Protein formulations for use at elevated temperatures

Information

  • Patent Grant
  • 7964561
  • Patent Number
    7,964,561
  • Date Filed
    Thursday, January 28, 2010
    14 years ago
  • Date Issued
    Tuesday, June 21, 2011
    13 years ago
Abstract
Liquid formulations of bone morphogenetic proteins are provided for prolonged use at elevated temperatures. More specifically, the invention relates to liquid formulations comprising rhGDF-5, trehalose, and one or more biocompatible excipients that provide stability to the protein for at least 30 days at temperatures up to body temperature.
Description
FIELD OF THE INVENTION

The invention relates to liquid formulations of bone morphogenetic proteins for prolonged use at elevated temperatures. More specifically, the invention relates to liquid formulations comprising rhGDF-5, trehalose, and one or more biocompatible excipients to provide formulations that are stable at temperatures up to 37° C. for 30 days or longer.


BACKGROUND OF THE INVENTION

GDF-5 is a member of the Bone Morphogenetic Proteins (BMP), which is a subclass of the TGF-β superfamily of proteins. GDF-5 includes several variants and mutants, including mGDF-5 first isolated from the mouse by Lee (U.S. Pat. No. 5,801,014). Other variants include MP52 (WO 95/04819), which is a human form of GDF-5, also known as hGDF-5 and LAP-4 (Triantfilou, et al. Nature Immunology 2, 338-345 (2001)); also CDMP-1, an allelic protein variant of hGDF-5 (WO 96/14335); also rhGDF-5, the recombinant human form manufactured in bacteria (EP 0955313); also rhGDF-5-Ala83, a monomeric variant of rhGDF-5; also BMP-14, a collective term for hGDF-5/CDMP-1 like proteins; also Radotermin, the international name designated by the World Health Organization; also HMW MP52's, high molecular weight protein variants of MP52; also C465A, a monomeric version wherein the cysteine residue responsible for the intermolecular cross-link is substituted with alanine; also other active single amino acid substitution mutants including N445T, L441P, R438L, and R438K. For the purposes of this application the term “GDF-5” is meant to include all variants and mutants of the protein, and rhGDF-5 is the exemplary member having 119 amino acids.


All members of the BMP family share common structural features including a carboxy terminal active domain and share a highly conserved pattern of cysteine residues that create 3 intramolecular disulfide bonds and one intermolecular disulfide bond. The active form can be either a disulfide-bonded homodimer of a single family member or a heterodimer of two different members (see Massague, et al. Annual Review of Cell Biology 6:957 (1990); Sampath, et al. Journal of Biological Chemistry 265:13198 (1990); Celeste et al. PNAS 87:9843-47 (1990); U.S. Pat. No. 5,011,691, and U.S. Pat. No. 5,266,683). The proper folding of the protein and formation of these disulfide bonds are essential to biological functioning, and misfolding leads to inactive aggregates and cleaved fragments.


The degradation and stabilization of proteins in general has been well described in the literature, and the use of excipients such as dextran, lactose, sorbitol, mannitol, sucrose and trehalose as cryoprotectants and osmoregulators are well documented (see for example reviews of protein stability by Arakawa et al., Advanced Drug Delivery Reviews, 46, 307-326 (2001), Wang, et al., International Journal of Pharmaceutics 185, 129-188 (1999), and on trehalose by Crowe, et al., Cryobiology 43, 89-105 (2001)). The use of excipients to protect lyophilized formulations of GDF-5 has also been described in USPAP 20040132653 by Ichikawa, et al., USPAP 20060286171 by Zhou, et al., and U.S. Patent Application Ser. No. 60/870,032 by Garigapati, et al. Lyophilization is a process that is commonly used and is comprised of freeze-drying a sample to remove water to yield a solid cake for storage, which can then be rehydrated at the time of use. For proteins such as GDF-5, the freezing, drying, and rehydration with water all represent separate insults and challenges to the structure and integrity of the protein.


The use of trehalose as a bulking agent in formulations for stabilizing solutions of the protein troponin has been described by Flaa, et al., in U.S. Pat. No. 6,165,981. The stabilization of antibodies using trehalose have also been described, such as by Lam, et al., in U.S. Pat. Nos. 6,171,586 and 6,991,790. These proteins share little structural similarity with GDF-5, and the use of formulations that are successful for other proteins are not necessarily predictable for use in stabilizing GDF-5.


