[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2013096835A1 - Formulations stables de protéine - Google Patents

Formulations stables de protéine Download PDF

Info

Publication number
WO2013096835A1
WO2013096835A1 PCT/US2012/071368 US2012071368W WO2013096835A1 WO 2013096835 A1 WO2013096835 A1 WO 2013096835A1 US 2012071368 W US2012071368 W US 2012071368W WO 2013096835 A1 WO2013096835 A1 WO 2013096835A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
formulation
concentration
polyol
formulations
Prior art date
Application number
PCT/US2012/071368
Other languages
English (en)
Inventor
Wolfgang Fraunhofer
Vineet Kumar
Original Assignee
Abbvie Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbvie Inc. filed Critical Abbvie Inc.
Publication of WO2013096835A1 publication Critical patent/WO2013096835A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • therapeutic proteins such as antibodies
  • stability e.g., suitability for administration, concentration, and manufacturability.
  • therapeutic proteins During manufacturing, storage, and delivery, therapeutic proteins have been known to undergo physical and chemical degradations. These instabilities can reduce the potency of the protein and increase the risk of adverse events in patients, and, therefore, significantly impact regulatory approval (see, e.g., Wang, et al. (2007) J Pharm Sci 96: 1).
  • a stable protein formulation is essential to the safety and efficacy of a successful therapeutic protein drug product.
  • concentration formulations are desirable as they can impact the mode ⁇ e.g., intravenous vs. subcutaneous) and frequency of administration of the drug to a subject (eg., daily vs. weekly). Furthermore, access to high concentrations can reduce the injection volume and thus foster feasibility of user-friendly delivery devices such as pens, autoinjectors, or micro-needle mediated transdermal delivery devices, and thus increase treatment adherence and patient convenience.
  • Timasheff and coworkers ⁇ see, e.g., Lee JC and Timasheff SN. J Biol Chem 256:7193-7201 (1982); Arakawa T. and Timasheff S.N. Biochemistry 21:6536-6544 (1982); Timasheff SN. Adv Protein Chem 51:355-432 (1998); Timasheff SN. Methods Mol Biol 40:253-269 (1995); and Arakawa T. and Timasheff S.N. Biophys J. 47:411-414 (1985)). Manning et al. further state that "the application of this approach as a general formulation strategy was only beginning to emerge at that time.
  • polyols such as sorbitol are often used to stabilize parenterals such as, for example, a number of lyophilized vaccine pharmaceuticals such as MumpsvaxTM, MeruvaxTM II and AttenuvaxTM or intravenous administrable solutions such as CardeneTM.
  • Stability of protein formulations is primarily determined by the conformational and the colloidal stability of the protein molecules. It is generally believed that the first step in non-native protein aggregation, which is the most prevalent form of aggregation, is a slight perturbation of the molecular structure, e.g., a partial unfolding of the protein, i.e., a conformational change. This is determined by the conformational stability of the protein. In the second step, the partially unfolded molecules then come into close proximity, being driven by diffusion and random Brownian motion, to form aggregates. This second step is primarily governed by the colloidal stability of the molecules (see Chi et al.
  • DSC Differential scanning calorimetry
  • DSF differential scanning fluorimetry
  • Polyols by raising the Tm of protein formulations as determined by DSC and DSF, are considered to be beneficial for enhancing the stability of the formulations during long-term, shelf stable storage conditions (see Burton et al., Use of Microcalorimetry and Its Correlation with Size Exclusion Chromatography for Rapid Screening of the Physical Stability of Large Pharmaceutical Proteins in Solution, Pharm Dev Technol, 2007; 12(3) 265-273).
  • the present invention is based, at least in part, on the surprising discovery that at particular protein concentrations, polyols are destabilizing to protein formulations.
  • the present invention provides a means to develop protein formulations with maximal stability, thus maintaining functionality of the pharmaceutical protein over the desired time frame and reducing patient risk and efficacy failures due to delivering suboptimal protein formulations.
  • the invention provides a protein formulation having enhanced shelf-life stability relative to a first protein formulation that comprises a polyol at a first polyol concentration, wherein the protein formulation comprises a protein at a concentration below a Reverse Stability Concentration for the protein in the first protein formulation; and no polyol or a polyol at a concentration below the first polyol concentration.
  • the protein concentration in the protein formulation is greater than the protein concentration in the first protein formulation.
  • the formulations are low ionic formulations, e.g., comprise a buffer, such as a phosphate buffer, a citrate buffer, a citrate and phosphate buffer, a histidine buffer, an acetate buffer, or a succinate buffer, and do not comprise additional ionic excipient(s), such as NaCl.
  • the formulation is substantially free of protein aggregation in a liquid form at 5°C for a period of time selected from the group consisting of at least 3 months, at least 6 months, at least 12 months, at least 18 months and at least 24 months.
  • aspects of the invention feature formulations with protein aggregation at a level less than 10% protein aggregation, less than 5% protein aggregation, less than 2% protein aggregation or less than 1% aggregation following storage at room temperature for at least 3 months.
  • the polyol concentration is below about 200 mg/ml, below about 120 mg/ml, below about 80 mg/ml or below about 40 mg/ml.
  • the Reverse Stability Concentration of the protein is between about 100 mg/mL and 150 mg/mL. In another embodiment, the Reverse Stability Concentration of the protein is between about 125 mg/mL and 135 mg/mL.
  • the protein concentration is at least about 25 mg/mL, at least about 50 mg/ml, at least about 100 mg/ml or at least about 150 mg.
  • Additional aspects of the invention include protein formulations having one or more distinct proteins, wherein at least one of the proteins is a therapeutic protein.
  • the protein is an antibody, or an antigen-binding portion thereof.
  • the antibody, or an antigen-binding portion thereof comprises a lambda light chain or a kappa light chain.
  • the protein formulation contains a polyol that is sucrose, trehalose, mannitol and/or sorbitol.
  • Additional aspects of the invention include methods of treating a disorder in a subject by administering to the subject a protein formulation of the invention.
  • a method of determining the Reverse Stability Concentration of a protein includes preparing a series of formulations comprising a protein and a polyol, wherein the protein concentration in the formulations is constant and the polyol concentration is varied over a range of
  • a method of detecting the Reverse Stability Concentration of a protein in a formulation comprising a polyol includes preparing a first series of formulations comprising a protein at a first protein concentration and a polyol, wherein the polyol concentration is varied over a range, preparing a second series of formulations comprising a protein at a second protein concentration that is higher than the first protein concentration and a polyol, wherein the polyol concentration is varied over a range, and wherein the
  • concentration of other solutes in the first series of formulations and second series of formulations is the same, exposing the first series of formulations and the second series of formulations to a test condition, measuring the level of protein aggregation in the first series of formulations and the second series of formulations after exposure to the test condition; and determining the change in the level of aggregation in each series of formulations from lowest to highest polyol concentration, wherein an increase in the level of aggregation in the first series of formulations and a decrease in the level of aggregation in the second series indicates that the Reverse Stability Concentration is between the first protein concentration and the second protein concentration for the protein in a protein formulation with a polyol.
  • Concentration of a protein in a formulation comprising a polyol at a given ionic strength includes preparing a first formulation and a second formulation, wherein the concentration of polyol is lower in the first formulation than in the second formulation, and wherein the concentration of the protein in the first formulation and second formulation is the same, exposing the first and second formulations to a test condition; and measuring protein aggregation in the first and second formulations after testing, wherein greater protein aggregation in the second formulation as compared to the first formulation indicates that a Reverse Stability Concentration exists for the protein in the formulation comprising the polyol.
  • the formulations are low ionic formulations, e.g., comprise a buffer, such as a phosphate buffer, a citrate buffer, a citrate and phosphate buffer, a histidine buffer, an acetate buffer, or a succinate buffer, and do not comprise additional ionic excipient(s), such as NaCl.
  • the test condition comprises storage at a temperature of 5°C, 40°C or 50°C. In one embodiment, the test condition comprises storage for a period of time of at least about 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.
  • protein aggregation is measured by size exclusion HPLC, UPLC, or nephelometry.
  • the protein is a therapeutic protein.
  • the protein is an antibody, or an antigen-binding portion thereof.
  • the antibody, or an antigen-binding portion thereof comprises a lambda light chain or a kappa light chain.
  • aspects of the invention include formulations where the protein concentration is below about 125 mg/ml. Aspects of the invention include formulations where the protein concentration is below about 100 mg/ml. In one embodiment, the protein concentration is below about 80 mg/ml. In one embodiment, the protein concentration is below about 60 mg/ml. In one embodiment, the protein concentration is below about 40 mg/ml. In one embodiment, the polyol is trehalose, mannitol, sucrose and/or sorbitol. In some embodiments, the formulations are low ionic formulations, e.g., comprise a buffer and do not comprise additional ionic excipient(s), such as NaCl.
  • Embodiments of the invention include methods for reducing protein aggregation in a formulation comprising a protein and a polyol, comprising determining a Reverse Stability Concentration for the protein in the formulation, and reducing the concentration of the polyol in the formulation if the protein concentration is lower than the Reverse Stability
  • a method for reducing protein aggregation in a formulation comprising a protein and a polyol comprises selecting a formulation comprising a protein and a polyol, identifying the protein concentration in the formulation as above or below the Reverse Stability Concentration for the protein in the formulation; and reducing the concentration of the polyol when the protein concentration is below the Reverse Stability Concentration for the protein in the formulation, thereby reducing protein aggregation in the formulation.
  • a method for preparing a stable formulation having increased protein concentration as compared to a first formulation comprises selecting a first formulation comprising a protein at a first protein concentration and a polyol at a first polyol concentration, determining a Reverse Stability Concentration for the protein in the first formulation; increasing the protein concentration in the formulation to a concentration that is above the first protein concentration and below the Reverse Stability Concentration; and reducing the concentration of the polyol in the formulation below the first polyol
  • the formulations are low ionic formulations, e.g., comprise a buffer, such as a phosphate buffer, a citrate buffer, a citrate and phosphate buffer, a histidine buffer, an acetate buffer, or a succinate buffer, and do not comprise additional ionic excipient(s), such as NaCl.
  • the polyol concentration is reduced to less than about 200 mg/ml. In some embodiments, the polyol concentration is reduced to less than about 120 mg/ml. In some embodiments, the polyol concentration is reduced to less than about 80 mg/ml. In some embodiments, the polyol concentration is reduced to less than about 40 mg/ml. In some embodiments, the polyol concentration is reduced to the polyol concentration is reduced to 0.
  • the protein formulation is substantially free of protein aggregation in a liquid form at 5°C for a period of time of at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.
  • the protein formulation comprises protein aggregation at a level of less than 10% protein aggregation following storage at room temperature for at least 3 months.
  • the protein formulation comprises protein aggregation at a level of less than 5% protein aggregation following storage at room temperature for at least 3 months.
  • the protein formulation comprises protein aggregation at a level of less than 2% protein aggregation following storage at room temperature for at least 3 months.
  • the protein formulation comprises protein aggregation at a level of less than 1% aggregation following storage at room temperature for at least 3 months.
  • Additional aspects of the invention include methods for reducing protein aggregation in a formulation comprising a protein and a polyol, comprising the steps of selecting a formulation comprising a protein and a polyol, identifying the protein concentration in the formulation as above the Reverse Stability Concentration for the protein in the formulation; and increasing the concentration of the polyol, thereby reducing protein aggregation in the formulation.
  • One embodiment of the invention includes methods for preparing a stable formulation having increased protein concentration as compared to a first formulation, the method comprising: selecting a first formulation comprising a protein at a first protein concentration and a polyol at a first polyol concentration, determining a Reverse Stability Concentration for the protein in the first formulation, increasing the protein concentration in the formulation to a concentration that is above the first protein concentration and above the Reverse Stability Concentration; and increasing the concentration of the polyol in the formulation above the first polyol concentration, thereby preparing a stable formulation having increased protein concentration as compared to the first formulation.
  • Figures 1A and B show the impact of sugar (80 mg/ml trehalose and 40 mg/ml mannitol) on the aggregation of ABT-736 at 100 mg/ml and 200 mg/ml, respectively, following 18m at 5 °C.
  • Figure 2A shows the impact of sucrose concentration (% sucrose) on aggregation at
  • FIG. 1 shows the impact of sucrose concentration (% sucrose) on aggregation at 40°C of ABT-874 formulations of different protein concentrations following 3 m storage. Note that the 1 mg/mL data has been taken from samples stored at 50°C for 14 days.
  • Figures 3A and B show data for ABT-736 and ABT-874, respectively, in terms of protein concentration (X axis) vs. slope (% aggregation vs. % sucrose, over varying sucrose concentrations at a fixed protein concentration, as depicted in Figures 2A and B).
  • Low ionic strength data is from Figures 2A and B while high ionic strength data was obtained from samples stored at 50°C for 9 days.
  • the present invention provides protein formulations, e.g., antibody formulations which are substantially free of protein aggregation and which are, thus, more stable and easier to administer to subjects.
  • the invention further provides methods of preparing stable protein formulations.
  • the present invention is based, at least in part, on the surprising discovery that polyols typically used in protein formulations to stabilize protein may act as effective destabilizers. It was unexpectedly found that below a certain concentration of the protein (i.e. , at protein concentrations below the Reverse Stability Concentration, or RSC), sugars and polyols increase aggregation (e.g. , in solutions of low ionic strength, e.g., solutions comprising a buffer but no additional ionic excipient(s)).
  • RSC Reverse Stability Concentration
  • pharmaceutical formulation and/or “pharmaceutical composition” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable material, composition or vehicle, suitable for administration to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or
  • encapsulating material involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be
  • “Pharmaceutically acceptable excipients” are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed or to maintain stability of the pharmaceutical composition.
  • composition refers to a composition, e.g., an aqueous formulation, that it is useful for treating a disease or disorder.
  • subject is intended to include living organisms, e.g. , prokaryotes and eukaryotes. Examples of subjects include mammals, e.g. , humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In specific embodiments of the invention, the subject is a human.
  • excipient refers to an agent which may be added to a formulation to provide a desired consistency, e.g., altering the bulk properties, to improve stability, and/or to adjust osmolality.
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • a commonly used excipient is a polyol.
  • a "polyol” is a substance with multiple hydroxyl groups, and includes sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. Preferred polyols herein have a molecular weight which is less than about 600 kD.
  • Non-limiting examples of polyols are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, threitol, sorbitol, glycerol, L-gluconate and metallic salts thereof.
  • a "reducing sugar” is one that contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a
  • nonreducing sugar is one that does not have these properties of a reducing sugar.
  • reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose.
  • Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol and glycerol are examples of sugar alcohols.
  • sugar acids these include L-gluconate and metallic salts thereof.
  • the polyol may also act as a tonicity agent.
