Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
  • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
  • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• the present invention relates to aqueous solutions which have high concentrations of therapeutical antibodies and to stable liquid formulations which are based on such aqueous solutions of antibodies.
• the present invention also relates to uses, such as medical uses, of the stable liquid formulations and processes for the production of the stable liquid formulations.
• Stable liquid formulations of antibodies are useful for parenteral administration, such as intravenous (i.v.), intramuscular (i.m.) or subcutaneous (s.c.) administration.
• parenteral administration such as intravenous (i.v.), intramuscular (i.m.) or subcutaneous (s.c.) administration.
• Such formulatioris must fulfill two key requirements: 1) the required drug concentration must be achieved, and, 2) the drug must be chemically and physically stable in order to have a sufficient shelf-life.
• a formulation For a protein to remain biologically active, a formulation must preserve intact the conformational integrity and at the same time the protein's multiple functional groups must be protected from degradation.
• Degradation pathways for proteins can involve chemical instability or physical instability. For example, chemical instability can result from deamidation, hydrolysis, oxidation, beta-elimination or disulfide exchange, while physical instability can result from denaturation, aggregation, precipitation or adsorption, for example. Aggregation is one of the most common protein degradation pathways.
• freeze-drying process is a costly and time consuming process, and it would be advantagenous if this step could be avoided when preparing a commercial antibody formulation.
• the present invention provides a stable aqueous solution comprising an antibody at a concentration of at least 50 mg/ml, and further comprising at least one acidic component.
• a suitable delivery system which contains the aqueous solution.
• aqueous solution in a nasal spray or a slow release formulation.
• aqueous solution in a drying or freeze-drying process.
• Stable aqueous solution are provided which can be used as an intermediate for the formulation of therapeutical formulations, e.g. further pharmaceutically acceptable components can be added to the aqueous solution in order to obtain the final therapeutical formulation.
• the stable aqueous solution of the invention can itself be used as a therapeutical formulation; i.e. including no or only few further additives.
• the stable aqueous solutions of the invention can be mere pharmaceutical additives which are not therapeutically active, or they can be therapeutically active substances. Also, by-products may or may not be present in the aqueous solutions of the invention. Accordingly, the stable aqueous solutions of the invention may either comprise, consist essentially of, or consist of an antibody at a concentration of at least 50 mg/ml and at least one acidic component.
• a process for the preparation of a therapeutical liquid formulation comprising an antibody, wherein in a first step an aqueous solution including an antibody at a concentration of at least 50 mg/ml and at least one acidic component is prepared; and, in a second step, at least one pharmaceutically acceptable additive is added to said aqueous solution.
• a process for the preparation of a therapeutical liquid formulation comprising an antibody at a concentration of more than 50 mg/ml, wherein in a first step an antibody solution in a suitable buffer is concentrated to between about 10 mg/ml and about 50 mg/ml; in a second step, the concentrated solution obtained in the first step is diafiltered with an aqueous solution of at least one acidic component, optionally containing MgCl 2 and/or CaCl 2 and/or further suitable additives; and, in a third step, the solution obtained in the second step is further concentrated to a concentration of more than 50 mg/ml.
• an antibody solution in a suitable buffer is concentrated to a concentration of between about 10 mg/ml and about 50 mg/ml;
• the concentrated solution obtained in the first step is diafiltered with an aqueous solution of at least one acidic component
• the solution obtained in the second step is further concentrated to an intermediate concentration of between about 100 and 200 mg/ml, preferably between about 100 and 150 mg/ml;
• the intermediate concentrated solution obtained in the third step is diafiltered with an aqueous solution of at least one acidic component and further containing MgCl 2 and/or CaCl 2 and/or further suitable additives,
• the solution obtained in the fourth step is further concentrated to a concentration of more than 150 mg/ml.
• the present invention provides highly concentrated aqueous solutions of antibody and liquid formulations based thereon.
• the concentrated aqueous solutions of the invention include a therapeutical antibody and at least one acidic component.
• the aqueous solutions therefore generally have a pH below pH 7.0. They may or may not include further salts or additives. They may be used as an intermediate in a process to obtain a therapeutical liquid formulation of the invention, but they also may be suitable therapeutical liquid formulations themselves, i.e. without the addition of further pharmaceutically acceptable additives.