In contrast, the preparation of a liquid solution of GDF-5 that is stable for prolonged periods of time at elevated temperatures, such as at room temperature, or even at body temperature, present a separate set of challenges that are distinctly different from those of freezing, drying, and rehydrating, as encountered in lyophilization and reconstitution. No longer are the biochemical insults to the protein structure derived from the removal of water, the crystallization of excipients, and the changes in the local microenvironment of the protein chains, their hydrogen and sulfide bonds, and their tertiary structure, but rather the challenge is from increased thermodynamic motion. This leads to an increased rate of oxidation, deamidation, hydrolysis, and cleavage of the amino acids of the protein as the predominant deactivation mechanisms, producing small fragments and an inactive parent molecule, with a lesser amount of aggregation than is commonly observed in lyophilization processes. Reversed phase high performance liquid chromatography (rp-HPLC) and size exclusion chromatography (SEC) appear to be more reliable indicators of protein purity and stability than other methods, such as electrophoresis.


Thus, the strategy and chemistry needed to stabilize a protein such as GDF-5 for a liquid solution at room temperature or above may require a different formulation than one for lyophilization. GDF-5 is not stable in solution for prolonged periods of time at 2-8° C., and is typically stored at temperatures between −60 to −80° C. GDF-5 is not soluble at neutral pH and is typically solubilized in acidic solutions, thereby increasing the potential for acid hydrolysis.


In view of the above-mentioned limitations and complications of preparing a stable GDF-5 liquid formulation for prolonged storage and use at elevated temperatures, new and effective formulations are needed.


SUMMARY OF THE INVENTION

The invention is a liquid protein formulation. The formulation comprises a BMP, at least a 50% w/v solution of trehalose, and at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate in an amount sufficient to stabilize the BMP as evidenced by retention of at least 80% of the main chromatography peak for at least 30 days storage at temperatures up to 37° C.


In another embodiment, the invention is a method of stabilizing a BMP solution having a BMP, at least a 50% w/v solution of trehalose, and at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate.


The protein formulations of the present invention can be used in all applications where the storage and use of bone morphogenetic proteins is desired. The storage and use of bone morphogenetic proteins at room temperature or body temperature present useful applications of these formulations. Table 1 shows a summary of the different formulations tested and the results.









TABLE 1





Summary of Formulations and Data























SEC
rp-HPLC
rp-HPLC







% Main Peak
% Main Peak
% Main Peak


Formulation
Day 30,
Day 30,
Day 60,

Trehalose
Taurine
Raffinose


#
37° C.
37° C.
37° C.
pH
w/v %
wt %
wt %





1

66

6.8
60




2

73

6.6
60


3

76

6.3
60


4

86

6.1
60


5

92

3.1
60


6

91

3.2
60


7

88

3.0
60


8

87

2.9
60


9

87

2.9
60


10

87

2.7
60

3


11

77

5.8
60

3


12

74

6.0
60

3


13

56

6.4
60

3


14

88

5.0
60



15

89

5.4
60



16

89

5.8
60



17

84

6.0
60



18
98
94
76
5.6
60



19
95
91
88
5.3
60
0.1


20

79
71
3.5
60
0.1


21
97
92
92
5.4
60


22

76
66
5.3
60


23
98
93
78
5.4
60


24

82
75
5.6
60


25

62
48
5.7
60


26

66
58
5.7
60


27

65
53
5.4
60


28

83
76
5.3
60


29

68
53
3.9
60


30

78
66
3.5
60


31

76
56
3.6
60


32

80
67
3.5
60


33

56
43
5.7
60


34

87
69
5.9
60


35

59
52
6.4
60


36

72
49
5.2
60


37
91
94
91
5.3
60




















Myo-







Potassium


Formulation
inositol
Betaine
HSP70
β-Alanine
TMAO
TMA
TEA
L-Proline
Aspartate


#
wt %
wt %
wt %
wt %
wt %
wt %
wt %
wt %
wt %





1




1


2




0.5


3




0.25


4




0.1


5


0.1


6


0.2


7





1


8





2


9





5


10
1


11
1



0.01


12
1



0.25


13
1



0.5


14



0.25


15



0.5


16



0.25
0.1


17



0.5
0.1


18



0.5

0.1


19



0.5


20

0.5


21



0.5


0.1


22



0.5


0.5


23

0.5




1
0.5
0.5


24

0.5

0.5


0.1
0.5
0.5


25

0.5

0.5



0.5
0.5


26



0.5



0.5
0.5


27



0.5



0.5


28

0.5





0.5
0.5


29

0.5





0.5


30

0.5


31







0.5


32

0.5


33



1


34



2


35



5


36



0.5

0.1


37



0.5


3












BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 shows the comparative stability of formulations #18, 19, 21, 23, and 37 by rp-HPLC after 30 and 60 days at 37° C. and also by SEC after 30 days at 37° C.



FIG. 2 shows the stability of formulation 18 by rp-HPLC after 30 days at 37° C. The preservation of the protein is shown to be 94% by the main peak and the absence of additional peaks.



