WO2008134310A1 - Stabilization of liquid solutions of recombinant protein for frozen storage - Google Patents

Stabilization of liquid solutions of recombinant protein for frozen storage Download PDF

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Publication number
WO2008134310A1
WO2008134310A1 PCT/US2008/061147 US2008061147W WO2008134310A1 WO 2008134310 A1 WO2008134310 A1 WO 2008134310A1 US 2008061147 W US2008061147 W US 2008061147W WO 2008134310 A1 WO2008134310 A1 WO 2008134310A1
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Prior art keywords
solution
recombinant protein
carbohydrate
liquid
concentration
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PCT/US2008/061147
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French (fr)
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WO2008134310A8 (en
Inventor
Nelly Tsvetkova
Omkar Joshi
Paul Wu
Degian Wang
Arnaud Desponds
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Bayer Healthcare Llc
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Priority to MX2009011367A priority Critical patent/MX2009011367A/en
Priority to BRPI0810500-6A2A priority patent/BRPI0810500A2/en
Priority to JP2010506432A priority patent/JP5401446B2/en
Priority to AU2008245821A priority patent/AU2008245821B2/en
Priority to CA2683317A priority patent/CA2683317C/en
Priority to US12/597,333 priority patent/US8187799B2/en
Priority to RU2009143493/15A priority patent/RU2469739C2/en
Priority to EP08746549.8A priority patent/EP2150264A4/en
Application filed by Bayer Healthcare Llc filed Critical Bayer Healthcare Llc
Priority to CN200880013538.9A priority patent/CN101678066B/en
Priority to KR1020157023163A priority patent/KR20150103333A/en
Publication of WO2008134310A1 publication Critical patent/WO2008134310A1/en
Priority to IL201508A priority patent/IL201508A0/en
Publication of WO2008134310A8 publication Critical patent/WO2008134310A8/en
Priority to US13/459,806 priority patent/US8536125B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K9/08—Solutions

