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WO1993005799A1 - Lyophilized stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor - Google Patents

Lyophilized stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor Download PDF

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Publication number
WO1993005799A1
WO1993005799A1 PCT/EP1992/002084 EP9202084W WO9305799A1 WO 1993005799 A1 WO1993005799 A1 WO 1993005799A1 EP 9202084 W EP9202084 W EP 9202084W WO 9305799 A1 WO9305799 A1 WO 9305799A1
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Prior art keywords
csf
composition according
anyone
freeze
composition
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PCT/EP1992/002084
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French (fr)
Inventor
Alessandra Cavallo
Rosanna Dalla Casa
Roberto Magrini
Lorena Mella
Original Assignee
Farmitalia Carlo Erba S.R.L.
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Application filed by Farmitalia Carlo Erba S.R.L. filed Critical Farmitalia Carlo Erba S.R.L.
Publication of WO1993005799A1 publication Critical patent/WO1993005799A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • Lyophilized stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor containing a granulocyte macrophage colony stimulating factor.
  • the present invention relates to freeze-dried (lyophilized) 5 compositions containing granulocyte-macrophage colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSF is a glycoprotein able to control the proliferation, maturation and differentiation of myeloid progenitor cells to form differentiated granulocytes, acrophages and certain 0 related hemopoietic cells.
  • GM-CSF also enhances the function of mature blood cells and stimulates the production of other cytokines such as, for example, interleukin 1 and M-CSF. It is known that it is very difficult to prepare stable 5pharmaceutical compositions containing proteins since these substances easily undergo processes of degradation with consequent decrease or loss of their pharmacological activity. Degradation pathways for proteins can be separated into two • distinct classes, involving both chemical and physical ⁇ instability.
  • chemical instability can include proteolysis, deamidation, oxidation, racemization and ⁇ -elimination.
  • Physical instability refers to processes such as aggregation, precipitation, denaturation and adsorption to surface. 5Temperature, light, and humidity are the most important factors responsible for the above mentioned drop in the activity of the proteins.
  • Freeze-drying also known as lyophilization
  • lyophilization is a process commonly used in the manufacture of protein products that are insufficiently stable for distribution and use in aqueous solution, even if frozen.
  • pharmaceutical protein products are not pure proteins, but are formulated products in which general chemical components have been added for specific purposes, e.g. to improve stability during the freeze-drying process and/or during subsequent storage. It would therefore be desirable to prepare a lyophilized composition containing GM-CSF with a long shelf life, able to endure physico- chemical and microbial degradations.
  • a lyophilized composition which comprises a granulocyte macrophage colony stimulating factor (GM-CSF) , a pharmaceutically acceptable bulking agent, a polyoxyethylene sorbitan fatty acid ester and a basic amino acid.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • said lyophilized compositions may also contain a suitable buffering agent such as, e.g. a monobasic alkali metal phosphate, preferably monobasic sodium phosphate.
  • a suitable buffering agent such as, e.g. a monobasic alkali metal phosphate, preferably monobasic sodium phosphate.
  • the GM-CSF contained in the pharmaceutical preparations of the present invention may be any GM-CSF molecule though it is, preferably, a recombinantly prepared GM-CSF, as obtained, for example, by expressing a recombinant DNA in an appropriate microbial host cell such as, e.g., a bacterial host, e.g. E. coli. a yeast or a mammalian cell.
  • the GM-CSF is preferably human GM-GSF.
  • GM-CSF a preferred one for use in the invention is the human GM-CSF whose amino acid sequence is shown in SEQ ID NO:l.
  • This CM-CSF is a preferred reconibinant GM-CSF.
  • a deletion, insertion, substitution or extension may be N-terminal, C-terminal or internal to the basic sequence and may comprise one or more amino acids.
  • GM-CSF may be present in a very small amount.
  • a pharmaceutical composition containing from 0. 1 to 5 mg of GM-CSF, preferably from 250 ⁇ g to 750 ⁇ g of GM-CSF may be administered.
  • the amount of GM-CSF in the composition of the present invention is preferably from 0. 1 to 5% , most preferably from 0. 1 to 1% , by weight of the bulking agent .
  • a pharmaceutically acceptable bulking agent may be • any bulking agent suitable for ' use in freeze-drying such as , for example , mannitol , lactose , polyvinylpyrrolidone (PVP) , dextran or glycine ; of these , mannitol is preferred.
  • any bulking agent suitable for ' use in freeze-drying such as , for example , mannitol , lactose , polyvinylpyrrolidone (PVP) , dextran or glycine ; of these , mannitol is preferred.
