U.S. patent application number 10/223682 was filed with the patent office on 2003-05-08 for pharmaceutical formulations for igf/igfbp.
This patent application is currently assigned to Insmed Inc.. Invention is credited to Danko, Stephen, Ogawa, Yasushi, Passmore, David.
Application Number | 20030087806 10/223682 |
Document ID | / |
Family ID | 22215473 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087806 |
Kind Code |
A1 |
Danko, Stephen ; et
al. |
May 8, 2003 |
Pharmaceutical formulations for IGF/IGFBP
Abstract
New pharmaceutical formulations for IGF/IGFBP complex are
disclosed. IGF/IGFBP complex, preferably rhIGF-I/IGFBP-3 complex is
formulated with bulking agents and optionally buffer salts, but
without added osmolyte salts. Also disclosed are lyophilized
formulations for IGF/IGFBP complex.
Inventors: |
Danko, Stephen; (San
Francisco, CA) ; Passmore, David; (Menlo Park,
CA) ; Ogawa, Yasushi; (Pacifica, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Insmed Inc.
|
Family ID: |
22215473 |
Appl. No.: |
10/223682 |
Filed: |
August 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10223682 |
Aug 20, 2002 |
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09089062 |
Jun 1, 1998 |
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6436897 |
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Current U.S.
Class: |
514/8.6 ;
514/8.5; 514/8.7 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/30 20130101; A61K 38/30 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/18 |
Claims
1. A pharmaceutical formulation for insulin-like growth factor
(IGF) and insulin-like growth factor binding protein (IGFBP)
complex; comprising: IGF/IGFBP complex; a bulking agent; and ph
buffer salts, wherein said formulation contains no added osmolyte
salts.
2. The formulation of claim 1, wherein said pH buffer salts
comprise sodium succinate.
3. The formulation of claim 2, wherein said pH buffer salts are at
a concentration of less than about 40 millimolar (mM).
4. The formulation of claim 3, wherein said ph buffer salts are at
a concentration of less than about 20 mM.
5. The formulation of claim 2, wherein said pH buffer salts are at
a concentration of less than 10 mM.
6. The formulation of claim 1, wherein said formulation has a pH of
about 5.5 to 6.5.
7. The formulation of claim 1, wherein said bulking agent comprises
mannitol.
8. The formulation of claim 1, wherein said bulking agent comprises
sorbitol.
9. The formulation of claim 1 wherein said bulking agent comprises
sucrose.
10. The formulation of claim 1 wherein said bulking agent comprises
mannitol and sucrose.
11. The formulation of claim 1, wherein said bulking agent is
present at about 6% (w/v).
12. The formulation of claim 11 wherein said mannitol is present at
about 3.6% (w/v) and said sucrose is present at about 2.4%
(w/v).
13. The formulation of claim 1 wherein said formulation further
comprises a non-ionic surfactant.
14. The formulation of claim 1 wherein said IGFBP is IGFBP-3.
15. The formulation of claim 1 wherein said IGF is IGF-I.
16. The formulation of claim 15 wherein said IGFBP is IGFBP-3.
17. The formulation of claim 1, wherein said formulation further
comprises a preservative.
18. The formulation of claim 17, wherein said preservative is
benzyl alcohol.
19. The formulation of claim 10, wherein said IGF/IGFBP is 50
milligrams per milliliter (mg/ml), said mannitol is 1.5% (w/v), and
said sucrose is 1% (w/v).
20. The formulation of claim 10, wherein said IGF/IGFBP is 100
mg/ml, said mannitol is 3% (w/v) and said sucrose is 2% (w/v).
21. A pharmaceutical formulation for insulin-like growth factor
(IGF) and insulin-like growth factor binding protein (IGFBP)
complex, comprising: IGF/IGFBP complex; and a bulking agent,
wherein said formulation contains no added osmolyte salts and no
added pH buffer salts.
22. The formulation of claim 21 wherein said bulking agent is
mannitol.
23. The formulation of claim 21 wherein said bulking agent is
sorbitol.
24. The formulation of claim 21 wherein said bulking agent is
sucrose.
25. The formulation of claim 21 wherein said bulking agent
comprises mannitol and sucrose.
26. The formulation of claim 21, wherein said bulking agent is
present at about 6% (w/v).
27. The formulation of claim 26 wherein said mannitol is present at
about 3.6% (w/v) and said sucrose is present at about 2.4%
(w/v).
28. The formulation of claim 21 wherein said formulation further
comprises a non-ionic surfactant.
29. The formulation of claim 21 wherein said IGF is IGF-I.
30. The formulation of claim 21 wherein said IGFBP is IGFBP-3.
31. The formulation of claim 29 wherein said IGFBP is IGFBP-3.
32. The formulation of claim 21, wherein said formulation further
comprises a preservative.
33. The formulation of claim 32, wherein said preservative is
benzyl alcohol.
34. The formulation of claim 1, wherein said formulation is
lyophilized.
35. The formulation of claim 21, wherein said formulation is
lyophilized.
36. A lyophilized pharmaceutical formulation for insulin-like
growth factor (IGF) and insulin-like growth factor binding protein
(IGFBP) complex, produced by: forming a mixture comprising IGF,
IGFBP, a bulking agent, and pH buffer salts, wherein said mixture
lacks any added osmolyte salts; and lyophilizing said mixture to
form a lyophilized formulation of IGF, IGFBP, a bulking agent, and
pH buffer salts.