Definitions

  • the invention relates to the freezing and storage of liquid solutions of recombinant protein, preferably bulk solutions.
  • Production of recombinant proteins in cell culture normally involves a series of purification steps, by which the desired protein product is recovered from recombinant host cells and/or the associated culture media. Many important recombinant proteins are produced on a large commercial scale. In the case of pharmaceutical proteins, for example, it is not uncommon for more than one purification stage to be used to achieve the desired ievel of product purity.
  • a protein-containing product of a recombinant fermentation reaction can be initially purified in an affinity or ion exchange column. After an initial pass through the column, the protein product is only partially purified, and the solution still contains contaminants such as remnants of the cell culture and other proteins. Prior to final formulation into a pharmaceutical product, the bulk solution must be further processed to obtain the protein in a satisfactory purity.
  • the solution e.g. elution buffer
  • the "bulk" solution recovered from first pass purification treatment can comprise a solution having a high concentration of monovalent salts, normally sodium chloride but potentially potassium chloride, or other salts.
  • the storage of a "bulk" solution of recombinant protein poses unique challenges due to the high salt concentration and very low protein concentration of the solution.
  • proteins are stored below the glass transition temperature to assure stability, since in the glassy state, protein inactivation and denaturation are extremely slow on a pharmaceutical time scale.
  • the presence of high salt concentration in a solution tends to depress its glass transition temperature, and in solutions with high salt concentration and low protein concentration, very low temperatures are needed to achieve this state,
  • the invention is a method for stabilizing a liquid solution of recombinant protein for frozen storage, which comprises: providing a solution of recombinant protein wherein said solution has a monovalent salt concentration, e.g. of NaCI and/or KCI, of at least 100 mWl; adding a carbohydrate to said solution in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 C or higher; and freezing said solution for storage.
  • a monovalent salt concentration e.g. of NaCI and/or KCI
  • the invention also provides a liquid solution of recombinant protein which is stabilized for frozen storage, which contains a carbohydrate in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 C or higher
  • Figure 1 shows the effect on preserving recombinant protein activity of adding carbohydrate, with or without other excipients as described in the text, to a bulk solution of recombinant Factor VIII.
  • the four different formulations are designated F1 , F2, F3 and F4.
  • Factor VMI activity was assayed after freezing and storage at-30 0 C 1 at different time points as shown, for up for 24 weeks.
  • the bulk solution contained approximately 60OmM NaCi, 1OmM CaCI 2 , 2OmM imidazole and 0.1% Triton X-100, except F3 which was diluted as described herein.
  • the graph plots time against coagulation potency (lU/mL).
  • Figure 2 shows loss of recombinant FVIlI activity after freezing and storage at -70 0 C and -30 0 C of the bulk Factor VIII solution used in the experiments illustrated in Figure 1 but without stabilization excipients.
  • the designation "LN 2 to -7O 0 C” indicates that the samples were frozen in liquid nitrogen prior to storage at -70 0 C
  • "LN 2 to -30 0 C” indicates that the samples were frozen in liquid nitrogen prior to storage at -30 °C.
  • EVA to -70 0 C indicates that the samples were frozen in polymer storage bags and stored at -70 0 C.
  • the liquid solution of recombinant protein may comprise a solution of any recombinant protein obtained from recombinant cell culture using affinity chromatography, ion-exchange chromatography, or the like.
  • the solution is a bulk solution, which comprises a solution which has been partially purified.
  • the liquid solution is a high-salt solution, preferably an aqueous solution.
  • Recombinant proteins include, for example and without limitation, coagulation factors, virus antigens, bacterial antigens, fungal antigens, protozoal antigens, peptide hormones, chemokines, cytokines, growth factors, enzymes, blood proteins such as hemoglobin, ⁇ -1 -antitrypsin, fibrinogen, human serum albumin, prothrombin/thrombin, antibodies, blood coagulation and/or clotting factors, and biologically active fragments thereof; such as Factor V, Factor Vl, Factor VII, Factor VlII and derivatives thereof such as B-domain deleted FVIII, Factor IX, Factor X, Factor Xl, Factor XII, Factor XIIi, Fletcher Factor, Fitzgerald Factor, and von Willebrand Factor; milk proteins such as casein, lactoferrin, lysozyme, ⁇ -1 antitrypsin, protein factors, immune proteins, and biologically active fragments thereof;
  • a "bulk” solution within the meaning of the present invention comprises a partially but not fully purified liquid solution of recombinant protein, which contains at least 100 mM monovalent salt.
  • the monovalent salt is preferably NaCI which is commonly used to elute recombinant proteins from purification columns.
  • the NaCI may be replaced, in whole or in part, with KCf.
  • the bulk solution may also contain varying amounts of other salts, such as divalent salts including calcium chloride.
  • liquid solution has been subjected to at least one purification step, but the liquid solution still contains sufficient residual impurities that at least one further purification step is required prior to final product formulation.
  • a "bulk" solution of recombinant Factor VlII must be further purified prior to final formulation, which in the case of Factor VIiI and other proteins may include lyophilization.
  • the liquid solution contains at least 100 mM monovalent salt, preferably 100 mM NaCI, more preferably at least 300 mM NaCI, more preferably at least 500 mM NaCI, more preferably at least 560 mM NaCI and still more preferably at least 600 mM NaCI. It is not uncommon for bulk solutions of recombinant protein to have this high monovalent salt concentration following an initial purification stage.