  • ionic excipient refers to an agent that has a net charge. In one embodiment, the ionic excipient has a net charge under certain formulation conditions, such as pH. Examples of an ionic excipient include, but are not limited to, histidine, arginine, and sodium chloride.
  • non-ionic excipient refers to an agent having no net charge. In one embodiment, the non-ionic excipient has no net charge under certain formulation conditions, such as pH.
  • non-ionic excipients include, but are not limited to, sugars (e.g., sucrose), sugar alcohols (e.g., mannitol), and non-ionic surfactants (e.g., polysorbate 80).
  • lyoprotectant includes agents that provide stability to a protein during water removal during the drying or lyophilisation process, for example, by maintaining the proper conformation of the protein.
  • lyoprotectants include saccharides, in particular di- or trisaccharides. Cryoprotectants may also provide lyoprotectant effects.
  • protein is meant to include a sequence of amino acids for which the chain length is sufficient to produce the higher levels of secondary and/or tertiary and/or quaternary structure.
  • the term protein is intended to include peptides that do not have such structure.
  • the proteins used herein have a molecular weight of at least about 1 kD, 5 kD, 10 kD, 20 kD, 30 kD, 40 kD or more. Examples of proteins encompassed within the definition used herein include therapeutic proteins.
  • a "therapeutically active protein” or “therapeutic protein” refers to a protein which may be used for therapeutic purposes, i.e. , for the treatment of a disorder in a subject.
  • therapeutic proteins may be used for treatment purposes, the invention is not limited to such use, as said proteins may also be used for in vitro studies.
  • the therapeutic protein is a fusion protein or an antibody, or antigen-binding portion thereof.
  • the methods and compositions of the invention comprise at least two distinct proteins, which are defined as two proteins having distinct amino acid sequences. Additional distinct proteins do not include degradation products of a protein.
  • buffer refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
  • the buffers of this invention have a pH ranging from about 4 to about 8; preferably from about 4.5 to about 7; and most preferably has a pH in the range from about 5.0 to about 6.5.
  • the surprising findings of this invention are the consequence of protein-polyol interactions or the interaction of effects exerted on the protein by the presence of a polyol in the composition, e.g. , effects on activity, osmolarity, density, etc.
  • the invention applies to protein formulations for any pH value at which the protein is formulated.
  • buffers that will control the pH in this range include, e.g. , phosphate, acetate (e.g. , sodium acetate), succinate (e.g. , sodium succinate), gluconate, glutamate, histidine, citrate and other organic acid buffers.
  • surfactant generally includes those agents which protect a protein in a formulation from air/solution interface-induced stresses and solution/surface induced- stresses.
  • a surfactant may protect the protein from aggregation.
  • Suitable surfactants may include, e.g. , polysorbates, polyoxyethylene alkyl ethers such as Brij 35.RTM, or poloxamer such as Tween 20, Tween 80, or poloxamer 188.
  • Preferred detergents are poloxamers, e.g. , Poloxamer 188, Poloxamer 407; polyoxyethylene alkyl ethers, e.g.
  • stabilizing agent refers to an excipient that improves or otherwise enhances stability.
  • Stabilizing agents include, but are not limited to, a-lipoic acid, a-tocopherol, ascorbyl palmitate, benzyl alcohol, biotin, bisulfites, boron, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid and its esters, carotenoids, calcium citrate, acetyl-L-camitine, chelating agents, chondroitin, chromium, citric acid, coenzyme Q-10, cysteine, cysteine hydrochloride, 3-dehydroshikimic acid (DHS), EDTA
  • the term "tonicity modifier” is intended to mean a compound or compounds that can be used to adjust the tonicity of a liquid formulation. Suitable tonicity modifiers include glycerin, lactose, mannitol, dextrose, sodium chloride, magnesium sulfate, magnesium chloride, sodium sulfate, sorbitol, trehalose, sucrose, raffinose, maltose and others known to those or ordinary skill in the art. In one embodiment, the tonicity of the liquid formulation approximates that of the tonicity of blood or plasma. As used herein, the term “acidic component” refers to an agent, including a solution, having an acidic pH, i.e., less than 7.0.
  • acidic components include phosphoric acid, hydrochloric acid, acetic acid, citric acid, oxalic acid, succinic acid, tartaric acid, lactic acid, malic acid, glycolic acid and fumaric acid.
  • the aqueous formulation of the invention does not include an acidic component.
  • basic component refers to an agent which is alkaline, i.e., pH greater than 7.0.
  • basic components include potassium hydroxide (KOH) and sodium hydroxide (NaOH)As used herein, the term “antioxidant” is intended to mean an agent which inhibits oxidation and thus is used to prevent the deterioration of preparations by the oxidative process.
  • Such compounds include by way of example and without limitation, acetone, sodium bisulfate, ascorbic acid, ascorbyl palmitate, citric acid, butylated
  • the term "bulking agent” is intended to mean a compound used to add bulk to the reconstitutable solid and/or assist in the control of the properties of the formulation during preparation.
  • Such compounds include, by way of example and without limitation, dextran, trehalose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol,
  • dimethylsulfoxide dimethylsulfoxide, glycerol, albumin, calcium lactobionate, and others known to those of ordinary skill in the art.
  • cryoprotectants generally includes agents, which provide stability to the protein from freezing-induced stresses.
  • cryoprotectants include polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants also contribute to the tonicity of the formulations.
  • a “stable" composition or formulation is one in which the protein, e.g., antibody, therein essentially retains its physical stability and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90.
  • the stability of the protein is determined according to the percentage of monomer protein in the solution, with a low percentage of degraded (e.g. , fragmented) and/or aggregated protein.
  • the formulation is stable at room temperature (about 30 °C) or at 40 °C for at least 1 month and/or stable at about 2-8° C for at least 6 months, or for at least 1 year or for at least 2 years.
  • the formulation is preferably stable following freezing (to, e.g. , -70 °C) and thawing of the formulation, hereinafter referred to as a
  • a protein e.g., an antibody
  • Aggregation is a process whereby individual protein molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the extent that a visible precipitate is formed.
  • Substantial protein aggregation refers to a level of protein aggregation in a protein formulation that is substantially greater than the level of protein aggregation in a first (e.g. , reference) protein formulation.
  • the first protein formulation may be the same protein formulation before a period of storage or before a treatment (e.g. , the first protein formulation may be a formulation of protein of time zero (TO) when the formulation is initially prepared or of time zero before the formulation is subjected to a destabilizing condition, such as elevated temperature, and/or to long term storage.).
  • TO protein of time zero
  • a composition which is substantially free of protein aggregation refers to protein formulations of the invention that do not have a significantly greater level or percentage of aggregated protein than a first (e.g. , reference) formulation.
  • this phrase refers to compositions in which the level of protein aggregation is less than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1%.
  • the level of protein aggregation is less than about 2%.
  • the level of protein aggregation in a composition may be determined using standard techniques known in the art, and as described in the Examples herein.
  • concentration of polyol in the composition that does not result in substantial protein aggregation refers to a concentration of polyol in the formulation that is sufficiently low at a temperature of, e.g., 5 °C, 25 °C or 40 °C such that a normal or acceptable level of aggregation present in the composition is observed, e.g., the aggregate level observed in a corresponding normal stressed lot, e.g., about 1.5% aggregation.
  • the concentration of polyol in the formulation is such that only less than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3%, 4%, 5%, 8%, 10% of aggregation is observed.
  • concentration of polyol in the formulation is such that only less than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3%, 4%, 5%, 8%, 10% of aggregation is observed.
  • the concentration of polyol in the formulation is such that only less than about 2% aggregation is observed.
  • Stability such as physical stability of a composition or formulation
  • a sample's apparent attenuation of light absorbance, or optical density
  • Such a measurement of light attenuation relates to the turbidity of a formulation.
  • the turbidity of a formulation is partially an intrinsic property of the protein dissolved in solution and is commonly determined by nephelometry, and measured in Nephelometric Turbidity Units (NTU).
  • NTU Nephelometric Turbidity Units
  • the degree of turbidity e.g., as a function of the concentration of one or more of the components in the solution, e.g., protein and/or salt concentration, is also referred to as the "opalescence" or "opalescent appearance" of a formulation.
  • the degree of turbidity can be calculated by reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the degree of turbidity for pharmaceutical compositions can be based on the European Pharmacopeia criteria (European Pharmacopoeia, Fourth Ed., Directorate for the Quality of Medicine of the Council of Europe (EDQM), France).
  • a clear solution is defined as one with a turbidity less than or equal to a reference suspension which has a turbidity of approximately 3 according to European Pharmacopeia standards.
  • Nephelometric turbidity measurements can detect Rayleigh scatter, which typically changes linearly with
  • a protein, e.g. , antibody "retains its chemical stability" in a pharmaceutical formulation, if the chemical stability at a given time is such that the protein, e.g. , antibody, is considered to still retain its biological activity (e.g. , as defined below for antibody).
  • Chemical stability can be assessed by, e.g. , detecting and quantifying chemically altered forms of the protein, e.g. , antibody.
  • Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix- assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.
  • Other types of chemical alteration include charge alteration (e.g. occurring as a result of deamidation or oxidation) which can be evaluated by ion-exchange chromatography, for example.
  • a protein, e.g. , antibody "retains its biological activity" in a pharmaceutical formulation, if the protein, e.g. , antibody, in a pharmaceutical formulation is biologically active for its intended purpose.
  • biological activity is retained if the biological activity of the protein, e.g., antibody, in the pharmaceutical formulation is within about 30%, about 20%, or about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared (e.g. , as determined in an antigen binding assay).
  • the stability of a protein formulation may be "improved" by removing or adding a component of the formulation, by changing the amount of the component in the formulation or the concentration of a component in the formulation.
  • an amount of a component (e.g., polyol) of a first protein formulation is reduced while keeping the amount of all other components the same, thereby improving the stability of the protein formulation.
  • shelf-life stability refers to the stability of a formulation under storage conditions that are substantially similar to conditions under which the formulation would be stored as a commercial product for use.
  • shelf-life stability is distinguished from stability of a formulation when stored or tested under conditions that promote the instability, i.e. unfolding or aggregating, of the protein(s) in the formulation.
  • Treatment in the context of a protein formulation refers to a process done to the formulation or condition under which the formulation is placed in order to test the effects of the components of the formulation on the stability of the protein of the formulation.
  • a "reverse stability concentration" of a protein or a “reverse stability protein concentration” is the concentration of the protein for any polyol concentration (polyol concentration at least in the pharmaceutically relevant range of 10-200 mg/ml) at which there is no net effect of any concentration of the the polyol (at least the concentration of the polyol of interest in pharmaceutical formulations, e.g., ⁇ 120 mg/ml for disaccharides and ⁇ 80 mg/ml for monosacharides) on the stability of protein in the protein formulation. Addition of polyol at a protein concentration higher than this would be stabilizing while addition of polyol at a protein concentration lower than this would be destabilizing.
  • the presence of a reverse stability concentration for a protein will be dependent on the ionic strength of the formulation (e.g., will exist in low ionic strength protein formulations). In some embodiments of the invention, increasing the ionic strength of a formulation will reduce the reverse stability concentration. In some embodiments of the invention, increasing the ionic strength of a formulation will eliminate the reverse stability concentration, i.e., by increasing the ionic strength of a formulation, the addition of a polyol will be stabilizing regardless of protein concentration. In one embodiment, the stability of the protein is measured in terms of protein aggregation, e.g., percentage of protein aggregation.
  • Examples of a reverse stability concentration of a protein include, e.g., 10 mg/ml, 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml or 200 mg/ml.
  • 10 mg/ml 10 mg/ml, 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml or 200 mg/ml.
  • a reverse stability concentration of a protein is, e.g., between 100-160 mg/ml, between 110-150 mg/ml, between 120-140 mg/ml or between 125-135 mg/ml. In one embodiment, a reverse stability concentration of a protein is about 130 mg/ml. In another embodiment, the reverse stability concentration of a protein in the protein formulation ⁇ e.g., formulations of Stelara or Simponi or high concentration protein formulations that contain polyols) is the concenctration below which a decrease in the polyol concentration would lead to an increase in stability of protein in the protein formulation at both recommended storage temperature and temperature of administration ⁇ e.g., 5°C, ambient, or 37°C).
  • substantially free of monomer loss refers to a percentage of protein that is in monomer form in a protein formulation that is, e.g., about 95%, 96%, 97%, 98%, 99% or greater.
  • Substantially free of fragments refers to a fragmented protein concentration in a protein formulation that is, e.g., about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml 2.0 mg/ml, 1.0 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml or less.
  • a "a protein concentration below the reverse stability concentration of a protein in the formulation” refers to a protein concentration in the formulations of the invention at which an increase in the polyol concentration of the formulation would reduce stability of the protein in the protein formulation.
  • a a protein concentration below the reverse stability concentration of a protein in the formulation is a protein concentration in the formulation at a concentration where a reduction in the polyol concentration of the formulation increases stability of the protein in the formulation.
  • a "a protein concentration below the reverse stability concentration of a protein in the formulation” is, e.g., at least about 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, or 180 mg/ml.
  • a “protein concentration above the reverse stability concentration of a protein in the formulation” refers to a protein concentration in the formulations of the invention at which an increase in the polyol concentration of the formulation would increase stability of the protein in the protein formulation.
  • a "protein concentration above the reverse stability concentration of a protein in the formulation” is, e.g., at least 80 mg/ml, 90 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, 180 mg/ml, 200 mg/ml, 220 mg/ml, 240 mg/ml, 260 mg/ml, 280 mg/ml or 300 mg/ml.
  • a "protein concentration above the reverse stability concentration of a protein in the formulation” is, e.g., at least about 120 mg/ml, 125 mg/ml, 130 mg/ml, 135 mg/ml, 140 mg/ml, or 150 mg/ml.
  • a “low ionic strength" or “low ionic” formulation refers to a formulation with an ionic strength such that a protein comprised by the formulation will have a reverse stability concentration, e.g. , at certain low protein concentrations, the presence of a polyol, e.g., mannitol, sucrose, trehalose, sorbitol, in the formulation will not have a stabilizing effect and at higher protein concentrations, the presence of a polyol in the formulation will have a stabilizing effect.
  • a polyol e.g., mannitol, sucrose, trehalose, sorbitol
  • a low ionic formulation of the invention is a protein formulation comprising a buffer (such as a phosphate buffer, a citrate buffer, a citrate and phosphate buffer, a histidine buffer, an acetate buffer, or a succinate buffer) and that does not contain additional ionic excipients, e.g., sodium chloride.
  • a "series of formulations” or “formulation series” refers to a set of two or more compositions comprising a protein, water, and other ingredients, wherein the concentration of ingredients in the compositions, other than the protein and any polyols, is fixed or constant.
  • a series of formulations comprises a "first formulation” and a "second formulation".
  • aspects of the invention include improvements to protein formulations that enhance stability and shelf-life. Accordingly, improvements in protein stability can be seen by comparing a first protein formulation (also known herein as a "reference formulation") to a second protein formulation, which may differ in terms of composition (e.g. , presence, amount, or concentration of one or more components, or their chemical, molecular or ionic form(s)), treatment or age.
