = no glass transition detectable.
[0059] The thermograms for the lyophilized formulations containing
either disaccharides alone or disaccharide/HES mixtures had a
single observable glass transition (FIG. 2). As expected, the
T.sub.g values for formulations with sucrose alone were
substantially lower than those for formulations with trehalose
alone (Table I above). (Duddu and Monte, Pharm. Res. 12, 1250-1259
(1997)). The T.sub.g values for trehalose formulations were well
above the temperatures (40 and 60.degree. C.) employed in the
storage stability described below. In contrast, the T.sub.g values
for the sucrose formulations were around 60.degree. C. The T.sub.g
values were based on the midpoint of the glass transition event in
the thermogram, which were determined on the timescale of minutes.
Clearly, the transition from the glassy to the rubbery states
occurs at a lower temperature, and hence, during long-term storage
at 60.degree. C., the formulations in sucrose alone will not be in
a glassy state. Thus, although both sucrose and trehalose
formulations meet the criterion for long-term storage stability of
prevention of protein unfolding during lyophilization, with the
sucrose formulation the second criterion for storage at a
temperature below T.sub.g will not be met during storage at
60.degree. C.
[0060] For the lyophilized rhIL-11 formulation with 2.5% HES alone
a glass transition could not be detected in the thermogram. HES is
a relatively large carbohydrate polymer (mean molecular weight of
200 kD) and is expected to have a T.sub.g near that of similarly
sized unmodified starches (e.g., >120.degree. C.) that is
substantially greater than that for sucrose or trehalose. (Hageman,
Water sorption and solid state stability of protein. In stability
of protein pharmaceutical. Part A. Chemical and physical pathways
of protein degradation; Aher, T. J. Manning, M. C., Eds.; Plenum
press: New York: pp 273-309 (1992) and To and Flink, J Food
Technol. 13, 567-581 (1978)). With both sucrose and trehalose
formulations, the presence of 2.5% HES raised the formulations'
T.sub.g about 15-20.degree. C., to well above the highest storage
temperature of 60.degree. C. The increased T.sub.g in disaccharide
formulations prepared in HES is because the T.sub.g of a large
polymer such as HES is substantially higher than that of a
disaccharide. Because these formulations also preserved the native
protein secondary structure after lyophilization, it is predicted
that they will provide the greatest stability, of the formulations
tested, to rhIL-11 during storage at both 40 and 60.degree. C.
Infrared Spectroscopy
[0061] Infrared spectroscopy was used to study protein secondary
structure in the dried formulations immediately after
lyophilization and as a function of storage in the dried solid at
40 and 60.degree. C. In a dry nitrogen-purged glove box, dry
protein samples (approximately 0.5 mg protein) were mixed with 300
mg KBr The mixture was ground and then pressed into a pellet at
12,500 PSI. This procedure for preparing KBr pellets does not alter
the structure of proteins in the dry solid. (Prestrelski et al.,
Biophys J. 65, 661-671 (1993)). Aqueous solutions of native rhIL-11
(20 mg/mL) were placed into a cell fitted with CaF.sub.2 windows
and path length of 6 .mu.m (Biotools, Ill.). Spectra were acquired
with a Bomem MB series spectrometer and processed as previously
described. (Prestrelski et al., Biophys J. 65, 661-671 (1993);
Allison et al., J. Pharm. Sci. 89, 199-214 (2000); Chang et al.,
Arch Biochem Biophys. 331, 249-258 (1996); Kreilgaard et al., J
Pharm Sci. 8, 281-290 (1999); Allison et al., Arch Biochem Biophys.
358, 171-181 (1998); and Allison et al., Arch Biochem Biophy. 365,
289-298 (1999)). Second-derivative spectra in the conformationally
sensitive amide I region were normalized for area and overlaid.
(Kendrick et al., J Pharm Sci. 85, 155-158 (1996)).