  • the liquid solution contains 100-200 mM NaCI, 100-300 mM NaCI, 200-30OmM NaCI, 100-400 mM NaCI, 100-500 mM NaCI, 100-600 mM NaCI, 100-800 mM NaCI, 300-500 mM NaCl, 300-600 mM NaCl , 300-800 mM NaCl, 400-600 mM NaCI, 400-800 mM NaCI, 500-600 mM NaCI, 560- 700 mM NaCI and 500-800 mM NaCI.
  • the "bulk” solutions of the invention are further characterized by their very low protein concentration.
  • the concentration of recombinant protein in the bulk solution can be as low as 0.0001 micromolar, 0.001 micromolar, or 0.01 micromolar.
  • the concentration of recombinant protein in the bulk solution can be as high as 10 micromolar, 1 micromoiar, or 0.1 micromoiar. Any concentration of protein falling within any combination of these upper and lower limits is an embodiment of a "bulk" solution within the meaning of the invention.
  • the carbohydrate is added to the liquid solution, prior to freezing, in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 0 C or higher, more preferably at least -34 0 C, or any temperature expressed by a whole or fractional number therebetween.
  • the normal glass transition temperature of a high salt, low protein bulk solution is substantially less than -56 0 C, e.g. -60 to -70 0 C.
  • the amount of added carbohydrate needed to elevate the glass transition temperature to -56 0 C should take into account, as one factor, the protein concentration. Higher protein concentrations tend to themselves elevate the glass transition temperature of a bulk solution.
  • the amount of carbohydrate should not excessively increase the viscosity of the soiution, and preferably the viscosity is maintained below about 9.0 cP.
  • the conductance of the solution can be changed by carbohydrate addition, and preferably, should be maintained below about 39 mS/cm.
  • Freezing the solution in the context of the present invention, means freezing the bulk liquid solution, and is to be distinguished from freeze-drying, which involves different technical considerations.
  • the carbohydrate can be the type of carbohydrate normally used in pharmaceutical formulations, including sugars and di- , oligo- and poly- saccharides. Examples include dextrans, cyclodextrans, chitosans, starches, halyuronic acids, cellulose, raffinose, maltose, lactose, stachyose, and combinations thereof. Preferred examples are carbohydrates which are approved for injection, which includes sucrose, trehalose, hydroxyethylstarch, dextran, or combinations thereof. Pharmaceutical grade carbohydrates are available commercially from a number of suppliers.
  • carbohydrate needed to protect the solution during freezing can be readily determined, for example by differential scanning calorimetry, and depends on the particular protein and the particular carbohydrate. Currently preferred amounts of carbohydrate are 8-25% (w/w) based on weight of liquid soiution. [024] In specific embodiments of the invention, the amounts of carbohydrate are about 8-15%, 12-20%, 16-20%, 15-25%, and 20-25% (w/w) based on weight of liquid solution,
  • ком ⁇ онентs from the initial purification may be present in a bulk solution, including a surfactant (e.g. Tween 80 or Triton-X), calcium chloride, or imidazole.
  • a surfactant e.g. Tween 80 or Triton-X
  • calcium chloride e.g. calcium chloride
  • imidazole e.g. imidazole
  • excipients can be added to the liquid solutions. As shown in the below formulations, additional surfactant may be added as an excipient. As a further excipient, an amino acid (e.g. glycine) may be added.
  • recombinant Factor VIII is illustrated using, as exemplary recombinant protein, recombinant Factor VIII.
  • Recombinant Factor VIH is produced using methods known in the art, for example as described in US Pat. Nos. 5,576,194; 5,804,420; and 5,733,873.
  • recombinant Factor VIII is produced in mammalian cells in large-scale fermentation reactors, in media which can be serum- free and/or protein free.
  • the recombinant Factor VIII is secreted into the media by the recombinant cells.
  • Recombinant Factor VMS (full length) was expressed from host cells and purified from clarified tissue culture fluid by membrane adsorber chromatography.
  • the membrane adsorber process isolates and concentrates recombinant Factor VlII from the tissue culture fluid by binding and elution (generally as described in Suck et ai., J. Biotechnology, 121 : 361-367, 2006.)
  • the eluate was divided into four batches and each batch was transferred into a sterile bottle (400 mL in each bottle).
  • a sterile bottle 400 mL in each bottle
  • the eluate (bulk solution) contained approximately 60OmM NaCI, 1OmM CaCI 2 , 2OmM imidazole and 0.1 % Triton X-100.
  • the concentration of recombinant Factor VIII in the eluate was approximately 0.067 micromolar.
  • Formulation 3 was prepared from the same eiuate but was diluted with a buffer containing 20 mM imidazole and 10 tnM CaCI 2 to decrease the NaCI concentration by half. This dilution was performed to examine the applicability of the process of the invention to solution having a lower, but stili relatively high, monovalent salt concentration.
  • the glass transition temperature of each sample was determined using Differential Scanning Caiorimetry (DuPont Modulated DSC).
  • the glass transition temperatures exhibited by Formulations I, 2, 3 and 4 were, respectively, -56 0 C, - 52.1 0 C, -34.9 0 C and -35.5 ° C, In each case, the glass transition temperature is significantly higher than the glass transition temperature observed in the absence of an added carbohydrate (which for the same bulk solution without carbohydrate was determined to be between -60 and -70 0 C).
  • the viscosities of the formulations were: Formulation 1 : 1.8428 cP; Formulation 2: 3.1089 cP; Formulation 3: 6.8076 cP; and Formulation 4: 7.2123 cP.
  • the conductivities of the formulations were: Formulation 1: 27.8 mS/cm; Formulation 2: 25.57 mS/cm; Formulation 3: 21.05 mS/cm; and Formulation 4: 32.1 mS/cm.
  • AtI of the formulations were found to be stable after frozen storage at -80, -30 -18 and -14 0 C up to 24 weeks, as determined by coagulation assay for Factor VIII activity at various time points, without significant loss of activity.