  • PVP polyvinylpyrrolidone
  • polyoxyethylene sorbitan fatty acid esters examples include partial C 12 -2 0 saturated or unsaturated fatty acid esters of sorbitol and its mono- and di-anhydrides copolymerised with ethylene oxide . Typically, from 10 to 40 , for example about 20 moles of ethylene oxide for each mole of sorbitol and its anhydrides will be present .
  • Polyoxyethlene sorbitan fatty acid esters are known generally as polysorbates . Examples of polysorbates include polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate, Chemical Abstracts C AS Reference No.
  • 9005-64-5 which is a mixture of partial lauric esters or sorbitol and its mono- and di-anhydrides copolymerized with approximately 20 moles of ethylene oxide for each mole of sorbitol and its anhydrides, polysorbate 40 ( polyoxyethylene 20 sorbitan monopal itate, CAS No. 9005-66-
  • polysorbate 60 polyoxyethylene 20 sorbitan monostearate CAS No. 9005-67-8
  • polysorbate 65 polyoxyethylene 20 sorbitan tristearate, CAS No. 9005-71-4
  • polysorbate 80 polyoxyethylene 20 sorbitan monoleate, CAS No. 9004-65-6)
  • polysorbate 85 polyoxyethylene 20 sorbitan trioleate, CAS No. 9005-70-3
  • polysorbate 80 also known as Tween 80.
  • the amount of polysorbate is generally from 0.01% to 25%, preferably from 0.1%-to 1%, by weight of the bulking agent.
  • Typical examples of basic amino acids for use in making the stable GM-CSF-containing pharmaceutical preparations of the present invention include lysine and arginine. These may be used either singly or in admixture.
  • the amino acids are preferably used in an amount ranging from 0.001% to 5%, most preferably from 0.1% to 2%, by weight of the bulking agent. .
  • the solution may also be buffered, e.g. to a pH of about 6.5, with a pharmaceutically acceptable buffering agent, such as monobasic sodium phosphate.
  • Compositions of the present invention will normally . be formulated in solution prior to freeze-drying. The solution may be freeze-dried in any quantity although preferably, the solution will be divided into aliquots containing 5 from 10 to 1000, for example from 100 to 500, most preferably 250, / ⁇ g of GM-CSF.
  • aliquots will be freeze-dried separately, e.g. in individual glass vials. Before the solution is freeze-dried, it may be sterilized by filtration. For example, a 0.22 ⁇ m polyvinylidenedifluoride membrane filter may be used for this purpose, to prevent adsorption of the molecules on the surface.
  • a typical freeze-drying cycle used for GM-CSF containing pharmaceutical preparation may be, e.g., as follows: (a) freeze at -45°C, and maintain this temperature for' four hours; (b) primary drying at -45°C to +10°C for approximately, twenty hours, with vacuum level less than 13.3 Pa (0.1 torr) and a condenser temperature of -60°C; and (c) secondary drying at 10°C to +25°C for approximately twenty-four hours, with the ' same vacuum and condenser temperature as described in (b) above. Variations of this protocol which do not substantially alter the stability of the GM-CSF may be made.
  • Aliquots of the composition of the present invention may be dispensed into sterile vials.
  • Sterile glass vials can be suitable.
  • the glass vials can be sealed with conventional rubber stoppers (chloro butyl rubber) because no losses of protein, due to adsorption of GM-CSF to the rubber surface, was observed.
  • the lyophilized composition of the present invention may be srored for example under an inert gas, e.g. nitrogen.
  • the freeze-dried product composition of the invention may be reconstituted using any aqueous physiologically acceptable sterile solvent.
  • the solvent used will provide a reconstituted solution with a pH between about 5 and about 7.0, most preferably about 6.5
  • a 0.9% aqueous solution of sodium chloride i.e. physiological saline
  • the solution may contain an effective amount of an anti-microbial preservative agent such as, for example, benzalkonium chloride, in order to inhibit the microbial activity in reconstituted solutions of the present invention.
  • an anti-microbial preservative agent such as, for example, benzalkonium chloride
  • the invention thus provides both a method for preparing a lyophilized GM-CSF composition according to the invention, which process comprises mixing, in aqueous solution, GM-CSF, a pharmaceutically acceptable bulking agent, a polyoxyethylene sorbitan fatty acid ester and a basic amino acid, and freeze-drying the resulting solution; '. and a method of preparing an aqueous injectable GM-CSF solution which comprises reconstituting the freeze-dried composition of the present invention with a physiologically acceptable sterile aqueous solvent.
  • the present invention also provides a kit containing the lyophilized compositions described above in a sterile vial and a physiologically acceptable sterile . aqueous solvent for reconstitution of the lyophilized composition.
  • compositions or kits according to the present invention are useful in a method of treatment of the human or animal body, e.g. in the treatment of neutropenic disorders of cancer patients after chemotherapy.