[0062] Infrared spectroscopy was used to assess the effects of the
excipients on lyophilization-induced protein unfolding. The second
derivative spectrum for native rhIL-11 in the
conformationally-sensitive amide I region is dominated by a band at
1656 cm.sup.-1 for .alpha.-helix (FIG. 1). In the spectrum for the
protein lyophilized in buffer alone, this band was reduced in
intensity concomitant with increased absorbance at around 1623 and
1690 cm.sup.-1, which is due to an increase in .beta.-sheet content
(FIG. 1A). These results document that in the absence of a
stabilizing excipient, rhIL-11's secondary structure was perturbed
during freeze-drying; native .alpha.-helix was lost and non-native
.beta.-sheet structure was formed. When the protein was lyophilized
in the presence of 2.5% (w/v) HES, lyophilization-induced unfolding
was only partially inhibited (FIG. 1A). This observation is
consistent with earlier results in which it was shown that another
carbohydrate polymer, dextran, could not fully inhibit protein
unfolding during freeze-drying. (Allison et al., J Pharm Sci. 89,
199-214 (2000); Kreilgaard et al., J Pharm Sci. 8, 281-290 (1999);
Prestrelski et al., Pharm Res. 12, 1250-1259 (1995) and Kreilgaard
et al., Arch Biochem Biophys. 360, 121-134 (1998)). In contrast,
second derivative infrared spectra of rhIL-11 lyophilized from
solutions of 2.5 or 5% (w/v) sucrose or trehalose (FIGS. 1B and C)
were native-like immediately after lyophilization. The major band
at 1656 cm.sup.-1 in the spectrum of the native protein in aqueous
solution was retained in the spectra of the protein lyophilized in
trehalose or sucrose. Furthermore, formulations lyophilized in the
presence of HES-disaccharide mixtures had infrared spectra that
were as similar to the spectrum of the native protein as were the
spectra of the protein lyophilized in disaccharide alone (FIGS. 1B
and C). Thus, even in the presence of HES, sucrose and trehalose
can inhibit lyophilization-induced unfolding of rhIL-11.
Example 2
Assessment of Short-Term Stability of Compositions Containing
IL-11, HES, and Sucrose or Trehalose
[0063] Compositions containing 2.5% HES and 0, 1, 2.5, 5, or 10%
sucrose or trehalose were prepared. The percentage of multimer
formation and Met.sub.58OX was determined after one or two weeks of
storage of the formulations at 60.degree. C.
[0064] Stored samples were rehydrated in 10 mM Tris buffer (pH 7.0)
to a final protein concentration of approximately 4.0 mg/ml. Size
exclusion chromatography (SEC) was used to measure the levels of
soluble aggregates. Analysis was performed using a Waters HPLC
system equipped with a TosoHaas TSK-2000 SW.sub.XL column
maintained at 5.degree. C. Isocratic elution was performed for 20
minutes at 1.0 mL/min. The mobile phase was 50 mM MES, 0.1 mM
Glycine, and 0.5M NaCl (pH 6.0). UV detection was performed at 225
nm. Peak areas in the chromatogram were used to quantify the
amounts of soluble aggregates in rehydrated samples
[0065] For 2.5 and 5% trehalose and sucrose formulations, protein
aggregates and cleavage products were not detected after storage at
40.degree. C. and rehydration (FIGS. 4 and 5). Aggregates were also
not detected in the trehalose formulations stored at 60.degree. C.,
but were noted in sucrose formulations (FIG. 4), consistent with
the structural perturbations arising in the dried solid. There was
only minor cleavage product generation during storage of the
disaccharide formulations at 60.degree. C.
[0066] Oxidation of Met-58 did not occur in 2.5 or 5% trehalose and
sucrose formulations during storage at 40.degree. C. (FIG. 6), but
was detected during storage at 60.degree. C. The amount of
oxidation occurring at 60.degree. C. did not appear to correlate
with the T.sub.g of the formulation, suggesting that mobility of
reactive oxygen species may not be coupled to the glassy state in
disaccharide-protein formulations.