Abstract

The invention relates to a method for stabilizing a bulk solution of recombinant protein for frozen storage, which comprises providing a partially-purified solution of recombinant protein which has a monovalent salt concentration of at least 100 mM, and adding a carbohydrate to said solution in an amount sufficient that, upon freezing, the solution has a glass transition temperature of -56 °C or higher.

Description

STABfLlZATlON OF LIQUID SOLUTIONS OF RECOMBINANT PROTEiN FOR
FROZEN STORAGE
[001] This application claims benefit of Provisional Patent application 60/926,698, filed April 26, 2007, the disclosure of which is incorporated by reference.
FIELD OF THE INVENTION
[002] The invention relates to the freezing and storage of liquid solutions of recombinant protein, preferably bulk solutions.
BACKGROUND OF THE INVENTION
[003] Production of recombinant proteins in cell culture normally involves a series of purification steps, by which the desired protein product is recovered from recombinant host cells and/or the associated culture media. Many important recombinant proteins are produced on a large commercial scale. In the case of pharmaceutical proteins, for example, it is not uncommon for more than one purification stage to be used to achieve the desired ievel of product purity.
[004] It can be necessary to store a bulk solution of recombinant protein which has been initially purified, but not finally purified, prior to final purification for formulation. For example, a protein-containing product of a recombinant fermentation reaction can be initially purified in an affinity or ion exchange column. After an initial pass through the column, the protein product is only partially purified, and the solution still contains contaminants such as remnants of the cell culture and other proteins. Prior to final formulation into a pharmaceutical product, the bulk solution must be further processed to obtain the protein in a satisfactory purity.
[005] Normally the solution, e.g. elution buffer, which is used to recover the protein from a first-pass purification treatment is a high salt solution. In the case of elution from a column, a high salt concentration is needed to release the protein from the column. Accordingly, the "bulk" solution recovered from first pass purification treatment can comprise a solution having a high concentration of monovalent salts, normally sodium chloride but potentially potassium chloride, or other salts. [006] The storage of a "bulk" solution of recombinant protein poses unique challenges due to the high salt concentration and very low protein concentration of the solution. Ideally, proteins are stored below the glass transition temperature to assure stability, since in the glassy state, protein inactivation and denaturation are extremely slow on a pharmaceutical time scale. On the other hand, the presence of high salt concentration in a solution tends to depress its glass transition temperature, and in solutions with high salt concentration and low protein concentration, very low temperatures are needed to achieve this state,
[007] Frozen storage at higher temperature is desirable for bulk solutions in large volume quantities for cost and efficiency reasons, but while preserving the stability and activity of the protein,
SUMMARY OF THE INVENTION
[008] The invention is a method for stabilizing a liquid solution of recombinant protein for frozen storage, which comprises: providing a solution of recombinant protein wherein said solution has a monovalent salt concentration, e.g. of NaCI and/or KCI, of at least 100 mWl; adding a carbohydrate to said solution in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 C or higher; and freezing said solution for storage.
[009] The invention also provides a liquid solution of recombinant protein which is stabilized for frozen storage, which contains a carbohydrate in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 C or higher
DESCRIPTION OF THE FIGURES
[010] Figure 1 shows the effect on preserving recombinant protein activity of adding carbohydrate, with or without other excipients as described in the text, to a bulk solution of recombinant Factor VIII. The four different formulations are designated F1 , F2, F3 and F4. Factor VMI activity was assayed after freezing and storage at-30 0C1 at different time points as shown, for up for 24 weeks. Before addition of carbohydrate, the bulk solution contained approximately 60OmM NaCi, 1OmM CaCI2, 2OmM imidazole and 0.1% Triton X-100, except F3 which was diluted as described herein. The graph plots time against coagulation potency (lU/mL). ton] Figure 2 shows loss of recombinant FVIlI activity after freezing and storage at -70 0C and -30 0C of the bulk Factor VIII solution used in the experiments illustrated in Figure 1 but without stabilization excipients. The designation "LN2 to -7O0C" indicates that the samples were frozen in liquid nitrogen prior to storage at -70 0C, and "LN2 to -300C" indicates that the samples were frozen in liquid nitrogen prior to storage at -30 °C. 0EVA to -700C" indicates that the samples were frozen in polymer storage bags and stored at -70 0C.
DESCRIPTION OF PREFERRED EMBODIMENTS
[012] The liquid solution of recombinant protein may comprise a solution of any recombinant protein obtained from recombinant cell culture using affinity chromatography, ion-exchange chromatography, or the like. In a preferred embodiment, the solution is a bulk solution, which comprises a solution which has been partially purified. In all embodiments, the liquid solution is a high-salt solution, preferably an aqueous solution.
[013] Recombinant proteins include, for example and without limitation, coagulation factors, virus antigens, bacterial antigens, fungal antigens, protozoal antigens, peptide hormones, chemokines, cytokines, growth factors, enzymes, blood proteins such as hemoglobin, α-1 -antitrypsin, fibrinogen, human serum albumin, prothrombin/thrombin, antibodies, blood coagulation and/or clotting factors, and biologically active fragments thereof; such as Factor V, Factor Vl, Factor VII, Factor VlII and derivatives thereof such as B-domain deleted FVIII, Factor IX, Factor X, Factor Xl, Factor XII, Factor XIIi, Fletcher Factor, Fitzgerald Factor, and von Willebrand Factor; milk proteins such as casein, lactoferrin, lysozyme, α-1 antitrypsin, protein factors, immune proteins, and biologically active fragments thereof; and antibodies, including monoclonal antibodies, single chain antibodies, antibody fragments, chimeric antibodies, humanized antibodies, and other antibody variant molecules which can be produced in recombinant cell culture. [014] A currently preferred recombinant protein is recombinant Factor VIH. Factor ViII as used herein includes engineered variants of Factor VIM, such as B-domain deleted variants of Factor VlII.
[015j A "bulk" solution within the meaning of the present invention comprises a partially but not fully purified liquid solution of recombinant protein, which contains at least 100 mM monovalent salt. The monovalent salt is preferably NaCI which is commonly used to elute recombinant proteins from purification columns. However, the NaCI may be replaced, in whole or in part, with KCf. The bulk solution may also contain varying amounts of other salts, such as divalent salts including calcium chloride.
[016] By "partially but not fully purified" is meant the liquid solution has been subjected to at least one purification step, but the liquid solution still contains sufficient residual impurities that at least one further purification step is required prior to final product formulation. For example, a "bulk" solution of recombinant Factor VlII must be further purified prior to final formulation, which in the case of Factor VIiI and other proteins may include lyophilization.