  • the GM-CSF is a reco binantly prepared human GM-CSF having the sequence shown in SEQ ID NO:l, which is prepared following the conventional recombinant techni ⁇ ues well known in the art
  • rh GM-CSF. 25 It is, e.g., obtained as a solid bulk at a concentration of active substance of approximately 880 ⁇ g/mg expressed as protein content measured by the biuret reaction. This solid bulk is stored at about -20'C. It was observed that thawing and diluting this bulk to a concentration of about 125 ⁇ g/ml using a 2.5% mannitol solution does not affect protein stability. HP C analysis of this solution shows that the active drug substance (rh GM-CSF) is quantitatively recovered.
  • a lyoprotectant is defined as a compound that stabilizes and prevents the degradation of the proteins both during freeze-drying and afterwards, during storage, whereas a cryoprotectant only infers protection from freezing damage.
  • lyoprotectants such as Arginine significantly improved protein stability.
  • Polysorbate 80 proved to be ineffective if used alone, but surprisingly this stabilizer worked well in combination, with arginine.
  • the low stabilizing activity of polysorbate 80 might be expected, due to the low coordination ' power of this additive towards the water molecules.
  • the synergistic effect of polysorbate 80 with arginine was quite unexpected.
  • composition of rh GM-CSF formulation stabilized with polysorbate 80 and Arginine.
  • Freeze-dried vials containing compositions according to the present invention comprising about 250 g of GM-CSF were examined for long term stability over various periods of time at different temperatures. The following parameters were examined and the acceptable standards are also given:
  • GM-CSF working standard Acetonitrile, HPLC grade Water, HPLC grade
  • Trifluoroacetic acid, analytical grade Phosphate buffer at pH 7.5 Transfer 7.3 g of sodium chloride and 3.2 g of Sodium dihydrogen phosphate in a 1000 ml volumetric flask.
  • Membrane filter 0.22 ⁇ m porosity, Millipore Durapore GVWP, or equivalent
  • Mobile phase (B) consisting of 95% acetonitrile-5% water containing 0.1% of trifluoroacetic acid (w/v) , filtered through the membrane filter and deaerated.
  • the standard solution must be freshly prepared and used within a working day.
  • the standard and sample solution are alternatively injected at least 2 times into the liquid chromatograph under the following experimental conditions:
  • Detector sensitivity the detector "computer” output is connected to integrator for maximum sensitivity Injection volume : 100 ⁇ l

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Abstract

The invention relates to a lyophilized composition which comprises a granulocyte macrophage colony stimulating factor (GM-CSF), a pharmaceutically acceptable bulking agent, a polyoxyethylene sorbitan fatty acid ester and a basic amino acid. The compositions according to the present invention are useful in a method of treatment of the human or animal body, e.g. in the treatment of neutropenic disorders of cancer patients after chemotherapy.

Description

Lyophilized stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor.
The present invention relates to freeze-dried (lyophilized) 5 compositions containing granulocyte-macrophage colony stimulating factor (GM-CSF).
GM-CSF is a glycoprotein able to control the proliferation, maturation and differentiation of myeloid progenitor cells to form differentiated granulocytes, acrophages and certain 0related hemopoietic cells.
GM-CSF also enhances the function of mature blood cells and stimulates the production of other cytokines such as, for example, interleukin 1 and M-CSF. It is known that it is very difficult to prepare stable 5pharmaceutical compositions containing proteins since these substances easily undergo processes of degradation with consequent decrease or loss of their pharmacological activity. Degradation pathways for proteins can be separated into two • distinct classes, involving both chemical and physical θinstability.
First, chemical instability can include proteolysis, deamidation, oxidation, racemization and β-elimination. Physical instability refers to processes such as aggregation, precipitation, denaturation and adsorption to surface. 5Temperature, light, and humidity are the most important factors responsible for the above mentioned drop in the activity of the proteins.
These molecules are also at risk of microbial degradation due to adventitious contaminations of the • solution during 0purification or storage. Freeze-drying (also known as lyophilization) is a process commonly used in the manufacture of protein products that are insufficiently stable for distribution and use in aqueous solution, even if frozen.
In general, pharmaceutical protein products are not pure proteins, but are formulated products in which general chemical components have been added for specific purposes, e.g. to improve stability during the freeze-drying process and/or during subsequent storage. It would therefore be desirable to prepare a lyophilized composition containing GM-CSF with a long shelf life, able to endure physico- chemical and microbial degradations.
According to the present invention there is provided a lyophilized composition which comprises a granulocyte macrophage colony stimulating factor (GM-CSF) , a pharmaceutically acceptable bulking agent, a polyoxyethylene sorbitan fatty acid ester and a basic amino acid.