  • First formulations can vary from second formulations based on one or more aspects of the formulations.
  • the first formulation contains one or more of the components of a second formulation at a different concentration as compared to the concentration of those components in the second formulation and one or more of the components of the second formulation at the same concentration as compared to the concentration of those components in the second formulation.
  • the first formulation and the second formulation comprise a protein and a polyol, and further comprise additional components which are present at the same concentration in each formulation.
  • all of the components of the first and second formulations have the same concentration, except for the concentration of a protein or the concentration of a polyol.
  • the concentration of all components in the first and second formulations are the same, except for the concentration of a protein and a polyol.
  • all of the components of the first and second formulations are the same, but the treatment (e.g. , storage conditions, testing conditions) and/or the amount of time that has elapsed since the formulations were made (i.e. , the age of the formulations) is/are different.
  • a "first protein formulation” refers to a protein formulation comprising the same protein as a second protein formulation but differing in other components (e.g. , presence, amount, or concentration of one or more components, or their chemical, molecular or ionic form(s)), protein concentration, treatment, manufacture and/or age.
  • a first protein formulation is compared to a second protein formulation with the same formulation that has been stored under different storage conditions.
  • a first protein formulation is compared to a second protein formulation that has a different concentration of the protein.
  • a first protein formulation is compared to a second protein formulation that has a specific polyol concentration, known as the "second formulation polyol concentration", which may be the same, about the same or significantly different from the polyol concentration of the first protein formulation.
  • reducing the concentration of a polyol refers to the reformulation of a composition to contain less polyol than a previous formulation of the composition. In one embodiment, “reducing the concentration of a polyol” refers to the reduction or removal of a polyol from a composition. In one embodiment, the reduction or removal of a polyol from a composition is performed with ultrafiltration or dialysis.
  • a “therapeutically effective amount” or “effective amount” of an antibody refers to an amount effective in the prevention or treatment or alleviation of a symptom of a disorder for the treatment of which the antibody is effective.
  • a “disorder” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those
  • antibody broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • Ig immunoglobulin
  • Such mutant, variant, or derivative antibody formats are known in the art, nonlimiting embodiments of which are discussed herein.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin molecules can be of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. , IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (US Patent Nos: 5,648,260 and 5,624,821).
  • the Fc portion of an antibody mediates several important effector functions, e.g.
  • IgG isotypes particularly IgGl and IgG3, mediate ADCC and
  • a half Ig molecule may have certain advantages in tissue penetration due to its smaller size than that of a regular antibody.
  • at least one amino acid residue is replaced in the constant region of the binding protein of the invention, for example the Fc region, such that the dimerization of the heavy chains is disrupted, resulting in half Ig molecules.
  • the light chain may be either a kappa or lambda type.
  • CDR refers to the complementarity determining region within a antibody variable sequence.
  • CDR1, CDR2 and CDR3 are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
  • the exact boundaries of these CDRs have been defined differently according to different systems.
  • the system described by Kabat (Id.) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al.
  • CDRs have been described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45. Still other CDR boundary definitions may not strictly follow one of the herein described systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.
  • antigen-binding portion of an antibody or “antibody portion” includes fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-12 and/or hIL-23). Such antibody embodiments may also be bispecific, dual specific, or multi- specific, e.g., it specifically binds to two or more different antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two
  • Fab fragments linked by a disulfide bridge at the hinge region (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546 ), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2: 1121- 1123).
  • Such antibody binding portions are known in the art
  • single chain antibodies also include "linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. (1995) Protein Eng.
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol.
  • Antibody portions such as Fab and F(ab') 2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and
  • immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • Preferred antigen binding portions are complete domains or pairs of complete domains.
  • bispecific antibody refers to full-length antibodies that are generated by quadroma technology (Milstein, C. and A.C. Cuello (1983) Nature 305(5934):537-40), by chemical conjugation of two different monoclonal antibodies (Staerz, U.D. et al. (1985) Nature 314(6012):628-31), or by knob-into-hole or similar approaches that introduce mutations in the Fc region (Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 8.18), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds.
  • dual-specific antibody refers to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (PCT Publication No. WO 02/02773). Accordingly, a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain.
  • Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
  • Two antibody domains are "complementary" where they belong to families of structures which form cognate pairs or groups or are derived from such families and retain this feature. For example, a VH domain and a VL domain of an antibody are complementary; two VH domains are not complementary, and two VL domains are not complementary.
  • Complementary domains may be found in other members of the immunoglobulin superfamily, such as the Vcc and ⁇ (or gamma and delta) domains of the T-cell receptor.
  • domain refers to a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • single antibody variable domain is meant a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • variable domains and modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least in part the binding activity and specificity of the full-length domain.
  • Variable domains of the invention may be combined to form a group of domains; for example, complementary domains may be combined, such as VL domains being combined with VH domains. Non-complementary domains may also be combined. Domains may be combined in a number of ways, involving linkage of the domains by covalent or non-covalent means.
  • a “dAb” or “domain antibody” refers to a single antibody variable domain (V H or V L ) polypeptide that specifically binds antigen.
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen.
  • the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibody is produced by hybridoma technology.
  • epitope is meant to refer to that portion of any molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen binding regions.
  • first and second “epitopes” are understood to be epitopes which are not the same and are not bound by a single monospecific antibody, or antigen-binding portion thereof.
  • recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g. , a mouse) that is transgenic for human immunoglobulin genes (see e.g. , Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of particular immunoglobulin gene sequences (such as human
  • immunoglobulin gene sequences to other DNA sequences.
  • recombinant antibodies include chimeric, CDR-grafted and humanized antibodies.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies used in the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. , mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Recombinant human antibodies of the invention have variable regions, and may also include constant regions, derived from human germline immunoglobulin sequences (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the
  • recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. In certain embodiments, however, such recombinant antibodies are the result of selective mutagenesis or backmutation or both.
  • backmutation refers to a process in which some or all of the somatically mutated amino acids of a human antibody are replaced with the corresponding germline residues from a homologous germline antibody sequence.
  • the heavy and light chain sequences of a human antibody of the invention are aligned separately with the germline sequences in the VBASE database to identify the sequences with the highest homology.
  • Differences in the human antibody of the invention are returned to the germline sequence by mutating defined nucleotide positions encoding such different amino acid.
  • the role of each amino acid thus identified as candidate for backmutation should be investigated for a direct or indirect role in antigen binding and any amino acid found after mutation to affect any desirable characteristic of the human antibody should not be included in the final human antibody.
  • those amino acid positions found to be different from the closest germline sequence but identical to the corresponding amino acid in a second germline sequence can remain, provided that the second germline sequence is identical and colinear to the sequence of the human antibody of the invention for at least 10, preferably 12 amino acids, on both sides of the amino acid in question. Backmuation may occur at any stage of antibody optimization.
  • chimeric antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g. , CDR3) has been replaced with human CDR sequences.
  • humanized antibody refers to an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g. , a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e. , more similar to human germline variable sequences.
  • a non-human species e.g. , a mouse
  • human CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized antibody is an antibody or a variant, derivative, analog or fragment thereof that specifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody.
  • An "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds the p40 subunit of IL-12/IL-23 is substantially free of antibodies that specifically bind antigens other than the p40 subunit of IL-12/IL-23).
  • an isolated antibody that specifically binds a target may, however, have cross-reactivity to other antigens, such as, e.g., the p40 subunit of IL-12/IL-23 molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • a “neutralizing antibody” as used herein is intended to refer to an antibody whose binding to antigen results in inhibition of the biological activity of the antigen (e.g., IL-12/IL- 23, amyloid beta).
  • This inhibition of the biological activity of the antigen can be assessed by measuring one or more indicators of the antigen's biological activity.
  • indicators of the biological activity of human IL-12 hIL-12
  • activity includes activities such as the binding specificity/affinity of an antibody for an antigen, for example, an anti-hIL-12 antibody that binds to an IL-12 antigen and/or the neutralizing potency of an antibody, for example, an anti-hIL-12 antibody whose binding to hIL-12 inhibits the biological activity of hIL-12, e.g. inhibition of PHA blast proliferation or inhibition of receptor binding in a human IL-12 receptor binding assay (see Example 3 of U.S. Patent No. 6,914,128).
  • surface plasmon resonance refers to an optical
  • K on is intended to refer to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex as is known in the art.
  • a binding protein e.g., an antibody
  • K 0 ff is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • Kd is intended to refer to the dissociation constant of a particular antibody-antigen interaction and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (k Q ff) by the association rate constant (k on ).
  • human interleukin 12 or “human IL-12” (abbreviated herein as hIL-12, or IL-12), as used herein, includes a human cytokine that is secreted primarily by
  • the term includes a heterodimeric protein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40) which are both linked together with a disulfide bridge.
  • the heterodimeric protein is referred to as a "p70 subunit".
  • the structure of human IL-12 is described further in, for example, Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl. Acad. Sci. 90: 10188-10192; Ling, et al. (1995) J. Exp Med. 154: 116-127; Podlaski, et al. (1992) Arch.
  • human IL-12 is intended to include recombinant human IL-12 (rh IL-12), which can be prepared by standard recombinant expression methods.
  • human interleukin 23 or "human IL-23” (abbreviated herein as hIL-23, or IL-23), as used herein, includes a human cytokine that is secreted primarily by
  • the term includes a heterodimeric protein comprising a 19 kD subunit (pi 9) and a 40kD subunit (p40) which are both linked together with a disulfide bridge.
  • the heterodimeric protein is referred to as a "p40/p 19" heterodimer.
  • the structure of human IL-23 is described further in, for example, Beyer et al. (2008) J. Mol. Biol. 382:942- 955; Lupardus et al. (2008) J. Mol. Biol. 382:931-941.
  • human IL-23 is intended to include recombinant human IL-23 (rhIL-23), which can be prepared by standard recombinant expression methods.
  • p40 subunit of human IL-12/IL-23 or "p40 subunit of human IL-12 and/or IL-23," or "p40 subunit” as used herein, is intended to refer to a p40 subunit that is shared by human IL-12 and human IL-23.
  • the structure of the p40 subunit of IL-12/IL-23 is described in, for example, Yoon et al. (2000) EMBO Journal 19(14): 3530-3541.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • the present invention features protein compositions and protein pharmaceutical formulations (e.g., antibody compositions and formulations) having improved properties as compared to art-recognized compositions and formulations.
  • Protein aggregation is a common problem in protein solutions, and often results from increased concentration of the protein.
  • the instant invention provides compositions containing proteins which are substantially free of protein aggregation.
  • the instant invention also provides reformulated compositions containing proteins which have substantially reduced amounts of protein aggregation.
  • the present invention is based on the surprising finding that for protein compositions having protein concentrations below the RSC, reductions in polyol concentrations leads to significantly reduced protein aggregation after long-term storage.
  • the stability of a protein e.g. , an antibody, or antigen-binding fragment thereof
  • the formulations of the invention maintain a low level of protein aggregation and/or fragmentation (e.g. , less than 2% or less than 1%).
  • the formulations of the invention e.g.
  • antibody formulations with antibody concentrations below the reverse stability concentration maintain solubility and maintain a low viscosity suitable for subcutaneous injection.
  • the formulations remain stable for extended periods of time (e.g. , 6 months, 12 months, 18 months or 24 months) and / or at elevated temperatures (e.g. , at 5°C, 25°C or 40°C).
  • the formulation comprises less than 14% protein aggregation after 6 months at elevated temperature (e.g. , at 40°C).
  • the formulations comprise less than 2.0% protein aggregation after 12 months at 5°C.
  • the formulations comprise less than 0.6% protein aggregation after 12 months at 5°C.
  • turbidity of the formulations is less than 100 NTU after a standard 48 hour stir-stress assay.
  • Protein aggregation is generally concentration dependent.
  • the mean-field lattice model predicts that proteins will aggregate at sufficiently high concentrations (Fields et ah, J. Phys. Chem. 96, 3974-3981 (1992)).
  • polyol excipients e.g. , sugar alcohol, which is known in the art to reduce protein aggregation in protein composition in general.
  • Increased turbidity is often associated with the formation of insoluble protein aggregates, precipitates, or protein particles (e.g., aggregation).
  • the present invention is based on the surprising discovery that protein concentrations below the reverse stability concentration, the addition of polyol excipients can lead to greater amounts of protein aggregation, and not reduced amounts.
  • formulations of the invention include protein concentrations below the reverse stability concentration such that the liquid formulation does not show significant opalescence, aggregation, or precipitation.
  • the methods of the invention provide a means of formulating a protein at protein concentrations below the reverse stability concentration with reduced amounts of excipients, or without excipients, that destabilize proteins at those concentrations.
  • the methods of the invention provide a means of increasing the protein stability at protein concentrations below the reverse stability concentration by reducing the amount of polyol in protein formulations.
  • the concentration of the protein in the formulations obtained using the methods of the invention can be any amount in accordance with the desired concentration.
  • the concentration of protein in a formulation made according to the methods herein is less than about 10 ⁇ g/mL; less than about 1 mg/mL; less than about 10 mg/mL; less than about 20 mg/mL; less than about 50 mg/mL; less than about 75 mg/mL; less than about 100 mg/mL; less than about 110 mg/mL; less than about 120 mg/mL; less than about 125 mg/mL; less than about 130 mg/mL; less than about 135 mg/mL; less than about 140 mg/mL; less than about 150 mg/mL; less than about 175 mg/mL; less than about 200 mg/mL; less than about 220 mg/mL; less than about 250 mg/mL; less than about 300 mg/mL; or less than about 300 mg/mL.
  • Ranges intermediate to the above recited concentrations are also intended to be encompassed by the invention.
