KR100408229B1 - Pharmaceutical compositions containing an mpl ligand - Google Patents

Abstract

The present invention provides a full length or truncated mpl ligand having an amino acid sequence corresponding to amino acids 7-151 to 1-332 of a natural human mpl ligand covalently bound to at least one water soluble polymer; Buffers selected from the group consisting of glutamate, phosphate, histidine, imidazole and acetate; Excipients selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and nonpolar amino acids; Optionally a detergent such as Tween; It optionally relates to a composition comprising an antioxidant or chelating agent selected from the group consisting of glutathione, methionine, citrate and EDTA and preferably having a pH in the range from 5.0 to 6.0 (total). Such compositions may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

Description

Pharmaceutical composition containing MPL ligand {PHARMACEUTICAL COMPOSITIONS CONTAINING AN MPL LIGAND}

Natural human mpl ligands are recently cloned cytokines that are key regulators of circulating platelet levels. Bartley (TD) group, Cell 77: 1117-1124 (1994); Lok, S. et al. , Nature 369: 565-568 (1994); De Sauvage (FJ) group, Nature 369: 533-538 (1994); Miyazake, H., Exp. Hematol. 22: 838 (1994); And Kuter, DJ, PNAS USA , 91: 11104-11108 (1994). Natural human mpl ligands, also referred to as thrombopoietin (TPO) and megapoietin, are proteins with a total of 332 amino acids.

Recombinant mpl ligands produced in both Chinese hamster ovary (CHO) and E. Coli cells have been shown to have biological activity that specifically stimulates or increases megakaryocytes and / or platelets in vivo in mice, mice and monkeys . See, eg, Hunt, P. et al., Blood 84 (10): 390A (1994). Human mpl ligands cleaved from the C-terminus to 181 amino acids retain biological activity in vivo. The resulting mpl ligand has a sequence corresponding to amino acids 1 to 151 to 1 to 331 of the human sequence of full length. The first six amino acids can be removed from the N-terminus of the human mpl ligand protein and maintained for biological activity. The biological activity thus appears to be contained in amino acids 7 to 151 of the mature amino acid sequence of the human mpl ligand.

Derivatives of the mpl ligands have been shown to have advantageous activity in stimulating the production of megakaryocytes and / or platelets in vivo. See PCT Application Publication WO 95/26746. In particular, mpl ligands derived from water-soluble polymers such as polyethylene glycol ('PEG') residues are of interest from a therapeutic point of view because of their long life and activity in vivo.

Compositions containing mpl ligands and related derivatives have been disclosed in a general sense. See PCT Application WO 95/26746, WO 95/21919, WO 95/18858 and WO 95/21920. However, no control experiments have been reported so far to determine that compositions containing mpl ligands are suitable for pharmaceutical use. Such compositions are important for the practical application of mpl ligands to patients such as humans. Thus, there has been a continuing need in the art for compositions for use in administering mpl ligands to patients to cause platelet increase.

Summary of the Invention

It is therefore an object of the present invention to provide a pharmaceutically acceptable stable composition comprising a mpl ligand.

Another object of the present invention is to provide a composition containing a mpl ligand for administration to a patient.

In one embodiment, the present invention provides a full length or truncated mpl having an amino acid sequence corresponding to amino acids 7-151 to 1-332 (total) of a natural human mpl ligand, optionally covalently bound to at least one water soluble polymer. Ligands; Buffers selected from the group consisting of glutamate, phosphate, histidine, imidazole and acetate; Excipients selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and nonpolar amino acids; Optionally a detergent such as Tween; Optionally a composition comprising an antioxidant or chelating agent selected from the group consisting of glutathione, methionine, citrate and EDTA and preferably having a pH in the range from 5.0 to 6.0. Such compositions may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

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

The present invention relates to a composition containing a mpl ligand, suitable for pharmaceutical administration.

1 depicts the natural human cDNA sequence of the mpl ligand and the corresponding protein (SEQ ID NOs: 1 and 2). These sequences include leader sequences (amino acids -21 to -1) that are cleaved from cDNA encoded proteins in vivo to produce mature proteins.

The present invention provides compositions comprising mpl ligands with other substances that result in stable and biologically active compositions suitable for administration to a patient such as a human.