Optionally said lyophilized compositions may also contain a suitable buffering agent such as, e.g. a monobasic alkali metal phosphate, preferably monobasic sodium phosphate. The GM-CSF contained in the pharmaceutical preparations of the present invention may be any GM-CSF molecule though it is, preferably, a recombinantly prepared GM-CSF, as obtained, for example, by expressing a recombinant DNA in an appropriate microbial host cell such as, e.g., a bacterial host, e.g. E. coli. a yeast or a mammalian cell. The GM-CSF is preferably human GM-GSF. Among the GM-CSFs a preferred one for use in the invention is the human GM-CSF whose amino acid sequence is shown in SEQ ID NO:l. This CM-CSF is a preferred reconibinant GM-CSF. The term GM-CSF, according to the invention, includes also muteins obtained by deletions, insertions or substitutions of aminoacid residues as well as extensions by way of aminoacid residues.. A deletion, insertion, substitution or extension may be N-terminal, C-terminal or internal to the basic sequence and may comprise one or more amino acids.
A further preferred embodiment of the present invention is the
23 mute in Leu GM-CSF, i.e . a mute in of human GM-CSF wherein the amino acid naturally present in the position 23 of the. human GM-CSF sequence shown in SEQ N0: 1 is substituted by leucyn.
In the compositions of the invention GM-CSF may be present in a very small amount. For example a pharmaceutical composition containing from 0. 1 to 5 mg of GM-CSF, preferably from 250 μg to 750 μg of GM-CSF, may be administered. The amount of GM-CSF in the composition of the present invention is preferably from 0. 1 to 5% , most preferably from 0. 1 to 1% , by weight of the bulking agent .
A pharmaceutically acceptable bulking agent may be • any bulking agent suitable for' use in freeze-drying such as , for example , mannitol , lactose , polyvinylpyrrolidone (PVP) , dextran or glycine ; of these , mannitol is preferred.
Examples of polyoxyethylene sorbitan fatty acid esters include partial C12-20 saturated or unsaturated fatty acid esters of sorbitol and its mono- and di-anhydrides copolymerised with ethylene oxide . Typically, from 10 to 40 , for example about 20 moles of ethylene oxide for each mole of sorbitol and its anhydrides will be present . Polyoxyethlene sorbitan fatty acid esters are known generally as polysorbates . Examples of polysorbates include polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate, Chemical Abstracts CAS Reference No. 9005-64-5) which is a mixture of partial lauric esters or sorbitol and its mono- and di-anhydrides copolymerized with approximately 20 moles of ethylene oxide for each mole of sorbitol and its anhydrides, polysorbate 40 (polyoxyethylene 20 sorbitan monopal itate, CAS No. 9005-66-
7) , polysorbate 60 (polyoxyethylene 20 sorbitan monostearate CAS No. 9005-67-8) , polysorbate 65 (polyoxyethylene 20 sorbitan tristearate, CAS No. 9005-71-4) , polysorbate 80 (polyoxyethylene 20 sorbitan monoleate, CAS No. 9004-65-6) and polysorbate 85 (polyoxyethylene 20 sorbitan trioleate, CAS No. 9005-70-3) . Of these the preferred polysorbate is polysorbate 80, also known as Tween 80. In the compositions of the invention the amount of polysorbate is generally from 0.01% to 25%, preferably from 0.1%-to 1%, by weight of the bulking agent.
Typical examples of basic amino acids for use in making the stable GM-CSF-containing pharmaceutical preparations of the present invention include lysine and arginine. These may be used either singly or in admixture. The amino acids are preferably used in an amount ranging from 0.001% to 5%, most preferably from 0.1% to 2%, by weight of the bulking agent. . As already said, if desired, the solution may also be buffered, e.g. to a pH of about 6.5, with a pharmaceutically acceptable buffering agent, such as monobasic sodium phosphate. Compositions of the present invention will normally . be formulated in solution prior to freeze-drying. The solution may be freeze-dried in any quantity although preferably, the solution will be divided into aliquots containing 5 from 10 to 1000, for example from 100 to 500, most preferably 250, /λg of GM-CSF.
These aliquots will be freeze-dried separately, e.g. in individual glass vials. Before the solution is freeze-dried, it may be sterilized by filtration. For example, a 0.22 μm polyvinylidenedifluoride membrane filter may be used for this purpose, to prevent adsorption of the molecules on the surface.
Using HPLC analysis carried out before and after such filtration, we have found that GM-CSF is consistently recovered on a quantitative basis.