FIG. 3 shows the stability of formulation 37 by rp-HPLC after 30 days at 37° C. The preservation of the protein is shown to be 93% by the main peak and the absence of additional peaks.



FIG. 4 shows the stability of formulation 35 by rp-HPLC after 30 days at 37° C. The preservation of the protein is shown to be 59% by the main peak, with the presence of additional peaks indicating degradation.



FIG. 5 shows the stability of formulation 21 by rp-HPLC after 60 days at 37° C. The preservation of the protein is shown to be 91% by the main peak and the absence of additional peaks.



FIG. 6 shows the stability of formulation 29 by rp-HPLC after 60 days at 37° C. The preservation of the protein is shown to be 53% by the main peak, with the presence of additional peaks indicating degradation.



FIG. 7 shows the stability of formulation 12 by SEC after 12 days at 37° C. The preservation of the protein is shown to be 82% by the main peak, with the presence of a small additional peak indicating slight degradation.



FIG. 8 shows the stability of formulation 18 by SEC after 30 days at 37° C. The preservation of the protein is shown to be 98% by the main peak and the absence of additional peaks.



FIG. 9 shows the stability of a GDF-5 sample with 60% w/v trehalose alone by rp-HPLC after 14 days at 37° C. The preservation of the protein is shown to be 75% by the main peak, with the presence of additional peaks indicating degradation.



FIG. 10 shows a chromatograph of a GDF-5 reference standard (no formulation)



FIG. 11 shows the correlation between the activity of a freshly prepared GDF-5 standard solution, a freshly prepared solution of formulation #18, and a solution of formulation #18 after 60 days exposure to 37° C. The preservation of the activity of the protein is shown by the comparative similarity of the curves.





DETAILED DESCRIPTION

The bone morphogenetic proteins that may be used in the present invention include, but are not limited to, BMP-2, BMP-4, BMP-6, BMP-7, BMP12, and BMP-14, and all variants and mutants thereof The preferred BMP is rhGDF-5, also known as MP52.


Here we provide several formulations that are useful as compositions providing stability of the BMP molecule in aqueous solution at elevated temperatures for prolonged periods of time. For the purposes of the present invention, the terms “room temperature” and “ambient temperature” are understood to be interchangeable and to mean the temperature of an ordinary office or laboratory having a temperature of between approximately 18 to 25° C.; the term “body temperature” means the normal body temperature of humans, being a temperature of approximately 37° C.; the term “refrigerated temperature” means a temperature of between approximately 2 to 8° C.; the term “frozen” means a temperature of between approximately −4° C. to −20° C.


The stability of BMP has been shown by various analytical methods such as rp-HPLC and SEC, and does not rely on vague biological assays for determining the chemical purity of the BMP molecule and thus the performance of the formulation. For the purposes of the present invention, the terms “stability” and “purity” are interchangeable and meant to describe the characterization of a BMP by rp-HPLC or SEC chromatography, and refer to the area under the curve of the main peak as a measure of the preservation of the parent molecule. A biological assay has also been performed to correlate the stability with the biological activity of the BMP (see FIG. 11).


A formulation of the present invention includes a BMP, at least a 50% w/v solution of trehalose in an acidic solution, and at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate. Trehalose is a well-known excipient that provides stability to proteins in solution and in lyophilized formulations, but it is not sufficient in itself to provide prolonged stability to liquid solutions of GDF-5 at body temperature. This is evidenced by the reduction in the main peak and the appearance of additional peaks in the rp-HPLC chromatograph of FIG. 9 after 14 days storage at a temperature of 37° C., as compared to the standard chromatograph shown in FIG. 10.


The solubility of trehalose has been reported to be 68.9 g/100 g H2O (Higashiyama, Pure Appl. Chem. 74, 1263-1269 (2002)). We used trehalose solutions of 50% and 60% w/v and investigated several excipients and combinations thereof to find a formulation that would provide for a stable liquid BMP solution to yield at least 80% retention of the protein purity, as evidenced by the main peak in an rp-HPLC or SEC chromatograph after storage for 30 days or longer at temperatures up to 37° C. After much experimentation, we have found certain combinations of excipients to be of value in meeting this goal. We also attempted to increase the pH of various formulations to minimize the potential for acid hydrolysis and cleavage of the protein. The desired pH range is from about 2.5 to about 7.0, and more preferably from about 4.0 to about 6.0. Lower pH values tend to lead to higher rates of acid hydrolysis of the protein, and higher pH values tend to cause insolubility of the protein. Hydrochloric acid is preferred due to its biocompatibility, and values of 0.5 to about 3 millimolar are preferred, although higher values of up to about 10 millimolar HCl are acceptable. It would be obvious to one of ordinary skill in the art that other biocompatible acids could also be used in the formulations of the present invention.


