WO1998014476A1

WO1998014476A1 - Pharmaceutical compositions containing an mpl ligand

Info

Publication number: WO1998014476A1
Application number: PCT/US1997/016196
Authority: WO
Inventors: David N. Brems; Michael J. Treuheit
Original assignee: Amgen Inc.
Priority date: 1996-10-04
Filing date: 1997-09-12
Publication date: 1998-04-09
Also published as: KR100408229B1; KR20000048905A; AU4344697A; JP2001501619A; HK1024490A1; CN1142940C; CN1239480A; TW561050B

Abstract

The subject invention relates to compositions of mpl ligands, comprising a full-length or truncated mpl ligand having a sequence of amino acids corresponding to amino acids 7-151 through 1-332, inclusive, of native human mpl ligand, optionally covalently linked to at least one water-soluble polymer; a buffering agent selected from glutamate, phosphate, histidine, imidazole, and acetate; an excipient selected from sorbitol, sucrose, mannitol, glycerol, polyethylene glycol, and non-polar amino acids; optionally, a detergent or lipid such as Tween; optionally, an antioxidant or chelating agent selected from glutathione, methionine, citrate and EDTA; and having a pH preferably ranging from 5.0 to 6.0 (inclusive). Such compositions may be liquid (preferably, aqueous), frozen (preferably, aqueous), or lyophilized.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING AN MPL LIGAND

Field of the Invention The present invention relates to compositions containing an mpl ligand, which compositions are suitable for pharmaceutical administration.

Background of the Invention

The native human mpl ligand is a recently cloned cytokine that appears to be the major regulator of circulating platelet levels. See Bartley, T.D. et al . , Cell 77:1117-1124 (1994); Lok, S. et al . , Nature 369:565-568 (1994); de Sauvage, F.J. et al . , Nature 369:533-538 (1994); Miyazake, H. et al . , Exp. Hematol . 22:838 (1994); and Kuter, D.J. et al., PNAS USA, 91:11104-11108 (1994). Native human mpl ligand, also referred to as thro bopoietin (TPO) and megapoietin, is a protein having 332 amino acids in total. Recombinant mpl ligand produced in both Chinese

Hamster Ovary (CHO) and E. coli cells has been demonstrated to have a biological activity of specifically stimulating or increasing megakaryocytes and/or platelets in vivo in mice, rats and monkeys. See e.g., Hunt, P. et al . , Blood 84(10) :390A (1994). Human mpl ligands that have been truncated from the C-terminus by up to 181 amino acids retain biological activity in vivo . The resulting mpl ligands have sequences that correspond to amino acids 1 - 151 up to 1 - 331 of the full-length human sequence. It is also possible to remove up to the first six amino acids at the N-terminus of the human mpl ligand protein and retain biological activity. Therefore, it appears that biological activity is retained within amino acids 7 to 151 (inclusive) of the mature amino acid sequence of human mpl ligand. Derivatives of mpl ligands have been demonstrated to have advantageous activity to stimulate production of megakaryocytes and/or platelets in in vivo tests. See published PCT Application WO 95/26746. In particular, mpl ligands derivatized with water soluble polymers such as polyethylene glycol ("PEG") moieties are of interest in a clinical setting because they are long-lived and active in vivo.

Compositions containing mpl ligands and related derivatives have been disclosed in a general sense. See published PCT Applications WO 95/26746, WO 95/21919, WO 95/18858, and WO 95/21920. However, controlled experiments resulting in a determination of which compositions containing mpl ligands are suitably stable for pharmaceutical use, as set forth herein, have not been previously reported. Such compositions are important for practical application of mpl ligands to patients such as humans. Thus, there continues to exist a need for such compositions in the art for use in administering mpl ligands to patients so as to result in an increase in platelets.

Summary of the Invention

Accordingly, it is an object of this invention to provide stable compositions that are pharmaceutically acceptable, which include mpl ligands.

It is another object of this invention to provide compositions that contain mpl ligands for administration to patients .

In one embodiment, the subject invention relates to compositions of mpl ligands, comprising a full-length or truncated mpl ligand having a sequence of amino acids corresponding to amino acids 7-151 through 1-332, inclusive, of native human mpl ligand, optionally covalently linked to at least one water-soluble polymer; a buffering agent selected from glutamate, phosphate, histidine, imidazole, and acetate; an excipient selected from sorbitol, sucrose, mannitol, glycerol, polyethylene glycol, and non-polar amino acids; optionally, a detergent such as Tween; optionally, an antioxidant or chelating agent selected from glutathione, methionine, citrate and EDTA; and having a pH preferably ranging from 5.0 to 6.0 (inclusive). Such compositions may be liquid (preferably, aqueous) , frozen (preferably, aqueous) , or lyophilized.

Other aspects of the present invention are set forth in the detailed disclosure provided hereinbelow.

Brief Description of the Drawings

FIG. 1 shows the sequences of the native human cDNA for mpl ligand and the corresponding protein (SEQ ID NOS : 1 and 2) . The sequences include a leader sequence (amino acids -21 through -1, inclusive) that is cleaved in vivo from the cDNA encoded protein to yield the mature protein.

Detailed Description of the Invention

The subject invention provides compositions including mpl ligands along with other agents that result in stable, biologically active compositions suitable for administration to subjects such as human beings.

In a first embodiment, the subject invention relates to compositions of an mpl ligand. By "mpl ligand" in its broadest sense is meant any proteinaceous molecule that has the ability to specifically bind to and activate the mpl receptor to result in the stimulation in vivo of megakaryocyte and/or platelet production. In a preferred embodiment, the mpl ligand has an amino acid sequence identical to one obtainable from a human, such as amino acids 1-332 of the native human sequence (SEQ ID NO: 2) . In another preferred embodiment, the mpl ligand has an amino acid sequence identical to at least amino acids 7-151 of SEQ ID NO: 2, preferably 1-171 ± 20 amino acids (i.e., amino acids 1-151 through 1-191) particularly preferably 1-161 ± 10 amino acids corresponding to SEQ ID NO: 2. Some specific preferred species of mpl ligands are the following: amino acids 1-151, 1-152, 1-153, 1-154, 1-163, 1-174, 1-191, 1-232, 1-244 of SEQ ID NO: 2. The most preferred species has amino acids 1-163 of SEQ ID NO: 2.