  • ranges of values using a combination of any of the above recited values (or values between the ranges described above) as upper and/or lower limits are intended to be included, e.g., 100 to 125 mg/mL, 113 to 125 mg/mL, 125 to 135 mg/mL, 127 to 133 mg/mL, and 126 to 200 mg/mL or more.
  • the methods of the invention provide a means of increasing the protein concentration in protein formulations having protein concentrations below the reverse stability concentration without raising aggregations levels, by simultaneously reducing the amount of polyol in the protein formulations.
  • the level of aggregates in the protein formulation with the higher protein concentration can be maintained or even lowered in comparison to the level of aggregates in the original protein formulation of lower protein concentration after longer-term storage or conditions of accelerated stability testing.
  • the formulations of the invention comprise a protein concentration below the reverse stability concentration of an antibody, or antigen-binding portion thereof (e.g. , Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®).
  • Such formulations comprise the antibody, or antigen -binding portion thereof, at concentrations including, for example, a concentration of about 100 mg/mL, about 150 mg/mL or about 200 mg/mL of an antibody, or antigen-binding fragment thereof.
  • the pharmaceutical formulation comprises an antibody concentration of about 100 mg/mL.
  • the pharmaceutical formulation comprises an antibody concentration of about 150 mg/mL.
  • the pharmaceutical formulation comprises an antibody concentration of about 200 mg/mL.
  • the pharmaceutical formulation comprises an antibody concentration below the Reverse Stability Concentration (RSC) of that protein in the formulation.
  • RSC Reverse Stability Concentration
  • the formulations are low ionic formulations, e.g., comprise a buffer and do not comprise additional ionic excipient(s), such as NaCl.
  • the pharmaceutical formulation comprises an antibody concentration of less than about 200mg/mL, less than about 150 mg/mL, less than about 140 mg/mL, or less than about 130 mg/mL.
  • the preferred embodiments of the invention are formulations comprising protein concentrations below the RSC, it is also contemplated that the formulations of the invention may comprise an antibody concentration between about 1 mg/mL and about 300 mg/mL, between about 50 mg/mL-250 mg/mL, between about 50 mg/mL- 150 mg/mL, or between about 50mg/mL- 130 mg/mL.
  • Concentrations and ranges intermediate to the above recited concentrations are also intended to be part of this invention (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
  • formulations of the invention provide the advantage that the resulting formulation has a low percentage of protein aggregates.
  • formulations of the invention comprising a reduced amount of polyol, or no polyol, and a protein (e.g., antibodies) concentration below the RSC for the formulation, contain less than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% protein aggregates.
  • the formulation comprises no more than about 7% aggregate protein; the formulation comprises no more than about 5% aggregate protein; the formulation comprises no more than about 4% aggregate protein; the formulation comprises no more than about 3% aggregate protein; the formulation comprises no more than about 2% aggregate protein; or the formulation comprising no more than about 1% aggregate protein.
  • the formulation comprises at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% monomer protein. Ranges intermediate to the above recited concentrations, e.g. , at least about 98.6%, no more than about 4.2%, are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the invention provides a composition comprising excipients, a surfactant, and a buffer system, in amounts sufficient to formulate a therapeutic protein, e.g. , an antibody, e.g. , briakinumab, for therapeutic use at a concentration of about, for example, 100 mg/mL.
  • a therapeutic protein e.g. , an antibody, e.g. , briakinumab
  • the pharmaceutical compositions do not comprise polyol.
  • the pharmaceutical compositions comprise an amount of polyol that is reduced in comparison with a standard composition.
  • the pharmaceutical compositions comprise an amount of polyol that does not cause or contribute to protein instability, e.g., protein aggregation and/or fragmentation.
  • the invention provides a pharmaceutical composition comprising a therpeutic protein, e.g., an antibody or antigen binding fragment thereof, (e.g., briakinumab), without the addition of polyol, in amounts sufficient to formulate a protein for therapeutic use.
  • a pharmaceutical composition comprising a therapeutic protein, e.g. , an antibody or antigen binding fragment thereof (e.g. , briakinumab), formulated to have low concentrations of polyol (e.g. , where the protein concentration is below the reverse stability concentration for the protein in the formulation), in amounts sufficient to formulate a protein for therapeutic use.
  • the present invention also provides formulations comprising a therapeutic protein, e.g, an antibody or antigen-binding fragment thereof (e.g. , Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®) and having a turbidity of less than about 60 NTU after a standard 24 hour stir-stress assay, with reduced or low levels of polyol or without the addition of polyol (e.g. , about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37.
  • a therapeutic protein e.g., an antibody or antigen-binding fragment thereof (e.g. , Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®) and having a turbidity of less than about 60 NTU after a standard 24 hour stir-s
  • the invention provides formulations comprising a therapeutic protein, e.g, an antibody or antigen-binding fragment thereof (e.g., Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®) having a turbidity of less than about 100 NTU after a standard 48 hour stir-stress assay, with reduced or low levels of polyol or without the addition of polyol (e.g.
  • a therapeutic protein e.g, an antibody or antigen-binding fragment thereof (e.g., Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®) having a turbidity of less than about 100 NTU after a standard 48 hour stir-stress assay, with reduced or low levels of polyol or without the addition of polyol (e.g.
  • the invention provides formulations comprising a therapeutic protein, e.g, an antibody or antigen- binding fragment thereof (e.g., Briakinumab®, Stelara®, Enbrel®, Avastin®, Herceptin®, Erbitux®, Remicade®, or Simponi®) having a turbidity of less than about 40 NTU after 3 months storage at 5°C, 25°C, or 40°C, with reduced or low levels of polyol or without the addition of polyol (e.g. , about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 NTU).
  • a therapeutic protein e.g, an antibody or antigen- binding fragment thereof (e.g., Briakinumab®, Stelara®, Enbrel®, Avast
  • a formulation comprising the protein in a pH-buffered solution.
  • the buffer of this invention has a pH ranging from about 4 to about 8, preferably from about 4.5 to about 7.0, more preferably from about 4.5 to about 6.0, even more preferably from about 4.8 to about 5.5, and most preferably has a pH of about 5.0 to about 6.4.
  • the pH of the formulation of the invention is about 5.2.
  • the pH of the formulation of the invention is about 6.0. Ranges intermediate to the above recited pH's are also intended to be part of this invention (e.g.
  • Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included, e.g., 5.2 - 5.8.
  • buffers that will control the pH within this range include phosphate, acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, glutamate, histidine, citrate and other organic acid buffers. In one embodiment, the buffer is histidine.
  • the formulation comprises a buffer system which contains citrate and/or phosphate to maintain the pH in a range of about 5.0 to about 6.4. In one embodiment, the pH of the formulation is about 5.2. In another
  • the pH of the formulation is about 6.0.
  • the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate.
  • the surprising findings of this invention are the consequence of protein- polyol interactions or the interaction of effects exerted on the protein by the presence of polyol in the composition , e.g. , effects on activity, osmolarity, density, etc.
  • the invention applies to protein formulations for any pH value at which the protein is formulated.
  • a detergent or surfactant may also be added to the therapeutic protein formulation of the invention.
  • exemplary detergents include nonionic detergents such as polysorbates (e.g. polysorbates 20, 80, etc.) or poloxamers (e.g. poloxamer 188).
  • the amount of detergent added is such that it reduces aggregation of the formulated therapeutic protein and/or minimizes the formation of particulates in the formulation and/or reduces adsorption.
  • the formulation includes a surfactant which is a polysorbate.
  • the formulation contains the detergent polysorbate 80.
  • the formulation contains between about 0.1 and about 2.0 mg/mL of polysorbate 80, e.g. , about 1 mg/mL.
  • the formulation of the invention consists essentially of a therapeutic protein, e.g, an antibody or antigen-binding fragment thereof, at a concentration of at least about 50 mg/mL or about 100 mg/mL, wherein the concentration is below the RSC, a surfactant (e.g., polysorbate 80), a buffering system, and a reduced or low level of polyol.
  • the formulation contains any one or more of the above-identified agents and is essentially free of preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium CI.
  • a preservative may be included in the formulation.
  • One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the formulation provided that they do not significantly adversely affect the desired characteristics of the formulation.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.
  • the formulation herein may also be combined with one or more other therapeutic agents as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect the therapeutic protein of the
  • Such therapeutic agents are suitably present in combination in amounts that are effective for the purpose intended. Additional therapeutic agents which can be combined with the formulation of the invention are further described in U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,914,128, each of which is incorporated herein by reference.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes prior to, or following, preparation of the formulation, or other methods know to those skilled in the art.
  • compositions or formulations of the invention are liquid compositions or formulations.
  • the liquid compositions/formulations of the invention have advantageous stability and storage properties.
  • Compositions/formulations of the invention include, but are not limited to, formulations which are frozen, lyophilized, spray-dried, or formulations in which the active ingredient is suspended. Stability can be measured at a selected temperature for a selected time period.
  • the protein in the liquid formulations is stable in a liquid form for at least about 3 months; at least about 4 months, at least about 5 months; at least about 6 months; at least about 12 months; at least about 18 months; at least 24 months; at least 36 months; at least 48 months; at least 60 months.
  • Values and ranges intermediate to the above recited time periods are also intended to be part of this invention, e.g. , about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 months.
  • ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the formulation is stable at room temperature (about 30°C) or at 40°C for at least about 1 month and/or stable at about 2-8°C for at least about 1 year, or more preferably stable at about 2-8°C for at least about 2 years.
  • the formulation is preferably stable following freezing (to, e.g. , -80°C) and thawing of the formulation, hereinafter referred to as a "freeze/thaw cycle.”
  • Stability of a protein in a liquid formulation may also be defined as the percentage of monomer, aggregate, or fragment, or combinations thereof, of the protein in the formulation.
  • a protein "retains its physical stability" in a formulation if it shows substantially no signs of aggregation, precipitation and/or denaturation, upon visual examination of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography.
  • a stable liquid formulation is a formulation having less than about 10%, less than about 5%, and preferably less than about 2% of the protein being present as aggregate in the formulation.
  • the physical stability of a liquid formulation is determined by determining turbidity of the formulation following a stir stress assay, e.g., 24 hour or 48-hour stir-stress assay.
  • a stir stress assay may be performed by placing a suitable volume of a liquid formulation in a beaker with a magnetic stirrer, e.g. , (multipoint HP, 550 rpm), removing aliquots at any suitable time, e.g. , at T0-T48 (hrs), and performing suitable assays as desired on the aliquots. Samples of a formulation under the same conditions but without stirring serve as control.
  • Turbidity measurements may be performed using a laboratory turbidity measurement system from Hach (Germany) and are reported as nephelometric units (NTU).
  • the concentration of protein in the formulations of the invention is not limited by the protein size and the formulation may include any size range of proteins. Included within the scope of the invention is a formulation comprising at least 50 mg/mL and as much as 200 mg/mL or more of a protein, which may range in size from 5 kDa to 150 kDa or more.
  • the protein in the formulation of the invention is at least about 15 kD in size, at least about 20 kD in size; at least about 47 kD in size; at least about 60 kD in size; at least about 80 kD in size; at least about 100 kD in size; at least about 120 kD in size; at least about 140 kD in size; at least about 160 kD in size; or greater than about 160 kD in size.
  • Ranges intermediate to the above recited sizes are also intended to be part of this invention.
  • ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • the formulation herein may also contain more than one protein.
  • an additional, distinct protein may be added as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect the other protein.
  • two or more antibodies that bind to different antigens may be combined in the one formulation.
  • Such proteins are suitably present in combination in amounts that are effective for the purpose intended.
  • examples of different types of antibodies, or antigen-binding fragments thereof, that may be used in the invention include, but are not limited to, a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).
  • the protein is a therapeutic protein.
  • proteins which may be included in the methods and compositions described herein include
  • mammalian proteins including recombinant proteins thereof. Proteins suitable for use in the compositions and formulations of the invention are discussed in further detail below in Section IV.
  • Turbidity measurements may be performed using a laboratory turbidity measurement system from Hach (Germany) and are reported as nephelometric units (NTU).
  • the liquid formulations of the invention also have advantageous tolerability properties. Tolerability is evaluated based on assessment of subject-perceived injection site pain using the Pain Visual Analog Scale (VAS).
  • VAS Pain Visual Analog Scale
  • VAS is a measurement instrument that measures pain as it ranges across a continuum of values, e.g. , from none to an extreme amount of pain.
  • a VAS is a horizontal line, about 100 mm in length, anchored by numerical and/or word descriptors, e.g. , 0 or 10, or 'no pain' or 'excruciating pain', optionally with additional word or numeric descriptors between the extremes, e.g., , mild, moderate, and severe; or 1 through 9) (see, e.g. , Lee JS, et al. (2000) Acad Emerg Med 7:550).
  • Draize Scale hereinafter, petechiae, erythema, edema, pruritus
  • a method for formulating a stable protein formulation with a protein concentration below the reverse stability concentration is provided.
  • the invention is based on the surprising discovery that at some concentrations of protein in some formulations, commonly found components of protein formulations can have a deleterious effect on the protein of the formulation.
  • polyols e.g. , sugars and sugar alcohols
  • polyols are commonly known in the art to enhance protein stability, by reducing protein aggregation and destabilization, thereby reducing turbidity and protein fragmentation.
  • polyols can actually have the opposite effect on protein stability, e.g., they can increase aggregation, turbidity and fragmentation at a variety of formulation conditions of frequent use with the pharmaceutical compositions.
  • non-native protein aggregates is considered to be a two-step process.
  • the first step is the occurrence of a permutation of protein structure, e.g. , a partial unfolding of the protein.
  • the second step is the association of two protein molecules with structural permutations to form an aggregated molecule.
  • This process of the formation of non-native protein aggregates can be rate-limited by either the first step or the second step, which depends on a number of factors, including protein concentration, excipient
  • polyols act as crowders, driving protein molecules together and promoting the second step of the aggregation process.
  • a higher concentration of polyols increases the crowding effect and accelerates the aggregate formation process.
  • one aspect of the invention features a method of determining the RSC for a protein in a particular formulation, i.e. , he protein concentration for a formulation below which the addition of a polyol (any polyol concentration of pharmaceutical interest and typically ⁇ 200 mg/ml) may be destabilizing for the protein formulation (e.g. , as detected by higher levels of aggregated protein in the formulation).
  • the invention features a method of determining the concentration of a protein in a protein formulation containing a given polyol, below which the net stabilizing effect of the polyol is replaced with a net destabilizing effect (e.g., as detected by higher levels of aggregated protein and/or protein fragments in the formulation).
  • the stability of a protein formulation can be improved (e.g. , a reduction in the protein aggregation and/or fragmentation of a formulation can be achieved), for example, by reducing the polyol concentration or eliminating the polyol from the formulation if the protein concentration is below the reverse stability concentration for the formulation.
