HK1024490A1 - Pharmaceutical compositions containing an mpl ligand - Google Patents

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 MPL ligands

Technical Field

The present invention relates to compositions comprising mpl ligands, the components of which are suitable for pharmaceutical use.

Background

The natural human mpl ligand is the most recently cloned cytokine and is the primary regulator of circulating platelet levels. See Bartley, t.d. et al, Cell (Cell) 77: 1117-; lok, s, et al, Nature 369 (Nature): 565-568 (1994); de Sauvage, f.j., et al, nature 369: 533, 538 (1994); miyazake, h, et al, exp. hemtool.22: 838 (1994); and Kuter, d.j. et al, PNAS USA, 91: 11104-11108(1994). Human endogenous mpl ligands, also known as Thrombopoietin (TPO) and megapoetin, are proteins that contain a total of 332 amino acids.

Recombinant mpl ligands prepared from Chinese Hamster Ovary (CHO) and e.coli cells have been shown to specifically stimulate or increase the biological activity of megakaryocytes and/or platelets in mice, rats and monkeys. See, e.g., Hunt, p, et al, Blood 84 (10): 390A (1994). Human mpl ligand truncated up to 181 amino acids from the C-terminus retains in vivo biological activity. The resulting mpl ligand corresponds to a fragment of amino acids 1-151 through 1-331 of the full-length sequence. Biological activity can also be retained by removing the first 6 amino acids at the N-terminus of the human mpl ligand protein. Thus, it was shown that biological activity was retained in amino acids 7-151 (inclusive) of the mature amino acid sequence of human mpl ligand.

In vivo assays demonstrate that derivatives of mpl ligand have beneficial activity in stimulating the production of megakaryocytes and/or platelets. See published PCT application WO 95/26746. In particular, mpl ligands derivatized with water soluble polymers such as polyethylene glycol ("PEG") moieties are clinically significant because they are long lived and active in vivo.

Compositions containing mpl ligands and related derivatives are disclosed in a general manner. See published PCT applications WO95/26746, WO95/21919, WO95/18858, and WO 95/21920. However, no reports have been reported for the control experiments described herein, which determined which compositions containing mpl ligand had pharmaceutically acceptable stability. Such compositions are of great interest for the practical use of mpl ligands in human patients. Thus, there is a need in the art for the use of such compositions to administer mpl ligands to patients to increase platelet levels.

Summary of The Invention

Accordingly, it is an object of the present invention to provide pharmaceutically stable compositions comprising mpl ligands.

It is another object of the present invention to provide compositions containing mpl ligands for administration to a patient.

In embodiments, the present invention relates to compositions comprising: a full-length or truncated mpl ligand having an amino acid sequence corresponding to amino acids 7-151 to 1-332 (inclusive) of natural human mpl ligand, optionally covalently linked to at least one water-soluble polymer; a buffer selected from glutamate, phosphate, histidine, imidazole and acetate; an excipient selected from the group consisting of 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; has a pH preferably in the range of 5.0-6.0 inclusive. Such compositions may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

Other aspects of the invention are set forth in the detailed description provided below.

Brief description of the drawings

FIG. 1 shows the cDNA sequence and corresponding protein sequence of the human endogenous mpl ligand (SEQ ID NOS: 1 and 2). They contain a leader sequence (amino acids-21 to-1) which is cleaved in vivo from the cDNA-encoded protein to yield the mature protein.

Detailed description of the invention

The present invention provides compositions comprising mpl ligands and other agents that are stable, biologically active and suitable for human consumption.

In a first embodiment, the present invention relates to a composition of mpl ligands. "mpl ligand" broadly refers to all protein molecules capable of specifically binding to and activating the mpl receptor, thereby stimulating megakaryocyte and/or platelet production in vivo. In a preferred embodiment, the mpl ligand has the same amino acid sequence as the ligand from human, e.g., 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 that is at least the same as the amino acid sequence of seq id no: SEQ ID NO: 2, preferably amino acids 7 to 151 of SEQ ID NO: 2 (i.e., amino acids 1-151 to 1-191), and particularly preferably 1-161. + -.10 amino acids. Some particularly preferred classes of mpl ligands are as follows: SEQ ID NO: 2, amino acids 1-151, 1-152, 1-153, 1-154, 1-163, 1-174, 1-191, 1-232, 1-244. The most preferred species has SEQ id no: 2, 1-163.

The mpl ligand 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 (e.g., physiological environment). Examples of water soluble polymers are given in published PCT application WO95/26746, which is incorporated herein by reference.

The water-soluble polymers may be attached using chemical reactions, for example those described in published PCT application WO 95/26746. Preferred linkage chemistries are acylation and alkylation. The mpl ligand derivatives of the present invention may link multiple polymer molecules, for example, there may be 2 to 6, preferably 2 to 5. The polymer group is typically attached to the protein at the alpha or epsilon amino group of the amino acid, but it is also contemplated that the polymer group may be attached to any amino group on the protein, provided that the reaction conditions are suitable and the reactivity is sufficient.

In a preferred embodiment, a single polymer molecule is linked to an mpl ligand. In such cases, the polymer reacted with the mpl ligand is modified with a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled.

The polymer may be branched or unbranched. Preferably, the polymer should be pharmaceutically acceptable for therapeutic use in the final product formulation. The water-soluble polymer may be selected from groups such as polyethylene glycol, monomethoxypolyethylene glycol, dextran, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g. glycerol) and polyvinyl alcohol. For the mpl ligand to be derivatized by acylation, the polymer chosen should have a single reactive ester group. For the mpl ligand to be derivatized by reductive alkylation, the polymer selected should have a single reactive aldehyde group. In general, water-soluble polymers of naturally occurring glycosyl residues should not be selected as they are generally easier to prepare via mammalian recombinant expression systems. The polymer may be of any molecular weight so long as it does not substantially interfere with or eliminate the biological activity of the resulting mpl ligand derivative.