Trimethylammonium N-Oxide dihydrate (TMAO) was tested in various concentrations in addition to trehalose, and in general found to have deleterious effects on the stability of the protein. While a 0.1% w/v addition of TMAO provided an acceptable 86% retention of the main peak, increasing concentrations of TMAO led to a reduction in protein stability (formulations 1-4). In comparing formulations 15 and 17, both containing a 0.5% w/v content of β-alanine, the addition of 0.1% w/v TMAO reduced the protein stability from 89% to 84%. In other formulations comprising raffinose and myo-inositol, the addition of TMAO also appeared to have a deleterious effect on the protein stability. While a formulation of 3% raffinose and 1% myo-inositol had satisfactory performance, the addition of increasing amounts of TMAO had increasingly deleterious effects on the stability of the protein (see formulations 10-13).


Heat shock proteins are known in the art and are capable of stabilizing some biological systems from thermal stress (see for example Nakamoto, et al. in Cell Mol Life Sci. February; 64(3): 294-306 (2007)). Heat shock protein 70 was tested at levels of 0.1% and 0.2% w/v in a 60% trehalose solution with GDF-5 and showed acceptable results yielding 92% and 91% retention of the main peak, respectively.


Trimethylammonium hydrochloride (TMA) is a well-known excipient and was tested in conjunction with trehalose in several formulations at various concentrations, and also in conjunction with β-alanine. TMA provided satisfactory results in all formulations tested except formulation 36, which had a 50% trehalose solution. Due to the strong undesirable odor of TMA, triethylammonium hydrochloride (TEA) was investigated for its potential as a suitable substitute for TMA. Direct comparison of formulations 18 and 21 show that TEA is in fact a suitable substitute for TMA and provides for superior protein stabilization, as evidenced by FIGS. 2 and 5. In fact, FIG. 5 shows that formulation 21 with TEA provided the best overall long-term performance of all formulations tested, with 91% main peak retention after 60 days at 37° C.


A betaine is any neutral chemical compound with a positively charged cationic functional group such as an ammonium ion or phosphonium ion, and also has a negatively charged functional group such as a carboxyl group. These compounds exist as zwitterions and serve as organic osmolytes in biological systems, and are useful excipients. Historically, the term betaine has been reserved for the compound trimethylglycine after its discovery in sugar beets. Trimethylglycine hydrochloride is an exemplary betaine of the present invention, and has shown acceptable performance in several formulations at a 0.5% w/v level in conjunction with trehalose and other excipients (see formulations 23, 24, 28, and 32).


Amino acids are known to provide buffering capacity and are contemplated for use as excipients in the present invention. Examples of suitable amino acids include, but are not limited to isomers of alanine, glycine, proline, lysine, arginine, and histidine as exemplary amino acids useful as buffering agents. The amino acids may be used individually or in combination to provide buffering capacity. β-alanine was tested as an amino acid buffer in several formulations, and in general found to have beneficial properties. Formulations with trehalose and β-alanine provided acceptable results with β-alanine concentrations of 0.25% up to 2% w/v (formulations 14, 15 and 34), but not in 1% and 5% solutions (formulations 33 and 35). Formulation 19 included 0.5% β-alanine and 0.1% taurine and yielded excellent results, showing greater than 90% stability after 30 days at 37° C. by both rp-HPLC and SEC, and 88% stability after 60 days. Formulation 23 included 0.5% betaine, 1% TEA, and 0.5% each of L-Proline and potassium aspartate, and yielded excellent results after 30 days at 37° C., showing greater than 90% stability, but after 60 days had 78% stability. Other formulations utilizing L-proline and potassium aspartate that yielded acceptable results include formulations 24 and 28, yielding 82% and 83% respectively after 30 days at 37° C.


To confirm the correlation between the protein stability as demonstrated by the rp-HPLC main peak and the biological activity of the GDF-5 protein, formulation 18 was compared with a standard GDF-5 protein solution after exposing the formulation to 37° C. for 60 days, and also with the formulation at time zero. The biologic activity of GDF-5 was measured using different concentrations of GDF-5 on a bone marrow stromal cell line W-20-17. Increasing the amount of GDF-5 increased the Alkaline Phosphatase (ALP) in W-20-17, as was determined by a colorimetric assay. In FIG. 11 the ALP bioassay of formulation #18 at time zero and after 60 days at 37° C. were compared with a freshly prepared standard GDF-5 solution without excipients. All three curves exhibit similar profiles, indicating that the formulation does not reduce the activity at time zero, and that the formulation provides for an active protein after 60 days at 37° C.