In one embodiment, the present invention relates to a composition of mpl ligands. 'mpl ligand' broadly refers to a proteinaceous molecule that has the ability to specifically bind to and activate mpl receptors that stimulate megakaryocytes and / or platelet production in vivo. In a preferred embodiment, the mpl ligand has the same amino acid sequence as that obtained from human, such as amino acids 1-332 (SEQ ID NO: 2) of a natural human sequence. In another preferred embodiment, the mpl ligand corresponds to at least amino acids 7-151, preferably 1-171 ± 20 amino acids (ie amino acids 1-151 to 1-191) of SEQ ID NO: 2, particularly preferably SEQ ID NO: 2. Have the same amino acid sequence as 1-161 ± 10 amino acids. Specific preferred species of mpl ligands are as follows: amino acids 1-151, 1-152, 1-153, 1-154, 1-163, 1-174, 1-191, 1-232, 1 of SEQ ID NO: 2 -244. Most preferred species have amino acids 1-163 of SEQ ID NO: 2.

The mpl ligand can be derived by one or more water soluble polymers, such as one or more polyethylene glycol (PEG) groups. The polymer selected is water soluble so that the attached mpl ligands should not precipitate in an aqueous environment, such as a physiological environment. Examples of water soluble polymers are disclosed in published PCT application WO 95/26746, which is incorporated herein by reference.

Water soluble polymers can be attached using chemical reactions such as those described in published PCT application WO 95/26746. Preferred adhesion chemistries are acylation and alkylation reactions. The mpl ligand derivatives of the present invention may be attached to a plurality of polymer molecules, for example they may contain 2 to 6, preferably 2 to 5 attached polymer groups. While these polymers are frequently attached to proteins at the alpha or epsilon amino acids of amino acids, it is contemplated that these polymer groups may be attached to any amino group attached to a protein that is sufficiently reactive to attach to the polymer group under suitable reaction conditions.

In a preferred embodiment, a single polymer molecule is attached to the mpl ligand. In this case, the polymer chosen to react with the mpl ligand must be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation.

The polymers may be branched or straight chain. Preferably, for the therapeutic use of the final product preparation, the polymer should be one that is pharmaceutically acceptable. Water soluble polymers include, for example, polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols ( For example, glycerol) and polyvinyl alcohol. For the induction of mpl ligands via acylation reactions, the selected polymer should have a single reactive aldehyde group. In general, water soluble polymers should not be selected from naturally occurring glycosyl residues because these residues can be made more conveniently by mammalian recombinant expression systems. This polymer can be of any molecular weight unless it substantially interferes with or eliminates the biological activity of the resulting mpl ligand derivative.

Particularly preferred water-soluble polymers for use herein are polyethylene glycol, abbreviated PEG. Polyethylene glycol or PEG is meant herein encompassing any form of PEG used to derive other proteins such as mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycols (see US Pat. No. 5,252,714).

PEGylation of the mpl ligand can be carried out by any PEGylation reaction known in the art. Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 164 316; EP 0401 384; And other documents cited in connection with PEGylation. Preferably, the PEGylation is carried out via acylation or alkylation with reactive polyethylene glycol molecules.

Thus, as a preferred feature, the present invention relates to pegylated mpl ligands, wherein PEG groups are attached via acyl or alkyl groups. As mentioned above, such products may be mono-pegylated or poly-pegylated (eg, may contain 2-6, preferably 2-5 PEG groups). PEG groups are generally attached to proteins at alpha or epsilon amino groups of amino acids, but PEG groups can be attached to any amino group attached to a protein that is sufficiently reactive to be able to attach to PEG groups under suitable reaction conditions.

Preferably, the PEG group is attached via a reductive alkylation process and has a molecular weight of 5 to 50 kd. In the most preferred embodiment, the PEG-mpl ligand has a PEG group having an average molecular weight of about 20 kd (eg 20 kd ± 2 kd).

Particularly preferred mpl ligand derivatives correspond to amino acids 1-163 of SEQ ID NO: 2 attached to a single PEG group in the alpha amino phase 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 hereinafter referred to as 'PEG-rHuMGDF'.

In a preferred embodiment, the mpl ligand is the expression product of an exogenous DNA sequence infected with a host cell. In other words, in a preferred embodiment, the mpl ligand is a 'recombinant mpl ligand'. Recombinant mpl ligands can be carried out in any cell known for this purpose, such as CHO cells. Preferred hosts are bacteria, particularly preferably E. coli cells. Recombinant mpl ligands can be advantageously produced according to the methods described in the literature cited herein with respect to cloning and expression of mpl ligands.