A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG. Is any form that includes PEG from which other proteins are derived, such as mono- (C1-C10) alkoxy-or aryloxy-polyethylene glycol (see us 5252714).

Pegylation of mpl ligands (pegy1ation) can be carried out by any reaction known in the art for pegylation. See, for example: focus on Growth Factor 3 (2): 4-10 (1992); EP 0154316; EP 0401384; and other publications cited herein relating to peg methylation. Preferably, pegylation is performed by acylation or alkylation with a reactive polyethylene glycol molecule.

Thus, preferably, the invention relates to PEG-based mpl ligands wherein the PEG groups are linked through an acyl or alkyl group. As discussed above, such products may be mono-PEG based or multi-PEG based (e.g. containing 2-6, preferably 2-5 PEG groups). The polymer group is typically attached to the protein at the alpha or epsilon amino group of the amino acid, but also includes that a PEG group can be attached to any amino group on the protein that is sufficiently reactive under suitable reaction conditions.

Preferably, PEG groups having a molecular weight of 5 to 50kd are attached by reductive alkylation. In a most preferred embodiment, the PEG group in the PEG-mpl ligand has an average molecular weight of about 20kd (i.e., 20kd + -2 kd).

Particularly preferred mpl ligand derivatives refer to SEQ ID NO: 2 to the amino group alpha to the first residue on the fragment of amino acids 1-163 and a PEG group, wherein the PEG is attached by reductive alkylation with a PEG aldehyde reactant. This type of mpl ligand is designated herein by the abbreviation "PEG-rHuMGDF".

In a preferred embodiment, the mpl ligand is the expression product of an exogenous DNA sequence that has been transfected into the host cell; that is, in a preferred embodiment, the mpl ligand is a "recombinant mpl ligand". The recombinant mpl ligand may be prepared in any cell known for this purpose, such as CHO cells. Preferred hosts are bacteria, particularly preferred are E.coli cells. Recombinant mpl ligands are readily prepared according to the methods described in the publications cited herein for the cloning and expression of mpl ligands.

Although the prior art has reported compositions involving the natural human mpl ligand (amino acids 1-332 of SEQ ID NO: 2), none have previously reported the extensive stability data presented herein as a function of composition. Thus, while the stability of mpl ligand compositions was previously predicted, until the present invention, compositions having the desired stability, particularly for truncated derivatized mpl ligands, have not been specifically disclosed.

Based on the data given below, the inventors have found that some stabilizing compositions include: a full-length or truncated mpl ligand having amino acid fragments corresponding to 7-151 through 1-332 (inclusive) of natural human mpl ligand covalently selectively linked to at least one water-soluble polymer; a buffer selected from glutamate, phosphate, histidine, imidazole and acetate; an excipient selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and non-polar amino acids; optionally, the

Detergents such as Tween; optionally, an antioxidant or chelating agent selected from glutathione, methionine, citrate and EDTA; has a pH preferably in the range of 5.0-6.0 inclusive. Such composition forms may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

The concentration of the protein (mpl ligand) in the final composition should generally be in the range of about 0.1mg/ml to 5mg/ml, preferably 0.2mg/ml to 3mg/ml, particularly preferably 0.3-1 mg/ml.

Preferably, the buffer is acetate at a concentration of 5-20mM, particularly preferably about 10. + -. 2 mM. A summary of specific buffers and concentrations is provided in table 1 below:

TABLE 1

Buffer solution	Preferred concentration range (mM)	Operative concentration Range (mM)	Assay concentration (mM)
				Acetic acid salt	5-20	8-12	10
Phosphate salts	5-20	8-12	10
				Histidine	5-20	8-12	10

The pH of the composition varies with the particular buffer and other factors. The preferred pH range for improved stability with an appropriate acidic buffer (e.g., acetate) is 4.0-6.0. The most preferred range is 4.5 to 5.5 with about 5.0 being the most preferred embodiment.

The composition should also contain excipients. Some exemplary excipients and representative concentrations are listed in table 2:

TABLE 2

Excipient	Preferred concentration Range (W/V)	Operating concentration Range (W/V)	Test concentration (W/V)
				Sorbitol	3-10％	4-6％	5％
Sucrose	5-10％	8-10％	9％
				Mannitol	3-10％	4-6％	5％

Excipients are generally added in amounts such that an isotonic solution is produced.

The compositions may further contain certain amino acids that will enhance stability in some cases. The 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.

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

The compositions of the present invention may also contain detergents or lipids. Some representative detergents are: tween brand of Polysorbate (e.g., Tween20 and Tween 80); brij 35; pluronics (e.g., F-127 and F-68); sodium lauryl sulfate; triton (e.g., X-100); dimyristoyl phosphatidylglycerol (DMPG); PEG castor oil (e.g., PEG-40); oleyl (oleth) -3-phosphate; diethanolamine oleyl (oleth) -10-phosphate; mixtures (e.g., 1: 1) of short-chain, long-chain unilamellar vesicles (SLUV) containing, for example, C8 (octan) and C14 (tetradec) lipids. The amount of these detergents/lipids should generally be sufficient to prevent the loss of mpl ligand due to surface adhesion or aggregation. Some exemplary detergent concentrations are 0.004mg/ml to 50 mg/mI; preferably 0.004mg/ml to 10 mg/ml; most preferably 0.006mg/ml to 0.060 mg/ml. The need to contain these detergents/lipids is greater when the concentration of mpl ligand is lower, such as, in particular, 0.2mg/ml or less of mpl ligand.