In one embodiment, the present invention comprises a formulation of at least 50% w/v trehalose in an acidic solution, a BMP, and a trialkylammonium salt present in a concentration of from about 0.1% to about 5% w/v. In preferred embodiments the trialkylammonium salt can be trimethylammonium hydrochloride, triethylammonium hydrochloride, or a combination thereof, although one skilled in the art will appreciate that minor substitutions or modifications of the alkyl groups, the salt, or both the alkyl groups and salt would be considered equivalents of the present invention.


In another embodiment, the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, and at least one amino acid present in an amount of from about 0.25% to about 5% w/v. In a preferred embodiment the at least one amino acid is β-alanine and is present in an amount of about 0.5% w/v. In another embodiment, the at least one amino acid is a combination of glycine and β-alanine, each present in an amount of from about 0.1% to about 2.5% w/v. In a preferred embodiment the β-alanine is present in an amount of about 0.25% and the glycine is present in an amount of about 0.25% w/v. In another embodiment the amino acid is a combination of L-glycine, L-proline, and L-alanine, each present in an amount of from about 0.1% to about 2.5% w/v.


In another embodiment the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, and a heat shock protein present in an amount of from about 0.1% to about 0.2% w/v. In a preferred embodiment the heat shock protein is heat shock protein-70 and is present in an amount of about 0.1% w/v.


In another embodiment, the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, β-alanine present in an amount of from about 0.25% to about 5% w/v, and taurine present in an amount of from about 0.01% to about 1% w/v. In a preferred embodiment the β-alanine is present in an amount of about 0.5% w/v and the taurine is present in an amount of about 0.1% w/v.


In another embodiment, the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, β-alanine present in an amount of from about 0.25% to about 5% w/v, and a trialkylammonium salt present in an amount of from about 0.1% to about 5% w/v. In a preferred embodiment the β-alanine is present in an amount of about 0.5% w/v and the trialkylammonium salt is triethylammonium hydrochloride and is present in an amount of about 0.1% w/v.


In another embodiment, the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, raffinose present in an amount of from about 1% to about 5% w/v, and myo-inositol present in an amount of from about 0.1% to about 3% w/v. In a preferred embodiment the raffinose is present in an amount of about 3% w/v and myo-inositol is present in an amount of about 1% w/v.


In another embodiment, the formulation comprises at least 50% w/v trehalose in an acidic solution, a BMP, β-alanine present in an amount of from about 0.25% to about 5% w/v, triethylammonium hydrochloride present in an amount of from about 0.1% to about 5% w/v, L-proline present in an amount of from about 0.1 to about 3% w/v, and potassium aspartate present in an amount of from about 0.1 to about 3% w/v. In a preferred embodiment the β-alanine is present in an amount of about 0.5% w/v, the triethylammonium hydrochloride is present in an amount of about 1% w/v, the L-proline is present in an amount of about 0.5% w/v, and the potassium aspartate is present in an amount of about 0.5% w/v.


The following examples illustrate some of the various embodiments and benefits of the present invention, however one skilled in the art will appreciate that other similar embodiments can be made without deviating from the scope and intent of the present invention.


EXAMPLES
Example 1
Preparation of Bulk Trehalose 60% w/v Solution

165.78 g of trehalose dihydrate (MW 378.34) were carefully weighed and transferred to a 250 ml size, clean volumetric flask, to which 1 mmol HCl was added slowly to just below the mark. The mixture was mixed thoroughly by shaking and warming in warm water at 60° C. The volume was brought to 250 ml by adding more 1 mmol HCl and letting all of the crystals completely dissolve; the solution was then filtered through a 0.2 um filter. This solution was used for the preparation of the formulations. In an analogous manner a 50% w/v solution was made for formulations 32 & 36 using a 50% trehalose solution.


Example 2

A non-limiting example of the preparation of a composition of the present invention is as follows (Formulation #19): 165.78 g of trehalose dihydrate (MW 378.34) were carefully weighed and transferred to a clean 250 ml volumetric flask, to which 1 mmol HCl was added slowly to just below the mark to produce a 60% w/v solution. The mixture was mixed thoroughly by shaking and warming in warm water at 60° C. The volume was brought to the mark by adding more 1 mmol HCl and ensuring that all of the trehalose crystals completely dissolved; the solution was then filtered through a 0.22 um filter. To 10 ml of the 60% trehalose solution, 51 mg of β-alanine and 10 mg of taurine were added. The mixture was swirled, to which 1000 ug (1 mg) of rhGDF-5 in solution was added. The protein concentration of the formulation was measured by UV light to ensure the desired concentration was achieved, and was adjusted by adding solvent or protein as needed.