[017] The liquid solution contains at least 100 mM monovalent salt, preferably 100 mM NaCI, more preferably at least 300 mM NaCI, more preferably at least 500 mM NaCI, more preferably at least 560 mM NaCI and still more preferably at least 600 mM NaCI. it is not uncommon for bulk solutions of recombinant protein to have this high monovalent salt concentration following an initial purification stage.
[018] In further embodiments of the invention, the liquid solution contains 100-200 mM NaCI, 100-300 mM NaCI, 200-30OmM NaCI, 100-400 mM NaCI, 100-500 mM NaCI, 100-600 mM NaCI, 100-800 mM NaCI, 300-500 mM NaCl, 300-600 mM NaCl , 300-800 mM NaCl, 400-600 mM NaCI, 400-800 mM NaCI, 500-600 mM NaCI, 560- 700 mM NaCI and 500-800 mM NaCI.
[019] The "bulk" solutions of the invention are further characterized by their very low protein concentration. In embodiments of the invention, the concentration of recombinant protein in the bulk solution can be as low as 0.0001 micromolar, 0.001 micromolar, or 0.01 micromolar. In embodiments of the invention, the concentration of recombinant protein in the bulk solution can be as high as 10 micromolar, 1 micromoiar, or 0.1 micromoiar. Any concentration of protein falling within any combination of these upper and lower limits is an embodiment of a "bulk" solution within the meaning of the invention.
{020J The carbohydrate is added to the liquid solution, prior to freezing, in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 0C or higher, more preferably at least -34 0C, or any temperature expressed by a whole or fractional number therebetween. The normal glass transition temperature of a high salt, low protein bulk solution is substantially less than -56 0C, e.g. -60 to -70 0C. The amount of added carbohydrate needed to elevate the glass transition temperature to -56 0C should take into account, as one factor, the protein concentration. Higher protein concentrations tend to themselves elevate the glass transition temperature of a bulk solution. As other factors, the amount of carbohydrate should not excessively increase the viscosity of the soiution, and preferably the viscosity is maintained below about 9.0 cP. The conductance of the solution can be changed by carbohydrate addition, and preferably, should be maintained below about 39 mS/cm.
[021] Freezing the solution, in the context of the present invention, means freezing the bulk liquid solution, and is to be distinguished from freeze-drying, which involves different technical considerations.
[022] The carbohydrate can be the type of carbohydrate normally used in pharmaceutical formulations, including sugars and di- , oligo- and poly- saccharides. Examples include dextrans, cyclodextrans, chitosans, starches, halyuronic acids, cellulose, raffinose, maltose, lactose, stachyose, and combinations thereof. Preferred examples are carbohydrates which are approved for injection, which includes sucrose, trehalose, hydroxyethylstarch, dextran, or combinations thereof. Pharmaceutical grade carbohydrates are available commercially from a number of suppliers.
[023] The precise amount of carbohydrate needed to protect the solution during freezing can be readily determined, for example by differential scanning calorimetry, and depends on the particular protein and the particular carbohydrate. Currently preferred amounts of carbohydrate are 8-25% (w/w) based on weight of liquid soiution. [024] In specific embodiments of the invention, the amounts of carbohydrate are about 8-15%, 12-20%, 16-20%, 15-25%, and 20-25% (w/w) based on weight of liquid solution,
[025] Other components from the initial purification (e.g. elution) may be present in a bulk solution, including a surfactant (e.g. Tween 80 or Triton-X), calcium chloride, or imidazole. Other excipients can be added to the liquid solutions. As shown in the below formulations, additional surfactant may be added as an excipient. As a further excipient, an amino acid (e.g. glycine) may be added.
EXAMPLES
[026] The invention is illustrated using, as exemplary recombinant protein, recombinant Factor VIII. Recombinant Factor VIH is produced using methods known in the art, for example as described in US Pat. Nos. 5,576,194; 5,804,420; and 5,733,873. In preferred embodiments, recombinant Factor VIII is produced in mammalian cells in large-scale fermentation reactors, in media which can be serum- free and/or protein free. Preferably the recombinant Factor VIII is secreted into the media by the recombinant cells.
[027] Recombinant Factor VMS (full length) was expressed from host cells and purified from clarified tissue culture fluid by membrane adsorber chromatography. The membrane adsorber process isolates and concentrates recombinant Factor VlII from the tissue culture fluid by binding and elution (generally as described in Suck et ai., J. Biotechnology, 121 : 361-367, 2006.) The eluate was divided into four batches and each batch was transferred into a sterile bottle (400 mL in each bottle). In addition to recombinant Factor VIiI and residua! impurities which remain, the eluate (bulk solution) contained approximately 60OmM NaCI, 1OmM CaCI2, 2OmM imidazole and 0.1 % Triton X-100. The concentration of recombinant Factor VIII in the eluate was approximately 0.067 micromolar.
[028] A carbohydrate or combination of carbohydrates, along with other excipients as indicated, were then added to each bottle at room temperature in the amounts shown as Formulations I, 2, 3 and 4 in Table 1 below. TABLE 1
Figure imgf000008_0001
[029] All components in Table 1 are shown in percent by weight based on weight of solution. Carbohydrates and other excipients were obtained commercially. Each fresh formulated batch was sampled. Samples from Formulations (1 ), (2), (3) and (4) were assessed for Factor VIII activity using a standard coagulation assay.
[030] Formulation 3 was prepared from the same eiuate but was diluted with a buffer containing 20 mM imidazole and 10 tnM CaCI2 to decrease the NaCI concentration by half. This dilution was performed to examine the applicability of the process of the invention to solution having a lower, but stili relatively high, monovalent salt concentration.
[031] The glass transition temperature of each sample was determined using Differential Scanning Caiorimetry (DuPont Modulated DSC). The glass transition temperatures exhibited by Formulations I, 2, 3 and 4 were, respectively, -56 0C, - 52.1 0C, -34.9 0C and -35.5 ° C, In each case, the glass transition temperature is significantly higher than the glass transition temperature observed in the absence of an added carbohydrate (which for the same bulk solution without carbohydrate was determined to be between -60 and -70 0C). The viscosities of the formulations were: Formulation 1 : 1.8428 cP; Formulation 2: 3.1089 cP; Formulation 3: 6.8076 cP; and Formulation 4: 7.2123 cP. The conductivities of the formulations were: Formulation 1: 27.8 mS/cm; Formulation 2: 25.57 mS/cm; Formulation 3: 21.05 mS/cm; and Formulation 4: 32.1 mS/cm. [032] AtI of the formulations were found to be stable after frozen storage at -80, -30 -18 and -14 0C up to 24 weeks, as determined by coagulation assay for Factor VIII activity at various time points, without significant loss of activity.
[033] As shown in Figure 1 , all four formulations in accordance with the invention maintained Factor VIiI coagulation activity at substantially the initial level after storage at -30 0C for up to 24 weeks,
[034] As shown in Figure 2, in the absence of the added excipients, the solution lost substantially all of its Factor VIII coagulation activity after only 1 day of storage at -30 0C.