Such compositions may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

With respect to lyophilized compositions, there is an increased likelihood of protein aggregation compared to liquid compositions. Particularly preferred lyophilized compositions contain a mixture of glutamate, sucrose and mannitol at a pH in the range of 4.0-6.0. A particularly preferred composition description is provided in table 3 below:

TABLE 3

Material	Range of	Examples
			Glutamate salts	5-20mM	10mM
Sucrose	2-10％(w/v)	6％(w/v)
			Mannitol	1-5％(w/v)	2％(w/v)
pH	4.0-6.0	5.0

The compositions of the present invention are "stable," meaning that at least about 87%, preferably about 90%, and most preferably about 93%, of the mpl ligand derivative remains intact after storage for 12 weeks at a temperature of 37 ℃ according to SEC-only chromatographic analysis (see Table 4). This degree of stability is of great practical significance, as less stability may result in unacceptable safety for the patient.

As used herein, a "therapeutically effective amount" is one that produces the appropriate biological activity in the patient, i.e., a therapeutic effect for the patient given the symptoms and prescription.

The compositions of the present invention may be administered systemically parenterally, intravenously or subcutaneously. In this case, the therapeutic composition used in the present invention may be a pyrogen-free, physiologically acceptable aqueous solution. The particular route chosen will depend on the condition to be treated. The required dose is an amount sufficient to raise the platelet and/or megakaryocyte levels in a patient and will vary depending on the severity of the condition to be 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 that involve the presence of megakaryocyte/platelet deficiency or the expected megakaryocyte/platelet deficiency (e.g., due to scheduled surgery). Such symptoms are often the result of a lack (temporary or permanent) of active mpl ligand in the body. The general term for platelet deficiency is thrombocytopenia, and thus the methods and compositions of the present invention are generally used to treat thrombocytopenia.

Thrombocytopenia (platelet deficiency) exists for a variety of reasons, including chemotherapy and other treatments with various drugs, radiation therapy, surgery, occasional blood loss, and other specific conditions. Exemplary specific conditions which relate to thrombocytopenia and which may be treated according to the invention are: aplastic anemia, spontaneous thrombocytopenia, metastases leading to thrombocytopenia, systemic lupus erythematosus, splenomegaly, fanconi syndrome, vitamin B12 deficiency, folate deficiency, May-Hegglin abnormalities, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Some wound healing disorders may also benefit from an increase in platelet count.

With respect to expected platelet deficiencies, for example, due to future surgery, mpl ligand analogs of the present invention may be administered days to hours before platelets are needed. For acute conditions, such as occasional and massive blood loss, mpl ligand analogs can be administered with blood or purified platelets.

The mpl ligand composition may also be administered to normal persons who are scheduled to provide platelets or other related cells. Administration of the compositions of the present invention increases the amount of platelets and/or associated cells that are given out by a patient at one time.

The dosage regimen involved in the method for the treatment of the above-mentioned conditions will depend on the various factors which are considered to modify the action of the drug, such as age, symptoms, body weight, sex and diet of the patient, severity of infection, time of administration and other clinical factors, according to the attending physician. In general, the daily dose for each kilogram of body weight should be in the range of 0.01-1000 micrograms mpl of ligand analogue.

The compositions of the present invention may also be used alone or in combination with other cytokines, soluble Mpl receptors (i.e., Mpl receptors), hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states characterized by other syndromes and platelet deficiencies. Such compositions are expected to prove useful in treating some cases of thrombocytopenia associated with common stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyte stimulating factors, i.e., meg-CSF, stem cell growth factor (SCF), Leukemia Inhibitory Factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity may also be used with the mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 α, IL-1 β, 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- α (IFN- α), IFN- β or IFN- γ. It is further useful to administer simultaneously or sequentially an effective amount of soluble mammalian Mpl receptor, indicating an effect that causes megakaryocytes to lyse into fragments to platelets once they reach a mature form. Thus, administration of PEG-Mpl ligand (to increase the number of mature megakaryocytes) followed by administration of soluble Mpl receptor (to inactivate the analog and allow the mature megakaryocytes to produce platelets) would be expected to be a particularly effective method of stimulating platelet production. Such additional ingredients in the therapeutic composition may be compensated for by adjusting the above dosages. The course of treatment of a patient may be monitored by conventional methods.

The following examples more fully illustrate the invention but are not to be considered as limiting its scope.

Example 1

Tables 4 and 5 below summarize the data provided in some of the examples below. In each of these examples, the mpl ligand tested was PEG-rHuMGDF, which contains the amino acid sequence of SEQ ID NO: 2, mono-peg-glycosylated with a polyethylene glycol group having an average molecular weight of about 20kDa at the alpha amino group of the N-terminal amino acid.

TABLE 4

Formulation of	Passage through SEC after 12 weeks at 37 ℃1Percent Main Peak determined
		Histidine, pH8.0, 5% sorbitol	70
Tris, pH8.0, 5% sorbitol	47
		Phosphate, pH7.0, 5% sorbitol	71
Histidine, pH7.0, 5% sorbitol	84
		Phosphate, pH6.0, 5% sorbitol	89
Glutamate/histidine, pH6.0, 5% sorbitol	92
		Histidine, pH6.0, 5% sorbitol	92
Imidazole, pH6.0, 5% sorbitol	92
		Glutamate, pH5.5, 5% sorbitol	91
Glutamate/histidineAcid, pH5.5, 5% sorbitol	93
		Acetate, pH5.0, 5% sorbitol	92
Glutamate/histidine, pH5.0, 5% sorbitol	93
		Glutamate, pH5.0, 5% sorbitol	91
Histidine, pH5.0, 5% sorbitol	89
		Succinate, pH5.0, 5% sorbitol	76
Glutamate, pH4.0, 5% sorbitol	87
		Succinate, pH4.0, 5% sorbitol	81
Acetate, pH4.0, 5% sorbitol	77
		Tartrate, pH4.0, 5% sorbitol	18
Succinate, pH3.5, 5% sorbitol	74

¹Additional stability index determination, in particular, reverse phase and cation exchange colors

Spectra, giving similar results for SEC.