[0067] Proteins reconstituted from 10% sucrose or 10% trehalose
were found to be unstable. Proteins reconstituted from 10% sucrose
showed higher levels of multimers (had lower Tg).
Example 3
Assessment of Long-Term Stability of Compositions
[0068] Formulations were prepared using the ingredients described
below and protein structure was analyzed after lyophilization and
storage for up to nine months at different temperatures (5.degree.
C., 40.degree. C., and 60.degree. C.) with infrared spectroscopy,
DSC, and after rehydration by SEC-HPLC and RP-HPLC.
Determination of T.sub.g of Compositions Including IL-11, a
Disaccharide and Hydroxyethyl Starch Using Differential Scanning
calorimetry.
[0069] DSC was performed on IL-11 containing formulations with the
excipients indicated in Table 2. T.sub.g is taken as the midpoint
of a glass transition region, and was determined by measuring the
change in heat flow T.sub.g is taken as the mid-point temperature
of a glass transition region (mW) at increasing temperatures. The
texture (glassy or non-glassy) of the formulation was also
noted.
TABLE-US-00002 TABLE 2 Excipient Added to T.sub.g Formulation
(.degree. C.) Glassy 2.5% Sucrose 65 Yes 5.0% Sucrose 65 Yes 2.5%
Trehalose 105 Yes 5.0% Trehalose 110 Yes 2.5% Hydroxyethyl Starch
ND -- 2.5% Sucrose/2.5% HES 80 Yes 5.0% Sucrose/2.5% HES 75 Yes
2.5% Trehalose/2.5% HES 125 Yes 5.0% Trehalose/2.5% HES 120 Yes
[0070] All freeze-dried formulations tested were observed to be in
a glassy state. The T.sub.g range extended from 60.degree. C. in
pure sucrose systems, to about 120.degree. C.-125.degree. C. in
IL-11 that was lyophilized in the presence of trehalose. No T.sub.g
was observed in IL-11 lyophilized in the presence of only HES. The
presence of HES along with a disaccharide increased the Tg of the
dried power relative to that measured for a formulation lyophilized
with only disaccharides (sucrose or trehalose). The T.sub.g for all
formulations did not change on storage for 9 months relative to
T.sub.g measured immediately after lyophilization (Table I), with
two exceptions. Glass transitions for the formulations containing
2.5% and 5.0% sucrose at 60.degree. C. were not observed during
storage and the cakes collapsed completely. DSC analysis of the
collapsed cakes showed a melting peak for crystalline sucrose.
Storage of the sucrose formulations at a temperature above T.sub.g
resulted in crystallization of the sugar.
Determination of IR-Spectra of Compositions Including IL-11, a
Disaccharide and Hydroxyethyl Starch
[0071] The structure of rhIL-11 in various compositions containing
a disaccharide and/or the carbohydrate polymer hydroxyethyl starch
(HES) was determined by examining the amide I band at 1600-1700
cm.sup.-1. Samples whose spectra were measured, and the
temperatures at which they were stored, included the following:
2.5% sucrose (40.degree. C. and 60.degree. C.); 2.5% trehalose
(40.degree. C. and 60.degree. C.); and 2.5% HES (40.degree.
C.).
Storage Stability of rhIL-11 Dried with HES Alone
[0072] rhIL-11 was freeze-dried with 2.5% (w/v) HES alone to test
the hypothesis that even if a polymeric carbohydrate excipient has
a high glass transition temperature it will not provide optimal
storage stability to a dried protein, if it does not also prevent
protein unfolding during lyophilization. First, to determine if
alterations in the protein's secondary structure were occurring
during storage in the dried solid, infrared spectra for rhIL-11 in
the dried formulations stored at 40 and 60.degree. C. were compared
to the spectrum for the formulation immediately after
lyophilization. Spectra of the protein in the samples stored for 3
months at both temperatures had a small decrease in the intensity
of the .alpha.-helix band, indicating a minor, further structural
perturbation of the protein during storage in the dried solid (FIG.