37. A lyophilized pharmaceutical formulation for insulin-like
growth factor (IGF) and insulin-like growth factor binding protein
(IGFBP) complex, produced by: forming a mixture comprising IGF,
IGFBP, and a bulking agent, wherein said mixture lacks any added
osmolyte salts; and lyophilizing said mixture to form a lyophilized
formulation of IGF, IGFBP, and a bulking agent.
Description
TECHNICAL FIELD
[0001] The invention relates generally to the field of formulation
of therapeutic proteins, and particularly to formulations for
complexes of insulin-like growth factor I (IGF-I) and insulin-like
growth factor binding protein 3 (IGFBP-3).
BACKGROUND ART
[0002] Growth factors are polypeptides that stimulate a wide
variety of biological responses (e.g. DNA synthesis, cell division,
expression of specific genes, etc.) in a defined population of
target cells. A number of different growth factor families have
been identified, including the transforming growth factor beta
family (TGF-.beta.S), epidermal growth factor and transforming
growth factor alpha (the TGF-.alpha.s), the platelet-derived growth
factors (PDGFs), the fibroblast growth factor family (FGFs) and the
insulin-like growth factor family (IGFs), which includes IGF-I and
IGF-II.
[0003] IGF-I and IGF-II (the "IGFs") are related in amino acid
sequence and structure, with each polypeptide having a molecular
weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the
major effects of growth hormone, and is thus the primary mediator
of growth after birth. IGF-I has also been implicated in the
actions of various other growth factors, since the treatment of
cells with such growth factors leads to increased production of
IGF-I. In contrast, IGF-II is believed to have a major role in
fetal growth. Both IGF-I and IGF-II have insulin-like activities
(hence their names), and are mitogenic (stimulate cell division)
for the cells in neural tissue.
[0004] Almost all IGF circulates in a non-covalently associated
complex of IGF-I, insulin-like growth factor binding protein 3
(IGFBP-3) and a larger protein subunit termed the acid labile
subunit (ALS), such that very little free IGF-I is detectable. The
ternary complex is composed of equimolar amounts of each of the
three components. ALS has no direct IGF-binding activity and
appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J.
Biol. Chem. 264(20):11843-11848, 1989), although some reports
suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee
et al., Endocrinology 136:4982-4989, 1995). The ternary complex of
IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa.
This ternary complex is thought to act "as a reservoir and a buffer
for IGF-I and IGF-II preventing rapid changes in the concentration
of free IGF" (Blum et al. (1991), "Plasma IGFBP-3 Levels as
Clinical Indicators" in MODERN CONCEPTS OF INSULIN-LIKE GROWTH
FACTORS, pp. 381-393, E. M. Spencer, ed., Elsevier, New York).
While there is essentially no excess (unbound) IGFBP-3 in
circulation, a substantial excess of free ALS does exist (Baxter,
J. Clin. Endocrinol. Metab. 67:265-272, 1988).
[0005] The complex of IGF-I and IGFBP-3 ("binary complex" or
"IGF-I/IGFBP-3") is considerably different from uncomplexed IGF-I,
both physically and chemically. The binary complex is approximately
5 times larger than uncomplexed IGF-I, has a different overall pI,
and has a different overall hydrophobicity. These differences cause
the binary complex to behave quite differently than IGF-I.
[0006] Due to its wide range of activities, IGF-I has been
developed as a treatment for a variety of conditions, including
amyotrophic lateral sclerosis (commonly known as Lou Gehrig's
disease) and diabetes. Unfortunately, the administration of IGF-I
is accompanied by a variety of undesirable side effects, including
hypoglycemia, edema (which can cause Bell's palsy, carpal tunnel
syndrome, and a variety of other deleterious conditions),
hypophosphatemia (low serum phosphorus), and hypernatermia
(excessive serum sodium). Administration of IGF-I as a complex of
IGF-I and IGFBP-3 can reduce or eliminate these undesirable side
effects (Adams et al., 1996, Prog. Growth Factor Res. 6:2-4)
[0007] While administration of IGF-I/IGFBP-3 complex may be
desirable, the complex, like many proteins, has very limited
stability (shelf life) in most formulations. A variety of
purportedly stable formulations have been disclosed for IGF-I,
either alone or in combination with another proteins (e.g., growth
hormone), but the formulations thus disclosed for IGF-I/IGFBP-3
have been unsatisfactory due to poor stability of the proteins.
These formulations for binary complex require that the protein be
frozen, frequently at very low temperatures (e.g., -70.degree. C.).
Freezers, particularly the ultra-low temperature freezers required
to maintain -70.degree. C., are uncommon outside of research
facilities and are also very expensive. Accordingly, formulations
which can be stored at normal refrigerator temperatures or higher
while still providing a long shelf life are critical to the
commercial development of IGF-I/IGFBP for use as a therapeutic.
[0008] A variety of formulations have been disclosed for IGF,
particularly IGF-I. For example, U.S. Pat. No. 5,681,814 discloses
an IGF-I formulation for use in subcutaneous administration which
comprises IGF-I, 2-50 mg/ml of an osmolyte (e.g., sodium chloride),
1-15 mg/ml of a preservative (e.g. benzyl alcohol or phenol) in a
buffered in solution at pH 5-5.5. International Patent Application
No. WO 97/07816 discloses a liquid IGF-I formulation which
comprises IGF-I and mannitol in a buffered solution. However, due
to the substantial physico/chemical differences between IGF-I and
IGF/IGFBP-3, there is no reasonable expectation that IGF-I
formulations will be suitable for IGFBP-3.