The mpl ligands may also be derivatized with one or more water soluble polymers, such as one or more polyethylene glycol (PEG) groups. The polymer selected should be water soluble so that the mpl ligand to which it is attached does not precipitate in an aqueous environment, such as a physiological environment . Examples of water soluble polymers are set forth in published PCT Application WO 95/26746, which is hereby incorporated by reference.

The water soluble polymers may be attached using chemical reactions such as those described in published PCT Application WO 95/26746. Preferred attachment chemistries are acylation and alkylation. The mpl ligand derivatives of this invention may be attached to multiple polymeric molecules, for example, they may contain 2-6, preferably 2-5, polymer groups attached. The polymer groups are usually attached to the protein at the alpha or epsilon amino groups of amino acids, but it is also contemplated that the polymer groups could be attached to any amino group attached to the protein which is sufficiently reactive to become attached to a polymer group under suitable reaction conditions.

In a preferred embodiment, a single polymer molecule is attached to the mpl ligand. In such cases, the polymer selected to react with the mpl ligand should be modified to have a single reactive group, such as an active -b-

ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled.

The polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, monomethoxy- polyethylene glycol, dextran, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers , a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol. For derivatization of the mpl ligand via an acylation reaction, the polymer (s) selected should have a single reactive ester group. For derivatization of the mpl ligand via a reductive alkylation reaction, the polymer (s) selected should have a single reactive aldehyde group. Generally, the water soluble polymer will not be selected from naturally-occurring glycosyl residues since these are usually made more conveniently by mammalian recombinant expression systems. The polymer may be of any molecular weight as long as it does not substantially interfere with or abolish biological activity of the resulting mpl ligand derivative .

A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol or PEG is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono- (C1-C10) alkoxy- or aryloxy- polyethylene glycol (see, U.S. Patent 5,252,714). Pegylation of an mpl ligand may be carried out by any of the pegylation reactions known in the art. See, for example: Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 154 316; EP 0 401 384; and the other publications cited herein that relate to pegylation. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule.

Thus, in a preferred aspect, the present invention relates to pegylated mpl ligand, wherein the PEG group (s) is (are) attached via acyl or alkyl groups. As discussed above, such products may be mono-pegylated or poly-pegylated (e.g., containing 2-6, preferably 2-5, PEG groups) . The PEG groups are generally attached to the protein at the alpha or epsilon amino groups of amino acids, but it is also contemplated that the PEG groups could be attached to any amino group attached to the protein, which is sufficiently reactive to become attached to a PEG group under suitable reaction conditions.

Preferably, the PEG group is attached via a reductive alkylation procedure and has a molecular weight of from 5 to 50 kd. In the most preferred embodiment, the PEG- mpl ligand has a PEG group that has an average molecular weight of approximately 20 kd (e.g., 20 kd ± 2 kd) .

A particularly preferred mpl ligand derivative is one corresponding to amino acids 1-163 of SEQ ID NO: 2 attached to a single PEG group on the alpha amino group of the first amino acid, wherein the PEG is attached via a reductive alkylation reaction with a PEG aldehyde reactant . This type of mpl ligand is referred to herein by the abbreviation "PEG-rHuMGDF" . In a preferred embodiment, the mpl ligand is the product of the expression of an exogenous DNA sequence that has been transfected into a host cell; that is, in a preferred embodiment the mpl ligand is a "recombinant mpl ligand" . Recombinant mpl ligand may be made in any cells known for this purpose, for example, CHO cells. The preferred host is bacterial, particularly preferably E. coli cells. Recombinant mpl ligand is advantageously produced according to the procedures described in the publications cited herein regarding cloning and expression of mpl ligand. Although previous workers have reported on compositions involving native human mpl ligand (amino acids 1-332 of SEQ ID NO: 2), no one has previously reported extensive stability data as a function of composition ingredients, as set forth herein. Thus, although predictions of stability of mpl ligand compositions have been made before, until the present invention, compositions having desirable stability have not been clearly established, especially for truncated, derivatized mpl ligands. Based on the data presented hereinbelow, the present inventors have discovered certain stable compositions which contain a full-length or truncated mpl ligand having a sequence of amino acids corresponding to amino acids 7-151 through 1-332, inclusive, of native human mpl ligand, optionally covalently linked to at least one water-soluble polymer; a buffering agent selected from glutamate, phosphate, histidine, imidazole, and acetate; an excipient selected from sorbitol, sucrose, mannitol, glycerol, polyethylene glycol, and non-polar amino acids; optionally, a detergent such as Tween; optionally, an antioxidant or chelating agent selected from glutathione, methionine, citrate and EDTA; and having a pH preferably ranging from 5.0 to 6.0 (inclusive) . Such compositions may be liquid (preferably, aqueous) , frozen (preferably, aqueous) , or lyophilized.

The concentration of the protein (mpl ligand) in the final compositions should generally range from about 0.1 mg/ml to 5 mg/ml, preferably 0.2 mg/ml to 3 mg/ml, particularly preferably 0.3 to 1 mg/ml. Preferably, the buffer will be acetate at a concentration of from 5 to 20 mM, particularly preferably, about 10 ± 2 mM. A summary of specific buffers and concentrations is provided in the following Table 1: TABLE 1

Buffer Preferred Cone . Working Cone Exemplary Range (mM) Range (mM) Cone. (mM)

Acetate 5-20 8-12 10

Phosphate 5-20 8-12 10

Histidine 5-20 8-12 10

The pH of the compositions will vary depending on the particular buffer and other factors. The preferred pH range for enhanced stability with appropriate acidic buffers (e.g., acetate) is 4.0-6.0. A more preferred range is 4.5- 5.5, with about 5.0 being a most preferred embodiment.

The compositions should also contain an excipient. Some exemplary excipients and representative concentrations are listed in Table 2 :

TABLE 2

Excipient Preferred Cone , Working Cone , Exemplary Range (W/V) Range (W/V) Cone. (W/V)

sorbitol 10% 6%

sucrose 10% 10% 9%

mannitol 3 - 10% 6% 5%

The excipients will generally be added in an amount so as to result in an isotonic solution.

The compositions may further contain an amino acid, which in some cases will enhance stability. Amino acids may be polar or non-polar, with non-polar amino acids being preferred. Exemplary polar amino acids are arginine and lysine, and exemplary non-polar amino acids are glycine, proline, and alanine.