A decrease in the percentage of the main peak indicates a preferred pH range of 4.0 to 6.0, preferably 5.0 to 6.0. In addition, the buffering effect in the pH range of 4.0-6.0 indicates that some buffers in this range are not preferred.

TABLE 5

Formulation of	SEC 12 weeks after 37 deg.C1Percent Main Peak determined
		Acetate, pH5.0, 5% isotonic polyol (sorbitol)	92
Acetate, pH5.0, isotonic saline, both monovalent and divalent (NaCl)	10
		Acetate, pH5.0, isotonic polar amino acid (lysine)	10
Acetate, pH5.0, isotonic nonpolar amino acid (glycine)	84

All polyols tested, including: sorbitol, sucrose, glycerol, mannitol and polyethylene glycol all gave similar results.

All salts (both monovalent and divalent) including: sodium chloride, calcium chloride, copper chloride, magnesium chloride, manganese chloride, nickel chloride, zinc chloride and iron dichloride, all gave similar results.

All tested polar amino acids, including: arginine and lysine, both of which gave similar results.

All non-polar amino acids tested, including: glycine, proline and alanine, all gave similar results.

All antioxidants tested, including: EDTA, ascorbic acid, glutathione, methionine + EDTA and citrate, did not show a substantial improvement in the stability of PEG-rHuMGDF over a50S (see defined examples).

Example 2

pH assessmentA. Starting materials: PEG-rhumgdfb. formulation: 10mM acetate, pH5.0, 5% sorbitol (A50S)10mM acetate, pH4.0, 5% sorbitol (A40S)10mM succinate, pH3.5, 5% sorbitol (S35S)10mM succinate, pH4.0, 5% sorbitol (S40S)10mM succinate, pH5.0, 5% sorbitol (S50S)10mM histidine, pH6.0, 5% sorbitol (H60S)10mM imidazole, pH6.0, 5% sorbitol (160S)10mM tartrate, pH4.0, 5% sorbitol (T40S)10mM glutamate, pH4.0, 5% sorbitol (E40S)10mM phosphate, pH6.0, 5% sorbitol (P60S)10mM phosphate, pH7.0, 5% sorbitol (P S)10mM acetate, pH 8.8.5% sorbitol (A50S)10mM acetate, pH4.0, 5% sorbitol (S40 365) 10mM histidine, pH 5% sorbitol (S60) 10mM histidine, pH7.0, 5% sorbitol (T40S)10mM glutamate, pH6.0, 5% sorbitol (E60) 10mM phosphate, pH7.0, 5% Tris 3680 mM phosphate, 5% histidine (3630, 3630% sorbitol (P S), ph7.0, 5% sorbitol (H70S)10mM histidine, ph8.0, 5% sorbitol (H80S) c. vial: concentrated at 0.5mg/ml proteinFill into 3cc vials 1 mL. D. Temperature and time point: 37 ℃; the time points are given in the table. E. And (3) analysis: HPLC: size Exclusion Chromatography (SEC), reverse phase chromatography (RP), ion exchange chromatography (IEX). F. Data of

Tables 6-11 show the percentage of major peaks determined by size exclusion, reverse phase and cation exchange chromatography after incubation at 37 ℃ for the indicated time.

pH assessment-incubation at 37 ℃ for a specified time followed by

Percentage of major peaks determined by size exclusion chromatography

TABLE 6

Form.*	Incubation time
							T＝0	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks	T is 17 weeks
	A40S	94.3994.9094.6394.2794.9994.9389.7194.90	90.8693.6592.5987.5394.3194.5643.4694.29	88.2090.2990.2085.2993.7794.2034.4493.65	82.9180.9586.1680.7791.7492.7724.4891.30	77.2174.2880.5176.4589.1391.4918.1086.84							70.1962.4372.3471.1285.9694.83-77.94
S35S
	S40S
S50S
	H60S
160S
	T40S
E40S

^*Form.

TABLE 7

Fom.*	Incubation time
									T＝0	T is 3 days	T is 7 days	T10 days	T2 weeks	T3 weeks	T is 6 weeks	T-12 weeks
	T80S	97.7097.5397.6297.6297.7097.6597.8197.72	93.5694.2097.0296.64-95.5596.4596.28	91.2894.2297.2496.7594.6896.8495.8495.43	88.1294.3797.0995.8693.2796.3594.8395.45	86.6394.5494.5095.3792.0396.3894.1295.63	80.2592.9496.1994.3389.3695.7791.7794.71	66.1891.7695.1290.9581.5293.7684.8193.97									46.5889.1891.6884.3469.6589.1671.1491.65
H50S
	H60S
H70S
	H80S
P60S
	P70S
A50S

pH assessment-incubation at 37 ℃ for a specified time followed by

Percent Main Peak determined by reverse phase chromatography

TABLE 8

Formulation of	Incubation time
							T＝0	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks	T is 17 weeks
	A40S	93.4993.7893.9193.4993.6593.6893.6593.67	91.6990.1091.6192.5990.5492.6188.0492.39	84.3480.8683.8988.9386.6987.6474.5186.03	78.8770.3677.1685.9686.4987.3466.0880.64	70.5559.35-79.3580.8884.1153.8771.50							64.2350.9859.2273.7176.1179.49-65.23
S35S
	S40S
S50S
	H60S
160S
	T40S
E40S