  • the stability of formulations with protein concentrations below the reverse stability concentration can be improved by reducing the polyol concentration.
  • the stability of formulations with protein concentrations below the reverse stability concentration can be improved by eliminating the polyol.
  • one aspect of the invention provides a method of determining, for a protein formulation containing a protein and a particular polyol, the reverse stability concentration for the protein in that formulation.
  • a group of two or more protein formulations comprising a buffer and having different protein concentrations (e.g. , two different protein concentrations equal to or greater than 100 mg/ml) are prepared, each identical except for having a different concentration of polyol.
  • Each of the protein formulations is optionally assayed prior to a treatment phase to determine the content of monomer, aggregated and/or fragmented protein, as described below in the Examples (e.g. , Table 2A).
  • each of the protein formulations undergoes a treatment phase.
  • each protein formulation may be stored for an extended period (e.g. , 12 months) at a specific temperature (e.g. , 40°C, or 5°C), and/or in the presence or absence of additional ionic excipients, e.g., NaCl.
  • the protein formulations undergo a physical stress test such as the stir-stress assay described herein.
  • the protein formulations undergo cycles of freezing and thawing.
  • samples of the same formulation receive differential treatment, e.g. , storage for a period of time in different temperatures.
  • the protein formulations are assayed to determine the content of protein monomer, aggregates and/or fragments.
  • the results between formulations containing different concentrations of the polyol e.g. , at one protein concentration, or between series of formulations at different protein concentrations, or in the presence and/or absence of additional ionic excipients, e.g., NaCl
  • results between formulations are additionally compared for each protein formulation before and after treatment.
  • the Reserve Stability Concentration of the protein can be identified by determing the protein concentration at which the presence of polyol has no net effect on the stability over a range of polyols.
  • Figures 1A and IB show that the level of protein aggregation is higher in the presence of polyols when compared to the formulation without polyol at 100 mg/mL. At 200 mg/ml protein concentration (e.g., a high protein concentration for this protein in this formulation) the effect is reversed.
  • Figure 2 shows results at 40°C for two different proteins, ABT-736 in Figure 2A and ABT-874 in Figure 2B, formulated with a range of protein concentrations. The results clearly show that the addition of sugar is deleterious at concentrations below the reverse stability concentration (e.g. , where the slope of the line is zero or positive). The addition of sugars is only useful at higher concentrations above the RSC(i.e. , where the slope of the line is negative).
  • the invention provides a method of determining the protein concentration for the protein (i.e. , the Reverse Stability Concentration) below which a polyol in any concentration of pharmaceutical interest is destabilizing for the formulation.
  • the protein concentration for the protein i.e. , the Reverse Stability Concentration
  • a comparison of stability upon long-term storage of protein formulations and having the same polyol concentrations but varying protein concentrations reveals both the stabilizing and destabilizing effects of polyols on protein formulations, as compared to each other, depending on the protein concentration.
  • the invention provides methods of analyzing formulations with concentrations of protein and polyols in which the presence of polyols has either a stabilizing effect or a destabilizing effect, to determine the concentration of protein wherein the effect of the polyol is neutral (i.e. , the Reverse Stability Concentration).
  • a stabilizing effect or a destabilizing effect
  • Proteins which may be used in the compositions, formulations and methods of the invention may be any size, i.e., molecular weight (M w ).
  • the protein may have a M w equal to or greater than about 1 kDa, a M w equal to or greater than about 10 kDa, a M w equal to or greater than about 47 kDa, a M w equal to or greater than about 57 kDa, a M w equal to or greater than about 100 kDa, a M w equal to or greater than about 150 kDa, a M w equal to or greater than about 200 kDa, or a M w equal to or greater than about 250 kDa.
  • proteins used in the invention may range in size from 57 kDa to 250 kDa, from 56 kDa to 242 kDa, from 60 kDa to 270 kDa, and so forth.
  • the methods of the invention include formulating protein compositions that comprise one protein, or at least two or more distinct proteins.
  • the protein composition may contain two or more types of antibodies directed to different molecules or different epitopes of the same molecule.
  • the protein composition may contain one therapeutic protein, e.g., a therapeutic antibody, and one or more additional non-therapeutic proteins.
  • the protein used in the composition is a therapeutic protein, including, but not limited to, fusion proteins and enzymes.
  • therapeutic proteins include, but are not limited to, Pulmozyme (Dornase alfa), Regranex (Becaplermin), Activase (Alteplase), Aldurazyme (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetin alfa), Becaplermin Concentrate, Betaseron (Interferon beta- lb), BOTOX (Botulinum Toxin Type A), Elitek (Rasburicase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel (Etanercept), Fabrazyme (Agalsidase beta), Infergen (Interferon alfacon-1), Intron A (Interferon alfa-2a), Kineret (Anakinra), MYOBLOC
  • proteins which may be included in the methods and compositions described herein, include mammalian proteins, including recombinant proteins thereof, such as, e.g. , growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone;
  • lipoproteins a- 1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue- type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor-cc and - ⁇ enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP- 1- ); serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin- associated peptide; DNase; inhibin
  • IGF-I and IGF-II des(l-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO);
  • TPO thrombopoietin
  • BMP bone morphogenetic protein
  • interferon such as interferon-cc, - ⁇ ., and - ⁇ .
  • colony stimulating factors e.g.
  • ILs interleukins
  • IL- 1 to IL- 10 superoxide dismutase
  • T-cell receptors surface membrane proteins
  • DAF decay accelerating factor
  • a viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the above-listed polypeptides.
  • the protein used in the invention may also be an antibody, or antigen-binding fragment thereof.
  • the antibody, or antigen-binding portion thereof is a therapeutic antibody.
  • antibodies that may be used in the invention include chimeric antibodies, non-human antibodies, human antibodies, humanized antibodies, and domain antibodies (dAbs).
  • the antibody, or antigen-binding fragment thereof is an anti-IL-12 or IL-23 antibody.
  • antibodies, or antigen-binding fragments thereof, which may be used in the methods and compositions of the invention include, but are not limited to, 1D4.7 (anti-IL-12/IL-23 antibody; Abbott Laboratories), 2.5(E)mgl (anti-IL-18; Abbott
  • Antibody 7 as described in International Application WO 2007/149032 (Cambridge Antibody Technology), the entire contents of which are incorporated by reference herein, the anti-IL-13 antibody CAT-354 (Cambridge Antibody Technology), the anti-human CD4 antibody CE9y4PE (IDEC-151, clenoliximab) (Biogen IDEC/Glaxo Smith Kline), the anti-human CD4 antibody IDEC CE9.
  • Polyclonal antibodies generally refer to a mixture of antibodies that are specific to a certain antigen, but bind to different epitopes on said antigen. Polyclonal antibodies are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant.
  • sc subcutaneous
  • ip intraperitoneal
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • a bifunctional or derivatizing agent for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R NCNR, where R and Ri are different alkyl groups.
  • a “monoclonal antibody” as used herein is intended to refer to a hybridoma-derived antibody (e.g., an antibody secreted by a hybridoma prepared by hybridoma technology, such as the standard Kohler and Milstein hybridoma methodology).
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al. , Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a hybridoma-derived dual- specificity antibody of the invention is still referred to as a monoclonal antibody although it has antigenic specificity for more than a single antigen.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • Antibodies and antibody fragments may also be isolated from yeast and other eukaryotic cells with the use of expression libraries, as described in U.S. Pat. Nos. 6,423,538; 6,696,251; 6,699,658; 6,300,065; 6,399,763; and 6,114,147.
  • Eukaryotic cells may be engineered to express library proteins, including from combinatorial antibody libraries, for display on the cell surface, allowing for selection of particular cells containing library clones for antibodies with affinity to select target molecules. After recovery from an isolated cell, the library clone coding for the antibody of interest can be expressed at high levels from a suitable mammalian cell line.
  • Additional methods for developing antibodies of interest include cell-free screening using nucleic acid display technology, as described in U.S. Pat. Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479, 6,518,018; 7,125,669; 6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804; 6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926; 6,416,950; 6,660,473; 6,312,927; 5,922,545; and 6,348,315.
  • nucleic acid that codes for the protein is also selected.
  • antibody sequences isolated from immune system cells can be isolated and partially randomized polymerase chain reaction mutagenesis techniques to increase antibody diversity. These partially randomized antibody genes are then expressed in a cell-free system, with concurrent physical association created between the nucleic acid and antibody.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immuno globulin polypeptide.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen- combining site of an antibody to create a chimeric bivalent antibody comprising one antigen- combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by substituting non-human (e.g., rodent) CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies.
  • Additional references which describe the humanization process include Sims et al, J. Immunol., 151:2296 (1993); Chothia et al, J. Mol. Biol., 196:901 (1987); Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol., 151:2623 (1993), each of which is incorporated by reference herein.
  • the formulations and methods of the invention comprise a humanized IgGl kappa monoclonal antibody, or antigen-binding fragment thereof, that selectively binds with high affinity to the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2).
  • HER2 human epidermal growth factor receptor 2 protein
  • the formulations and methods of the invention comprise a humanized IgGl kappa monoclonal antibody, or antigen -binding fragment thereof, that binds to and inhibits the biologic activity of human vascular endothelial growth factor.
  • Human antibodies comprising a humanized IgGl kappa monoclonal antibody, or antigen -binding fragment thereof, that binds to and inhibits the biologic activity of human vascular endothelial growth factor.
  • transgenic animals ⁇ e.g., mice
  • transgenic animals that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • J H antibody heavy-chain joining region
  • Human antibodies can also be derived from phage-display libraries (Hoogenboom et ah, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581-597 (1991)).
  • the formulation of the invention comprises an antibody, or antigen-binding portion thereof, which binds human IL-12, including, for example, the antibody J695 (Abbott Laboratories; also referred to as ABT-874) (U.S. Patent No.
  • J695 is a fully human monoclonal antibody designed to target and neutralize interleukin-12 and interleukin-23.
  • the antibody, or antigen-binding fragment thereof has the following characteristics: it dissociates from human IL-lcc with a K D of 3 x 10 "7 M or less; dissociates from human IL- ⁇ with a K D of 5 x 10 ⁇ 5 M or less; and does not bind mouse IL- 1 a or mouse IL- 1 ⁇ . Examples and methods for making human, neutralizing antibodies which have a high affinity for human IL-12, including sequences of the antibody, are described in U.S. Patent No. 6,914,128, incorporated by reference herein.
  • the formulation of the invention comprises an antibody, or antigen-binding portion thereof, which binds human IL-18, including, for example, the antibody 2.5(E)mgl (Abbott Bioresearch; also referred to as ABT-325) (see U.S. Patent Application No. 2005/0147610, incorporated by reference herein).
  • the formulation of the invention comprises an anti-IL-12 / anti-IL- 23 antibody, or antigen-binding portion thereof, which is the antibody 1D4.7 (Abbott).
  • the formulation of the invention comprises an anti-IL-13 antibody, or antigen-binding portion thereof, which is the antibody 13C5.5 (Abbott Laboratories; also referred to as ABT-308) (see. PCT/US2007/ 19660 (WO 08/127271), incorporated by reference herein).
  • the formulation of the invention comprises an antibody, or antigen-binding portion thereof, which is the antibody 7C6, an anti-amyloid ⁇ antibody (Abbott Laboratories; see PCT publication WO 07/062852, incorporated by reference herein).
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Such antibodies can be derived from full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et ah, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690 published Mar. 3, 1994. For further details of generating bispecific antibodies see, for example, Suresh et ah, Methods in Enzymology, 121:210 (1986).
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies from antibody fragments
  • the following techniques can also be used for the production of bivalent antibody fragments which are not necessarily bispecific.
  • Fab' fragments recovered from E. coli can be chemically coupled in vitro to form bivalent antibodies. See, Shalaby et al, J. Exp. Med., 175:217-225 (1992).
  • bivalent heterodimers have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • the "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH heavy-chain variable domain
  • VL light- chain variable domain
  • Another strategy for making bispecific/bivalent antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et ah, J.
  • the formulation of the invention comprises an antibody which is bispecific for IL-1 (including IL-lcc and IL- ⁇ ).
  • IL-1 including IL-lcc and IL- ⁇ .
  • Examples and methods for making bispecific IL-1 antibodies can be found in U.S. Provisional Appln. No. 60/878165, filed December 29, 2006.
  • antibodies that can be used in the formulations of the invention include antibodies directed against the p40 subunit of IL-12 and/or IL-23, including the p40 subunit of human IL-12 and/or IL-23, and against amyloid beta. Specific examples of such antibodies are described in further detail below
  • the formulation contains an antibody, e.g., human antibody, that binds to an epitope of the p40 subunit of IL-12/IL-23.
  • the antibody binds to the p40 subunit when the p40 subunit is bound to the p35 subunit of IL-12.
  • the antibody binds to the p40 subunit when the p40 subunit is bound to the pl9 subunit of IL-23.
  • the antibody binds to the p40 subunit when the subunit is bound to the p35 subunit of IL-12 and also when the p40 subunit is bound to the pl9 subunit of ⁇ -23.
  • the antibody, or antigen-binding portion thereof is an antibody like those described in U.S. Patent No. 6,914,128, the entire contents of which are incorporated by reference herein.
  • the antibody binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of Y61 and J695, as described in U.S. Patent No. 6,914,128, binds.
  • human antibodies Especially preferred among the human antibodies is J695 as described in U.S. Patent No. 6,914,128.
  • Other antibodies that bind IL-12 and/or IL-23 and which can be used in the formulations of the invention include the human anti-IL-12 antibody C340, as described in U.S. Patent No. 6,902,734, the entire contents of which are incorporated by reference herein.
  • the formulation of the invention includes a combination of antibodies (two or more), or a "cocktail" of antibodies.
  • the formulation can include the antibody J695 and one or more additional antibodies.
  • the formulation of the invention contains J695 antibodies and antibody portions, J695-related antibodies and antibody portions, and other human antibodies and antibody portions with equivalent properties to J695, such as high affinity binding to hlL- 12/IL-23 with low dissociation kinetics and high neutralizing capacity.
  • the formulation contains a human antibody, or antigen-binding portion thereof, that dissociates from the p40 subunit of human IL-12/IL-23 with a of 1.34 x 10 " 10 M or less or with a K 0 ff rate constant of 1 x 10 ⁇ 3 s ⁇ l or less, as determined by surface plasmon resonance.
  • the antibody, or antigen-binding portion thereof dissociates from the p40 subunit of human IL-12/IL-23 with a k Q ff rate constant of 1 x 10 "4 s "1 or less, and more preferably with a k Q ff rate constant of 1 x 10 ' V 1 or less, or with a K d of 1 x 10 "10 M or less, and more preferably with a K d of 9.74 x 10 "11 M or less.
  • the dissociation rate constant (K 0 ff ) of an IL-12/IL-23 antibody can be determined by surface plasmon resonance.
  • surface plasmon resonance analysis measures realtime binding interactions between ligand (recombinant human IL-12 immobilized on a biosensor matrix) and analyte (antibodies in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, NJ).
  • Surface plasmon analysis can also be performed by immobilizing the analyte (antibodies on a biosensor matrix) and presenting the ligand (recombinant IL-12/IL-23 in solution) (see, for example, assays described in Example 5 of US 6,914,128, the contents of which are incorporated by reference herein).
  • Neutralization activity of IL-12/IL-23 antibodies, or antigen binding portions thereof, can be assessed using one or more of several suitable in vitro assays (see for example, assays described in Example 3 of US 6,914,128, the contents of which are incorporated by reference herein).