An antioxidant or chelating agent may also be included in the compositions of this invention. Preferred antioxidants are: EDTA, ascorbic acid, glutathione, methionine and citrate. Combinations of these agents are also contemplated, for example, citrate plus EDTA. Such agents are included in an amount suitable to reduce or eliminate oxidation of the mpl ligand. Exemplary concentrations are: 0.1 - 10 mM, preferably, 0.5 - 5 mM, typically 1 - 3 mM.

A detergent or lipid may also be included in the compositions of this invention. Some representative detergents are: Tween brand of polysorbate (e.g., Tween 20 and Tween 80); Brij 35; Pluronics (e.g., F-127 and F-68); sodium dodecyl sulfate; Triton (e.g., X-100); dimyristoyl phosphatidyl glycerol (DMPG) ; PEG castor oil (e.g., PEG-40); oleth-3 -phosphate; diethanolamme oleth-10-phosphate; and a mixture (e.g., 1:1) of short, long chain unilamellar vesicles

(SLUV) containing, e.g. , C8 (caprylic) and C14 (myristic) lipids. These detergents/lipids are generally included in an amount sufficient to prevent loss of mpl ligand due to sticking to surfaces or aggregation. Some exemplary detergent concentrations are 0.004 mg/ml - 50 mg/ml; preferably, 0.004 mg/ml - 10 mg/ml; most preferably, 0.006 - 0.060 mg/ml. The need to include these detergents/lipids will be greater when the concentration of mpl ligand is lower, such as especially <_. 0.2 mg/ml of mpl ligand. Such compositions may be liquid (preferably, aqueous) , frozen (preferably, aqueous) , or lyophilized.

With regards to lyophilized compositions, there is the possibility of increased protein aggregation as compared to liquid compositions. A particularly preferred lyophilized composition contains a combination of glutamate, sucrose and mannitol at a pH within the range of 4.0-6.0. A list of particularly preferred compositions is provided in the following Table 3 :

TABLE 3

Material Range Example

Glutamate 5-20 mM 10 mM

Sucrose 2-10% (w/v) 6% (w/v) Mannitol 1-5% (w/v) 2% (w/v) pH 4.0-6.0 Ξ O

The compositions of this invention are "stable", by which is meant that they retain at least about 87 %, preferably about 90 %, most preferably about 93 %, of intact mpl ligand derivative after storage for 12 weeks at a temperature of 37°C as analyzed by SEC chromatography only (see Table 4) . This degree of stability is important in a practical sense because less stability would result in unacceptable safety concerns for patients.

A "therapeutically effective amount" as used herein refers to that amount which provides a suitable biological effect in a subject, usually a therapeutic effect for a given condition and administration regimen in a patient. The present compositions can be systemically administered parenterally, intravenously or subcutaneously. When systemically administered, the therapeutic compositions for use in this invention may be in the form of a pyrogen- free, physiologically acceptable aqueous solution. The specific route chosen will depend upon the condition being treated. The required dosage will be in amounts sufficient to raise the platelet and/or megakaryocyte levels of patients and will vary depending upon the severity of the condition being treated, the method of administration used and the like. The conditions to be treated by the methods and compositions of the present invention are generally those which involve an existing megakaryocyte/platelet deficiency or an expected megakaryocyte/platelet deficiency in the future (e.g., because of planned surgery). Such conditions will usually be the result of a deficiency (temporary or permanent) of active mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally useful for treating thrombocytopenia.

Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocytopenia, metastatie tumors which result in thrombocytopenia, systemic lupus erythematosus , splenomegaly, Fanconi ' s syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria . Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT) . Certain wound healing disorders might also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to future surgery, an mpl ligand analog of the present invention could be administered several days to several hours prior to the need for platelets . With regard to acute situations, e.g., accidental and massive blood loss, an mpl ligand analog could be administered along with blood or purified platelets.

Mpl ligand compositions may also be administered to normal human subjects who plan to donate platelets or other related cells in the future. Administration of a composition of this invention would increase the amount of platelets and/or related cells that the patient could donate at one time.

The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of

0.01-1000 micrograms of mpl ligand analog per kilogram of body weight .

The compositions of the present invention may also be employed, alone or in combination with other cytokines, soluble Mpl (i.e., mpl ligand) receptor, hematopoietic factors, interleukins , growth factors or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. It is anticipated that such compositions will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor (SCF) , leukemia inhibitory factor (LIF) , oncostatin M (OSM) , or other molecules with megakaryocyte stimulating activity may also be employed with mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2 , IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1) , GM-CSF, granulocyte colony stimulating factor (G-CSF) , EPO, interferon-alpha (IFN-alpha) , IFN-beta, or IFN-gamma. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of PEG-mpl ligand (to enhance the number of mature megakaryocytes) followed by administration of the soluble Mpl receptor (to inactivate the analog and allow the mature megakaryocytes to produce platelets) is expected to be a particularly effective means of stimulating platelet production. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by conventional methods.

The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1

The following Tables 4 and 5 are a summary of data provided in some of the following examples. In each of these examples, the mpl ligand tested was PEG-rHuMGDF, which contains amino acids 1-163 of SEQ ID NO: 2, mono-pegylated at the alpha amino group of the N-terminal amino acid with a polyethylene glycol group having an average molecular weight of about 20 kDa.

TABLE 4

Formulation Percent Main Peak as Measured by

SEC¹ after 12 weeks at 37°C

Histidine, pH 8.0, 5% Sorbitol 70

Tris, pH 8.0, 5% Sorbitol 47

Phosphate, pH 7.0, 5% Sorbitol 71

Histidine, pH 7.0 , 5% Sorbitol 84

Phosphate, pH 6.0 , 5% Sorbitol 89

Glutamate/Histidine, pH 6.0, 5% Sorbitol 92

Histidine, pH 6.0 , 5% Sorbitol 92

Imidazole, pH 6.0, 5% Sorbitol 92

Glutamate, pH 5.5, 5% Sorbitol 91

Glutamate/Histidine, pH 5.5, 5% Sorbitol 93

Acetate, pH 5.0, 5% Sorbitol 92

Glutamate/Histidine, pH 5.0 , 5% Sorbitol 93

Glutamate, pH 5.0, 5% Sorbitol 91

Histidine, pH 5.0 , 5% Sorbitol 89

Succinate, pH 5.0 , 5% Sorbitol 76

Glutamate, pH 4.0, 5% Sorbitol 87

Succinate, pH 4.0 , 5% Sorbitol 81

Acetate, pH 4.0 , 5% Sorbitol 77

Tartrate, pH 4.0, 5% Sorbitol 18

Succinate, pH 3.5, 5% Sorbitol 74

^Additional stability indicating assays, specifically, reversed phase and cation exchange chromatography, gave similar results to SEC.