Claims

What is claimed is:
1. A method for stabilizing a liquid solution of recombinant protein for frozen storage, which comprises: a. providing a liquid solution of recombinant protein wherein said solution has an NaC! and/or KC! concentration of at least 10O mM; b. adding a carbohydrate to said solution in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 0C or higher; and c. freezing said solution for storage.
2. A method as claimed in claim 1 , wherein the liquid solution of recombinant protein is a bulk solution.
3. A method as claimed in claim 1 , wherein the solution has an NaCI concentration of at least 100 mM.
4. A method as claimed in claim 1 , wherein the carbohydrate is added in an amount of 8-25% by weight, based on weight of solution.
5. A method as claimed in claim 1 , wherein the carbohydrate is selected from the group consisting of sucrose, trehalose, hydroxyethylstarch, dextran, and combinations thereof.
6. A method as claimed in claim 1 , wherein the bulk solution has an NaCI concentration of at least 300 mM.
7. A method as claimed in ciaim 1 , wherein the bulk solution has an NaCl concentration of at least 600 mM.
8. A method as claimed in claim 1 , wherein the recombinant protein is Factor VIII.
9. A method as claimed in claim 1 , wherein the glass transition temperature is between -56 0C and -35 0C.
10. A method as claimed in claim 1 , further comprising adding an amino acid and/or a surfactant.
11. A liquid solution of recombinant protein which is stabilized for frozen storage, and which has an NaCI and/or KCI concentration of at least 100 mM, which contains a carbohydrate in an amount sufficient to provide the solution, upon freezing, with a glass transition temperature of -56 0C or higher.
12. The liquid solution of recombinant protein as claimed in claim 1 1 , which comprises a bulk solution.
13. The liquid soiution of claim 11 , having an NaCI concentration of at least 100 mM.
14. A liquid solution of recombinant protein as claimed in claim 11 , containing carbohydrate in an amount of 8-25% by weight, based on weight of solution.
15. A liquid soiution of recombinant protein as claimed in claim 1 1 , wherein the carbohydrate is selected from the group consisting of sucrose, trehalose, hydroxyethy! starch, dextran, and combinations thereof.
16. A liquid solution of recombinant protein as claimed in claim 11 , wherein the liquid solution has an NaCI concentration of at least 300 mM.
17. A liquid solution of recombinant protein as claimed in claim 11 , wherein the liquid solution has an NaCI concentration of at least 600 mM.
18. A liquid solution of recombinant protein as claimed in claim 11 , wherein the recombinant protein is Factor VIII.
19. A liquid solution of recombinant protein as claimed in claim 11 , wherein the glass transition temperature is between -56 0C and -35 0C.
20. A liquid solution of recombinant Factor VIII, which contains an NaCi concentration of at least 100 mM, contains recombinant Factor VIII at a concentration between 0.0001 micromolar and 10 micromolar, and is stabilized for frozen storage by addition of a carbohydrate.
PCT/US2008/061147 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage WO2008134310A1 (en)