A typical freeze-drying cycle used for GM-CSF containing pharmaceutical preparation may be, e.g., as follows: (a) freeze at -45°C, and maintain this temperature for' four hours; (b) primary drying at -45°C to +10°C for approximately, twenty hours, with vacuum level less than 13.3 Pa (0.1 torr) and a condenser temperature of -60°C; and (c) secondary drying at 10°C to +25°C for approximately twenty-four hours, with the ' same vacuum and condenser temperature as described in (b) above. Variations of this protocol which do not substantially alter the stability of the GM-CSF may be made.
Aliquots of the composition of the present invention may be dispensed into sterile vials. Sterile glass vials can be suitable.
It is clearly known that proteins adhere to glass surface, and we have found that, when the freeze-dried product of the present invention is reconstituted in a glass vial, some loss of protein, possibly due to adhesion on the glass surface, occurs. However, we have found that coating the glass vials with silicon, in order to minimise sticking, successfully overcomes this problem.
The glass vials can be sealed with conventional rubber stoppers (chloro butyl rubber) because no losses of protein, due to adsorption of GM-CSF to the rubber surface, was observed. The lyophilized composition of the present invention may be srored for example under an inert gas, e.g. nitrogen. The freeze-dried product composition of the invention may be reconstituted using any aqueous physiologically acceptable sterile solvent. Preferably, the solvent used will provide a reconstituted solution with a pH between about 5 and about 7.0, most preferably about 6.5 Preferably, a 0.9% aqueous solution of sodium chloride (i.e. physiological saline) is used as the reconstitution solvent.
Optionally, the solution may contain an effective amount of an anti-microbial preservative agent such as, for example, benzalkonium chloride, in order to inhibit the microbial activity in reconstituted solutions of the present invention.
The invention thus provides both a method for preparing a lyophilized GM-CSF composition according to the invention, which process comprises mixing, in aqueous solution, GM-CSF, a pharmaceutically acceptable bulking agent, a polyoxyethylene sorbitan fatty acid ester and a basic amino acid, and freeze-drying the resulting solution; '. and a method of preparing an aqueous injectable GM-CSF solution which comprises reconstituting the freeze-dried composition of the present invention with a physiologically acceptable sterile aqueous solvent. 5 The present invention also provides a kit containing the lyophilized compositions described above in a sterile vial and a physiologically acceptable sterile . aqueous solvent for reconstitution of the lyophilized composition.
10 The compositions or kits according to the present invention are useful in a method of treatment of the human or animal body, e.g. in the treatment of neutropenic disorders of cancer patients after chemotherapy.
The Examples which follow illustrate aspects of the
15 present invention without limiting its scope.
In the following Examples, the GM-CSF is a reco binantly prepared human GM-CSF having the sequence shown in SEQ ID NO:l, which is prepared following the conventional recombinant techniσues well known in the art
?_Z .-ΪNA.6(3), 221-229, 1987, Current Microbiology 17, 321-332, 1988).
•Similar techniques may be followed for preparing any GM-CSF molecule . according to the invention.
This compound will be referred to in the present specification as rh GM-CSF. 25 It is, e.g., obtained as a solid bulk at a concentration of active substance of approximately 880 μg/mg expressed as protein content measured by the biuret reaction. This solid bulk is stored at about -20'C. It was observed that thawing and diluting this bulk to a concentration of about 125 μg/ml using a 2.5% mannitol solution does not affect protein stability. HP C analysis of this solution shows that the active drug substance (rh GM-CSF) is quantitatively recovered.
At first, studies were conducted to choose the best bulking agent suitable for the formulation.
EXAMPLE 1
Solutions containing rh GM-CSF (125 μg/ml) , mannitol, lactose, polyvinyl yrrolidone (PVP) , were prepared aseptically, filled into vials (nominal volume 2-0 ml) and freeze-dried. The appearance of the reconstituted solution and the e fect of storage on th_, protein potency in the final freeze-dried formulation, were checked through accelerated stability studies (25*-35'C). The results, as summarized in Table 1 , demonstrate that, among the test substances, the most suitable bulking agent for the pharmaceutical compositions of the present invention is mannitol.
Figure imgf000011_0001
Figure imgf000011_0002
Example 2
According to the literature (P.P. De Luca and M. . Townsend J. Par. Sci. and Teen. Vol. 42 No. 6, pag. 190), a lyoprotectant is defined as a compound that stabilizes and prevents the degradation of the proteins both during freeze-drying and afterwards, during storage, whereas a cryoprotectant only infers protection from freezing damage.