  • the formulation contains an antibody (e.g., human antibody), or antigen-binding portion thereof, that neutralizes the biological activity of the p40 subunit of human IL-12/IL-23.
  • the antibody, or antigen-binding portion thereof neutralizes the biological activity of free p40, e.g., monomer p40 or a p40 homodimer, e.g., a dimer containing two identical p40 subunits.
  • the antibody, or antigen-binding portion thereof neutralizes the biological activity of the p40 subunit when the p40 subunit is bound to the p35 subunit of 11-12 and/or when the p40 subunit is bound to the pl9 subunit of IL-23.
  • the antibody, or antigen- binding portion thereof inhibits human IL-12-induced phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC 50 of 1 x 10 - " 7 M or less, preferably with an IC 50 of 1 x 10 - " 8 M or less, more preferably with an IC 50 of 1 x 10 "9 M or less, even more preferably with an IC 50 of 1 x 10 "10" M or less, and most preferably with an IC 50 of 1 x 10 "n” M or less.
  • the antibody, or antigen-binding portion thereof inhibits human IL-12-induced human IFNy production with an IC 50 of 1 x 10 "10 M or less, preferably with an IC 50 of 1 x 10 "
  • the formulation contains a human antibody, or antigen-binding portion thereof, which has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2.
  • the human antibody, or antigen binding portion thereof further has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3 and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4.
  • the human antibody, or antigen binding portion thereof further has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5 and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6.
  • the antibody, or antigen binding portion thereof has heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
  • the antibody, or antigen binding portion thereof, of the formulations of the invention can comprise a heavy chain constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions.
  • the antibody heavy chain constant region is IgGl.
  • the antibody, or antigen binding portion thereof is a Fab fragment, a F(ab')2 fragment, or a single chain Fv fragment.
  • the formulation contains an antibody, e.g., humanized antibody, that binds to an epitope of the amyloid beta protein.
  • the antibody binds to oligomers of the amyloid beta protein.
  • the antibody binds to a epitope comprising the globulomer epitope of the amyloid beta protein, e.g., ⁇ (20- 42), ⁇ (20-40), ⁇ (12-42), ⁇ (12-40), ⁇ (1-42) and ⁇ (1-40).
  • the antibody, or antigen-binding portion thereof is an antibody like those described in U.S. Patent Application No.
  • the antibody comprises an antigen-binding domain which binds to amyloid-beta (20-42) globulomer, as described in Patent Application No. 2009/0175847 Al .
  • the humanized antibodies is 5F7, as described in Patent Application No. 2009/0175847 Al .
  • Other antibodies that bind amyloid beta and which can be used in the formulations of the invention include the humanized anti-amyloid beta antibody 7C6, as described in U.S. Patent Application No. 2009/0175847 Al .
  • the formulation of the invention includes a combination of antibodies (two or more), or a "cocktail" of antibodies.
  • the formulation can include the antibody 5F7 and one or more additional antibodies.
  • the formulation of the invention contains 5F7 antibodies and antibody portions, 5F7-related antibodies and antibody portions, and other humanized antibodies and antibody portions with equivalent properties to 5F7, such as high affinity binding to amyloid- beta with low dissociation kinetics and high neutralizing capacity.
  • the formulation contains a humanized antibody, or antigen- binding portion thereof, that dissociates from the amyloid-beta (20-42) globulomer with a of 1 x 10-6 M or less.
  • the formulation contains an antibody (e.g. , human antibody), or antigen-binding portion thereof, that neutralizes the biological activity of the amyloid-beta.
  • the antibody, or antigen-binding portion thereof binds to an ⁇ (20-42) globulomer with a K D in the range of 1 x 10 "6 M to 1 x 10 "12 M, more preferably with a K D of 1 x 10 " M or greater affinity, even more preferably with a K D of 3 x
  • the formulation contains a human antibody, or antigen-binding portion thereof, which has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.
  • the human antibody, or antigen binding portion thereof further has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11 and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12.
  • the human antibody, or antigen binding portion thereof further has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13 and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.
  • the antibody, or antigen binding portion thereof has heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
  • the antibody, or antigen binding portion thereof, of the formulations of the invention can comprise a heavy chain constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions.
  • the antibody heavy chain constant region is IgGl.
  • the antibody, or antigen binding portion thereof is a Fab fragment, a F(ab')2 fragment, or a single chain Fv fragment.
  • compositions and formulations of the invention may be used both therapeutically or prophylactic ally, i.e., in vivo, or as reagents for in vitro or in situ purposes.
  • the methods of the invention may also be used to make a stable protein formulation comprising a protein at a concentration below the reverse stability concentration and having characteristics which are advantageous for therapeutic use.
  • the stable protein formulation having a protein concentration below the reverse stability concentration may be used as a pharmaceutical formulation to treat a disorder in a subject.
  • the formulation of the invention may be used to treat any disorder for which the therapeutic protein is appropriate for treating.
  • a "disorder" is any condition that would benefit from treatment with the protein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • a therapeutically effective amount of the antibody may be administered to treat a neurological disorder, such as multiple sclerosis,an autoimmune disease, such as rheumatoid arthritis, an intestinal disorder, such as Crohn's disease, or a skin disorder, such as psoriasis.
  • disorders in which the formulation of the invention may be used to treat include cancer, including breast cancer, leukemia, lymphoma, and colon cancer.
  • subject is intended to include living organisms, e.g. , prokaryotes and eukaryotes.
  • subjects include mammals, e.g. , humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder, as well as those in which the disorder is to be prevented.
  • the stable protein formulations comprising a protein at a concentration below the reverse stability concentration may be administered to a mammal, including a human, in need of treatment in accordance with known methods of administration.
  • methods of administration include intravenous administration, such as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, intradermal, transdermal, oral, topical, or inhalation administration.
  • a formulation of the invention can be administered to a human subject for therapeutic purposes (discussed further below).
  • the liquid pharmaceutical formulation is easily administratable, which includes, for example, a formulation which is self-administered by the patient.
  • the formulation of the invention is administered through subcutaneous injection, preferably single use.
  • a formulation of the invention can be administered to a non-human mammal expressing a target antigen with which the antibody cross-reacts (e.g. , a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g. , testing of dosages and time courses of administration).
  • One advantage of the formulations of the invention is the ability to prepare formulations comprising high concentrations of therapeutic protein without increasing the viscosity of the formulation. Therefore, the new formulations permit administration of high amounts (e.g. , effective amounts) of therapeutic protein in smaller volumes as compared to prior commercial formulations, thereby decreasing pain.
  • formulation of the invention may be used to deliver a high concentration of a protein, e.g. , a therapeutic protein, e.g. , an antibody, or antigen- binding portion, (e.g. , briakinumab) to a subject subcutaneously.
  • a protein e.g. , a therapeutic protein, e.g. , an antibody, or antigen- binding portion, (e.g. , briakinumab)
  • formulations of the invention are delivered to a subject subcutaneously.
  • the subject administers the formulation to himself/herself.
  • the stable protein formulations comprising a protein at a concentration below the reverse stability concentration is administered to the mammal by subcutaneous administration.
  • the formulation may be injected using a syringe, as well as other devices including injection devices (e.g. , the Inject-ease and Genject devices); injector pens (such as the GenPen); needleless devices (e.g. , MediJector and
  • the device e.g., a syringe, autoinjector pen
  • the needle gauge ranges in size from 25G to 33 G (including ranges intermediate thereto, e.g., 25sG, 26, 26sG, 27G, 28G, 29G, 30G, 31G, 32G, and 33G).
  • the smallest needle diameter and appropriate length is chosen in accordance with the viscosity characteristics of the formulation and the device used to deliver the formulation of the invention.
  • One advantage of the methods/compositions of the invention is that they provide large concentrations of a protein in a solution which may be ideal for administering the protein to a subject using a needleless device.
  • a needleless device allows for dispersion of the protein throughout the tissue of a subject without the need for an injection by a needle.
  • needleless devices include, but are not limited to, Biojectorr 2000 (Bioject Medical
  • delivery devices that house the stable protein formulations comprising a protein at a concentration below the reverse stability concentration.
  • delivery devices include, but are not limited to, a syringe, a pen (such as an autoinjector pen), an implant, an inhalation device, a needleless device, and a patch.
  • a syringe such as an autoinjector pen
  • an implant such as an autoinjector pen
  • an inhalation device such as a needleless device
  • patch such as an autoinjection pen
  • An example of an autoinjection pen is described in US Appln. No. 11/824516, filed June 29, 2007.
  • the invention also includes methods of delivering the formulations of the invention by inhalation and inhalation devices containing said formulation for such delivery.
  • the stable protein formulations comprising a protein at a concentration below the reverse stability concentration is administered to a subject via inhalation using a nebulizer or liquid inhaler.
  • nebulizers use compressed air to deliver medicine as wet aerosol or mist for inhalation, and, therefore, require that the drug be soluble in water.
  • Types of nebulizers include jet nebulizers (air-jet nebulizers and liquid-jet nebulizers) and ultrasonic nebulizers.
  • nebulizers examples include AkitaTM (Activaero GmbH) (see US2001037806, EP1258264).
  • AkitaTM is a table top nebulizer inhalation system (Wt: 7.5 kg , BxWxH: 260 x 170 x 270) based on Pari's LC Star that provides full control over patient's breathing pattern.
  • the device can deliver as much as 500 mg drug in solution in less than 10 min with a very high delivery rates to the lung and the lung periphery. 65% of the nebulized particles are below 5 microns and the mass median aerodynamic diameter (MMAD) is 3.8 microns at 1.8 bar.
  • the minimum fill volume is 2 mL and the maximum volume is 8 mL.
  • the inspiratory flow (200 mL/sec) and nebulizer pressure (0.3-1.8 bar) are set by the smart card.
  • the device can be individually adjusted for each patient on the basis of a lung function test.
  • the Aeroneb ® nebulizer is based on OnQTM technology, i.e., an electronic micropump (3/8 inch in diameter and wafer-thin) comprised of a unique dome-shaped aperture plate that contains over 1,000 precision-formed tapered holes, surrounded by a vibrational element.
  • Aeroneb ® Go is a portable unit for home use
  • Aeroneb ® Pro is a reusable and autoclavable device for use in hospital and ambulatory clinic
  • Aeroneb ® Lab is a device for use in pre-clinical aerosol research and inhalation studies.
  • the features of the systems include optimization and customization of aerosol droplet size; low- velocity aerosol delivery with a precisely controlled droplet size, aiding targeted drug delivery within the respiratory system; flexibility of dosing;
  • AerocurrentTM (AerovertRx corp) may also be used with compositions of the invention (see WO2006006963).
  • This nebulizer is a portable, vibrating mesh nebulizer that features a disposable, pre-filled or user filled drug cartridge.
  • StaccatoTM may also be used with compositions of the invention (see WO03095012).
  • the key to StaccatoTM technology is vaporization of a drug without thermal degradation, which is achieved by rapidly heating a thin film of the drug. In less than half a second, the drug is heated to a temperature sufficient to convert the solid drug film into a vapor.
  • the inhaler consists of three core components: a heating substrate, a thin film of drug coated on the substrate, and an airway through which the patient inhales. The inhaler is breath- actuated with maximum dose delivered to be 20-25 mg and MMAD in the 1-2 micron range.
  • AERx ® (Aradigm) may also be used with compositions of the invention (see
  • AERx ® is a hand held battery operated device which utilizes a piston mechanism to expel formulation from the AERx ® Strip.
  • the device monitors patients inspiratory air flow and fires only when optimal breathing pattern is achieved.
  • the device can deliver about 60% of the dose as emitted dose and 50-70% of the emitted dose into deep lung with ⁇ 25% inter-subject variability.
  • Respimat ® is a multi-dose reservoir system that is primed by twisting the device base, which is compressed a spring and transfers a metered volume of formulation from the drug cartridge to the dosing chamber.
  • the spring is released, which forces a micro-piston into the dosing chamber and pushes the solution through a uniblock; the uniblock consists of a filter structure with two fine outlet nozzle channels.
  • the MMAD generated by the Respimat ® is 2 um, and the device is suitable for low dose drugs traditionally employed to treat respiratory disorders.
  • compositions of the invention may also be delivered using the Collegium NebulizerTM (Collegium Pharma), which is a nebulizer system comprised of drug deposited on membrane.
  • Collegium NebulizerTM Cold Pharma
  • the dosage form is administered to a patient through oral or nasal inhalation using the Collegium Nebulizer after reconstitution with a reconstituting solvent.
  • a nebulizer device which may also be used with compositions of the invention includes the Inspiration ® 626 (Respironics).
  • the 626 is a compressor based nebulizer for home care.
  • the 626 delivers a particle size between 0.5 to 5 microns.
  • Nebulizers which can be used with compositions of the invention may include Adaptive Aerosol Delivery ® technology (Respironics), which delivers precise and
  • AAD ® systems incorporate electronics and sensors within the handpiece to monitor the patient's breathing pattern by detecting pressure changes during inspiration and expiration. The sensors determine when to pulse the aerosol delivery of medication during the first part of inspiration. Throughout the treatment, the sensors monitor the preceding three breaths and adapt to the patient's inspiratory and expiratory pattern.
  • AAD ® systems only deliver medication when the patient is breathing through the mouthpiece, these devices allow the patient to take breaks in therapy without medication waste.
  • AAD ® system nebulizers include the HaloLite ® AAD ® , ProDose ® AAD ® , and I-Neb ® AAD ® .
  • the HaloLite ® Adaptive Aerosol Delivery (AAD) ® (Respironics) is a pneumatic aerosolisation system powered by a portable compressor.
  • the AAD ® technology monitors the patient's breathing pattern (typically every ten milliseconds) and, depending upon the system being used, either releases pulses of aerosolized drug into specific parts of the inhalation, or calculates the dose drawn during inhalation from a "standing aerosol cloud" (see EP 0910421, incorporated by reference herein).
  • the ProDos AAD ® (Respironics) is a nebulizing system controlled by "ProDose DiscTM" system. (Respironics).
  • ProDos AAD ® is a pneumatic aerosol system powered by a portable compressor, in which the dose to be delivered is controlled by a microchip- containing disc inserted in the system that, among other things, instructs the system as to the dose to deliver.
  • the ProDose DiscTM is a plastic disc containing a microchip, which is inserted into the ProDose AAD ® System and instructs it as to what dose to deliver, the number of doses, which may be delivered together with various control data including drug batch code and expiry date (see EP1245244, incorporated by reference herein).
  • Promixin ® can be delivered via Prodose AAD ® for management of pseudomonas aeruginosa lung infections, particularly in cystic fibrosis.
  • Promixin ® is supplied as a powder for nebulization that is reconstituted prior to use.
  • the I-neb AAD ® is a handheld AAD ® system that delivers precise and reproducible drug doses into patients' breathing patterns without the need for a separate compressor ("I- Neb").
  • the I-neb AAD ® is a miniaturized AAD ® inhaler based upon a combination of electronic mesh-based aerosolisation technology (Omron) and AAD ® technology to control dosing into patients' breathing patterns.
  • the system is approximately the size of a mobile telephone and weighs less than 8 ounces.
  • I-neb A AD ® has been used for delivery of
  • Ventavis ® (iloprost) (CoTherix / Schering AG).
  • AriaTM Chrysalis
  • Aria is based on a capillary aerosol generation system. The aerosol is formed by pumping the drug formulation through a small, electrically heated capillary. Upon exiting the capillary, the formulation rapidly cooled by ambient air to produce an aerosol with MMAD ranging from 0.5-2.0 um.
  • the TouchSprayTM nebulizer may be used to deliver a composition of the invention.