Example 3

In a preferred embodiment, a formulation of the present invention is prepared as follows (Formulation #21): 165.78 g of trehalose dihydrate (MW 378.34) were carefully weighed and transferred to a 250 ml size, clean volumetric flask, to which 1 mmol HCl was added slowly to just below the mark to produce a 60% w/v solution. The mixture was mixed thoroughly by shaking and warming in warm water at 60° C. The volume was brought to the mark by adding more 1 mmol HCl and ensuring that all of the trehalose crystals were completely dissolved; the solution was then filtered through a 0.22 um filter. To 10 ml of the 60% trehalose solution, 50 mg of β-alanine and 10 mg of triethylamine hydrochloride (TEA) were added. The mixture was swirled, to which 1000 ug (1 mg) of rhGDF-5 in solution was added. The protein concentration of the formulation was measured by UV light to ensure the desired concentration was achieved, and was adjusted by adding solvent or protein as needed.


Example 4

In a preferred embodiment, a formulation of the present invention is prepared as follows (Formulation #32): 55.27 g of trehalose dihydrate (MW 378.34) were carefully weighed and transferred to a 100 ml size, clean volumetric flask, to which 1 mmol HCl was added slowly to just below the mark to produce a 50% w/v solution. The mixture was mixed thoroughly by shaking and warming in warm water at 60° C. The volume was brought to the mark by adding more 1 mmol HCl and ensuring that all of the trehalose crystals were completely dissolved; the solution was then filtered through a 0.22 um filter. To 10 ml of the 50% trehalose solution, 50 mg of betaine were added. The mixture was swirled, to which 1000 ug (1 mg) of rhGDF-5 in solution was added. The protein concentration of the formulation was measured by UV light to ensure the desired concentration was achieved, and was adjusted by adding solvent or protein as needed.


Materials and Equipment Used




  • 1.1 Trehalose dihydrate, Ferro-Pfanstiehl #T-104-1-MC

  • 1.2 Glycine, ultrapure grade, J. T. Baker #4059-00

  • 1.3 β-alanine, 99%, Aldrich #239720

  • 1.4 12 M HCl, EM Science #HX0603P/5 (concentrated stock reagent)

  • 1.5 Trimethylamine N-Oxide Dihydrate (TMAO), Sigma #T0514

  • 1.6 Trimethylammonium hydrochloride (TMA), 98%, Aldrich #T72761

  • 1.7 Triethylammonium hydrochloride (TEA), Fluka #90350

  • 1.8 Taurine, 99%, Sigma #T0625

  • 1.9 Betaine, Sigma #B3501

  • 1.10 Myo-inositol, 99% Sigma-Aldrich #15125

  • 1.11 D-(+)-Raffinose Penta Hydrate, 98%, Sigma #R0250

  • 1.12 HSP70, Sigma-Aldrich #H7283-1MG

  • 1.13 Filtration units, 250 mL, 0.22 micron membrane, Nalgene #568-0020

  • 1.14 Sterile, 250 mL, square PETG media bottles, Nalgene #2019-0250

  • 1.15 UV-VIS spectrophotometer, Beckman-Coulter DU800, ID #494203

  • 1.16 rhGDF-5: thawed at 2 to 8° C. prior to use

  • 1.17 Water for injection, Baxter #2B0306

  • 1.18 rp-HPLC: Waters model 2596, Vydac 218TP52, C18 column, eluted with 0.15% (v/v) TFA in water and 0.15% (v/v) TFA in acetonitrile at 0.3 ml/min. The eluted peaks were monitored at 214 nm.

  • 1.19 SEC: Waters model 2596, TOSOH Bioscience, Cat #08540, eluted with 0.1% (v/v) TFA and 45% (v/v) acetonitrile in water at 0.5 ml/min. The protein peaks were monitored at 280 nm.



The formulations listed in table 1 were prepared in an analogous manner to the methods described in the examples above. The formulations were evaluated for their ability to stabilize the rhGDF-5 protein molecule over extended periods of time at elevated temperatures, as characterized by rp-HPLC and in some select samples also by SEC.