TABLE 9

Formulation of	Incubation time
								Time0	T is 3 days	T is 7 days	T10 days	T2 weeks	T is 6 weeks	T-12 weeks
	T80S	95.9696.1495.8895.7895.8896.4996.0496.02	93.7595.3894.3292.6694.1194.6294.9895.13	92.6895.5294.7793.8493.2394.8294.1495.57	88.5794.7094.6590.7290.9494.1892.4194.68	87.9897.0894.0890.9589.6693.4494.3796.35	68.5892.1890.20-77.8286.2582.6792.78								48.8488.4584.8070.8367.6481.0971.1189.30
H50S
	H60S
H70S
	H80S
P60S
	P70S
A50S

pH assessment-incubation at 37 ℃ for a specified time followed by

Percent Main Peak determined by cation exchange chromatography

Watch 10

Formulation of	Incubation time
							T＝0	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks	T is 17 weeks
	A40S	81.1782.6482.1682.8682.3283.7279.2382.37	73.8472.9774.5475.3079.6080.2030.2575.53	69.6667.1070.9567.9175.5777.9527.5869.71	53.9652.3653.8158.1470.7772.1519.3759.80	43.0945.8444.6349.4865.4764.0511.9250.91							41.1437.7034.9641.49-66.01-47.72
S35S
	S40S
S50S
	H60S
160S
	T40S
E40S

TABLE 11

Formulation of	Incubation time
									Time0	T is 3 days	T is 7 days	T10 days	T2 weeks	T3 weeks	T is 6 weeks	T-12 weeks
	T80S	87.4484.4287.8682.9984.0885.6887.6083.00	80.3883.7086.8183.3384.7284.5878.7084.54	78.0483.8685.4081.3581.4983.7382.5081.65	76.4183.2384.5681.0182.1783.2284.0483.81	72.7682.1682.0079.5577.2879.1578.9981.54	71.2878.5278.9072.7064.2175.8777.8577.68	54.4976.0780.6467.9568.2173.3878.9079.50									-70.8573.7156.9154.7960.9369.1872.22
H50S
	H60S
H70S
	H80S
P60S
	P70S
A50S

Example 3

Assessment of mpl ligand concentrationA. Starting materials: PEG-rhumgdfb. formulation: 10mM acetate, pH5.0, 5% sorbitol, 2.0mg/ml (20A5S)10mM acetate, pH5.0, 5% sorbitol, 1.0mg/ml (10A5S)10mM acetate, pH5.0, 5% sorbitol, 0.5mg/ml (05A5S)10mM acetate, pH5.0, 5% sorbitol, 0.2mg/ml (02A5S) C. Vial: 1mL in a 3cc vial was filled at the indicated protein concentration. D. Temperature and time point: 37 ℃; the time points are given in the table. E. And (3) analysis: HPLC: SEC, RP, IEX. F. Data of

Tables 12-14 give the percent of the major peaks determined by size exclusion, reverse phase and cation exchange chromatography after incubation at 37 ℃ for the indicated time.

mpl ligand concentration assessment-percentage of main peak after incubation at 37 ℃ for a specified time

TABLE 12

Size exclusion chromatography

Formulation of	Incubation time
								T＝0	T1 week	T2 weeks	T3 weeks	T-4 weeks	T-8 weeks	T-12 weeks
	02A5S05A5S10A5S20A5S	94.4494.1193.9693.81	94.8693.7892.6091.50	93.8492.8691.6990.25	94.5393.2591.7490.20	94.5193.4091.3289.55	93.9091.5589.2086.89								92.7490.1186.8983.59

Watch 13

Reversed phase chromatography

Formulation of	Incubation time
								T＝0	T1 week	T2 weeks	T3 weeks	T-4 weeks	T-8 weeks	T-12 weeks
	02A5S05A5S10A5S20A5S	94.5394.7394.8694.67	93.4194.4494.5694.12	93.2893.3093.2593.07	92.1692.4192.3992.47	91.0591.9791.9891.79	88.8989.1888.0688.37								87.9085.9787.6287.07

TABLE 14

Cation exchange chromatography

Formulation of	Incubation time
							T＝0	T1 week	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks
	02A5S05A5S10A5S20A5S	79.4078.22-79.52	65.7567.4466.1876.71	78.5680.2680.8082.09	79.8081.1082.0281.38	74.4276.2775.8174.26							59.7461.3960.7260.89

Example 4

Evaluation of excipientsA. Starting materials: PEG-rhumgdfb. formulation: 10mM acetate, pH5.0, 5% sorbitol

5mM EDTA (A5SE)10mM acetate, pH5.0, 2% alanine (A5A)10mM acetate, pH5.0, 1.6% glycine (A5G)10mM acetate, pH5.0, 2.7% proline (A5P)10mM acetate, pH5.0, 3.5% lysine (A5K)10mM acetate, pH5.0, 4.3% arginine (A5R)10mM glutamate, pH5.0, 9.3% sucrose (E5Su)10mM glutamate, pH5.0, 5% sorbitol (E5S) C. Vial: 1mL of the protein was loaded into a 3cc vial at a concentration of 0.5 mg/mL. D. Temperature and time point: 37 ℃; the time points are given in the table. E. And (3) analysis: HPLC: SEC, RP, IEX. F. Data of

Tables 15-17 show the percentage of major peaks determined by size exclusion, reverse phase and cation exchange chromatography after incubation at 37 ℃ for the indicated time.