  • the TouchSprayTM nebulizer is a hand-held device which uses a perforate membrane, which vibrates at ultrasonic frequencies, in contact with the reservoir fluid, to generate the aerosol cloud. The vibration action draws jets of fluid though the holes in the membrane, breaking the jets into drug cloud. The size of the droplets is controlled by the shape/size of the holes as well as the surface chemistry and composition of the drug solution. This device has been reported to deliver 83% of the metered dose to the deep lung. Details of the TouchSprayTM nebulizer are described in US Patent No, 6659364, incorporated by reference herein.
  • nebulizers which are portable units which maximize aerosol output when the patient inhales and minimize aerosol output when the patient exhales using two one-way valves (see PARI nebulizers (PARI GmbH). Baffles allow particles of optimum size to leave the nebulizer. The result is a high percentage of particles in the respirable range that leads to improved drug delivery to the lungs.
  • nebulizers may be designed for specific patient populations, such a patients less than three years of age (PARI BABYTM) and nebulizers for older patients (PARI LC PLUS ® and PARI LC STAR ® ).
  • An additional nebulizer which may be used with compositions of the invention is the e-Flow ® nebulizer (PARI GmbH) which uses vibrating membrane technology to aerosolize the drug solution, as well as the suspensions or colloidal dispersions (, TouchSprayTM ;ODEM (United Kingdom)).
  • An e-Flow ® nebulizer is capable of handling fluid volumes from 0.5 ml to 5 ml, and can produce aerosols with a very high density of active drug, a precisely defined droplet size, and a high proportion of respirable droplets delivered in the shortest possible amount of time.
  • Drugs which have been delivered using the e-Flow ® nebulizer include aztreonam and lidocaine. Additional details regarding the e-Flow nebulizer are described in US 6962151, incorporated by reference herein.
  • nebulizers which may be used with compositions of the invention include a Microair ® electronic nebulizer (Omron) and a MysticTM nebulizer (Ventaira).
  • the Microair ® nebulizer is extremely small and uses Vibrating Mesh Technology to efficiently deliver solution medications.
  • the Microair device has 7 mL capacity and produces drug particle MMAD size around 5 microns.
  • the MysticTM nebulizer uses strong electric field to break liquid into a spray of nearly monodispersed, charged particles.
  • the MysticTM system includes a containment unit, a dose metering system, aerosol generation nozzles, and voltage converters which together offer multi-dose or unit- dose delivery options.
  • the MysticTM device is breath activated, and has been used with Coras 1030TM (lidocaine HC1), Resmycin ® (doxorubicin hydrochloride), Acuair (fluticasone propionate), NCE with ViroPharm, and NCE with Pfizer. Additional details regarding the MysticTM nebulizer may be found in U.S. Patent No. 6397838, incorporated by reference herein.
  • the appropriate dosage (therapeutically effective amount) of the protein will depend, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the discretion of the attending physician.
  • the protein is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards.
  • the protein may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
  • the formulations of the invention overcome the common problem of protein aggregation, and, therefore, provide a new means by which therapeutic proteins with low levels of protein aggregation may be administered to a patient.
  • the effective amount of therapeutic protein may be determined according to a strictly weight based dosing scheme (e.g., mg/kg) or may be a total body dose (also referred to as a fixed dose) which is independent of weight.
  • an effective amount of the formulation is 0.8 mL of the formulation containing a total body dose of about 80 mg of therapeutic protein (i.e., 0.8 mL of a 100 mg/mL therapeutic protein formulation of the invention).
  • an effective amount of the formulation is 0.4 mL of the formulation of the invention containing a total body dose of about 40 mg of therapeutic protein (i.e., 0.4 mL of a 100 mg/mL therapeutic protein formulation of the invention).
  • an effective amount of the formulation is twice 0.8 mL of the formulation containing a total body dose of about 160 mg of therapeutic protein (i.e., two units containing 0.8 mL each of a 100 mg/mL therapeutic protein formulation of the invention).
  • an effective amount of the formulation is 0.2 mL of the formulation of the invention containing a total body dose of about 20 mg of therapeutic protein (i.e., 0.2 mL of a 100 mg/mL therapeutic protein formulation of the invention).
  • an effective amount may be determined according to a weight-based fixed dosing regimen (see, e.g., WO
  • a liquid pharmaceutical formulation of the invention may be administered to a subject via a prefilled syringe, an autoinjector pen, or a needle-free administration device.
  • the invention features a delivery device comprising a dose of the formulation comprising 100 mg/mL a therapeutic protein, e.g, a human anti-IL-12 antibody, or antigen-binding portion thereof, e.g., an autoinjector pen or prefilled syringe comprises a dose of about 19 mg, 20, mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg,
  • the stable protein formulations comprising a protein at a concentration below the reverse stability concentration provided by the invention may also be used for non-therapeutic uses, i.e., in vitro purposes.
  • Stable protein formulations comprising a protein at a concentration below the reverse stability concentration described herein may be used for diagnostic or experimental methods in medicine and biotechnology, including, but not limited to, use in genomics, proteomics, bioinformatics, cell culture, plant biology, and cell biology.
  • stable protein formulations comprising a protein at a concentration below the reverse stability concentration described herein may be used to provide a protein needed as a molecular probe in a labeling and detecting methods.
  • An additional use for the formulations described herein is to provide supplements for cell culture reagents, including cell growth and protein production for manufacturing purposes.
  • Stable protein formulations comprising a protein at a concentration below the reverse stability concentration described herein could be used in protocols with reduced concern regarding how an excipient in the formulation may react with the experimental environment, e.g., interfere with another reagent being used in the protocol.
  • stable formulations containing protein concentrations below the reverse stability concentration may be used as a reagent for laboratory use.
  • Thee formulation may be used to deliver high concentrations of a desired protein, such as a nutritional supplement, to a food item, without the presence of polyols, e.g., sugar alcohols, which pass undigested into the large intestine.
  • a desired protein such as a nutritional supplement
  • polyols e.g., sugar alcohols
  • whey- and soy-derived proteins are lending versatility to foods as these proteins have an ability to mimic fat's mouthfeel and texture.
  • whey- and soy-derived proteins may be added to foods to decrease the overall fat content, without sacrificing satisfaction.
  • a stable protein formulation comprising suitable amounts of whey- and soy-derived proteins without polyols may be formulated and used to supplement food products.
  • an article of manufacture which contains the aqueous formulation of the present invention and provides instructions for its use.
  • the article of manufacture comprises a container.
  • Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as dual chamber syringes), autoinjector pen containing a syringe, and test tubes.
  • the container may be formed from a variety of materials such as glass, plastic or polycarbonate.
  • the container holds the aqueous
  • formulation and the label on, or associated with, the container may indicate directions for use.
  • the label may indicate that the formulation is useful or intended for
  • the container holding the formulation may be a multi-use vial, which allows for repeated administrations (e.g., from 2-6 administrations) of the aqueous formulation.
  • the article of manufacture may further comprise a second container.
  • the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • Size exclusion HPLC was used to determine the purity of the protein solution samples. The assay was performed with the parameters detailed below.
  • a TSK gel guard (cat. no. 08543, 6.0 mm x 4.0 cm, 7 ⁇ ), was combined with a TSK gel G3000SW (cat. no. 08541, 7.8 mm x 30 cm, 5 ⁇ ) and run with an upper column pressure limit of 70 bar.
  • the mobile phase consisted of 100 mM Na 2 HP0 4 / 200 mM Na 2 S0 4 , pH 7.0.
  • This buffer was created by dissolving 49.68 g anhydrous disodium hydrogen phosphate and 99.44 g anhydrous sodium sulfate in approximately 3300 mL Milli-Q water, adjusting the pH to 7.0 using 1 M phosphoric acid, increasing the buffer volume to 3500 mL with Milli-Q water and filtering the solution through a membrane filter.
  • Detection was done using a diode array detector using a 214 nm wavelength (> 0.1 min peak width and 8 nm band width) and a 360 nm reference wavelength (100 nm band width).
  • Ultra performance liquid chromatography was also used to determine the purity of the protein solution samples. Samples have a protein concentration of 1 mg/ml were prepared and analyzed to calibrate the retention and run times. The experimental parameters were as follows:
  • the antibody stock formulations were prepared in the appropriate buffer solutions by applying DF/UF step.
  • the solutions were sterile filtered through 0.22 micron Millipore syringe filters ( Millipore, SLGV 013 SL) and filled in 0.5 mL vials
  • the sugar/polyols used in the study were: sucrose (J. T. Baker, 4074-05, Lot# H52479), Trehalose (Merck, K32496116, Lot# 747), or Mannitol (Mallinckrodt, 6208-04, Lot# 48626).
  • Methionine, pH 6.0 The ABT-736 stock solutions were formulated in 15 mM Histidine, pH 6.0. Samples were prepared at 1, 100 and 200 mg/mL for all three antibodies containing 0, 25, 50, 75, 100, 125, 150, 175 or 200 mg/mL sugar/polyols.
  • Fragmentation and aggregation are important degradation pathways influencing protein stability during long-term storage of liquid protein formulations. Especially aggregation is a degradation pathway that very often is observed in protein formulations, and the use of polyol excipients to increase protein stability in liquid protein formulations during shelf-life is a very established and accepted stabilization strategy formulators successfully apply-
  • trehalose a sugar routinely used in protein formulation to increase protein stability
  • ABT-736 samples were formulated at 200 mg/mL and 100 mg/mL in 15 mM histidine, pH 6 buffer, with (80 mg/mL trehalose) and without (0 mg/mL trehalose) trehalose, and all formulations were exposed to real-time stability studies (see Table 2A).
  • Histidine + 100 mM NaCl buffer, pH 6) is provided in Table 2D.
  • Real-time long-term stability (two months) of ABT-736 at 5oC was also determined for samples formulated in 15 mM histidine and higher ionic strength conditions (50 mM NaCl) with and without 80 mg/ml sucrose.
  • the results of these studies are provided in Table 2E. All of the data (as shown in Table 2 and in Figures 1 and 2) show that aggregation stability (i.e. physical stability of protein) can be dependent on protein concentration. At lower concentrations aggregation is increased by sugars/polyols (in concentrations of pharmaceutical interest) while at higher concentrations aggregation may be decreased. This result was very surprising, since this is exactly opposite to the crowding hypothesis and results in literature available today.
  • Samples were filled into 0.5 mL repositories and stored under controlled conditions (in temperature chambers and in the absence of light) at 5°C, 40°C and 50°C. At predefined points of time, samples of prepared solutions were pulled for analysis according to the sample pull scheme provided at the top of Table 1. Symbols refer to number of vials that were stored/pulled. Data is provided in Table 2.
  • Table 1 Sample pull schemes for Table 2A:
  • Table 2A Impact of trehalose concentration on stability of ABT-736 at 5C (100 and 200 mg mL, 15 mM Histidine buffer, pH 6). Average of duplicates.
  • Table 2D Impact of sucrose concentration on stability of ABT-736 at 50C (9 days) (15 mM Histidine + 100 Mm NaCl buffer, pH 6).
  • Table 2E Impact of sucrose concentration on stability of ABT-736 at 5°C (two months) (15 mM Histidine pH 6.0 and 15 mM Histidine + 50 Mm NaCl buffer, pH 6).
  • ABT-736 stability decreased over time at 5°C conditions, and both aggregation and fragmentation was observed.
  • the sugars had a destabilizing influence at 100 mg/mL.
  • ABT-736 liquid formulations e.g., at 100 mg/mL protein
  • results in ABT-736 formulations with decreased stability i.e., levels of aggregate are higher and levels of monomer are lower in ABT-736 formulations that contain trehalose than in formulations without trehalose.
  • Example 3 shows that the intrinsic stability of ABT-736 in pH 6, 15 mM histidine buffer formulation is higher, the more sucrose is present in the formulation. According to the current understanding of DSC/DSF experiments and how to apply those data in a protein formulation laboratory, higher intrinsic stability of ABT-736 as determined in characterization studies such as DSF or DSC should be reflected in higher stability and decreased aggregate levels during accelerated stability studies. Thus, these results of ABT-736 liquid formulation stability studies were surprising.
  • Example 2 Impact of Sucrose Concentration on ABT-874 Stability during Accelerated And Long-Term Storage (Table 3)
  • ABT-874 samples were formulated with and without sucrose, and all formulations were exposed to real-time stability studies (see Table 3D).
  • ABT- 874 formulations were subjected to short-term storage at elevated temperatures (40°C) in order to relatively quickly gain insight in the sucrose stabilization potential for long-term storage at lower temperatures (often at 2-8°C) (Table 3A).
  • Storage stability under accelerated conditions at 50°C was also evaluated to gain data additional (Table 3B).
  • Storage stability under accelerated conditions (50°C) for ABT-874 in higher ionic strength conditions (15mM Histidine + 100 mM NaCl buffer, pH 6) is provided in Table 3C.
  • Samples were filled into 0.5 mL repositories and stored under controlled conditions (in temperature chambers and in the absence of light) at 40°C and 50°C. At predefined points of time, samples of prepared solutions were pulled for analysis according to the sample pull scheme provided in Table 1 (above). Numbers refer to number of vials that were
  • Differential scanning fluorescence was used to measure the thermal stability of ABT- 736 and 874 in the absence and presence of sugars.
  • the assay was performed with the parameters detailed in the method section. As shown in Table 4, an increase in polyol concentration lead to a progressive increase in intrinsic stability/unfolding temperature of ABT-736 and 874 at all concentrations. This was found to be true for all sugars tested i.e. sucrose, trehalose, mannitol and sorbitol.
  • the sugar concentration was varied from 0 to 120 mg/ml except for mannitol wherein the solubility restricted us to 150 mg/mL.
  • Example 4 Impact of Sugars on Anti-HER2 Antibody Stability during Long-Term Storage
  • a sugar routinely used in protein formulations to increase protein stability
  • additional experiments were carried out with a humanized IgGl kappa monoclonal antibody that selectively binds with high affinity to the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2).
  • HER2 human epidermal growth factor receptor 2 protein
  • Samples comprising the anti-HER2 antibody were formulated with and without sucrose and with and without NaCl, and all formulations were exposed to real-time stability studies (see Table 5).
  • VEGF vascular endothelial growth factor
  • Antibody samples were formulated with and without sucrose and with and without NaCl, and all formulations were exposed to real-time stability studies (see Table 6).
  • the data show that aggregation stability (i.e. , physical stability of the protein) is dependent on protein concentration. At lower concentrations of the antibody aggregation is increased by sugars/polyols (in concentrations of pharmaceutical interest) while at higher concentrations of the antibody (e.g., in low ionic formulations) aggregation is decreased by sugars/polyols (in concentrations of pharmaceutical interest). This is exactly opposite to results reported in the literature. Table 6. Impact of sucrose on anti-VEGF antibody in 15mM Histidine at pH 6 and in 15mM Histidine + 50 mM NaCl at pH 6.
  • Example 6 Determining Reverse Stability Concentrations
  • polyols e.g., sugars
  • This perturbation results in the destabilization effect that is surprising.
  • the destabilization of lower protein concentration formulations by polyols is a new, surprising finding.
  • Conformational (thermal) and stability studies may be conducted for various concentrations of polyols and/or proteins for various periods of time.
  • formulations comprising about 1-200 mg/mL concentrations of protein (e.g. , ABT-736 and 874) in combination with 0, 30, 60, 90, 120 mg/mL concentrations of sucrose are used in conformational and stability studies.
  • These different formulations are subjected to stability studies at various temperatures (e.g., 5, 25 and 40°C).
  • the formulations are also investigated for thermal stability using dynamic scanning fluorescence in order to determine the stabilization potential of the sugars. Size exclusion chromatography is used to monitor protein aggregation in the formulations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Oncology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une formulation de protéine comprenant un polyol, de stabilité accrue. L'invention concerne également des procédés de stabilisation d'une formulation de protéine par détermination de la Concentration de Stabilité Inverse pour une protéine dans une formulation comprenant un polyol. L'invention concerne en outre des procédés de stabilisation de formulations de protéine par réduction de la quantité de polyol dans la composition. L'invention concerne des formulations d'anticorps ayant une stabilité à long terme et des caractéristiques avantageuses pour une administration sous-cutanée.
PCT/US2012/071368 2011-12-23 2012-12-21 Formulations stables de protéine WO2013096835A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161580046P 2011-12-23 2011-12-23
US61/580,046 2011-12-23