The prior art has reported on compositions comprising natural human mpl ligands (amino acids 1-332 of SEQ ID NO: 2), but nobody has reported extensive stability as a function of the composition components as in the present invention. Thus, although predictions about the stability of mpl ligand compositions have been made before, compositions having the desired stability, particularly for cleaved and induced mpl ligands, have not been clearly established prior to the present invention.

Based on the data presented below, the present inventors have determined that full lengths have amino acid sequences corresponding to imino acids 7-151 to 1-332 (overall) of natural human mpl ligands optionally covalently bonded to at least one water soluble polymer. Or a cleaved mpl ligand; Buffers selected from the group consisting of glutamate, phosphate, histidine, imidazole and acetate; Excipients selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and nonpolar amino acids; Optionally a detergent such as Tween; Certain suitable compositions have been found, optionally comprising an antioxidant or chelating agent selected from the group consisting of glutathione, methionine, citrate and EDTA and preferably having a pH in the range from 5.0 to 6.0. Such compositions may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

The concentration of protein (mpl ligand) in the final composition should generally be in the range of 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 do.

Preferably, the buffer is acetate having a concentration of 5-20 mM, particularly preferably about 10 ± 2 mM. An overview of specific buffers and concentrations is shown in Table 1 below:

The pH value of the composition depends on the particular buffer and other factors. Preferred pH ranges from 4.0 to 6.0 when treated with an appropriate acid buffer (eg acetate) for improved stability. A more preferred range is 4.5 to 5.5, with about 5.0 being the most preferred embodiment.

The composition should also include excipients. Some examples and representative concentrations of excipients are shown in Table 2:

The excipient is usually added in an amount such that it becomes an isotonic solution.

The composition may also contain amino acids that enhance stability. The amino acids can be polar or nonpolar, with nonpolar amino acids being preferred. Examples of polar amino acids are arginine and lysine and examples of nonpolar amino acids are glycine, proline and alanine.

Antioxidants or chelating agents may also be included in the compositions of the present invention. Preferred antioxidants are: EDTA, ascorbic acid, glutathione, methionine and citrate. It is also contemplated to combine such agents, such as citrate + EDTA. Such agents are included in amounts suitable to reduce or eliminate the oxidation of mpl ligands. Exemplary concentrations are as follows: 0.1 to 10 mM, preferably 0.5 to 5 mM, typically 1 to 3 mM.

Detergents or lipids may also be included in the compositions of the present invention. Representative detergents include the following: Twin brand polysorbates (eg, Tween 20 and Tween 80); Brij 35; Pluronics (eg, F-127 and F-68); Sodium dodecyl sulfate; Tritons (eg, X-100); Dimyristoyl phosphatidyl glycerol (DMPG); PEG castor oil (such as PEG-40); Oleth-3-phosphate; Diethanolamine oleth-10-phosphate; And 1: 1 mixtures of short, long chain monolayer vacuoles (SLUV), including for example C8 (caprylic) and C14 (mystic). The detergent / lipid is usually included in an amount sufficient to prevent a decrease in mpl ligand due to sticking or aggregation to the surface. Exemplary concentrations of detergents are 0.004 mg / ml to 50 mg / ml, preferably 0.004 mg / ml to 10 mg / ml, most preferably 0.006 to 0.060 mg / ml. The lower the concentration of the mpl ligand, the greater the required amount of detergent / lipids, especially when below 0.2 mg / ml.

The composition may be liquid (preferably aqueous), frozen (preferably aqueous) or lyophilized.

In the case of lyophilized compositions, there is a possibility of increased protein aggregation compared to liquid compositions. Particularly preferred lyophilized compositions comprise a combination of glutamate, sucrose and mannitol at a pH in the range of 4.0 to 6.0. Particularly preferred compositions are shown in Table 3 below:

The composition of the present invention retained at least about 87%, preferably at least about 90% and most preferably at least about 93% of a complete mpl ligand derivative after storage at 37 ° C. for 12 weeks and analyzed by SEC chromatography. Stable '(see Table 4). The stability is important in terms of practicality because the low stability brings uncomfortable anxiety.

As used herein, a 'therapeutically effective amount' is an amount that gives the patient an appropriate biological effect, usually a therapeutically effective amount for a given condition and dosage regimen in the patient.