• the invention provides a stable aqueous solution comprising an antibody at a concentration of at least 50 mg/ml, and further comprising at least one acidic component.
• concentrations of the antibody of at least 80 mg/ml, 100 mg/ml, 140 mg/ml, 160 mg/ml, 180 mg/ml, 200 mg/ml, 220 mg/ml, 250 mg/ml or even 300 mg/ml.
• the high viscosity of protein solutions has been identified as a major obstacle.
• antibody solutions such as for example solutions of monoclonal antibody E25
• the viscosity increases with protein concentration.
• the high viscosity of antibody solutions is a disadvantage from a medical point of view as, for example, reconstitution times may be as long as 30min for an antibody lyophilizate. Further, after reconstitution and injection of a dry formulation about 30% of an antibody may be left in the vial and in the syringe, which severely increases the treatment cost.
• the present invention now provides means to obtain a stable liquid pharmaceutical formulation comprising antibodies, such as anti-IgE antibodies, with a high protein concentration and a low viscosity.
• Antibody aggregates can be soluble or insoluble and both forms of aggregates can be covalent or non-covalent.
• the aggregates can give opalescent solutions, but there can also be non-visible aggregation which only can be shown chemically.
• aggregation can be detrimental in several ways.
• covalent aggregation in protein formulations may be essentially irreversible and could result in the production of inactive species, which in addition also may be immunogenic.
• Non-covalent aggregation can lead to loss of activity due to precipitation.
• a “stable” aqueous solution or liquid formulation within the meaning of the invention is one in which the antibody therein essentially retains its physical and chemical stability and integrity upon storage.
• Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected time period. For rapid screening, a formulation may be kept at 40° C. for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8° C., generally the formulation should be stable at 30° C. or 40° C. for at least 1 month and/or stable at 2-8° C. for at least 1 year.
• the aqueous solution of the invention has a stability of at least 1 year at about 4° C.
• a “stable” formulation may be one wherein less than about 10% and, preferably, less than about 5%, preferably less than about 2%, or even less than about 1% of the protein is present as an aggregate in the formulation. Aggregation can, for example, be measured by size exclusion chromatography.
• the solutions of the invention are stable not only with regard to aggregation but also with regard to the chemical stability of the antibody.
• Chemical stability may, for example, be measured by hydrophobic interaction chromatography (HIC), for example by HIC-HPLC after papain digestion.
• HIC-HPLC hydrophobic interaction chromatography
• the peak representing unmodified antibody in HIC-HPLC after papain digestion decreases no more than 20%, preferably no more than 10%, more preferably no more than 5% or even no more than 1%, as compared to the antibody solution prior to storage.
• Chemical instability can impair the activity of the antibody in question.
• Examples of chemical instability are degradation of the antibody or changes in tertiary and/or quaternary structure of antibody molecules.
• the solutions and formulations of the invention lose less than 50%, preferably less than 30%, preferably less than 20%, more preferably less than 10% or even less than 5% or 1% of the antibody activity within 1 year storage under suitable conditions at about 4° C.
• the activity of an antibody can be determined by a suitable antigen-binding assay for the respective antibody.
• the ability of an acidic component to produce a stable liquid antibody solution at high protein concentration can be determined by making up a solution including the acidic component to be tested and storing it for 24 hours at 22° C. For example, if after this time the solution remains clear the acidic component has stabilized the antibody and is one suitable for the use in an aqueous solution according to the present invention.
• the degree of stability achieved depends on the acid used and on its concentration, the antibody concentration, and on the storage temperature. In general, the higher the concentration of the antibody and the higher the storage temperature, the shorter the time before aggregation occurs. In general higher antibody concentrations require higher concentrations of the acidic component.
• Such reduced viscosity allows for a aqueous solution or liquid formulation of the invention having a higher concentration of the respective antibody.
• the same amount of antibody may be administered in a smaller volume.
• such smaller volume advantageously, may allow to produce pre-filled delivery devices that include the entire therapeutical dosage of the respective antibody.
• a liquid formulation need not necessarily be isotonic to avoid pain to the patient.
• the aqueous solution of the invention is isotonic.
• isotonic it is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
• the acidic component and the amount of acid being used is so chosen as to achieve the desired viscosity and stability of the high concentrated protein solution.