The percent decrease in main peak indicates a preferred pH range of 4.0 - 6.0, preferably 5.0 - 6.0. In addition, buffer effects within the pH range 4.0 - 6.0 indicate that certain buffers within this range are not preferred.

TABLE 5

Formulation Percent Main Peak as Measured by SEC¹ after 12 weeks at

37°C

Acetate, pH 5.0, 5% isotonic 92 polyols (Sorbitol)

Acetate, pH 5.0, isotonic 10 saline, both mono- and divalent (NaCl)

Acetate, pH 5.0, isotonic 10 polar amino acids (Lysine)

Acetate, pH 5.0, isotonic 84 nonpolar amino acids (Glycine)

All polyols tested, which include: sorbitol, sucrose, glycerol, mannitol and polyethylene glycol, showed similar results.

All salts (both monovalent and divalent) , which include: NaCl, CaCl2, CuCl2 , MgCl2, MnCl2 , NiCl2 , ZnCl2 and FeCl2 , showed similar results.

All polar amino acids tested, which include: arginine, and lysine, showed similar results.

All nonpolar amino acids tested, which include: glycine, proline, and alanine, showed similar results.

All antioxidants tested which include: EDTA, ascorbic acid, glutathione, methionine, methionine + EDTA and citrate, did not show substantial increases in the stability of PEG-rHuMGDF beyond A50S (see Example 2 for definition) . EXAMPLE 2

pH Evaluation

Starting Material : PEG-rHuMGDF

B . Formulations : lOmM Acetate pH 5.0, 5% Sorbitol (A50S) lO M Acetate pH 4.0, 5% Sorbitol (A40S) lOmM Succinate pH 3.5, 5% Sorbitol (S35S) lOmM Succinate pH 4.0, 5% Sorbitol (S40S) lOmM Succinate pH 5.0 5% Sorbitol (S50S) lOmM Histidine pH 6.0, 5% Sorbitol (H60S) lOmM Imidazole pH 6.0, 5% Sorbitol (I60S) lOmM Tartrate pH 4.0, 5% Sorbitol (T40S) lOmM Glutamate pH 4.0, 5% Sorbitol (E40S) lOmM Phosphate pH 6.0, 5% Sorbitol (P60S) lOmM Phosphate pH 7.0, 5% Sorbitol (P70S) lOmM Tris pH 8.0 5% Sorbitol (T80S) lOmM Histidine pH 5.0, 5% Sorbitol (H50S) lOmM Histidine pH 6.0 , 5% Sorbitol (H60S) lOmM Histidine pH 7.0, 5% Sorbitol (H70S) lOmM Histidine pH 8.0, 5% Sorbitol (H80S)

C. Vials: ImL in 3cc vials filled at a protein concentration of 0.5 mg/mL

D. Temp & Time points: 37°C; time points indicated on Tables

E. Analyses: HPLC: Size exclusion chromatography (SEC) reverse phase chromatography (RP) , ion exchange chromatography (IEX) Data

Tables 6-11 show the percent main peak by size exclusion, reversed phase and cation exchange chromatography after incubation at 37°C for the times indicated.

pH Evaluation - Percent Main Peak by Size Exclusion

Chromatography After Incubation at 37°C for the Time

Indicated

TABLE 6

Incubation Time

Form. * τ=o T=2 T=4 T=8 T=12 T=17 Weeks weeks weeks Weeks Weeks

A40S 94.39 90.86 88.20 82.91 77.21 70.19

S35S 94.90 93.65 90.29 80.95 74.28 62.43

S40S 94.63 92.59 90.20 86.16 80.51 72.34

S50S 94.27 87.53 85.29 80.77 76.45 71.12

H60S 94.99 94.31 93.77 91.74 89.13 85.96

I60S 94.93 94.56 94.20 92.77 91.49 94.83

T40S 89.71 43.46 34.44 24.48 18.10

E40S 94.90 94.29 93.65 91.30 86.84 77.94

*Form. Formulation

TABLE 7

Incubation Time

Form. * τ=o T=3 T=7 T=10 T=2 T=3 T=6 T=12 Days Days Days Weeks Weeks Weeks Weeks

T80S 97.70 93.56 91.28 88.12 86.63 80.25 66.18 46.58

H50S 97.53 94.20 94.22 94.37 94.54 92.94 91.76 89.18

H60S 97.62 97.02 97.24 97.09 94.50 96.19 95.12 91.68

H70S 97.62 96.64 96.75 95.86 95.37 94.33 90.95 84.34

H80S 97.70 94.68 93.27 92.03 89.36 81.52 69.65

P60S 97.65 95.55 96.84 96.35 96.38 95.77 93.76 89.16

P70S 97.81 96.45 95.84 94.83 94.12 91.77 84.81 71.14

A50S 97.72 96.28 95.43 95.45 95.63 94.71 93.97 91.65

pH Evaluation - Percent Main Peak by Reversed Phase Chromatography After Incubation at 37C for the Time Indicated