The composition of the present invention is administered throughout the body parenterally, intravenously or subcutaneously. When administered throughout the body, the therapeutic composition used in the present invention may be in the form of a physiologically acceptable aqueous solution free of pyrogen. The particular route chosen depends on the conditions to be processed. The dosage required will depend on the severity of the condition to be treated in an amount sufficient to elevate the patient's platelet and / or megakaryocyte levels, the mode of administration used and the like.

The conditions to be treated by the methods and compositions of the present invention are usually those in which there is a megakaryocytic / platelet deficiency or the expected megakaryocytic / platelet deficiency (eg, due to planned surgery). These conditions usually arise due to (temporary or permanent) deficiency of the active mpl ligand in vivo. The generic name for thrombocytopenia is thrombocytopenia, and therefore the methods and compositions of the present invention are usually useful for treating thrombocytopenia.

Thrombocytopenia is caused by a number of conditions, including chemotherapy and various medications, radiation therapy, surgery, blood loss from accidents, and certain other disease conditions. Examples of specific disease conditions associated with thrombocytopenia and which can be treated in accordance with the present invention are: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors leading to thrombocytopenia, systemic lupus erythematosus, splenomegaly, FA Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome and paroxysmal nocturnal hemochromatosis. Thrombocytopenia may also develop in AIDS treatment (eg AZT). Certain wound healing disorders can also be treated by increasing platelet count.

For example, in anticipated platelet deficiency due to future surgery, the mpl ligand analogs of the present invention may be administered several days to hours before platelets are needed. In serious situations, such as, for example, accidental large blood loss, mpl ligand analogs may be administered with blood or purified platelets.

The Mpl ligand composition may also be administered to normal humans who plan to donate platelets or other related cells in the future. Administering the composition of the present invention increases the amount of platelets and / or related cells to the extent that a patient can donate once.

Dosages associated with the methods of treating the above-described conditions may be determined by a physician, taking into account various factors that may control the action of the drug, such as the age, condition, weight, sex and health of the patient, extent of infection, time of administration and other clinical factors. Decide Typically, daily dosages range from 0.01 to 1000 micrograms of mpl ligand analog per kilogram of body weight.

The compositions of the present invention may also be used alone or in combination with other cytoplasmic, soluble Mpl (such as mpl ligand) receptors, hematopoietic elements, internal leukemia, growth factors or thrombocytopenia as well as other antibodies for disease treatment characterized by other symptoms. Can be used. The composition is very useful for treating thrombocytopenia in combination with general stimulants of hematopoiesis such as IL-3 or GM-CSF. Other megakaryocyte stimulating factors such as meg-CSF, hepatocellular factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other substances with megakaryocyte stimulating activity may also be used with the mpl ligand. Additional examples of cytoplasmic or hematopoietic factors for co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulation Factor-1 (CSF-1), GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma. The above example is also useful for simultaneous or continuous administration of an effective amount of soluble mammalian Mpl receptor, so that it may also be advantageous to administer an effective amount of soluble mammalian Mpl receptor at the same time or continuously with the effect that the megakaryocytes break into platelets when they reach a mature form. .

Thus, after PEG-mpl ligand administration (to improve the number of mature megakaryocytes), administration of soluble Mpl receptors (inactivating mpl ligand analogs and allowing mature megakaryocytes to produce platelets) is recommended to stimulate platelet production. It will be a very effective means. The dosages described above may be adjusted to supplement additional components in the composition for treatment. The progress of the treated patient can be monitored in a conventional manner.

The following examples illustrate the invention in more detail, but do not limit the scope of the invention.

Example 1

Tables 4 and 5 below summarize the values used in some of the examples below. In each example, the mpl ligand tested is PEG-rHuMGDF, containing amino acids 1-163 of SEQ ID NO: 2, mono-pegylated with polyethylene glycol groups having an average molecular weight of about 20 kDa at the alpha amino group of the N-terminal amino acid.

^{As a} result of the analysis, the additional stability showing reverse phase and cation exchange chromatography in detail shows similar results to SEC.

The main peak percentage reduction represents a preferred pH range of 4.0 to 6.0, more preferably 5.0 to 6.0. In addition, the buffer effect in the pH range 4.0 to 6.0 indicates that the buffer in this range is not preferable.

All test polyols, including the following, show similar results: sorbitol, sucrose, glycerol, mannitol and polyethylene glycol.