[0009] It should be noted that, while IGFBP-3 is the most abundant
of the IGF binding proteins ("IGFBPs"), at least five other
distinct IGFBPs have been identified in various tissues and body
fluids. Although these proteins bind IGFs, they originate from
separate genes and have distinct amino acid sequences. Unlike
IGFBP-3, other circulating IGFBPs are not saturated with IGFs.
IGFBP-3 is the only IGFBP which can form the 150 kDa ternary
complex with IGF and ALS. However, some of the other IGFBPs have
also been suggested for use in combination with IGF-I as
therapeutics.
[0010] However, despite the advantages of administering IGF-I as a
complex with IGFBP-3, little has been disclosed regarding
formulations useful for pharmaceutical applications. Bagi et al.
(J. Bone Mineral Res. 9(8):1301-1311, 1994) disclose the
administration of IGF-I/IGFBP-3 to ovariectomized rats. The
IGF-I/IGFBP-3 complex was formulated in simple phosphate buffered
saline (PBS). Celtrix Pharmaceuticals, Inc. has disclosed the use
of IGF-I/IGFBP-3 formulated in acetate buffer (pH 5.5) with 105 mM
sodium chloride (NaCl) as the osmolyte. However, this formulation
is not ideal for a commercial pharmaceutical formulation, as it
does not permit lyophilization of the product.
[0011] Lyophilization (freeze drying under controlled conditions)
is commonly used for long term storage of proteins. The lyophilized
protein is substantially resistant to degradation, aggregation,
oxidation, and other degenerative processes while in the freeze
dried state. The lyophilized protein is normally reconstituted with
water optionally containing a bacteriostatic preservative (e.g.,
benzyl alcohol). Unfortunately, many preservatives (e.g., benzyl
alcohol) are not compatible with proteins, or at least reduce
stability. However, the addition of a preservative is currently
recommended for drugs which will be administered for periods of 24
hours or more, and this recommendation may become a requirement for
drugs sold in the U.S.
[0012] Acceptable commercial lyophilized pharmaceutical products
must form an acceptable "lyo cake" (mass of lyophilized product).
Preferably the lyo cake has a smooth surface and uniform
appearance. Lyophilized protein alone rarely makes an acceptable
lyo cake, so suitable bulking agents must be added. Generally,
carbohydrates such as mannitol, sorbitol, and sucrose are used as
bulking agents in lyophilized pharmaceutical products.
Additionally, a buffering agent is normally added, particularly in
pharmaceutical formulations for proteins such as growth factors and
cytokines. The buffering agent is used to control the pH of the
formulation when it is in a liquid state (i.e., before
lyophilization and after reconstitution) because proteins are
normally particularly sensitive to pH fluctuations or extremes.
[0013] Accordingly, there is a need in the art for pharmaceutically
acceptable formulations that provide high stability for
IGF-I/IGFBP-3 drug products.
DISCLOSURE OF THE INVENTION
[0014] The inventors have created novel formulations for
IGF-I/IGFBP-3 which provide long term stability for IGF-I/IGFBP-3
complex. The formulations of the instant invention are
pharmaceutically acceptable.
[0015] The inventors have surprisingly found that pharmaceutical
formulations of IGF-I/IGFBP-3 complex with very low levels of
osmolyte salts are more stable than formulations with high levels
of added salts. Additionally, the inventors have found the
surprising and unexpected result that omission of pH buffer salts
further increases the stability of IGF-I/IGFBP-3 formulations.
[0016] In a further surprising and unexpected discovery, the
inventors have found that IGF-I/IGFBP-3 formulations with high
protein concentrations and having low osmolyte salts and no added
pH buffer salts have high stability.
[0017] In one embodiment, the formulations of the invention
comprise IGF-I/IGFBP-3 complex, a bulking agent, and pH buffer
salt. No added osmolyte salt is present in the formulations of this
embodiment.
[0018] In a further embodiment, the formulations of the instant
invention comprise IGF-I/IGFBP-3 complex and a bulking agent. No
added osmolyte salts or pH buffer salts are present in the
formulations of this embodiment. The formulations of this
embodiment are particularly advantageous because they allow the
preparation of pharmaceutical formulations which contain very high
protein concentrations.
[0019] The formulations of the instant invention may be liquid
formulations or lyophilized formulations. Optionally, they may also
contain a non-ionic surfactant. Liquid formulations may optionally
contain a preservative for reducing or eliminating bacterial
growth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the amino acid sequence (single letter amino
acid code) of mature human IGFBP-3 (Ala.sub.5) variant.
BEST MODE FOR CARRYING OUT THE INVENTION
[0021] The inventors have made a number of surprising and
unexpected discoveries which allow the production of commercially
and pharmaceutically acceptable, stable IGF/IGFBP formulations. The
inventors have found the surprising and unexpected result that
elimination of added osmolyte salts increases the stability of
IGF-I/IGFBP-3 formulations. Further, and more unexpectedly, the
inventors have found that elimination of pH buffer salts increases
the stability of IGF-I/IGFBP-3 formulations. The inventors have
also surprisingly found that formulations having low levels of
osmolyte salts and no added pH buffer salts have increased
stability in the presence of benzyl alcohol, a commonly used
pharmaceutical preservative which frequently promotes aggregation
of proteins. Formulations having low osmolyte salts with or without
an added pH buffer can be created with high concentrations of
IGF-I/IGFBP complex without substantial loss of protein.