TABLE 8

Incubat. Lon Time

Form. * T=0 T=2 T=4 T=8 T=12 T=17 Weeks weeks weeks Weeks Weeks

A40S 93.49 91.69 84.34 78.87 70.55 64.23

S35S 93.78 90.10 80.86 70.36 59.35 50.98

S40S 93.91 91.61 83.89 77.16 59.22

S50S 93.49 92.59 88.93 85.96 79.35 73.71

H60S 93.65 90.54 86.69 86.49 80.88 76.11

I60S 93.68 92.61 87.64 87.34 84.11 79.49

T40S 93.65 88.04 74.51 66.08 53.87

E40S 93.67 92.39 86.03 80.64 71.50 65.23

TABLE 9

Incubation Time

Form. * Time T=3 T__7 T=10 T=2 T=6 T=12 Zero Days Days Days Weeks Weeks Weeks

T80S 95.96 93.75 92.68 88.57 87.98 68.58 48.84

H50S 96.14 95.38 95.52 94.70 97.08 92.18 88.45

H60S 95.88 94.32 94.77 94.65 94.08 90.20 84.80

H70S 95.78 92.66 93.84 90.72 90.95 70.83

H80S 95.88 94.11 93.23 90.94 89.66 77.82 67.64

P60S 96.49 94.62 94.82 94.18 93.44 86.25 81.09

P70S 96.04 94.98 94.14 92.41 94.37 82.67 71.11

A50S 96.02 95.13 95.57 94.68 96.35 92.78 89.30

pH Evaluation - Percent Main Peak by Cation Exchange

Chromatography After Incubation at 37C for the Time Indicated

TABLE 10

Incubat. Lon Time

Form. * T=0 T=2 T=4 T=8 T=12 T=17 Weeks weeks weeks Weeks Weeks

A40S 81.17 73.84 69.66 53.96 43.09 41.14

S35S 82.64 72.97 67.10 52.36 45.84 37.70

S40S 82.16 74.54 70.95 53.81 44.63 34.96

S50S 82.86 75.30 67.91 58.14 49.48 41.49

H60S 82.32 79.60 75.57 70.77 65.47

I60S 83.72 80.20 77.95 72.15 64.05 66.01

T40S 79.23 30.25 27.58 19.37 11.92

E40S 82.37 75.53 69.71 59.80 50.91 47.72

TABLE 11

Incubation Time

Form. * Time T=3 T=7 T=10 T=2 T=3 T=6 T=12 Zero Days Days Days Weeks Weeks Weeks Weeks

T80S 87.44 80.38 78.04 76.41 72.76 71.28 54.49

H50S 84.42 83.70 83.86 83.23 82.16 78.52 76.07 70.85

H60S 87.86 86.81 85.40 84.56 82.00 78.90 80.64 73.71

H70S 82.99 83.33 81.35 81.01 79.55 72.70 67.95 56.91

H80S 84.08 84.72 81.49 82.17 77.28 64.21 68.21 54.79

P60S 85.68 84.58 83.73 83.22 79.15 75.87 73.38 60.93

P70S 87.60 78.70 82.50 84.04 78.99 77.85 78.90 69.18

A50S 83.00 84.54 81.65 83.81 81.54 77.68 79.50 72.22

EXAMPLE 3

Mpl ligand Concentration Evaluation

Starting Material : PEG-rHuMGDF

B. Formulations : lOmM Acetate pH 5.0 , 5% Sorbitol, 2.0 mg/mL (20A5S) lOmM Acetate pH 5.0, 5% Sorbitol, 1.0 mg/mL (10A5S) lOmM Acetate pH 5.0, 5% Sorbitol, 0.5 mg/mL (05A5S) lOmM Acetate pH 5.0 , 5% Sorbitol, 0.2 mg/mL (02A5S)

Vials: ImL in 3cc vials filled at the protein concentrations indicated

D. Temp & Time points: 37°C; time points indicated on Tables

E . Analyses : HPLC: SEC, RP, IEX F . Data

Tables 12-14 show the percent main peak by size exclusion, reversed phase and cation exchange chromatography after incubation at 37°C for the times indicated.

Mpl Ligand Concentration Evaluation - Percent Main Peak After Incubation at 37°C for the Time Indicated

TABLE 12 Size Exclusion Chromatography

Form. * T=0 T=l T=2 T=3 T=4 T=8 T=12

Week Weeks Weeks Weeks Weeks Weeks

02A5S 94.44 94.86 93.84 94.53 94.51 93.90 92.74

05A5S 94.11 93.78 92.86 93.25 93.40 91.55 90.11

10A5S 93.96 92.60 91.69 91.74 91.32 89.20 86.89

20A5S 93.81 91.50 90.25 90.20 89.55 86.89 83.59

*Form. = Formulation

TABLE 13 Reversed Phase Chromatography

Incubation Time

Form. * T=0 T=l T=2 T=3 T=4 T=8 T=12

Week Weeks Weeks Weeks Weeks Weeks

02A5S 94.53 93.41 93.28 92.16 91.05 88.89 87.90

05A5S 94.73 94.44 93.30 92.41 91.97 89.18 85.97

10A5S 94.86 94.56 93.25 92.39 91.98 88.06 87.62

20A5S 94.67 94.12 93.07 92.47 91.79 88.37 87.07 TABLE 14 Cation Exchange Chromatography

Incubation Time

Form. * τ=o T=l T=2 T=4 T=8 T=12

Week Weeks Weeks Weeks Weeks

02A5S 79.40 65.75 78.56 79.80 74.42 59.74

05A5S 78.22 67.44 80.26 81.10 76.27 61.39

10A5S - 66.18 80.80 82.02 75.81 60.72

20A5S 79.52 76.71 82.09 81.38 74.26 60.89

EXAMPLE 4

Excipient Evaluation

A. Starting Material: PEG-rHuMGDF

B. Formulations:

lOmM Acetate pH 5.0, 5% Sorbitol,

5 mM EDTA (A5SE) lOmM Acetate pH 5.0, 2% Alanine (A5A) lOmM Acetate pH 5.0, 1.6% Glycine (A5G) lOmM Acetate pH 5.0 2.7% Proline (A5P) lOmM Acetate pH 5.0 , 3.5% Lysine (A5K) lOmM Acetate pH 5.0, 4.3% Arginine (A5R) lOmM Glutamate pH 5.0, 9.3% Sucrose (E5Su) lOmM Glutamate pH 5.0 , 5% Sorbitol (E5S)

Vials: ImL in 3cc vials filled at a protein concentration of 0.5 mg/ml

D. Temp & Time points: 37°C; time points indicated on Tables Analyses : HPLC: SEC, RP, IEX

Data

Tables 15-17 show the percent main peak by size exclusion, reversed phase and cation exchange chromatography after incubation at 37°C for the times indicated.

Excipient Evaluation - Percent Main Peak After Incubation at

37C for the Time Indicated.