Claims
  • 1. A composition comprising GDF-5 and excipients in an acidic solution, wherein the excipients are trehalose present in the amount of 50% w/v-60% w/v, and at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate.
  • 2. The composition of claim 1 wherein the amino acid is selected from the group consisting of β-alanine, L-glycine, and L-proline.
  • 3. The composition of claim 2 wherein the amino acid is comprised of β-alanine present in an amount of from about 0.25% to about 5% w/v.
  • 4. The composition of claim 3 wherein the β-alanine is present in an amount of about 0.5% w/v.
  • 5. The composition of claim 1 wherein the trialkylammonium salt is present in an amount of from about 0.1% to about 3% w/v.
  • 6. The composition of claim 5 wherein the trialkylammonium salt is triethylammonium hydrochloride.
  • 7. The composition of claim 1 wherein the amino acid is present in an amount of from about 0.25% to about 5% w/v and the trialkylammonium salt is present in an amount of from about 0.1% to about 3% w/v.
  • 8. The composition of claim 7 wherein the amino acid is β-alanine present in an amount of about 0.5% w/v and the trialkylammonium salt is triethylammonium hydrochloride present in an amount of about 0.1% w/v.
  • 9. The composition of claim 1 wherein the heat shock protein is HSP 70 present in an amount of from about 0.1% to about 0.2% w/v.
  • 10. The composition of claim 1 wherein the at least one additional excipient is comprised of β-alanine present in an amount of about 0.5% w/v and taurine present in an amount of about 0.1% w/v.
  • 11. The composition of claim 1 wherein the at least one additional excipient is a betaine.
  • 12. A method for stabilizing a GDF-5 solution comprising providing GDF-5 in an acidic solution, adding an amount of trehalose to provide 50% w/v-60% w/v solution of trehalose, and adding at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate.
RELATED APPLICATION

This application is a divisional application of U.S. patent application Ser. No. 12/145,016, filed on Jun. 24, 2008, which is now U.S. Pat. No. 7,678,764, which claims priority from U.S. Patent Application No. 60/947,092, filed on, Jun. 29, 2007.