[0022] Definitions
[0023] "Insulin-like growth factor" or "IGF " comprises a family of
factors, including, but not limited to, IGF-I and IGF-II. The IGFs
are polypeptides with molecular weights of about 7.5 kDa. IGF
includes naturally occurring IGF-I or IGF-II, analogs or variants
thereof (e.g., variants in which one or more of IGF-I's tyrosine
residues (i.e., residues 24, 31, or 60) are replaced with
non-aromatic residues (i.e., other than tyrosine, phenylalanine or
tryptophan), mutants where amino acid residues 49, 50, 51, 53, 55
and 56 are altered (for example, where residues 49-51 are altered
to Thr-Ser-Ile or where residues 55-56 are altered to Tyr-Gln) and
fusions between IGF-I or IGF-II and other amino acid sequences. IGF
may be obtained from natural sources or prepared by recombinant
means.
[0024] "Insulin-like growth factor binding protein" or "IGFBP", as
used herein, is a family of insulin-like growth factor binding
proteins which comprises, but is not limited to, IGFBP-1, IGFBP-2,
IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. IGFBP may be obtained from
natural or recombinant sources.
[0025] "Insulin-like growth factor binding protein 3" or "IGFBP-3"
refers to one of the members of the IGFBP family. The mature
protein is 264 amino acids and, in humans, comprises at least two
naturally occurring allelic variant proteins, wherein the fifth
amino acid residue of the mature protein is either glycine or
alanine (referred to as Gly.sub.5 IGFBP-3 and Ala.sub.5 IGFBP-3,
respectively). When produced by human and other mammalian cells,
the protein is post-translationally modified by up to three
N-linked glycosylations at three separate sites. When produced in
bacteria, the protein is not glycosylated. IGFBP-3 also includes
variants of the protein, for example those variants in which the
amino acid sites of the normal N-linked glycosylation are altered
to another amino acid (sequence variants will be notated throughout
the specification as X#Y, where X is the single letter amino acid
code for the amino acid residue in the native protein, # is the
residue number in the mature protein sequence, and Y is the amino
acid to which the residue is changed), particularly aspartate, such
as N89D; N109D; N172D; N89D,N109D; N89D,N172D; N109D,N172D; and
N89D,N109D,N172D variants or N89X; N109X; N172X; N89X,N109X;
N89X,N172X; N109X,N172X; and N89X,N109X,N172X variants. Other
variants include alterations at position 116 and 135 where the
native sequence aspartate is altered to glutamate (e.g., D116E,
D135E and D116E,D135E) or to any other amino acid (e.g., D116X,
D135X and D116X,D135X) and variants in IGFBP-3's nuclear localizing
sequence (NLS), such as K228E, R230G and K228E,R230G and/or
alterations at residues 215, 216 and/or 231. Of course, variant
IGFBP-3 may contain more than one variation (e.g., a variant
IGFBP-3 may include hydrolysis-resistance variations as well as NLS
variations). IGFBP-3 may be produced by purification from natural
sources or recombinantly in prokaryotic or eukaryotic host cells,
although variants other than naturally occurring allelic variant
proteins are preferably produce by recombinant means.
[0026] The term "bulking agent" refers to a compound which is
pharmaceutically acceptable and which adds bulk to a lyo cake.
Acceptable bulking agents include, but are not limited to,
carbohydrates such as simple sugars such as dextrose, ribose,
fructose and the like, alcohol sugars such as mannitol, inositol
and sorbitol, disaccharides including trehalose, sucrose and
lactose, naturally occurring polymers such as starch, dextrans,
chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin)
and glycogen, and synthetic monomers and polymers. Bulking agents
for use in the instant invention preferably also act as osmolytes
(i.e., aid in making the liquid form of the formulation isotonic
with normal human serum).
[0027] As used herein, the term "osmolyte salt" means salts which
are added for the purpose of helping a formulation to become
isotonic with normal human serum. Osmolyte salts are normally
compounds which are generally regarded as safe for administration
to humans, and include sodium chloride, calcium chloride, potassium
chloride and the like. Pharmaceutically acceptable osmolyte salts
may generally found in the USP (UNITED STATES PHARMACOPEIA, United
States Pharmacopeial Convention, Inc., Rockville, Md., 1995).
[0028] A "preservative" is a bacteriostatic, bacteriocidal,
fungistatic or fungicidal compound which may be added to the
formulations of the instant invention to retard or eliminate growth
of bacteria or other contaminating microorganisms in the
formulations. The preservative should be pharmaceutically
acceptable and generally regarded as safe for administration to
humans. Examples of preservatives useful in the formulations of the
instant invention include benzyl alcohol, phenol, benzalkonium
chloride, m-cresol, thimerosol, chlorobutanol, methylparaben,
propylparaben and the like. Pharmaceutically acceptable
preservatives may generally be found in the USP (Id). Benzyl
alcohol at 0.9-1% (v/v) is a preferred preservative for liquid
formulations and reconstituted lyophilized formulations.
[0029] As used herein, the term "non-ionic surfactant" refers to a
compound which reduces the surface tension of water. Surfactants
are sometimes helpful in a number of ways, such as reduction of
protein binding to storage and administration devices, for
reduction of aggregate formation by proteins and as an aid for the
resolubilization of proteins during reconstitution of lyophilized
formulations. A surfactant useful in the instant invention will not
promote protein denaturation. Examples of surfactants acceptable
for use in the instant invention include, but are not limited to
polyoxyethylenesorbitan monolaurate (Tween 20),
polyoxyethylenesorbitan monooleate (Tween 80). dodecyl
poly(oxyethyleneglycol ether).sub.23 (Brij 35) and octylphenol
poly(ethyleneglycol ether).sub.10 (Triton.RTM. X-100). Generally,
non-ionic surfactants acceptable for use in the compositions of the
invention may be found in the USP (Id.).