TABLE 15

Size Exclusion Chromatography

Incubation Time

Formulation τ=o T=2 T=4 T=8 T=12 Weeks Weeks Weeks Weeks

A5R 84.71 21.83 19.01 12.30

E5Su 94.30 91.40 91.95 88.36 86.52

A5G 94.57 91.22 91.43 92.26 84.50

A5K 86.75 17.24 15.02 9.76

A5SE 93.08 60.01 54.53 43.22 37.75

A5P 94.66 91.97 92.16 87.63 85.05

E5S 94.78 93.07 93.55 90.68 92.28

A5A 94.55 91.57 92.18 88.13 85.89

TABLE 16

Cation Exchange Chromatography

Incubation Time

Formulation T=0 T=2 T=4 T=8 T=12 Weeks Weeks Weeks Weeks

A5R 82.26 28.41 21.32 11.71

E5Su 84.93 86.22 88.62 72.16 65.04

A5G 86.17 81.90 66.81 18.95 26.45

A5K 80.88 20.18 14.23 8.75

A5SE 84.56 62.75 56.40 36.31 30.38

A5P 86.73 87.58 86.22 69.34 65.76

E5S 87.49 90.23 88.83 75.55 70.01

A5A 86.28 81.31 86.10 70.29 63.69

TABLE 17

Reversed Phase Chromatography

Incubat.on Time

Formulation τ=o T=2 T=4 T=12 Weeks Weeks Weeks

A5R 95.96 92.35 85.68

E5Su 96.10 94.57 91.59 77.92

A5G 95.71 69.61 39.86

A5K 95.81 88.93 81.30

A5SE 95.98 93.60 90.01

ASP 95.67 92.98 89.96 78.85

E5S 95.64 93.98 90.61 83.90

A5A 95.78 90.38 86.52 70.00

EXAMPLE 5

Isotonicity Evaluation

Starting Material: PEG-rHuMGDF B . Formulations :

lOmM Acetate pH 5.0, 9.3% Sucrose (A5SU) lOmM Acetate pH 5.0, 5% Mannitol (A5MA) lOmM Acetate pH 5.0, 140 mM NaCl (A5N) lOmM Acetate pH 5.0, 2% PEG 8000 (A5P8) lOmM Acetate pH 5.0, 2.5% Glycerol (A5G) lOmM Acetate pH 5.0, 5% Sorbitol,

.01% Tween 20 (A5ST) lOmM Histidine pH 6.0 , 5% Sorbitol (H6S) lOmM Histidine pH 6.0, 5% Sorbitol,

.001% Ascorbic Acid (H6AA)

C. Vials: ImL in 3cc vials filled at a protein concentration of 0.5 mg/ml

D. Temp & Time points: 37°C; time points indicated on Tables

Analyses : HPLC: SEC, RP, IEX

Data

Tables 18-20 show the percent main peak by size exclusion, reversed phase and cation exchange chromatography after incubation at 37°C for the times indicated.

Isotonicity Evaluation - Percent Main Peak After Incubation at

37^c for the Time Indicated. TABLE 18

Size Exclusion Chromatography

Incubation Time

Form. * T=0 T=3.5 T=l T=2 T=3 T=7 T=12 Days Week Weeks Weeks Weeks Weeks

H6S 96.70 96.23 96.30 95.95 95.77 93.37 91.03

H6Aa 96.75 95.66 96.03 95.72 95.29 93.32 90.61

A5N 83.54 30.42 28.04 24.64 20.19 17.62

A5Ma 96.55 95.14 95.22 95.02 95.13 93.08 91.42

A5Su 96.36 95.18 95.37 95.16 95.60 93.49 91.89

A5G 96.46 95.29 95.49 95.20 95.59 93.23 91.68

A5P8 94.88 92.89 92.89 92.61 91.62 92.78 82.05

A5ST 96.25 94.83 94.50 94.58 94.37 91.16 87.68

*Form.= Formulation

TABLE 19

Reversed Phase Chromatography

Incubation Time

Form. * T=0 T=3.5 T=l T=2 T=7 T=12 Days Week Weeks Weeks Weeks

H6S 95.49 94.39 94.69 85.55 87.60 85.62

H6Aa 95.64 92.00 90.67 93.02 74.22 66.22

A5N 94.94 93.99 92.19 89.52 68.67

A5Ma 95.90 95.05 95.29 94.34 90.13 87.22

A5Su 95.55 95.39 94.78 93.47 88.19 82.88

A5G 95.57 94.78 95.51 94.88 90.04 88.64

A5P8 94.75 91.31 87.07 76.93 13.41

A5ST 94.57 93.24 93.90 82.75 87.61 85.45

TABLE 20

Cation Exchange Chromatography

Incubation Time

Form. * T=0 T=3.5 T=l T=2 T=3 T=7 T=12 Days Week Weeks Weeks Weeks Weeks

H6S 87.13 85.79 84.67 82.66 82.77 79.32 59.35

H6Aa 85.12 80.75 79.31 75.99 75.23 64.62 45.93

A5N 79.98 39.01 29.14 25.03 20.77 14.67

A5Ma 84.73 84.37 84.43 81.92 83.07 78.17 58.52

A5Su 82.98 84.83 83.46 80.76 81.09 76.61 56.85

A5G 82.79 84.69 85.09 81.73 81.61 76.98 59.03

A5P8 85.65 82.26 81.74 77.41 74.77 53.74 4.80

A5ST 85.02 83.83 83.95 80.02 79.79 74.56 55.85

EXAMPLE 6

Anti-oxidant/Chelating Agent Evaluation

A. Starting Material: PEG-rHuMGDF

B. Formulations:

10 mM Acetate pH 5.0, 5% Sorbitol

3 mM glutathione (A5S GT)

10 mM Acetate pH 5.0, 5% Sorbitol

5 mM methionine (A5S M)

10 mM Acetate pH 5.0, 5% Sorbitol 5 mM methionine, 1 mM EDTA (A5S ME)

10 mM Acetate pH 5.0, 5% Sorbitol

1 mM citrate (A5S C)

10 mM Acetate pH 5.0, 5% Sorbitol 0.5 mM citrate (A5S 05C) 10 mM Acetate pH 5.0, 5% Sorbitol

1 mM EDTA (A5S IE)

10 mM Acetate pH 5.0, 5% Sorbitol

0.5 mM EDTA (A5S E)

C. Vials: ImL in 3cc vials filled at a protein concentration of

0.5 mg/ml

D. Temp & Time points: 37°C; time points indicated on Tables

E. Analyses: HPLC: SEC, RP, IEX F . Data

Tables 21-23 show the percent main peak by size exclusion, reversed phase and cation exchange chromatography after incubation at 37°C for the times indicated.