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RU2009143493/15A RU2469739C2 (en) 2007-04-26 2008-04-22 Stabilisation of liquid solutions of recombinant protein for storing in frozen state
JP2010506432A JP5401446B2 (en) 2007-04-26 2008-04-22 Stabilization of recombinant protein solutions for cryopreservation
AU2008245821A AU2008245821B2 (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage
CA2683317A CA2683317C (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage
US12/597,333 US8187799B2 (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage
MX2009011367A MX2009011367A (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage.
EP08746549.8A EP2150264A4 (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage
BRPI0810500-6A2A BRPI0810500A2 (en) 2007-04-26 2008-04-22 STABILIZATION OF RECOMBINANT PROTEIN LIQUID SOLUTIONS FOR FREEZING STATE
CN200880013538.9A CN101678066B (en) 2007-04-26 2008-04-22 Stabilization of liquid solutions of recombinant protein for frozen storage
KR1020157023163A KR20150103333A (en) 2007-04-26 2008-04-22 STABILIZATlON OF LIQUID SOLUTIONS OF RECOMBINANT PROTEIN FOR FROZEN STORAGE
IL201508A IL201508A0 (en) 2007-04-26 2009-10-14 Stabilization of liquid solutions of recombinant protein for frozen storage
US13/459,806 US8536125B2 (en) 2007-04-26 2012-04-30 Stabilization of liquid solutions of recombinant protein for frozen storage

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160108553A (en) 2014-01-20 2016-09-19 옥타파마 아게 A process for manufacturing Factor VIII having an improved ratio of FVIII:C/FVIII:Ag

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8883146B2 (en) 2007-11-30 2014-11-11 Abbvie Inc. Protein formulations and methods of making same
NZ586383A (en) * 2007-12-28 2012-11-30 Baxter Int Recombinant vwf formulations
US11197916B2 (en) 2007-12-28 2021-12-14 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
EP2601932A1 (en) * 2008-10-21 2013-06-12 Baxter International Inc. Lyophilized recombinant VWF formulations
JP2012526121A (en) * 2009-05-04 2012-10-25 アボツト・バイオテクノロジー・リミテツド Stable high protein concentration formulation of human anti-TNF alpha antibody
MX344727B (en) 2010-11-11 2017-01-05 Abbvie Biotechnology Ltd IMPROVED HIGH CONCENTRATION ANTI-TNFa ANTIBODY LIQUID FORMULATIONS.
JP5892303B1 (en) 2014-06-13 2016-03-23 凸版印刷株式会社 Manufacturing method of needle-shaped body

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5278289A (en) * 1991-11-12 1994-01-11 Johnson Alan J Antihemophilic factor stabilization
US5576194A (en) 1986-07-11 1996-11-19 Bayer Corporation Recombinant protein production
US5733873A (en) 1992-10-02 1998-03-31 Pharmacia & Upjohn Ab Composition comprising coagulation factor VIII formulation, process for its preparation and use of a surfactant as stabilizer
US5804420A (en) 1997-04-18 1998-09-08 Bayer Corporation Preparation of recombinant Factor VIII in a protein free medium
US20040116345A1 (en) 1999-02-22 2004-06-17 Marc Besman Novel albumin-free factor VIII formulations
US20040248793A1 (en) * 2002-06-21 2004-12-09 Jensen Michael Bech Stabilised solid compositions of factor VII polypeptides
US20040248778A1 (en) * 2001-10-05 2004-12-09 Oliver Gloger Stable galenic freeze-dried pharmaceutical preparation of recombined carbohydrate-binding polypeptides