[0030] The term "stable", as used herein in relation to a
particular formulation, means that the formulation meets minimum
acceptable standards for purity after storage for a specified time
under specified conditions. This means that the IGF/IGFBP in the
formulation preferably has less than a 30, more preferably less
than a 15, percentage point increase in aggregation over the course
of one year under normal storage conditions (i.e., approximately
20.degree. C. for a lyophilized formulation, or refrigerated,
frozen or 20.degree. C. for liquid formulations), as measured by
size exclusion chromatography (the material is analyzed by SEC and
percent aggregation is measured by taking ratio of the peak area of
material outside of the main IGF/IGFBP peak to the total peak
area). A stable formulation also preferably has less than a 10,
more preferably five, percentage point increase degradation over
the course of one year under normal storage conditions, as measured
by reverse phase HPLC (the material is analyzed by RP-HPLC and
percent degradation is measured by taking ratio of the peak area of
material outside of the main IGF and IGFBPs peak to the total peak
area).
[0031] Preferred IGFs include wild-type IGF-I (most preferably
recombinant human IGF-I, rhIGF-I) and the variant IGFs, which may
be produced by any method known in the art. Preferably, the rhIGF-I
is produced recombinantly, utilizing the technology disclosed in
International Patent Applications Nos. WO 94/04076 and WO 96/40722.
Preferred IGFBPs include recombinant wild-type human IGFBP-3,
including naturally occurring allelic variants (particularly the
Gly.sub.5 and Ala.sub.5 allelic variants of wild-type human
IGFBP-3) and variants (e.g., the variants at positions 89, 109,
116, 135, 172, 228 and 230) of human IGFBP-3. IGFBPs useful in the
instant invention may be produced by any method known in the art,
and are preferably produced recombinantly, utilizing the fusion
protein technology disclosed in International Patent Applications
Nos. WO 94/04076 and WO 96/40722.
[0032] Formation of IGF/IGFBP complex is preferably acomplished by
simply mixing IGF and IGFBP. In the case of IGF-I and IGFBP-3, the
complex forms quickly without any further manipulation. If so
desired, the complex may be further purified following complex
formation. Such purification may be accomplished by any technique
known in the art.
[0033] Preferably, the IGF/IGFBP comples for use in the instant
formulations has less than 5%, degradation products and less than
15% aggregates.
[0034] Normally, the complex is formed with the IGF and IGFBP in an
aqueous solution including pH buffer salts and dissolved osmolyte
salts (e.g., NaCl). For formation of the formulations of the
invention, the dissolved salts, and optionally the pH buffer salts
must be removed from the solution. This may be accomplished by any
buffer exchange technique known in the art, including, but not
limited to, diafiltration, dialysis, reverse osmosis and other
ultrafiltration techniques and desalting by size exclusion
chromatography. The protein solution may be directly exchanged into
the formulations of the instant invention, or, preferably, it is
exchanged into pure water. If the protein solution is exchanged
into pure water, the other components of the formulation are added
to the water/protein solution and thoroughly mixed. The components
of the formulation (e.g., bulking agent) may be added as dry
chemicals (which is the form in which most bulking agents and some
surfactants are supplied by the manufacturer) or as liquid
concentrates.
[0035] The formulations of the instant invention contain no added
osmolyte salts. Because it is nearly impossible to completely
remove salts which are added to buffer solutions during production
and purification of IGF and IGFBP, particularly when the
formulations are produced in a commercial process, the formulations
may not be completely free of osmolyte salts. However, the
concentration of osmolyte salts in the formulations of the
invention is low, preferably less than 12.5 mM, more preferably
less than 2.5 mM, and most preferably less than 1 mM.
[0036] In one preferred embodiment, IGF/IGFBP complex is formulated
in a pH buffer (i.e., a solution containing buffer salts which can
buffer against changes in pH). The pH buffer preferably has a pH of
about 5.0 to 7.0, more preferably about 5.5 to 6.5. The IGF/IGFBP
may be buffer exchanged into water, followed by the addition of a
concentrated solution of pH buffer salts of the desired pH or the
addition of dry pH buffer salts to the IGF/IGFBP solution.
Alternately, the IGF/IGFBP may be directly buffer exchanged into a
pH buffer. Preferably, the IGF/IGFBP is directly buffer exchanged
into a pH buffer. The buffer salts may be any buffer salts that are
pharmaceutically acceptable, such as sodium phosphate, potassium
phosphate, sodium acetate, sodium citrate, and sodium succinate.
Preferred buffer salts are sodium citrate and sodium succinate,
more preferably sodium succinate. In stability testing experiments,
Applicants have surpisingly found that pharmaceutical formulations
comprising IGF-I/IGFBP-3 with a pH buffer but lacking added
osmolyte salts are more stable than formulations containing added
osmolyte salts. In further surprising results, Applicants have
found that formulations comprising a succinate buffer at pH 5.5 are
more stable than formulations containing citrate and acetate
buffers.