Vehicle assessment-percentage of major peak after incubation at 37 ℃ for a specified time

Watch 15

Size exclusion chromatography

Formulation of	Incubation time
						T＝0	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks
	A5R	84.7194.3094.5786.7593.0894.6694.7894.55	21.8391.4091.2217.2460.0191.9793.0791.57	19.0191.9591.4315.0254.5392.1693.5592.18	12.3088.3692.269.7643.2287.6390.6888.13						-86.5284.50-37.7585.0592.2885.89
E5Su
	A5G
A5K
	A5SE
A5P
	E5S
A5A

TABLE 16

Cation exchange chromatography

Formulation of	Incubation time
						T＝0	T2 weeks	T-4 weeks	T-8 weeks	T-12 weeks
	A5R	82.2684.9386.1780.8884.5686.7387.4986.28	28.4186.2281.9020.1862.7587.5890.2381.31	21.3288.6266.8114.2356.4086.2288.8386.10	11.7172.1618.958.7536.3169.3475.5570.29						-65.0426.45-30.3865.7670.0163.69
E5Su
	A5G
A5K
	A5SE
A5P
	E5S
A5A

TABLE 17

Reversed phase chromatography

Formulation of	Incubation time
					T＝0	T2 weeks	T-4 weeks	T-12 weeks
	A5R	95.9696.1095.7195.8195.9895.6795.6495.78	92.3594.5769.6188.9393.6092.9893.9890.38	85.6891.5939.8681.3090.0189.9690.6186.52					-77.92---78.8583.9070.00
E5Su
	A5G
A5K
	A5SE
A5P
	E5S
A5A

Example 5

Isotonicity assessmentA. Starting materials: PEG-rhumgdfb. formulation: 10mM acetate, pH5.0, 9.3% sucrose (A5SU)10mM acetate, pH5.0, 5% mannitol (A5MA)10mM acetate, pH5.0, 140mM sodium chloride (A5N)10mM acetate, pH5.0, 2% PEG8000 (A5P8)10mM acetate, pH5.0, 2.5% glycerol (A5G)10mM acetate, pH5.0, 5% sorbitol,

0.01% Tween20 (A5ST)10mM histidine, pH6.0, 5% sorbitol (H6S)10mM histidine, pH6.0, 5% sorbitol,

0.001% ascorbic acid (H6AA) c. vial: 1mL of the protein was loaded into a 3cc vial at a concentration of 0.5 mg/mL. D. Temperature and time point: 37 ℃; the time points are given in the table. E. And (3) analysis: HPLC: SEC, RP, IEX. F. Data of

Tables 18-20 show the percentage of major peaks determined by size exclusion, reverse phase and cation exchange chromatography after incubation at 37 ℃ for the indicated time.

Isotonicity assessment-percentage of main peak after incubation at 37 ℃ for a specified time

Watch 18

Size exclusion chromatography

Formulation of	Incubation time
								T＝0	T is 3.5 days	T1 week	T2 weeks	T3 weeks	T7 weeks	T-12 weeks
	H6S	96.7096.7583.5496.5596.3696.4694.8896.25	96.2395.6630.4295.1495.1895.2992.8994.83	96.3096.0328.0495.2295.3795.4992.8994.50	95.9595.7224.6495.0295.1695.2092.6194.58	95.7795.2920.1995.1395.6095.5991.6294.37	93.3793.3217.6293.0893.4993.2392.7891.16								91.0390.61-91.4291.8991.6882.0587.68
H6Aa
	A5N
A5Ma
	A5Su
A5G
	A5P8
A5ST

Watch 19

Reversed phase chromatography

Formulation of	Incubation time
							T＝0	T is 3.5 days	T1 week	T2 weeks	T7 weeks	T-12 weeks
	H6S	95.4995.6494.9495.9095.5595.5794.7594.57	94.3992.0093.9995.0595.3994.7891.3193.24	94.6990.6792.1995.2994.7895.5187.0793.90	85.5593.0289.5294.3493.4794.8876.9382.75	87.6074.2268.6790.1388.1990.0413.4187.61							85.6266.22-87.2282.8888.64-85.45
H6Aa
	A5N
A5Ma
	A5Su
A5G
	A5P8
A5ST

Watch 20

Cation exchange chromatography

Formulation of	Incubation time
								T＝0	T is 3.5 days	T1 week	T2 weeks	T3 weeks	T7 weeks	T-12 weeks
	H6S	87.1385.1279.9884.7382.9882.7985.6585.02	85.7980.7539.0184.3784.8384.6982.2683.83	84.6779.3129.1484.4383.4685.0981.7483.95	82.6675.9925.0381.9280.7681.7377.4180.02	82.7775.2320.7783.0781.0981.6174.7779.79	79.3264.6214.6778.1776.6176.9853.7474.56								59.3545.93-58.5256.8559.034.8055.85
H6Aa
	A5N
A5Ma
	A5Su
A5G
	A5P8
A5ST

Example 6

Antioxidant/chelating agent evaluationA. Starting materials: PEG-rhumgdfb. formulation: 10mM acetate, pH5.0, 5% sorbitol 3mM glutathione (A5S GT)10mM acetate, pH5.0, 5% sorbitol 5mM methionine (A5S M)10mM acetate, pH5.0, 5% sorbitol 5mM methionine 1mM EDTA (A5S ME)10mM acetate, pH5.0, 5% sorbitol 1mM citrate (A5S C)10mM acetate, pH5.0, 5% sorbitol 0.5mM citrate (A5S 05C)10mM acetate, pH5.0, 5% sorbitol 1mM EDTA (A5S 1E)10mM acetate, pH5.0, 5% sorbitol 0.5mM EDTA (A5S E) C. Vial: 1mL of the protein was loaded into a 3cc vial at a concentration of 0.5 mg/mL. D. Temperature and time point: 37 ℃; the time points are given in the table. E. And (3) analysis: HPLC: SEC, RP, IEX. F. Data of

Tables 21-23 show the percent of major peaks determined by size exclusion, reverse phase and cation exchange chromatography after incubation at 37 ℃ for the indicated times.