Anti-Oxidant/Chelating Agent Evaluation - Percent Main Peak After Incubation at 37°C for the Time Indicated.

TABLE 21 Size Exclusion Chromatography

Incubation Time

Formulation τ=o T=2 T=4 T=9 Weeks Weeks Weeks

A5SGT 16.69 12.32 8.03 5.91

A5SM 94.53 91.03 89.72 89.00

A5SME 94.88 92.15 90.73 85.74

A5SC 94.30 90.83 88.14 79.61

A5S05C 93.96 90.95 88.98

A5S1E 94.88 90.75 89.15 80.63

A5SE 94.80 91.72 89.89 82.06

TABLE 22 Reversed Phase Chromatography

Incubation Time

Formulation T=0 T=2 T=4 T=9 T=12 Weeks Weeks Weeks Weeks

A5SGT 5.69 2.37 2.80 2.60 2.50

A5SM 94.70 91.59 88.67 88.55 86.83

A5SME 94.54 87.66 91.36 81.97 80.39

A5SC 93.98 81.13 90.33 68.01 62.91

A5S05C 94.43 87.01 84.30 79.83 76.35

A5S1E 94.36 86.84 76.43 89.93 84.56

A5SE 94.75 90.27 85.64 81.20 78.96 TABLE 23 Cation Exchange Chromatography

Incubation Time

Formulation τ=o T=2 T=4 T=9 Weeks Weeks Weeks

A5SGT 4.66 5.15 0.0

A5SM 84.69 81.18 80.96 72.95

A5SME 84.93 78.36 78.26 69.48

A5SC 85.23 72.11 66.46 54.68

A5S05C 86.06 78.00 72.53 66.06

A5S1E 85.38 77.00 76.42 66.21

A5SE 86.51 80.07 77.93 69.79

EXAMPLE 7

Detergent Evaluation

A. Starting Material: PEG-rHuMGDF

B. Formulations:

All forms contain lOmM acetate at pH 5.0, with 5% sorbitol and 0.050 mg/ml PEG-rHuMGDF .

004T20 0 004 mg/ml Tween- -20

006T20 0 006 mg/ml Tween- -20

010T20 0 010 mg/ml Tween- -20

040T20 0 040 mg/ml Tween- -20

060T20 0 060 mg/ml Tween- -20

004T80 0 004 mg/ml Tween- -80

006T80 0 006 mg/ml Tween- -80

010T80 0 010 mg/ml Tween- -80

040T80 . 0 .040 mg/ml Tween- -80

060T80 : 0 .060 mg/ml Tween- -80 C . Data :

Table 24 shows the results of reverse phase HPLC purity based on the percent main peak:

TABLE 24

INCUBATION TIME AT 37 DEGREES CELSIUS

FORMULATION T=0 T=2 T=4 T=6 T=12

WEEKS WEEKS WEEKS WEEKS WEEKS

A5S 94.66 94.05 90.67 90.90 89.07

004T20 94.68 94.79 91.25 91.02 88.49

006T20 94.69 93.99 91.48 90.46 88.43

010T20 94.89 94.30 91.33 90.76 88.47

040T20 94.59 92.95 90.63 89.88 87.58

060T20 94.61 92.62 90.57 89.49 86.72

004T80 94.44 94.35 90.91 90.80 88.48

006T80 94.43 94.05 91.01 90.18 88.08

010T80 94.89 94.22 90.69 90.22 88.04

040T80 94.45 92.64 89.74 89.26 86.56

060T80 94.19 88.38 88.67 87.64 84.53

D. Results:

Detergents such as Tween may be included in PEG- rHuMGDF formulations to enhance physical stability and recovery without detrimental effects to chemical stability. Tween-20 and Tween-80 may be added to PEG-rHuMGDF formulations up to final concentrations of about 0.060 mg/ml without causing excessive methionine oxidation. Tween-20 and Tween-80 are most effective in the concentration range of 0.006 mg/ml to 0.060 mg/ml. EXAMPLE 8

Lyophilized Compositions

The following Table 25 is a summary of data obtained for lyophilized compositions including an mpl ligand. In each of these examples, the mpl ligand tested was PEG-rHuMGDF, which contains amino acids 1-163 of SEQ ID NO: 2, mono-PEGylated at the alpha amino group of the N-terminal amino acid with a polyethylene glycol group having an average molecular weight of about 20 kDa. For Tables 25-27, the lyophilized PEG-rHuMGDF was reconstituted with about 1 ml of water for injection prior to analysis and the percent main peak represents the recovery of PEG-rHuMGDF as a consequence of lyophilization.

TABLE 25

Formulation % main peak by SEC lOmM histidine, 88 5% mannitol lOmM histidine, 93

4% mannitol, 1% sucrose

Note: Mpl ligand concentration was 0.5 mg/ml,

For the lyophilized samples no additional physical stability was achieved in pH's 6, 7 and 8 as measured by size exclusion. Range of sucrose concentrations studied:

TABLE 26

Range of buffers (lOmM concentration) studied:

TABLE 27

All stabilizing agents such as amino acids (e.g. isotonic arginine, lysine, proline and histidine) and amorphous agents (e.g., trehalose and PEG) did not show improved stability during lyophilization.

Formulation with best main peak recovery and lowest levels of aggregation:

lOmM glutamate, 6% sucrose, 2% mannitol, pH 5.0.

While the invention has been described in what is considered to be its preferred embodiments, it is not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims, which scope is to be accorded the broadest interpretation so as to encompass all such modifications and equivalents.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: AMGEN INC.

(ii) TITLE OF INVENTION: PHARMACEUTICAL COMPOSITIONS CONTAINING AN MPL LIGAND

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: AMGEN INC.

(B) STREET: 1849 DeHavilland Drive

(C) CITY: Thousand Oaks

(D) STATE: California

(E) COUNTRY: US

(F) ZIP: 91320-1789

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (Not Yet Assigned)

(B) FILING DATE: 04-OCT-1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: COOK Ph.D., Robert R.