All salts (monovalent and divalent) including the following show similar results: NaCl, CaCl ₂ , CuCl ₂ , MgCl ₂ , MnCl ₂ , NiCl ₂ , ZnCl ₂ and FeCl ₂ .

Test polar amino acids, including the following, show similar results: arginine and lysine.

Test non-polar amino acids, including the following, show similar results: glycine, proline and aniline.

Test antioxidants, including the following, do not sufficiently increase the stability of PEG-rHuMGDF above A50S (see Table 2): EDTA, ascorbic acid, glutathione, methionine, methionine + EDTA and citric acid.

Example 2

pH evaluation

A. Starting Material: PEG-rHuMGDF

B. Formulations:

10 mM acetate pH 5.0, 5% sorbitol (A50S)

10 mM acetate pH 4.0, 5% sorbitol (A40S)

10 mM succinic acid pH 3.5, 5% sorbitol (S35S)

10 mM succinic acid pH 4.0, 5% sorbitol (S40S)

10 mM succinic acid pH 5.0, 5% sorbitol (S50S)

10 mM histidine pH 6.0, 5% sorbitol (H60S)

10 mM imidazole pH 6.0, 5% sorbitol (I60S)

10 mM Tartrate pH 4.0, 5% Sorbitol (T40S)

10 mM glutamate pH 4.0, 5% sorbitol (E40S)

10 mM phosphate pH 6.0, 5% sorbitol (P60S)

10 mM phosphate pH 7.0, 5% sorbitol (P70S)

10 mM Tris pH 8.0, 5% Sorbitol (T80S)

10 mM histidine pH 5.0, 5% sorbitol (H50S)

10 mM histidine pH 6.0, 5% sorbitol (H60S)

10 mM histidine pH 7.0, 5% sorbitol (H70S)

10 mM histidine pH 8.0, 5% sorbitol (H80S)

C. Vials: 1 mL filled with protein concentration of 0.5 mg / mL in 3 cc vials.

D. Temperature and time: 37 ° C., time is shown in the table.

E. Assay: HPLC: Size Exclusion Chromatography (SEC),

Reverse phase chromatography (RP),

Ion Exchange Chromatography (IEX)

F. Resources

Tables 6 to 11 show the main peak percentages by size exclusion, reverse phase and cation exchange chromatography after incubation for 37 hours at 37 ° C.

pH evaluation—main peak percentage by size exclusion chromatography after incubation for a specified time at 37 ° C.

pH evaluation- main peak percentage by reverse phase chromatography after incubation for a specified time at 37 ° C.

pH evaluation- main peak percentage by cation exchange chromatography after incubation for a specified time at 37 ° C.

Example 3

Mpl Ligand Concentration Assessment

A. Starting Material: PEG-rHuMGDF

B. Formulations:

10 mM acetate pH 5.0, 5% sorbitol, 2.0 mg / mL (20A5S)

10 mM acetate pH 5.0, 5% sorbitol, 1.0 mg / mL (10A5S)

10 mM acetate pH 5.0, 5% sorbitol, 0.5 mg / mL (05A5S)

10 mM acetate pH 5.0, 5% sorbitol, 0.2 mg / mL (02A5S)

C. Vials: Fill a 3 cc vial with 1 mL of the indicated protein concentration.

D. Temperature and time point: 37 ° C .; When indicated in the table

E. Assay: HPLC: SEC, RP, IEX

F. Data

Tables 12-14 below show the main peak percentages by size exclusion, reverse phase and cation exchange chromatography after incubation for the time indicated at 37 ° C.

Mpl Ligand Concentration Assessment-Main peak percentage after incubation for the indicated time at 37 ° C.

Example 4

Excipient Evaluation

A. Starting Material: PEG-rHuMGDF

B. Formulations:

10 mM acetate pH 5.0, 5% sorbitol, 5 mM EDTA (A5SE)

10 mM acetate pH 5.0, 2% alanine (A5A)

10 mM acetate pH 5.0, 1.6% glycine (A5G)

10 mM acetate pH 5.0, 2.7% proline (A5P)

10 mM acetate pH 5.0, 3.5% lysine (A5K)

10 mM acetate pH 5.0, 4.3% arginine (A5R)

10 mM glutamate pH 5.0, 9.3% sucrose (E5Su)

10 mM glutamate pH 5.0, 5% sorbitol (E5S)

C. Vials: 3 cc vials filled with 1 mL of 0.5 mg / ml protein.

D. Temperature and time point: 37 ° C .; When indicated in the table

E. Assay: HPLC: SEC, RP, IEX

F. Data

Tables 15-17 below show the percent main peaks by size exclusion, reverse phase and cation exchange chromatography after incubation for the time indicated at 37 ° C.