Antioxidant/chelating agent evaluation-percentage of main peak after incubation at 37 ℃ for a specified time

TABLE 21

Size exclusion chromatography

Formulation of	Incubation time
					T＝0	T2 weeks	T-4 weeks	T-9 weeks
	A5SGT	16.6994.5394.8894.3093.9694.8894.80	12.3291.0392.1590.8390.9590.7591.72	8.0389.7290.7388.1488.9889.1589.89					5.9189.0085.7479.61-80.6382.06
A5SM
	A5SME
A5SC
	A5S05C
A5S1E
	A5SE

TABLE 22

Reversed phase chromatography

Formulation of	Incubation time
						T＝0	T2 weeks	T-4 weeks	T-9 weeks	T-12 weeks
	A5SGT	5.6994.7094.5493.9894.4394.3694.75	2.3791.5987.6681.1387.0186.8490.27	2.8088.6791.3690.3384.3076.4385.64	2.6088.5581.9768.0179.8389.9381.20						2.5086.8380.3962.9176.3584.5678.96
A5SM
	A5SME
A5SC
	A5S05C
A5S1E
	A5SE

TABLE 23

Cation exchange chromatography

Formulation of	Incubation time
					T＝0	T2 weeks	T-4 weeks	T-9 weeks
	A5SGT	4.6684.6984.9385.2386.0685.3886.51	5.1581.1878.3672.1178.0077.0080.07	0.080.9678.2666.4672.5376.4277.93					-72.9569.4854.6866.0666.2169.79
A5SM
	A5SME
A5SC
	A5S05C
A5S1E
	A5SE

Example 7

Evaluation of detergentsA. Starting materials: PEG-rhumgdfb. formulation: all forms contained 10mM acetate, pH5.0, and 5% sorbitol and 0.050mg/nl PEG-rHuMGDF.

004T20：0.004mg/ml Tween-20

006T20：0.006mg/ml Tween-20

010T20：0.010mg/ml Tween-20

040T20：0.040mg/ml Tween-20

060T20：0.060mg/ml Tween-20

004T80：0.004mg/ml Tween-80

006T80：0.006mg/ml Tween-80

010T80：0.010mg/ml Tween-80

040T80：0.040mg/ml Tween-80

060T 80: 0.060mg/ml Tween-80℃ data

Table 24 gives the results of reverse phase HPLC purity based on percent of main peak.

Watch 24

Reversed phase chromatography

Formulation of	Incubation time at 37 degrees Celsius
						T＝0	T2 weeks	T-4 weeks	T ═ 6 starPeriod of time	T-12 weeks
	A5S004T20006T20010T20040T20060T20004T80006T80010T80040T80060T80	94.6694.6894.6994.8994.5994.6194.4494.4394.8994.4594.19	94.0594.7993.9994.3092.9592.6294.3594.0594.2292.6488.38	90.6791.2591.4891.3390.6390.5790.9191.0190.6989.7488.67	90.9091.0290.4690.7689.8889.4990.8090.1890.2289.2687.64						89.0788.4988.4388.4787.5886.7288.4888.0888.0486.5684.53

D. As a result:

detergents such as Tween may be included in the PEG-rHuMGDF formulation to improve physical stability and recovered without adversely affecting chemical stability. Tween-20 and Tween-80 may be added to the PEG-rHuMGDF formulation at a final concentration of up to about 0.060mg/ml without causing oxidation of excess methionine. Tween-20 and Tween-80 were most effective in the concentration range of 0.006mg/ml to 0.060 mg/ml.

Example 8

Lyophilized compositions

Table 25 below summarizes the data obtained for lyophilized compositions containing mpl ligand. In each of these examples, the mpl ligand tested was PEG-rHuMGDF, which comprises 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, lyophilized PEG-rHuMGDF was reconstituted with approximately 1ml of water for injection prior to analysis, and the main peak percentages represent the recovery of PEG-rHuMGDF as a result of lyophilization.

TABLE 25

Formulation of	Percent Main Peak determined by SEC%
		10mM histidine, 5% mannitol	88
10mM histidine, 4% mannitol, 1% sucrose	93

Note: the mpl ligand concentration was 0.5 mg/ml.

For the lyophilized samples, there was no increase in physical stability at pH6, 7, and 8 as determined by size exclusion.

Range of sucrose concentrations studied:

watch 26

Concentration of sucrose	Percent Main Peak determined by SEC%
		2％	91
4％	94
		5％	97
6％	95

Range of buffer studied (10mM concentration):

watch 27

Formulation of	SEC, percentage of main peak by HPLC%
		Histidine, 3.8% mannitol, 2% sucrose, pH5	87
Citrate, 3.8% mannitol, 2% sucrose, pH5	87
		Acetate, 3.8% mannitol, 2% sucrose, pH5	79
Succinate, 3.8% mannitol, 2% sucrose, pH5	88
		MES, 3.8% mannitol, 2% sucrose, pH5	88
Phosphate, 3.8% mannitol, 2% sucrose, pH7	87
		Phosphate, 3.8% mannitol, 0.5% glycine, pH7	80

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

The formulation with the best main peak recovery and lowest level of agglomeration:

10mM glutamate, 6% sucrose, 2% mannitol, pH5.0

While the invention has been described in terms of preferred embodiments, it is not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalent arrangements 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 equivalent.

Sequence listing (1) general information:

(i) the applicant: AMGEN INC.

(ii) The invention provides a subject: pharmaceutical compositions containing mpl ligands

(iii) Sequence number: 2

(iv) Contact address:

(A) and (3) address: AMGEN INC.