(B) REGISTRATION NUMBER: 31,602

(C) REFERENCE/DOCKET NUMBER: A-412

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1342 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 36..1097 ( ix ) FEATURE :

(A) NAME/KEY: mat_peptide

(B) LOCATION: 99..1097

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 36.-98

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAGGGAGCCA CGCCAGCCAA GACACCCCGG CCAGA ATG GAG CTG ACT GAA TTG 53

Met Glu Leu Thr Glu Leu -21 -20

CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC 101 Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser -15 -10 -5 1

CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT 149 Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg 5 10 15

GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC 197 Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His 20 25 30

CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA 245 Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly 35 40 45

GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA 293 Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp lie Leu Gly 50 55 60 65

GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG 341 Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu 70 75 80

GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC 389 Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val 85 90 95

CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT 437 Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro 100 105 110

CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG 485 Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala lie Phe Leu 115 120 125 AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA 533 Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val 130 135 140 145

GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC 581 Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val 150 155 160

CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG 629 Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg 165 170 175

ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT 677 Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr 180 185 190

GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG ATT CCT 725 Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly Phe Arg Ala Lys lie Pro 195 200 205

GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC 773 Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu Asp Gin lie Pro Gly Tyr 210 215 220 225

CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA ACT CGT GGA CTC TTT CCT 821 Leu Asn Arg lie His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro 230 235 240

GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA 869 Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp lie Ser Ser Gly Thr 245 250 255

TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT 917 Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gin Pro Gly Tyr Ser Pro 260 265 270

TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT ACG CTC TTC CCT CTT CCA 965 Ser Pro Thr His Pro Pro Thr Gly Gin Tyr Thr Leu Phe Pro Leu Pro 275 280 285

CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC 1013 Pro Thr Leu Pro Thr Pro Val Val Gin Leu His Pro Leu Leu Pro Asp 290 295 300 305

CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC ACA TCC 1061 Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser 310 315 320

TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GGG TAA GGTTCTCAGA 1107

Tyr Thr His Ser Gin Asn Leu Ser Gin Glu Gly * 325 330

CACTGCCGAC ATCAGCATTG TCTCGTGTAC AGCTCCCTTC CCTGCAGGGC GCCCCTGGGA 1167 GACAACTGGA CAAGATTTCC TACTTTCTCC TGAAACCCAA AGCCCTGGTA AAAGGGATAC 1227 ACAGGACTGA AAAGGGAATC ATTTTTCACT GTACATTATA AACCTTCAGA AGCTATTTTT 1287 TTAAGCTATC AGC ATACTC ATCAGAGCAG CTAGCTCTTT GGTCTATTTT CTGCA 1342

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 354 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala -21 -20 -15 -10

Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val -5 1 5 10

Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 15 20 25

Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 30 35 40

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55

Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 60 65 70 75

Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90

Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 100 105

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120

Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135

Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala 140 145 150 155 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160 165 170

Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 175 180 185

Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly 190 195 200

Phe Arg Ala Lys lie Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 205 210 215

Asp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn Gly 220 225 230 235

Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250

Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 260 265

Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 270 275 280

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 295

His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315

Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 320 325 330

Gly

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an mpl ligand, a buffer selected from the group consisting of glutamate, phosphate, histidine, imidazole, and acetate; an excipient selected form the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol, and a non-polar amino acid; and having a pH ranging from 5.0 to 6.0, inclusive.

2. A composition according to Claim 1, wherein the mpl ligand comprises at least amino acids 7-151 of SEQ ID . NO : 2.

3. A composition according to Claim 1, wherein the mpl ligand consists of amino acids 1-171 ± 20 amino acids Of SEQ ID NO: 2.

4. A composition according to Claim 1, wherein the mpl ligand consists of amino acids 1-161 ± 10 amino acids of SEQ ID NO: 2.

5. A composition according to Claim 1, wherein the mpl ligand consists of amino acids 1-151 amino acids of SEQ ID NO: 2.

6. A composition according to Claim 1, wherein the mpl ligand consists of amino acids 1-163 of SEQ ID NO: 2.

7. A composition according to Claim 1, wherein the mpl ligand is attached to a water soluble polymer selected from the group consisting of polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers , a polypropylene oxide/ethylene oxide co- polymer, polyoxyethylated polyols.

8. A composition according to Claim 7, wherein the water soluble polymer is polyethylene glycol .

9. A composition according to Claim 6, which comprises acetate as the buffer, sorbitol as the excipient, and has a pH of about 5.0, in an aqueous medium.

10. A composition according to Claim 1, wherein the nonpolar amino acid is selected form the group consisting of glycine, proline, and alanine

11. A composition according to Claim 1, further comprising an antioxidant.

12. A composition according to Claim 11, wherein the antioxidant is selected from the group consisting of EDTA, ascorbic acid, glutathione, methionine, and citrate.

13. A composition according to Claim 1, further comprising a detergent or a lipid.

14. A composition according to Claim 13, wherein the detergent is selected from the group consisting of Tween;

Brij 35; Pluronics; sodium dodecyl sulfate; Triton; dimyristoyl phosphatidyl glycerol (DMPG) ; PEG-40 castor oil; oleth-3-phosphate; diethanolamme oleth-10-phosphate; and a mixture of short and long chain unilamellar vesicles (SLUV) containing C8 (caprylic) and C14 (myristic) lipids .

15. A composition according to Claim 1, wherein the composition is in an aqueous medium.

16. A composition according to Claim 1, wherein the composition is in a lyophilized form.

17. A composition according to Claim 1, which comprises phosphate buffer, 5% sorbitol and has a pH of about 6.0, in an aqueous medium.

18. A composition according to Claim 1, which comprises histidine buffer, and about 5% sorbitol in an aqueous medium.

19. A composition according to Claim 1, which comprises imidazole buffer, and about 5% sorbitol in an aqueous medium.

20. A composition according to Claim 1, which comprises glutamate buffer, and about 5% sorbitol, in an aqueous medium.

21. A composition according to Claim 1, which comprises glutamate buffer, 5% sorbitol and has a pH of about 5.0, in an aqueous medium.

22. A composition according to Claim 1, which comprises glutamate buffer, about 6% sucrose, about 2% mannitol, a pH of about 5.0 in a lyophilized form.