Publications (1)

Publication Number Publication Date
WO2013096835A1 true WO2013096835A1 (fr) 2013-06-27

Family

ID=48669556

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/071368 WO2013096835A1 (fr) 2011-12-23 2012-12-21 Formulations stables de protéine

Country Status (1)

Country Link
WO (1) WO2013096835A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015084883A2 (fr) 2013-12-02 2015-06-11 Abbvie, Inc. Compositions et méthodes pour traiter l'arthrose
US9340612B2 (en) 2001-03-19 2016-05-17 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
CN110559435A (zh) * 2013-09-11 2019-12-13 伊戈尔生物药品股份有限公司 包含离子性液体的液体蛋白质制剂
WO2020056008A1 (fr) 2018-09-12 2020-03-19 Silverback Therapeutics, Inc. Compositions pour le traitement d'une maladie avec des conjugués immunostimulants
WO2020257407A1 (fr) 2019-06-19 2020-12-24 Silverback Therapeutics, Inc. Anticorps anti-mésothéline et immunoconjugués de ceux-ci
US10961301B2 (en) 2011-04-01 2021-03-30 Yale University Cell-penetrating anti-DNA antibodies and uses thereof inhibit DNA repair
US11071782B2 (en) 2016-04-20 2021-07-27 Coherus Biosciences, Inc. Method of filling a container with no headspace
US11179473B2 (en) 2020-02-21 2021-11-23 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
US11229702B1 (en) 2015-10-28 2022-01-25 Coherus Biosciences, Inc. High concentration formulations of adalimumab
US11541126B1 (en) 2020-07-01 2023-01-03 Silverback Therapeutics, Inc. Anti-ASGR1 antibody TLR8 agonist comprising conjugates and uses thereof
US11590242B2 (en) 2016-06-15 2023-02-28 Yale University Antibody-mediated autocatalytic, targeted delivery of nanocarriers to tumors

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5804557A (en) * 1993-02-23 1998-09-08 Genentech, Inc. Excipient stabilization of polypeptides treated with organic solvents
US20090291062A1 (en) * 2007-11-30 2009-11-26 Wolfgang Fraunhofer Protein formulations and methods of making same
US20090298768A1 (en) * 2006-07-06 2009-12-03 Daewoong Co., Ltd. Stable liquid formulation of human growth hormone
US20100278822A1 (en) * 2009-05-04 2010-11-04 Abbott Biotechnology, Ltd. Stable high protein concentration formulations of human anti-tnf-alpha-antibodies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5804557A (en) * 1993-02-23 1998-09-08 Genentech, Inc. Excipient stabilization of polypeptides treated with organic solvents
US20090298768A1 (en) * 2006-07-06 2009-12-03 Daewoong Co., Ltd. Stable liquid formulation of human growth hormone
US20090291062A1 (en) * 2007-11-30 2009-11-26 Wolfgang Fraunhofer Protein formulations and methods of making same
US20100278822A1 (en) * 2009-05-04 2010-11-04 Abbott Biotechnology, Ltd. Stable high protein concentration formulations of human anti-tnf-alpha-antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AHAMED ET AL.: "Phase Behavior of an Intact Monoclonal Antibody.", BIOPHYS J, vol. 93, no. 2, 15 July 2007 (2007-07-15), pages 610 - 619, XP002614660 *

Cited By (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9340612B2 (en) 2001-03-19 2016-05-17 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10961301B2 (en) 2011-04-01 2021-03-30 Yale University Cell-penetrating anti-DNA antibodies and uses thereof inhibit DNA repair
US10286071B2 (en) 2012-09-07 2019-05-14 Coherus Biosciences, Inc. Syringe containing stable aqueous formulations of adalimumab
US10786566B2 (en) 2012-09-07 2020-09-29 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9382317B2 (en) 2012-09-07 2016-07-05 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9682145B2 (en) 2012-09-07 2017-06-20 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9707293B2 (en) 2012-09-07 2017-07-18 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9724414B2 (en) 2012-09-07 2017-08-08 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9724415B2 (en) 2012-09-07 2017-08-08 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9731009B2 (en) 2012-09-07 2017-08-15 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9731008B2 (en) 2012-09-07 2017-08-15 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9737600B2 (en) 2012-09-07 2017-08-22 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9757454B2 (en) 2012-09-07 2017-09-12 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9770507B2 (en) 2012-09-07 2017-09-26 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9782480B2 (en) 2012-09-07 2017-10-10 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9782479B2 (en) 2012-09-07 2017-10-10 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9789185B2 (en) 2012-09-07 2017-10-17 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9808525B2 (en) 2012-09-07 2017-11-07 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10286072B2 (en) 2012-09-07 2019-05-14 Coherus Biosciences, Inc. Methods of manufacturing stable aqueous formulations of adalimumab
US10155039B2 (en) 2012-09-07 2018-12-18 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10159733B2 (en) 2012-09-07 2018-12-25 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10159732B2 (en) 2012-09-07 2018-12-25 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10195275B2 (en) 2012-09-07 2019-02-05 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10207000B2 (en) 2012-09-07 2019-02-19 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9340611B2 (en) 2012-09-07 2016-05-17 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10799585B2 (en) 2012-09-07 2020-10-13 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9861695B2 (en) 2012-09-07 2018-01-09 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US9346880B2 (en) 2012-09-07 2016-05-24 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10688183B2 (en) 2012-09-07 2020-06-23 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10716854B2 (en) 2012-09-07 2020-07-21 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10716852B2 (en) 2012-09-07 2020-07-21 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10716853B2 (en) 2012-09-07 2020-07-21 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10722579B2 (en) 2012-09-07 2020-07-28 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10772959B2 (en) 2012-09-07 2020-09-15 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10772960B2 (en) 2012-09-07 2020-09-15 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
US10780163B2 (en) 2012-09-07 2020-09-22 Coherus Biosciences, Inc. Stable aqueous formulations of adalimumab
CN110559435B (zh) * 2013-09-11 2023-10-20 伊戈尔生物药品股份有限公司 包含离子性液体的液体蛋白质制剂
CN110559435A (zh) * 2013-09-11 2019-12-13 伊戈尔生物药品股份有限公司 包含离子性液体的液体蛋白质制剂
US11986526B2 (en) 2013-09-11 2024-05-21 Eagle Biologics, Inc. Liquid protein formulations containing 4-ethyl-4-methylmorpholinium methylcarbonate (EMMC)
WO2015084883A2 (fr) 2013-12-02 2015-06-11 Abbvie, Inc. Compositions et méthodes pour traiter l'arthrose
US11229702B1 (en) 2015-10-28 2022-01-25 Coherus Biosciences, Inc. High concentration formulations of adalimumab
US11071782B2 (en) 2016-04-20 2021-07-27 Coherus Biosciences, Inc. Method of filling a container with no headspace
US11576971B2 (en) 2016-04-20 2023-02-14 Coherus Biosciences, Inc. Method of filling a container with no headspace
US11590242B2 (en) 2016-06-15 2023-02-28 Yale University Antibody-mediated autocatalytic, targeted delivery of nanocarriers to tumors
WO2020056008A1 (fr) 2018-09-12 2020-03-19 Silverback Therapeutics, Inc. Compositions pour le traitement d'une maladie avec des conjugués immunostimulants
WO2020257407A1 (fr) 2019-06-19 2020-12-24 Silverback Therapeutics, Inc. Anticorps anti-mésothéline et immunoconjugués de ceux-ci
US11179473B2 (en) 2020-02-21 2021-11-23 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
US11541126B1 (en) 2020-07-01 2023-01-03 Silverback Therapeutics, Inc. Anti-ASGR1 antibody TLR8 agonist comprising conjugates and uses thereof

Similar Documents

Publication Publication Date Title
US11191834B2 (en) Protein formulations and methods of making same
US8420081B2 (en) Antibody formulations and methods of making same
WO2013096835A1 (fr) Formulations stables de protéine
US20130195888A1 (en) Ultrafiltration and diafiltration formulation methods for protein processing
AU2017203012A1 (en) Protein formulations and methods of making same
US20230007847A1 (en) Protein formulations and methods of making same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12859298

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12859298

Country of ref document: EP

Kind code of ref document: A1