Based on these theoretical considerations, experimental work was thus undertaken to determine the protective capacity on GM-CSF of a number of compounds which might act as lyoprotectants. ■Polysorbate 80 (Tween 80), sodium carboxymethyl cellulose (NaCMC), sodium chloride, arginine (Arg), lysine (Lys), aspartic acid (Asp) and meglumine were tested as possible protective agents. Solutions containing rh GM-CSF (125 μg/ml), mannitol (50 mg/ml) as bulking agent, monobasic sodium phosphate (pH 6.5) as buffering agent and a suitable concentration of each potential stabilizer, were prepared aseptically, filled into vials (nominal volume 2.0 ml) and then freeze-dried. The effect of storage on the protection potency was checked through accelerated stability studies {35βC). Basic experimental results are summarized in Table 2. The freeze-dried formulation containing only mannitol as bulking agent underwent about 10% potency loss after one week of storage.
The presence of lyoprotectants such as Arginine significantly improved protein stability.
Other tested compounds such as Lysine, Aspartic Acid, meglumine proved to be ineffective as stabilizers. Data are presented in Table 2 only for Asp, but also the other tested compounds behaved similarly.
Polysorbate 80 proved to be ineffective if used alone, but surprisingly this stabilizer worked well in combination, with arginine. The low stabilizing activity of polysorbate 80 might be expected, due to the low coordination' power of this additive towards the water molecules. On the contrary, the synergistic effect of polysorbate 80 with arginine was quite unexpected.
TABLB 2 Recombinant human GM-CSF (rh GM-CSF) preformulation studies. Accelerated stability results of different freeze-dried formulations, containing GM-CSF (250 mcg/ l), Mannitol (50 mg/ml) and Monobasic Sodium Phosphate (pH 6.5).
Figure imgf000013_0001
. . . Example 3
Composition of rh GM-CSF formulation stabilized with polysorbate 80 and Arginine.
per vial *.** per 2000 vials rh GM-CSF 0.2875 mg* 575 mg* Mannitol 52.5000 mg 105 g
Polysorbate 80 0.0525 mg 105 mg
L-Arginine 1.05 mg 1.1 g .,
Monobasic Sodium 2.898 mg 5.8 g
Phosphate Sodium hydroxide qs to 6.5 pH qs to pH 6.5
Water for Injection ** qs to 2.0 ml qs to 4.0 1 * Including 10% overage to compensate for losses during- manufacture
** During freeze-drying water for injections is removed
*** a 5% overfill of the rh GM-CSF/Mannitol/Polysorbate 80/L-Arginine/Monobasic Sodium Phosphate solution is included. The formulation was freeze-dried and individual vials were sealed under nitrogen. 93/05799
1 2
EXAMPLE 4
Stability of compositions of the invention
Freeze-dried vials containing compositions according to the present invention comprising about 250 g of GM-CSF were examined for long term stability over various periods of time at different temperatures. The following parameters were examined and the acceptable standards are also given:
- Appearance: colourless glass vials, containing a compact, white, freeze-dried cake or mass, determined by visual inspection
- Identification: Same retention time as rh GM-CSF working standard (HPLC method as illustrated below) - RP-HPLC assay: 85-115% of the label chain - Water: not more than 3% - Appearance after: clear and clean colourless solution, reconstitution* essentially free from visible particles of foreign matter - pH after reconstitution*: 6-7 * The contents of the vials are dissolved in 2 ml of the required solvent (0.9% Sodium Chloride Injection, BP) . The HPLC methodology employed is as follows: Materials
GM-CSF, working standard Acetonitrile, HPLC grade Water, HPLC grade
Trifluoroacetic acid, analytical grade Phosphate buffer at pH 7.5 : Transfer 7.3 g of sodium chloride and 3.2 g of Sodium dihydrogen phosphate in a 1000 ml volumetric flask.
Dissolve with about 800 ml of distilled water and bring the pH to 7.5 with 2N sodium hydroxide. Fill to the mark with distilled water.
Equipment Liquid chromatograph Milton Roy model CM 4000, or equivalent, equipped with:
- chromatographic column : (length 150 mm, internal diameter 4.6 mm) filled with PLRP-^-S 300 A (average particle size 8 μm) , supplied by Polymer Laboratories Ltd, Shropshire, U.K. or equivalent
- injection valve: Rheodyne model 7125, or equivalent, fitted with a 100 μl sample loop
- detector: Shi adzu model SPD 6A, or equivalent
- integrating recorder: SP 4270 (Spectra-Physics) , or equivalent
Membrane filter, 0.22 μm porosity, Millipore Durapore GVWP, or equivalent
High precision laboratory glassware
Solutions Mobile phase (A) consisting of water, containing 0.1% of trifluoroacetic acid (w/v) , filtered through the membrane filter and deaerated.
Mobile phase (B) consisting of 95% acetonitrile-5% water containing 0.1% of trifluoroacetic acid (w/v) , filtered through the membrane filter and deaerated.