US Referenced Citations (111)
Number Name Date Kind
2931802 Touey Apr 1960 A
4120810 Palmer Oct 1978 A
4891319 Roser Jan 1990 A
5011691 Oppermann Apr 1991 A
5013649 Wang May 1991 A
5202311 Folkman Apr 1993 A
5231169 Constantz Jul 1993 A
5266683 Oppermann Nov 1993 A
5318898 Israel Jun 1994 A
5385887 Yim Jan 1995 A
5411941 Grinna May 1995 A
5455231 Constantz Oct 1995 A
5516654 Israel May 1996 A
5658882 Celeste Aug 1997 A
5747058 Tipton May 1998 A
5770700 Webb Jun 1998 A
5776193 Kwan Jul 1998 A
5801014 Lee Sep 1998 A
5804557 Cleland Sep 1998 A
5866165 Liu Feb 1999 A
5955448 Colaco Sep 1999 A
5968542 Tipton Oct 1999 A
5972385 Liu Oct 1999 A
5985320 Edwards Nov 1999 A
6051558 Burns Apr 2000 A
6071428 Franks Jun 2000 A
6165981 Flaa Dec 2000 A
6171584 Hotten Jan 2001 B1
6171586 Lam Jan 2001 B1
6187742 Wozney Feb 2001 B1
6207718 Papadimitriou Mar 2001 B1
6281195 Rueger Aug 2001 B1
6284872 Celeste Sep 2001 B1
6288043 Spiro Sep 2001 B1
6340369 Ferree Jan 2002 B1
6344058 Ferree Feb 2002 B1
6352557 Ferree Mar 2002 B1
6407060 Charette Jun 2002 B1
6419702 Ferree Jul 2002 B1
6454804 Ferree Sep 2002 B1
6551801 Andou Apr 2003 B1
6645247 Ferree Nov 2003 B2
6648918 Ferree Nov 2003 B2
6648919 Ferree Nov 2003 B2
6648920 Ferree Nov 2003 B2
6656492 Kajiyama Dec 2003 B2
RE38385 Franks et al. Jan 2004 E
6685695 Ferree Feb 2004 B2
6719968 Celeste Apr 2004 B2
6723170 Ohashi Apr 2004 B2
6755863 Ferree Jun 2004 B2
6764994 Hotten Jul 2004 B1
6780324 Le Garrec Aug 2004 B2
6911411 Cox Jun 2005 B2
6936582 Charette Aug 2005 B1
6991790 Lam Jan 2006 B1
6992065 Okumu Jan 2006 B2
7060268 Andya Jun 2006 B2
RE39497 Franks et al. Feb 2007 E
7235527 Makishima Jun 2007 B2
7318840 McKay Jan 2008 B2
7323445 Zhang Jan 2008 B2
7375077 Mao May 2008 B2
7435260 Ferree Oct 2008 B2
7572440 Vukicevic Aug 2009 B2
7678764 Garigapati et al. Mar 2010 B2
20010024823 Vukicevic Sep 2001 A1
20020032155 Ferree Mar 2002 A1
20020128718 Ferree Sep 2002 A1
20020165542 Ferree Nov 2002 A1
20020173770 Flory Nov 2002 A1
20030026788 Ferree Feb 2003 A1
20030185812 Ferree Oct 2003 A1
20030192554 Ferree Oct 2003 A1
20040022771 Ferree Feb 2004 A1
20040024471 Ferree Feb 2004 A1
20040028733 Tracy Feb 2004 A1
20040132653 Ichikawa Jul 2004 A1
20040146923 Celeste Jul 2004 A1
20040197324 Liu Oct 2004 A1
20050069571 Slivka Mar 2005 A1
20050119754 Trieu Jun 2005 A1
20050191248 Hunter Sep 2005 A1
20060024346 Brody Feb 2006 A1
20060088565 Kohnert Apr 2006 A1
20060121113 Bartholomaeus Jun 2006 A1
20060223120 Kim Oct 2006 A1
20060286171 Zhou Dec 2006 A1
20060286289 Prajapati Dec 2006 A1
20060287676 Prajapati Dec 2006 A1
20070053871 Li et al. Mar 2007 A1
20070098756 Behnam May 2007 A1
20070172479 Warne et al. Jul 2007 A1
20070178159 Chen Aug 2007 A1
20080098614 Tchessalov May 2008 A1
20080147077 Garigapati et al. Jun 2008 A1
20080234727 Garigapati Sep 2008 A1
20080311078 Gokarn Dec 2008 A1
20090004048 Elliott Jan 2009 A1
20090030483 Risi Jan 2009 A1
20090043078 Daniel Feb 2009 A1
20090048412 Soula Feb 2009 A1
20090060976 Rueger Mar 2009 A1
20090099089 Zhang Apr 2009 A1
20090259023 Su Oct 2009 A1
20090286764 Kipp Nov 2009 A1
20090291062 Fraunhofer Nov 2009 A1
20100015230 Ron Jan 2010 A1
20100041870 Tchessalov Feb 2010 A1
20100144631 Ron Jun 2010 A1
20100255100 Margolin Oct 2010 A1
Foreign Referenced Citations (65)
Number Date Country
955313 Nov 1999 EP
957943 May 2003 EP
1350525 Oct 2003 EP
1448246 Aug 2004 EP
1462126 Sep 2004 EP
1274459 Nov 2005 EP
1604693 Dec 2005 EP
1604963 Dec 2005 EP
0955313 May 2006 EP
955313 May 2006 EP
1915986 Apr 2008 EP
1932519 Jun 2008 EP
957943 Nov 2008 EP
WO 8800205 Jan 1988 WO
WO 9011366 Oct 1990 WO
WO 9118098 Nov 1991 WO
WO 9200382 Jan 1992 WO
WO 9309229 May 1993 WO
WO 9316099 Aug 1993 WO
WO 9410203 May 1994 WO
WO 9415949 Jul 1994 WO
WO 9415965 Jul 1994 WO
WO 9415966 Jul 1994 WO
WO 9421681 Sep 1994 WO
WO 9426892 Nov 1994 WO
WO 9426893 Nov 1994 WO
WO 9501801 Jan 1995 WO
WO 9504819 Feb 1995 WO
WO 9510539 Apr 1995 WO
WO 9510802 Apr 1995 WO
WO 9516035 Jun 1995 WO
WO 9533830 Dec 1995 WO
WO 9601316 Jan 1996 WO
WO 9601845 Jan 1996 WO
WO 9614335 May 1996 WO
WO 9636710 Nov 1996 WO
WO 0178683 Oct 2001 WO
WO 03000282 Jan 2003 WO
WO 03030923 Apr 2003 WO
WO 03043673 May 2003 WO
WO 03066120 Aug 2003 WO
WO 2004037265 May 2004 WO
WO 2004052336 Jun 2004 WO
WO 2005060989 Jul 2005 WO
WO 2005100399 Oct 2005 WO
WO 2005107765 Nov 2005 WO
WO 2005115438 Dec 2005 WO
WO 2006138099 Dec 2006 WO
WO 2006138099 Dec 2006 WO
WO 2006138181 Dec 2006 WO
WO 2007025441 Mar 2007 WO
WO 2008009419 Jan 2008 WO
WO 2008045498 Apr 2008 WO
WO 2008049588 May 2008 WO
WO 2008079672 Jul 2008 WO
WO 2008082563 Jul 2008 WO
WO 2008099190 Aug 2008 WO
WO 2008099198 Aug 2008 WO
WO 2008143867 Nov 2008 WO
WO 2009006097 Jan 2009 WO
WO 2009006301 Jan 2009 WO
WO 2009015736 Feb 2009 WO
WO 2009016131 Feb 2009 WO
WO 2009016333 Feb 2009 WO
WO 2009020744 Feb 2009 WO
Related Publications (1)
Number Date Country
20100130730 A1 May 2010 US
Provisional Applications (1)
Number Date Country
60947092 Jun 2007 US
Divisions (1)
Number Date Country
Parent 12145016 Jun 2008 US
Child 12695357 US