[0037] In another preferred embodiment of the invention, IGF/IGFBP
complex is buffer exchanged into pure water. A bulking agent or
agents may be added to the IGF/IGFBP solution to make it isotonic
with normal human serum if necessary. Preferably, the bulking agent
is mannitol, sorbitol, sucrose, inositol, lactose, dextrose or a
mixture of bulking agents. In one preferred embodiment, the bulking
agents are mannitol and sucrose, and the bulking agents are added
to a total of 6% (w/v), with a preferred ratio of mannitol to
sucrose of 3:2 (i.e., 3.6% (w/v) mannitol and about 2.4% (w/v)
sucrose). Also contemplated are formulations which are made
hypotonic, lyophilized, then reconstituted with a reduced volume of
water to create a isotonic formulation of increased protein
concentration. For example, IGF-I/IGFBP-3 complex may be formulated
in 1.5% mannitol, 1% sucrose at 50 mg/ml protein, lyophilized, then
reconstituted to 0.5 times the original volume, to give a
reconstituted formulation of 100 mg/ml that is isotonic with human
serum. Neither osmolyte salts nor buffer salts are added to the
formulations of this embodiment. Applicants have surprisingly found
that pharmaceutical formulations comprising IGF-I/IGFBP-3 in
mannitol and sucrose which lack any added pH buffer or osmolyte
salts are more stable than formulations which contain pH buffers
and osmolyte salts. Additionally, Applicants have found that
formulations of this embodiment are particularly advantageous
because formulations containing very high protein concentrations
can be made (see Example 5).
[0038] The formulations of the instant invention may be kept as
liquid formulations or they may be lyophilized. Liquid formulations
are preferably frozen for long term storage. Frozen liquid
formulations may be stored in ultra-low freezers (i.e., less than
about -70.degree. C.), non-defrosting freezers (i.e., about
-20.degree. C.) or-defrosting freezers (i.e., cycling between about
5.degree. C. and -15.degree. C.). Preferably, liquid formulations
are stored in ultra-low freezers, but storage in non-defrosting
freezers or defrosting freezers is acceptable.
[0039] Lyophilized formulations are fist prepared as liquids, then
frozen and lyophilized. The lyophilization process is well known to
those of skill in the art, and involves the sublimation of water
from the frozen formulation under controlled conditions.
Lyophilized formulations may be stored refrigerated or at normal
room temperature (e.g., approximately 20.degree. C.). Lyophilized
formulations are reconstituted for use by addition of an aqueous
solution to redissolve the formulation. Preferably the
reconstitution solution is water (e.g., USP WFI, or water for
injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl
alcohol), although solutions containing buffers or other excipients
may also be used. Water and bacteriostatic water are preferred
reconstitution solutions. Other preservatives may be added to the
reconstitution solution, including phenol (preferably about 0.2 to
0.3%), m-cresol (preferably about 0.25-0.3%), thimerosal
(preferably about 0.25-0.3%), methylparaben (preferably about
0.25-0.3%), propylparaben (preferably about 0.25-0.3%),
chlorobutanol (preferably about 0.5%), and the like.
EXAMPLES
Example 1
Comparison of pH Buffers
[0040] Frozen recombinant human (rh) IGF-I/IGFBP-3 (at 10 mg/ml in
50 mM acetate, pH 5.5, and 105 mM NaCl) was thawed and divided into
3 ml samples. One sample was dialyzed against three 500 ml changes
of 20 mM sodium succinate 3% mannitol, 2% sucrose, pH 5.5. The
second sample was dialyzed against three 500 ml changes of 20 mM
sodium citrate, 3% mannitol, 2% sucrose, pH 5.5. After dialysis was
complete, the samples were readjusted to 10 mg/ml. The samples were
placed in a shelf lyophilizer and allowed to equilibrate at
18.degree. C. for approximately 10 minutes, after which the
temperature was reduced to 5.degree. C. for approximately 18
minutes. After equilibration at 5.degree. C., the temperature was
quickly reduced to -15.degree. C., where it was held for
approximately 12 minutes, then reduced to -35.degree. C., at which
point the pressure was reduced in the lyophilizer (200-300
millitorr) and the samples were lyophilized for four hours. The
temperature was increased (under vacuum) to 20.degree. C. over 6
hours, then held at 20.degree. C. (under vacuum) for an additional
28 hours.
[0041] The two reconstituted samples, plus a control sample of 10
mg/ml IGF-I/IGFBP-3 in 50 mM sodium acetate, 105 mM NaCl, pH 5.5,
were incubated at 37.degree. C. for 10 days. At the end of 10 days,
the samples were visually inspected, then analyzed by reverse phase
high performance liquid chromatography (RP-HPLC) and size exclusion
chromatography (SEC). RP-HPLC was performed using a Vydac
4.6.times.250 mm C.sub.18 column (5 .mu.m bead size) loaded in 5%
acetonitrile, 0.1% trifluoracetic acid (TFA) and eluted with a
26%-34% gradient over 40 minutes. SEC analysis was performed with a
Pharmacia Superdex 75 HR 10/30 column run in 50 mM potassium
phosphate, 0.5 M NaCl, pH 7.0 at a flow rate of 0.5 ml/min. A
sample of rhIGF-I/IGFBP-3 at 10 mg/ml in 50 mM acetate, pH 5.5, and
105 mM NaCl which was kept at -80.degree. C. during the experiment
was thawed and used as a control.
[0042] RP-HPLC analysis measures the degradation of IGF-I and
IGFBP-3 by measuring the ratio of material outside of the main
IGF-I and IGFBP-3 peaks to the total material (expressed as percent
degradation). The citrate and succinate buffers were approximately
equivalent in this test, and also approximately equivalent to the
control. Acetate buffer (control) gave 3.6% degradation, while the
citrate and succinate formulations were 3.5% and 4.1%,
respectively.
[0043] SEC analysis showed a great difference between the three
samples. SEC analysis is used to measure the formation of protein
aggregates, which is expressed at percent aggregate by comparing
the material outside of the main IGF-I/IGFBP-3 peak to the total
material in the sample. The control had the highest level of
aggregate, 6%, while citrate was 5%. Succinate was surprisingly
better than the other formulations, with only 2.5% aggregation
after 10 days at 37.degree. C.