Standard solution
Dissolve about 6 mg, exactly weighed, of GM-CSF in 50 ml of phosphate buffer at pH 7.5.
The standard solution must be freshly prepared and used within a working day.
Sample solution
Prepare the sample solution using at least five freeze- dried vials.
The content of each vial dosed at 250 μg of GM-CSF is dissolved in 2.0 ml of phosphate buffer at pH 7.5, then a pool is made with all prepared solutions.
Chromatoσraphic (HPLC) conditions
The standard and sample solution are alternatively injected at least 2 times into the liquid chromatograph under the following experimental conditions:
Column temperature : room temperature (22 + 2'C)
Mobile phase flow-rate : 1 ml/min Analytical wavelength : 215 + 1 nm Gradient conditions
Figure imgf000017_0001
Detector sensitivity : the detector "computer" output is connected to integrator for maximum sensitivity Injection volume : 100 μl
Integrating recorder attenuation : 1024
Chart speed : 0.5 cm/min
The results obtained from studies of accelerated stability, for the formulation illustrated in Example 3 are reported in the following Tables 3 to 8 with reference to two different batches.
TABLE 3 - Accelerated stability data of rh GM-CSF freeze-dried
- Batch No. TF/23765
Active drug substance Batch No.: OP52 COMPOSITION of Example 3
35βC
Figure imgf000018_0001
TABLE 4 - Long terra stability data of rh GM-CSF freeze-dried vials - Batch No. TF/23765 Active drug substance Batch No: OP.52
COMPOSITION of Example 3
25°C
h-1
Figure imgf000019_0001
TABLE 4 - Long term stability data of rh GM-CSF freeze-dried vials - Batch No. TF/23765 Active drug substance Batch No: OP.52
COMPOSITION of Example 3
4°C
Figure imgf000020_0001
Figure imgf000020_0002
TABLE 6 - Accelerated stability data of rh. GM-CSF freeze-dried vials - Batch No. NP8730/29F Active drug substance Batch No: OP44/A
COMPOSITION of Example 3
35°C
Figure imgf000021_0001
Water 1.5 n.d, n.d. n.d,
Appearance Complies Unchanged- (reconstituted solution)
PH 6.78 6.74 6.74 6.73
(reconstituted solution)
TABLE 7 - Long term stability data of rh GM-CSF freeze-dried vials - Batch No. NP8730/29F Active drug substance Batch No:OP44/A
COMPOSITION of Example 3
25°C
Tests Initial 2 weeks 4 weeks 2 mos 6 mos control
Appearance Complies Unchanged-
RP-HPLC assay 10. mcg/vial 258 . % initial 100
Figure imgf000022_0001
Water 1.5 n.d, n.d, n.d.
Appearance Complies Unchanged- (reconstituted 15 solution)
pH 6.78 6.74 6.74 6.76
(reconstituted solution)
TABLE 4 - Long term stability data of rh GM-CSF freeze-dried vials - Batch No . NP 8730/29F Active drug substance Batch No : OP 44/A
COMPOSITION of Example 3
4°C
Figure imgf000023_0001
SEQUENCE LISTING
(1) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Pro Ala Arg Ser Pro Ser Pre* Se Thr Gin Pro Trp Glu His Va 1 5 - 10 15
Asn Ala lie Gin Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Th 20 25 30
Ala Ala Glu Met Asn Glu Thr Val Glu Val lie Ser Glu Met Phe As 35 40 45
Leu Gin Glu Pro Thr Cys Leu Gin Thr Arg Leu Glu Leu Tyr Lys Gi 50 55 60
Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Me 65 70 75 80
Ala Ser His Tyr Lys Glη His Cys Pro Pro Thr Pro Glu Thr Ser Cys
85 90 95
Ala Thr Gin Thr lie Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110
phe Leu Leu Val lie Pro Phe Asp Cys Trp Glu Pro Val Gin Glu 115 120 125

Claims

1. A lyophilized composition which comprises a granulocyt macrophage colony stimulating factor (GM-CSF), a pharma ceutically acceptable bulking agent, a polyoxyethylen
5 sorbitan fatty acid ester and a basic amino acid.
2. A composition according to claim 1 which additionall comprises a buffering agent.
3. A composition according to claim 2 in which the bufferin agent is monobasic sodium phosphate.
104. A composition according to anyone of the preceding claim in which the bulking agent is mannitol.
5. A composition according to anyone of the preceding claim in which the polyoxyethylene sorbitan fatty acid ester is polysorbate 80.
156. A composition according to anyone of the preceding claim in which the basic amino acid is arginine.
7. A composition according to anyone of the preceding claims in which the GM-CSF is a recombinant human GM-CSF havin the amino acid sequence s owπ. in SEQ ID.N0:1.