(B) Street: 1849 DeHavilland Drive

(C) City: thousand Oaks

(D) State: california

(E) The state is as follows: united states of America

(F) Composed of braided (ZIP): 91320-1789

(V) computer readable form:

(A) floppy disk

(B) A computer: IBM PC compatibility

(C) Operating the system: PC-DOS/MS-DOS

(D) Floppy disk: patent In Release #1.0, Version #1.30

(Vi) current application data:

(A) application No.: US (not yet registered)

(B) Application date: 04-10-1996

(C) And (4) classification:

(Viii) agent/firm information:

(A) name: COOK ph.d., Robert R.

(B) Registration number: 31602

(C) Reference/record number: a-412(2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 1342 base pairs

(B) Type (2): nucleic acids

(C) Number of chains: is unknown

(D) Topology: is unknown

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) noun/keyword: CDS

(B) Position: 36..1097

(ix) Is characterized in that:

(A) noun/keyword: mat-peptides

(B) Position: 99..1097

(ix) Is characterized in that:

(A) noun/keyword: sig-peptides

(B) Position: the sequence of claim 36..98 (xi): SEQ ID NO: 1: CAGGGAGCCA CGCCAGCCAA GACACCCCGG CCAGA ATG GAG CTG ACT GAA TTG 53

Met Glu Leu Thr Glu Leu

-21 -20CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC 101Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser-15 -10 -5 1CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT 149Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg

5 10 15GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC 197Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His

20 25 30CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA 245Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly

35 40 45GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA 293Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly50 55 60 65GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG 34lAla Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu

70 75 80GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC 389Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val

85 90 95CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT 437Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro

100 105 110CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG 485Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu

115 120 125AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA 533Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val130 135 140 145GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC 581Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val

150 155 160CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG 629Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg

165 170 175ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT 677Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr

180 185 190GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG ATT CCT 725Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro

195 200 205GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC 773Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr210 215 220 225CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA ACT CGT GGA CTC TTT CCT 821Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro

230 235 240GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA 869Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr

245 250 255TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT 917Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro

260 265 270TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT ACG CTC TTC CCT CTT CCA 965Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro

275 280 285CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC 1013Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro Asp290 295 300 305CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC ACA TCC 1061Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Lau Leu Asn Thr Ser

310 315 320TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GGG TAA GGTTCTCAGA 1107Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly *

325330 CACTGCCGAC ATCAGCATTG TCTCGTGTAC AGCTCCCTTC CCTGCAGGGC GCCCCTGGGA 1167GACAACTGGA CAAGATTTCC TACTTTCTCC TGAAACCCAA AGCCCTGGTA AAAGGGATAC 1227ACAGGACTGA AAAGGGAATC ATTTTTCACT GTACATTATA AACCTTCAGA AGCTATTTTT 1287TTAAGCTATC AGCAATACTC ATCAGAGCAG CTAGCTCTTT GGTCTATTTT CTGCA 1342(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 354 amino acids

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: 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-51510 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 25Gln Cys Pro Glu Val His Pro Leu pro Thr Pro Val Leu Leu Pro Ala

30 35 40Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Mec60 65 70 75Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly

80 85 90Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 130 135Arg Phe Leu Mec Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala140 145 150 155Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu

160 165 170Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr

175 180 185Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly

190 195 200Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

205 210 215Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly220 225 230 235Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro

240 245 250Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu

255 260 265Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr

270 275 280Thr Leu phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu

285 290 295His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser300 305 310 315Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu

320 325 330Gly ^*

Claims (21)

1. A stable pharmaceutical composition comprising an mpl ligand derivative wherein a polyethylene glycol is linked to an mpl ligand; a buffer selected from glutamate, phosphate, histidine, imidazole and acetate; an excipient selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and non-polar amino acids; does not contain lipid; has a pH range of 5-6.

2. The composition of claim 1, wherein the mpl ligand comprises at least the amino acid sequence of SEQ ID NO: 2, amino acid fragment 7-151.

3. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, 1-151 to 1-191.

4. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, 1-151 to 1-171.

5. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, 1-151.

6. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, 1-163.

7. The composition of claim 6, which comprises acetate as a buffer, sorbitol as an excipient, and has a pH of 5 in an aqueous medium.

8. The composition of claim 1, wherein the non-polar amino acid is selected from the group consisting of glycine, proline and alanine.

9. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, and 1-191, said composition further comprising an antioxidant.

10. The composition of claim 9, wherein the antioxidant is selected from the group consisting of EDTA, ascorbic acid, glutathione, methionine and citrate.

11. The composition of claim 1, further comprising a detergent.

12. The composition of claim 11, wherein the detergent is selected from the group consisting of Tween; brij 35; pluronics; sodium lauryl sulfate; triton; PEG-40 castor oil; oleyl-3-phosphate; diethanolamine oleyl-10-phosphate.

13. The composition of claim 1, wherein the composition is in an aqueous medium.

14. The composition of claim 1, wherein the composition is in a lyophilized form.

15. The composition of claim 1, comprising phosphate buffer, 5% sorbitol, in an aqueous medium, at a pH of 6.

16. The composition of claim 1, wherein the aqueous medium comprises histidine buffer, and 5% sorbitol.

17. The composition of claim 1, comprising an imidazole buffer, and 5% sorbitol in an aqueous medium.

18. A composition according to claim 1, comprising glutamate buffer, and 5% sorbitol in the aqueous medium.

19. The composition of claim 1, comprising glutamate buffer, 5% sorbitol, in an aqueous medium, and having a pH of 5.

20. The composition of claim 1, comprising in lyophilized form glutamate buffer, 6% sucrose, 2% mannitol, and a pH of 5.

21. The composition of claim 1, wherein the mpl ligand consists of SEQ ID NO: 2, amino acids 1-332.