Example 2
pH Optimization for Formulations Containing pH Buffers
[0044] 5 ml samples of 10 mg/ml rhIGF-I/IGFBP-3 were dialyzed
against 20 mM sodium succinate buffer containing 3% mannitol, 2%
sucrose (three changes of 500 ml over 24 hours) at pH 4.5, 5.0,
5.5, 6.0 or 6.5. Following dialysis, protein concentrations were
checked by measuring OD.sub.276, and concentrations were adjusted
as necessary to make the samples 10 mg/ml. The pH of each sample
was checked to ensure that the pH was within 0.1 pH point of the
intended pH, then the samples were sterile filtered and lyophilized
as described in Example 1. Sample pH was checked after
reconstitution, then the samples were sterile filtered again and
aliquots of each were placed at 5.degree. C. and 37.degree. C. for
ten days. Stability of the formulation was assayed by RP-HPLC and
SEC.
[0045] SEC analysis showed that increased aggregation was
associated with increased pH. pH 4.5 buffer gave 3.5% aggregation,
pH 5 gave 4%, pH 5.5 gave 4.2%, while pH 6 and 6.5 resulted in 5.3%
and 7.2% aggregation, respectively.
[0046] RP-HPLC analysis showed an opposite trend, with increased
degradation generally associated with decreased pH. pH 4.5 gave the
highest degradation (6.2%), while the pH 5, 5.5, 6 and 6.5 resulted
in 4%, 3.8%, 3.5% and 4.2%, respectively.
[0047] Based on these results, pH 5.5 was selected as the best pH
for formulations containing a pH buffer.
Example 3
Optimization of pH Buffer Concentration for Formulations Containing
pH Buffers
[0048] 5 ml samples of rhIGF-I/IGFBP-3 were dialyzed against
solutions containing 3% mannitol (w/v), 2% sucrose (w/v) and
various concentrations of sodium succinate, pH 5.5 (0, 5 mM, 10 mM,
20 mM and 40 mM). Samples were dialyzed for 48 hours in three 500
ml changes of buffer. Following dialysis, protein concentrations
were checked by measuring OD.sub.276, and concentrations were
adjusted as necessary to make the samples 10 mg/ml. The pH of each
sample was checked to ensure that the pH was within 0.1 pH point of
the intended pH, then the samples were sterile filtered and
lyophilized as described in Example 1. Sample pH was checked after
reconstitution, then the samples were sterile filtered again and
aliquots of each were placed at 5.degree. C. and 37.degree. C. for
ten days. Stability of the formulation was assayed by RP-HPLC and
SEC.
[0049] SEC analysis showed a direct correlation between aggregation
and succinate buffer concentration. 40 mM succinate resulted in
5.5% aggregation, while the 20 mM, 10 mM, 5 mM and 0 samples
resulted in 4.5%, 3.6%, 3.2% and 2.4%, respectively.
[0050] RP-HPLC analysis showed that all the samples were equivalent
with regards to degradation (3.5-4% degradation), with the
exception of the 5 mM sample, which had 7.5% degradation. This
"spike" in degradation may be due to a salt optimum for whatever
process or enzyme is involved in IGFBP-3 degradation.
[0051] These results show that lyophilized formulations without
added pH buffer are more stable than formulations with a pH
buffer.
Example 4
Formulations Containing High Concentrations of IGF-I/IGFBP-3
[0052] 30 ml of a 10 mg/ml solution of rhIGF-I/IGFBP-3 was
exhaustively dialyzed against 3.6% (w/v) mannitol, 2.4% (w/v)
sucrose (formulation solution) then concentrated to 10, 20, and 40
mg/ml by first concentrating the solution by ultrafiltration using
an Amicon Centricon.RTM. 10 centrifugal ultrafiltration device,
testing the concentration by OD.sub.276, then adjusting the
concentration by addition of formulation solution. 10 mg/ml, 20
mg/ml and 40 mg/ml samples of rhIGF-I/IGFBP-3 in formulation
solution were lyophilized as described in Example 1, then
reconstituted with water or water plus 0.9% benzyl alcohol and
sterile filtered. The samples were placed a 37.degree. C. for seven
days, then assayed for stability by RP-HPLC and SEC. Assay results
are shown in Table 1.
[0053] RP-HPLC analysis shows that increasing protein concentration
has a minor effect on protein degradation, and that addition of
benzyl alcohol does not appear to affect degradation.
[0054] SEC analysis showed that increasing protein concentration
increased the level of aggregation in the sample. Interestingly,
the addition of benzyl alcohol, which normally increases
aggregation of protein, and particularly IGF-I/IGFBP-3, had no real
effect on aggregation in the mannitol/sucrose formulations without
added osmolyte salts or pH buffer.
1 TABLE 1 Sample Degradation Aggregation 10 mg/ml 1.4% 1.2% 10
mg/ml + benzyl alcohol 1.2% 1.2% 20 mg/ml 1.9% 2.1% 20 mg/ml +
benzyl alcohol 1.7% 2.1% 40 mg/ml 1.9% 4.2% 40 mg/ml + benzyl
alcohol 1.3% 6.1%
[0055] A further experiment was performed, using rhIGF-I/IGFBP-3
concentrated to 75 mg/ml. The protein was first dialyzed
extensively against 3.6% mannitol/2.4% sucrose, then concentrated
using a stirred cell ultrafiltration device with a 10 kDa cutoff
filter. The solution was sterilized by sterile filtration,
lyophilized as described above, then reconstituted. The
reconstituted protein was assayed to be 75 mg/ml by OD.sub.276.