3). Further alterations in the structure of the dried protein were
not apparent during subsequent analysis of stored dried samples at
6 and 9 months (FIG. 3). The cause for the change in structure that
appears to be restricted to the early phase of the storage period
is not known. Speculatively, there may be a structural relaxation
in the glassy matrix that is complete within the earlier phases of
storage and to which a perturbation of protein secondary structure
is coupled.
[0073] In contrast to the minor secondary structural changes
arising during storage, there was great degradation of the stored
protein detected after rehydration. Soluble aggregates and cleavage
product levels slowly increased in samples stored at 40.degree. C.
In samples stored at 60.degree. C., these degradation products
reached high levels after only one month of storage. Smaller
increases were noted with up to six months of storage, after which
degradation appeared to reach a plateau. Levels of oxidized
methionine-68 did not increase in samples stored at 40.degree. C.,
but did increase linearly with time in samples stored at 60.degree.
C.
[0074] The failure of HES alone to provide storage stability to
lyophilized rhIL-11 is consistent with earlier studies that showed
that dextran (another high molecular carbohydrate polymer) also did
not inhibit lyophilization-induced unfolding (Allison et al., J
Pharm Sci. 89, 199-214 (2000); Kreilgaard et al., J Pharm Sci. 8,
281-290 (1999); Prestrelski et al., Pharm Res. 12, 1250-1259 (1995)
and Kreilgaard et al., Arch Biochem Biophys. 360, 121-134 (1998))
and protein degradation reactions during storage in the dried
solid. (Allison et al., J Pharm Sci. 89, 199-214 (2000); Kreilgaard
et al., J Pharm Sci. 8, 281-290 (1999); Prestrelski et al., Pharm
Res. 12, 1250-1259 (1995); Kreilgaard et al., Arch Biochem Biophys.
360, 121-134 (1998) and Pikal et al., Pharm. Res. 8, 427-436
(1991)). Overall, the results of the current and previous studies
document that storage of an unfolded protein in a glassy polymeric
carbohydrate matrix alone is not sufficient to assure inhibition of
degradation in the dried solid.
Storage Stability of rhIL-11 Dried with Sucrose or Trehalose
Alone
[0075] Infrared spectra of rhIL-11 lyophilized in the presence of
either concentration (2.5% or 5%) of trehalose showed only trivial
changes during 9 months of storage at 40 or 60.degree. C. (FIG. 7).
With sucrose similar results were noted during storage at
40.degree. C. (FIG. 8). Thus, native rhIL-11 secondary structure,
which in these formulations was preserved during lyophilization,
was not altered during storage in dried solid.
[0076] In contrast, during storage of the sucrose formulations at
60.degree. C. there was a significant perturbation of secondary
structure (FIG. 8) and the cakes collapsed. The infrared spectrum
showed a reduction in the intensity of the native a-helix band at
1656 cm.sup.-1 and an increase in absorbance at ca. 1618 cm.sup.-1,
which is indicative of protein aggregation. The poor storage
stability noted in sucrose at 60.degree. C. may have been due to
the crystallization of the sugar. (Kreilgaard et al., J Pharm Sci.
8, 281-290 (1999); To and Flink, J Food Technol. 13, 567-581 (1978)
and Hancock and Zografi, J Pharm Sci. 86, 1-12 (1997). Crystalline
sucrose is anhydrous. Thus, removal of sucrose from the amorphous
phase reduces the solid mass of the glass, but not the absolute
amount of water present. The resulting increase in percentage water
content not only reduces the glass transition temperature but also
may directly contribute to protein degradation. (Hancock and
Zografi, Pharm. Res. 11, 471-477 (1994)). Protein damage might also
be fostered by the formation of new interfaces between sucrose
crystals and the protein in the amorphous phase. (Randolph, J Pharm
Sci. 86, 1198-1203 (1997)).