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4457916A (en) * 1982-08-31 1984-07-03 Asahi Kasei Kogyo Kabushiki Kaisha Method for stabilizing Tumor Necrosis Factor and a stable aqueous solution or powder containing the same
JPS59134730A (en) * 1983-01-20 1984-08-02 Green Cross Corp:The Heat treatment of viii factor of blood clotting
US5605884A (en) * 1987-10-29 1997-02-25 Rhone-Poulenc Rorer Pharmaceuticals Inc. Factor VIII formulations in high ionic strength media
CA1329760C (en) * 1987-10-29 1994-05-24 Ted C. K. Lee Plasma and recombinant protein formulations in high ionic strength media
US4877608A (en) * 1987-11-09 1989-10-31 Rorer Pharmaceutical Corporation Pharmaceutical plasma protein formulations in low ionic strength media
DE4111393A1 (en) * 1991-04-09 1992-10-15 Behringwerke Ag STABILIZED FACTOR VIII PREPARATIONS
US5576291A (en) * 1993-09-13 1996-11-19 Baxter International Inc. Activated factor VIII as a therapeutic agent and method of treating factor VIII deficiency
IL113010A (en) * 1994-03-31 1999-10-28 Pharmacia & Upjohn Ab Pharmaceutical formulation comprising factor viii with an activity of at least 500iu/ml and an enhancer for improved subcutaneous intramuscular or intradermal administration
US6818439B1 (en) * 1994-12-30 2004-11-16 Chiron Corporation Methods for administration of recombinant gene delivery vehicles for treatment of hemophilia and other disorders
US6790439B1 (en) * 1995-06-07 2004-09-14 Zymogenetics, Inc. Thrombopoietin compositions
SE9503380D0 (en) * 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
US5770700A (en) 1996-01-25 1998-06-23 Genetics Institute, Inc. Liquid factor IX formulations
US5763401A (en) * 1996-07-12 1998-06-09 Bayer Corporation Stabilized albumin-free recombinant factor VIII preparation having a low sugar content
AT407255B (en) * 1997-06-20 2001-02-26 Immuno Ag RECOMBINANT CELL CLONE WITH INCREASED STABILITY IN SERUM- AND PROTEIN-FREE MEDIUM AND METHOD FOR OBTAINING THE STABLE CELL CLONE
BR0012429A (en) * 1999-07-15 2002-09-17 Genetics Inst Formulations for il-11
JP2005501851A (en) 2001-08-13 2005-01-20 ハーグリーブス リレイ，マイケル Composition for removing toxins
KR100560697B1 (en) 2003-08-06 2006-03-16 씨제이 주식회사 Formulation of albumin-free erythropoietin

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576194A (en) 1986-07-11 1996-11-19 Bayer Corporation Recombinant protein production
US5278289A (en) * 1991-11-12 1994-01-11 Johnson Alan J Antihemophilic factor stabilization
US5733873A (en) 1992-10-02 1998-03-31 Pharmacia & Upjohn Ab Composition comprising coagulation factor VIII formulation, process for its preparation and use of a surfactant as stabilizer
US5804420A (en) 1997-04-18 1998-09-08 Bayer Corporation Preparation of recombinant Factor VIII in a protein free medium
US20040116345A1 (en) 1999-02-22 2004-06-17 Marc Besman Novel albumin-free factor VIII formulations
US20040248778A1 (en) * 2001-10-05 2004-12-09 Oliver Gloger Stable galenic freeze-dried pharmaceutical preparation of recombined carbohydrate-binding polypeptides
US20040248793A1 (en) * 2002-06-21 2004-12-09 Jensen Michael Bech Stabilised solid compositions of factor VII polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP2150264A4
SUCK ET AL., J. BIOTECHNOLOGY, vol. 121, 2006, pages 361 - 367

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160108553A (en) 2014-01-20 2016-09-19 옥타파마 아게 A process for manufacturing Factor VIII having an improved ratio of FVIII:C/FVIII:Ag
US10822393B2 (en) 2014-01-20 2020-11-03 Octapharma Ag Process for manufacturing factor VIII having an improved ratio of FVIII:C/FVIII/AG

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US8187799B2 (en) 2012-05-29
CN101678066A (en) 2010-03-24
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US20100113744A1 (en) 2010-05-06
CN101678066B (en) 2014-09-24
AU2008245821B2 (en) 2013-07-04
CA2683317C (en) 2014-12-16
CA2683317A1 (en) 2008-11-06
HK1198815A1 (en) 2015-06-12
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ZA200907247B (en) 2010-08-25
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