2C
8. A composition according to claims 1-6 in which the GKt-CSF is the
23 mutein- Leu GM-CSF.
9. A composition according to anyone of the preceding claims in a sealed sterile glass vial.
10. Method for preparing a lyophilized composition according 5 to claim 1 which comprises mixing, in aqueous solution.,
GM-CSF, a pharmaceutically acceptable bulking agent a polyoxyethylene sorbitan fatty acid ester and .a basic amino acid, and freeze-drying the resulting solution.
11. A method for preparing an aqueous GM-CSF solution which comprises reconstituting a freeze-dried composition
» according to anyone of claims 1 to 8 with a physiologi¬ cally acceptable aqueous sterile solvent.
512- A kit comprising a) a composition according to anyone of claims 1 to 8 and b) a physiologically acceptable aqueous sterile solvent for reconstituting said composition.
PCT/EP1992/002084 1991-09-24 1992-09-10 Lyophilized stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor WO1993005799A1 (en)

Applications Claiming Priority (2)

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GB919120304A GB9120304D0 (en) 1991-09-24 1991-09-24 Stable pharmaceutical compositions containing a granulocyte macrophage colony stimulating factor

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EP0852951A1 (en) * 1996-11-19 1998-07-15 Roche Diagnostics GmbH Stable lyophilized monoclonal or polyclonal antibodies containing pharmaceuticals
KR19990009888A (en) * 1997-07-12 1999-02-05 성재갑 Stable Solution Formulation of Colony Stimulating Factors
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
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US7060268B2 (en) 1995-07-27 2006-06-13 Genentech, Inc. Protein formulation
US7666413B2 (en) 2000-10-12 2010-02-23 Genetech, Inc. Method of reducing viscosity of high concentration protein formulations
US8318161B2 (en) 2009-03-06 2012-11-27 Genentech, Inc. Anti-oxidized LDL antibody formulation
EP1908482A4 (en) * 2005-06-10 2012-12-19 Chugai Pharmaceutical Co Ltd STABILIZER FOR PROTEIN PREPARATION CONTAINING MEGLUMIN AND USE THEREOF
US8703126B2 (en) 2000-10-12 2014-04-22 Genentech, Inc. Reduced-viscosity concentrated protein formulations
US8961964B2 (en) 2003-04-04 2015-02-24 Genentech, Inc. High concentration antibody and protein formulations
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
WO2020002650A1 (en) * 2018-06-29 2020-01-02 Targovax Asa A formulation

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US7060268B2 (en) 1995-07-27 2006-06-13 Genentech, Inc. Protein formulation
US9180189B2 (en) 1995-07-27 2015-11-10 Genentech, Inc. Treating a mammal with a formulation comprising an antibody which binds IgE
EP0920314A4 (en) * 1996-05-22 2001-08-16 Smithkline Beecham Corp Non-peptide g-csf mimetics
US8758747B2 (en) 1996-11-19 2014-06-24 Roche Diagnostics Gmbh Stable lyophilized pharmaceutical preparations of monoclonal or polyclonal antibodies
WO1998022136A3 (en) * 1996-11-19 1998-08-20 Boehringer Mannheim Gmbh Stable lyophilized pharmaceutical substances from monoclonal or polyclonal antibodies
EP0852951A1 (en) * 1996-11-19 1998-07-15 Roche Diagnostics GmbH Stable lyophilized monoclonal or polyclonal antibodies containing pharmaceuticals
KR100514207B1 (en) * 1996-11-19 2005-09-13 로셰 디아그노스틱스 게엠베하 Stable lyophilized pharmaceutical substances from monoclonal or polyclonal antibodies
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US6908610B1 (en) 1999-03-01 2005-06-21 Chugai Seiyaku Kabushiki Kaisha Long-term stabilized formulations
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US8703126B2 (en) 2000-10-12 2014-04-22 Genentech, Inc. Reduced-viscosity concentrated protein formulations
US8142776B2 (en) 2000-10-12 2012-03-27 Genentech, Inc. Reduced-viscosity concentrated protein formulations
US7666413B2 (en) 2000-10-12 2010-02-23 Genetech, Inc. Method of reducing viscosity of high concentration protein formulations
US10166293B2 (en) 2000-10-12 2019-01-01 Genentech, Inc. Reduced-viscosity concentrated protein formulations
US8961964B2 (en) 2003-04-04 2015-02-24 Genentech, Inc. High concentration antibody and protein formulations
US10034940B2 (en) 2003-04-04 2018-07-31 Genentech, Inc. High concentration antibody and protein formulations
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
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US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
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