Storage Stability of rhIL-11 Dried with Mixtures of Disaccharides
and HES
[0077] In the formulations with HES and trehalose, there was not a
detectable change in rhIL-11 secondary structure during storage at
either 40 or 60.degree. C. This result is expected because the
formulations with trehalose alone were also stable during storage
at these temperatures. The formulations with sucrose and HES were
also resistant to protein structural perturbations during storage
at 40 or 60.degree. C. HES increased the formulation T.sub.g
sufficiently high that sucrose crystallization did not occur during
storage at 60.degree. C., and protein structural perturbation was
avoided.
[0078] During storage of disaccharide/HES formulations at
40.degree. C., aggregates, oxidized Met-58 and cleavage products
did not increase. During storage at 60.degree. C., aggregate
formation was almost completely inhibited and oxidation of Met-68
was greatly reduced. The formation of cleavage products was also
low during storage of these formulations at 60.degree. C.
Determination of Soluble Aggregates and Degradation Products
[0079] The ability of a disaccharide and/or HES to prevent rhIL-11
aggregate formation following long-term storage was determined.
Also examined was the ability of a disaccharide and/or HES to
prevent oxidation of the methionine residue at position 58 of
rhIL-11 and degradation of the rhIL-11 polypeptide.
[0080] Compositions were prepared containing rhIL-11 and either
2.5% HES, 2.5% sucrose, 5% sucrose, 2.5% trehalose, or 5%
trehalose. Compositions were also prepared containing rhIL-11 along
with 2.5% HES and 2.5% sucrose, 5% sucrose, 2.5% trehalose, or 5%
trehalose. Samples were stored at 40.degree. C. or 60.degree. C.,
and the percentage of aggregate formation was examined after 1, 3,
6, and 9 months. The percent of soluble aggregates and breakdown
products was determined using size exclusion high performance
liquid chromatography (SEC-HPLC) and reverse phase high performance
liquid chromatography (RP-HPLC).
[0081] Soluble aggregates accounted for less than 4% of all
protein, with the exception of the rhIL-11 protein stored in 2.5%
HES alone at 60.degree. C. These results demonstrate that
formulations of rhIL-11 lyophilized with the disaccharides alone or
combined with HES showed a protective effect against soluble
aggregates formation at either storage temperature.
[0082] The chemical stability of rhIL-11 during long-term storage
of the proteins was examined by measuring the level of Met-58
oxidation in the preparations at the indicated temperatures. At
40.degree. C., less than 4% of the methionine residues were
oxidized for all formulations tested. At 60.degree. C., sucrose
formulations were not found to protect against Met-58 oxidation.
Formulations prepared with trehalose and HES showed reduced Met-58
oxidation as compared to sucrose. Formulations of rhIL-11
lyophilized with sucrose and HES showed lower levels of Met-58
oxidation than rhIL-11 formulated with trehalose-HES formulations.
HES by itself was not found to prevent Met-58 oxidation. Met-58
oxidation in HES-only formulations was higher in samples stored at
60.degree. C. than in samples stored at 40.degree. C.
[0083] The chemical stability of rhIL-11 during long-term storage
was also examined by assaying the level of IL-11-related cleavage
products. Levels of IL-11-related species were less than 3% and 10%
for the disaccharide formulations stored at 40.degree. C. or
60.degree. C., respectively. The levels of cleavage products were
slightly higher in rhIL-11 from trehalose-HES formulations as
compared to cleavage products from rhIL-11 from sucrose-HES
formulations. rhIL-11 formulated in 2.5% HES alone showed 12.0% and
45.0% related species after nine months storage at 40.degree. C.
and 60.degree. C., respectively.
[0084] These data demonstrate that optimal stability was found with
formulations that included both HES and either sucrose or
trehalose. Including HES along with the disaccharides did not
affect protein structure, but its presence did prevent aggregate
formation. The disaccharides alone were more effective than HES
alone at inhibiting lyophilization-induced protein unfolding.
Other Embodiments
[0085] Other embodiments are within the claims.
* * * * *