Samples with and without 0.9% benzyl alcohol were incubated at
37.degree. C. for seven days, then assayed by RP-HPLC and SEC
(results are shown below in Table 2).
[0056] Extremely high protein concentrations result in increased
protein aggregation. Interestingly, although benzyl alcohol had no
effect on aggregation or degradation at low protein concentrations,
addition of benzyl alcohol at this very high protein concentration
did enhance aggregation but not degradation.
2 TABLE 2 Sample Aggregation Degradation 75 mg/ml 15% 3.4% 75 mg/ml
+ benzyl alcohol 27% 3.0%
Example 5
Formulations Containing Very High Concentrations of
IGF-I/IGFBP-3
[0057] rhIGF-I/IGFBP-3 was exhaustively dialyzed against one of
four different formulations: (1) 3.6% mannitol, 2.4% sucrose; (2)
1.5% mannitol, 1% sucrose; (3) 0.525% mannitol, 0.35% sucrose; or
(4) water. After dialysis, the solutions were concentrated using a
stirred cell concentrator to 50 mg/ml protein concentration.
[0058] Samples (1 ml of formulation 1, 2 ml of formulation 2, 4 ml
of formulation 3 and 10 ml of formulation 4) were transferred to
vials and lyophilized as described in Example 1. The different
formulations were designed to yield nominal rhIGF-I/IGFBP-3
concentrations of 50, 100, 200, and 500 mg/ml (calculated final
concentrations were actually 49, 96, 187 and 495 mg/ml),
respectively. Samples were reconstituted to a net weight of 1 g
each and protein concentration was determined by measuring
OD.sub.276, except for formulation 4, which formed a thick syrup
which could not be assayed. Purity was measured by SEC in the
presence of Brij 35 (the standard, a sample of rhIGF-I/IGFBP-3
which had not been lyophilized, assayed at 98.57% pure in this
assay). Results are shown in Table 3
3TABLE 3 Nominal Concen- Expected Measured Purity by Formulation
tration Concentration Concentration SEC 1 50 mg/ml 49 mg/ml 50
mg/ml 98.29% 2 100 mg/ml 96 mg/ml 96 mg/ml 98.31% 3 200 mg/ml 187
mg/ml 186 mg/ml 97.57% 4 500 mg/ml 495 mg/ml not measurable not
measurable
[0059] pH values were also measured before lyophilization and after
reconstitution. Results are shown in Table 4.
4TABLE 4 Nominal pH Before pH After Formulation Concentration
Lyophilization Reconstitution 1 50 mg/ml not determined 6.88 2 100
mg/ml 6.91 6.87 3 200 mg/ml 6.62 6.80 4 500 mg/ml 6.60 not
determined
[0060] Osmolality was also measured before lyophilization and after
reconstitution using an osmometer. Normal laboratory osmolality
values for blood, plasma and serum range from 280-296 mOsm/kg
(Merck Manual of Diagnosis and Therapy, R. Berkow ed., 16th
edition, at 2581, 1992). Results are shown in Table 5.
5TABLE 5 Nominal Predicted Measured Formulation Concentration
Osmolality Osmolality 1 50 mg/ml 304 mOsm/kg 309.5 mOsm/kg 2 100
mg/ml 293 mOsm/kg 297 mOsm/kg 3 200 mg/ml 294 mOsm/kg 290
mOsm/kg
[0061] In a second experiment to evaluate stability of lyophilized
protein in these formulations, rhIGF-I/IGFBP-3 was exhaustively
dialyzed against formulation 1, 2 or 3 and lyophilized as described
in Example 1. After lyophilization, the samples were (a)
reconstituted to 50, 100, or 200 mg/ml ("Time 0") or (b) held at
22.degree. C. or 37.degree. C. for one month ("22.degree. C." and
"37.degree. C.", respectively), then reconstituted to 50, 100 or
200 mg/ml nominal concentration. Purity of the samples was assayed
by SEC or RP-HPLC (as described in Example 1). rhIGF-I/IGFBP-3
stored at -80.degree. C. served as the control. The results (shown
in Tables 6 and 7 for SEC and RP-HPLC, respectively) indicate that
formulations 1, 2 and 3 are stable under these conditions, but
formulations with low bulking agent concentration (e.g.,
formulation 3) were slightly less stable due to des(Gly-Pro)IGF-I
formation.
6TABLE 6 SEC Purity SEC Purity SEC Purity Nominal (%) (%) (%)
Formulation Concentration Time 0 1 mo. 22.degree. C. 1 mo.
37.degree. C. Control 10 mg/ml 96.7 97.5* 97.5* 1 50 mg/ml not 97.9
97.5 determined 2 100 mg/ml 98.4 96.5 97.8 3 200 mg/ml 98.3 98.3
97.3 *Control samples were held at -80.degree. C. during the
incubation period.
[0062]
7TABLE 7 HPLC Purity HPLC Purity Nominal HPLC Purity (%) (%)
Formulation Concentration (%) Time 0 1 mo. 22.degree. C. 1 mo.
37.degree. C. Control 10 mg/ml 99.3 99.6* 99.6* 1 50 mg/ml not 99.5
99.6 determined 2 100 mg/ml 99.4 99.4 99.6 3 200 mg/ml 99.4 99.3
99.4 *Control samples were held at -80.degree. C. during the
incubation period.
[0063] The patents, patent applications, and publications cited
throughout the disclosure are incorporated herein by reference in
their entirety.
[0064] The present invention has been detailed both by direct
description and by example. Equivalents and modifications of the
present invention will be apparent to those skilled in the art, and
are encompassed within the scope of the invention.
* * * * *