Excipient Evaluation-Main peak percentage after incubation for the indicated time at 37 ° C.

Example 5

Isotonic assessment

A. Starting Material: PEG-rHuMGDF

B. Formulations:

10 mM acetate pH 5.0, 9.3% sucrose (A5Su)

10 mM acetate pH 5.0, 5% mannitol (A5MA)

10 mM acetate pH 5.0, 140 mM NaCl (A5N)

10 mM acetate pH 5.0, 2% PEG 8000 (A5P8)

10 mM acetate pH 5.0, 2.5% glycerol (A5G)

10 mM acetate pH 5.0, 5% sorbitol,

0.01% Tween 20 (A5ST)

10 mM histidine pH 6.0, 5% sorbitol (H6S)

10 mM histidine pH 6.0, 5% sorbitol,

0.001% ascorbic acid (H6AA)

C. Vials: 3 cc vials filled with 1 mL of 0.5 mg / ml protein.

D. Temperature and time point: 37 ° C .; When indicated in the table

E. Assay: HPLC: SEC, RP, IEX

F. Data

Tables 18 to 20 below show incubation for the time indicated at 37 ° C., then size exclusion

Percent peak peaks by phase and cation exchange chromatography.

Isotonicity assessment-percent main peak after incubation for the indicated time at 37 ° C.

Example 6

Antioxidant / 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 1E)

10 mM acetate pH 5.0, 5% sorbitol,

0.5 mM EDTA (A5S E)

C. Vials: 3 cc vials filled with 1 mL of 0.5 mg / ml protein.

D. Temperature and time point: 37 ° C .; When indicated in the table

E. Assay: HPLC: SEC, RP, IEX

F. Data

Tables 21 to 23 below show the percent main peaks by size exclusion, reverse phase and cation exchange chromatography after incubation for the time indicated at 37 ° C.

Antioxidant / chelating agent evaluation—percentage peak peak after incubation for the indicated time at 37 ° C.

Example 7

Tax assessment

A. Starting Material: PEG-rHuMGDF

B. Formulations: All formulations contain 10 mM 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 main peak percent.

D. Results:

Detergents such as Tween can be included in the PEG-rHuMGDF blend to enhance physical stability and recovery without adversely affecting chemical stability. It can be added to the PEG-rHuMGDF formulation up to a final concentration of about 0.060 mg / ml without causing excessive methionine oxidation. Tween-20 and Tween-80 are most effective at concentrations ranging from 0.006 mg / ml to 0.060 mg / ml.

Example 8

Lyophilic composition

Table 25 below summarizes the data obtained for the lyophilic composition comprising the mpl ligand. In this example, the mpl ligand tested was SEQ ID NO; PEG-rHuMGDF which is amino-PEGylated with a polyethylene glycol group containing amino acid l-163 of 2 and having an average molecular weight of about 20 kDa in the alpha amino group of the N-terminal amino acid. In Tables 25 to 27 below, lyophilic PEG-rHuMGDF is reconstituted with about 1 ml of infusion before analysis and the main peak percentage means recovery of PEG-rHuMGDF as a result of lyophilic.

Note: Mpl ligand concentration is 0.5 mg / ml.

As measured by size exclusion, no further physical stability was achieved for the lyophilic samples at pH 6, 7 and 8.

Range of target sucrose concentration

Range of buffer of interest (concentration 10 mM):

All stabilizers and amorphous agents (such as trihalose and PEG), such as amino acids (eg isotonic arginine, lysine, proline and histidine), did not improve stability during the lyophilic process.

The combination with the best and lowest aggregate peak recovery is 10 mM glutamate, 6% sucrose, 2% mannitol, pH 5.0.

Although the present invention has been described in terms of preferred embodiments, it is not limited to the disclosed embodiments and encompasses various modifications and similar forms within the spirit and scope of the claims, with the scope being interpreted broadly and all such modifications And similar forms.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: AMGEN INC.

(ii) TITLE OF OF: PHARMACEUTICAL COMPOSITIONS CONTAINING

AN MPL LIGAND

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: AMGEN INC.

(B) STREET: One Amgen Center Drive

(C) CITY: Thousand Oaks

(D) STATE: California

(E) COUNTRY: US

(F) ZIP: 91320-1799

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patent In Release # 1.0, Version # 1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT / US97 / 16196

(B) FILING DATE: 12-SEP-1997

(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: 1:

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 Gln 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 Gln Met Glu Glu Thr Lys Ala Gln Asp Ile 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 Gln 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 Gln Leu Ser Gly Gln 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 Gln Ser Leu Leu Gly Thr Gln Leu Pro

100 105 110

CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG 485

Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu

115 120 125

AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA 533

Ser Phe Gln 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 Gln Gln Gly Phe Arg Ala Lys Ile 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 Gln Thr Ser Arg Ser Leu Asp Gln Ile 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 Ile 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 Ile 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 Gln 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 Gln 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 Gln 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 Gln Asn Leu Ser Gln 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 AGCAATACTC 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

Gln 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 Gln Met Glu Glu Thr Lys

45 50 55

Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

60 65 70 75

Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly

80 85 90

Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105

Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120

Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Gln Gly

190 195 200

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

205 210 215

Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile 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 Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu

255 260 265

Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr

270 275 280

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln 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 Gln Asn Leu Ser Gln Glu

320 325 330

Gly *

Claims (23)

Thrombopoietin derivatives wherein polyethylene glycol is attached to thrombopoietin; Buffers selected from the group consisting of glutamate, phosphate, histidine, imidazole and acetate; A composition comprising an excipient selected from the group consisting of sorbitol, sucrose, mannitol, glycerol, polyethylene glycol and nonpolar amino acids, and stimulating megakaryocyte or platelet production having a pH in the range of 5.0 to 6.0. The composition of claim 1, wherein the thrombopoietin comprises amino acids 7-151 of SEQ ID NO: 2. The composition of claim 1, wherein the thrombopoietin consists of amino acids 1-171 ± 20 amino acids of SEQ ID NO: 2. The composition of claim 1, wherein the thrombopoietin consists of amino acids 1-161 ± 10 amino acids of SEQ ID NO: 2. The composition of claim 1, wherein the thrombopoietin is comprised of amino acids 1-151 amino acids of SEQ ID NO: 2. The composition of claim 1, wherein the thrombopoietin consists of amino acids 1-163 of SEQ ID NO: 2. delete delete 7. The composition of claim 6 comprising acetate as an aqueous medium as a buffer, sorbitol as an excipient, further comprising polysorbate as a detergent and having a pH of 5.0 to 6.0. The composition of claim 1 wherein the nonpolar amino acid is selected from the group consisting of glycine, proline and alanine. The composition of claim 1 further comprising an antioxidant. The composition of claim 11 wherein the antioxidant is selected from the group consisting of EDTA, ascorbic acid, glutathione, methionine and citrate. The composition of claim 1, further comprising a detergent or a lipid. The method of claim 13, wherein the detergent is selected from polysorbates such as Tween; Polyethylene glycol ethers such as Brij 35; Polyoxypropylene polymers such as Pluronics; Sodium dodecyl sulfate; Tritons; Dimyristoyl phosphatidyl glycerol (DMPG); PEG-40 castor oil; Oleth-3-phosphate; Diethanolamine oleth-10-phosphate; And a mixture of short and long chain monolayer vacuoles (SLUV) containing C8 (caprylic) and C14 (mystic) lipids. The composition of claim 1, wherein the composition is an aqueous medium. The composition of claim 1, wherein the composition is in lyophilized form. The composition of claim 15 comprising phosphate buffer, 3-10% sorbitol in an aqueous medium and having a pH of 5.0-6.0. The composition of claim 15 comprising the histidine buffer and 3-10% sorbitol in an aqueous medium. The composition of claim 15 comprising imidazole buffer and 3-10% sorbitol in an aqueous medium. The composition of claim 15 comprising glutamate buffer and 3-10% sorbitol in an aqueous medium. The composition of claim 15 comprising glutamate buffer, 3-10% sorbitol in an aqueous medium and having a pH of 5.0-6.0. The composition of claim 16 having a pH of glutamate buffer, 2-10% sucrose, 1-5% mannitol, 5.0-6.0 in lyophilized form. The composition of claim 1, wherein the thrombopoietin consists of amino acids 1-332 of SEQ ID NO: 2. 8.