• Suitable acids that may be chosen include organic and inorganic acids.
• Organic acids of the invention may be carboxylic acids, such as monocarboxylic, dicarboxylic, tricarboxylic, tetracarboxylic, hydrocarboxylic acids or phenols.
• Weak organic acids are preferred acids of the present invention, for example monocarboxylic organic acids having a pK-value between 3.0 and 6.0, preferably between 4.5 and 5.0.
• acidic components of the invention are acetic acid, citric acid, oxalic acid, succinic acid, tartaric acid, lactic acid, malic acid, glycolic acid and fumaric acid.
• the acidic component included in the aqueous solution is acetic acid.
• the pH of said aqueous solution or liquid formulation is above pH 3, for example between pH 3 and pH 7, more preferably it is between pH 3 and pH 6, more preferably between pH 4 and pH6, or even between pH 5 and pH 6.
• the pH is about pH 5.0 or about pH 6.0.
• Certain pH ranges are particularly preferred, for example, preferred is a pH below pH 6.0, or below pH 5.8, or below pH 5.6 or below pH 5.4, and a pH that is above pH 4.0, or above pH 4.2, or above pH 4.4, or above pH 4.6 or above pH 4.8, or above pH 5.0.
• the acidic component of the invention such as acetic acid
• the acidic component of the invention is present in a final concentration of at least 0.001%, preferably at least 0.01%, more preferably between 0.01% -0.2%.
• no additional buffering agent is present in the aqueous solution or liquid formulation of the invention.
• no sodium salt such as for example sodium acetate is present in the aqueous solution or liquid formulation of the invention.
• the concentration of the antibody is above 50mg/ml, for example it may be between 100 and 200 mg/ml and can go up to 300 mg/ml. Preferred is a concentration of at least 80, 100, 140, 160, 180, 200, 220, 250 or even 300 mg/ml. One preferred range is between 100 and 220 mg/ml for injectable solutions. If a protein shall be delivered via the nasal or even the oral route, preferred concentrations are at least 250 mg/ml or even 300 mg/ml, as high concentrations are particularly desirable for the delivery via the nasal or oral route.
• the aqueous solution or liquid formulation of the invention may also contain more than one antibody as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect the other antibody.
• the aqueous solution or liquid formulation herein may also include an additional therapeutical protein which is not an antibody.
• Such antibodies or proteins are suitably present in combination in amounts that are effective for the purpose intended.
• the total protein concentration should be taken into account when choosing the concentration of the acidic component.
• the present invention also provides for a stable aqueous solution consisting merely of an antibody at a concentration of at least 50 mg/ml and an acidic component.
• the stable aqueous solution may also consist essentially of an antibody at a concentration of at least 50 mg/ml and an acidic component, in particular it may further include by-product or therapeutically inactive additives.
• the aqueous solution or liquid formulation of the invention further includes CaCl 2 and/or MgCl 2 .
• concentration of CaCl 2 is within the range of 50-200 mM, more preferably within 50-130 mM, preferably 100-130 mM, most preferably about 100 mM.
• concentration of MgCl 2 is within the range of 50-200 mM, more preferably within 50-130 mM, preferably 100-130 mM, most preferably about 100 mM.
• Stable aqueous solutions or liquid formulations including MgCl 2 are a particularly preferred embodiment of the present invention. In a further preferred embodiments these aqueous solutions or liquid formulations further include a detergent and/or a sugar.
• antibody is used in a broad sense.
• the term “antibody” specifically covers monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single- chain molecules, as well as antibody fragments and/or derivatives such as, for example, Fab, F(ab′) 2 , and Fv fragments or other antigen-binding fragments.
• an antibody derivative may be a PEGylated form of an antibody or antibody fragment.
• the antibody used in the aqueous solution of the invention has an isoelectric point between pH 6 and pH 8.
• the term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method or may be made by recombinant DNA methods.
• the “monoclonal antibodies” may also be isolated from phage antibody libraries.
• the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
• “Humanized” forms of non-human antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof, such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies, which contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species.
• CDR complementarity determining region
• Fv framework region residues of the human immunoglobulin are replaced by corresponding non-human residues.
• complementarity determining region (CDR) residues originating from the non-human species may be replaced by corresponding human residues.
• humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the antibody or antibody derivative is selected from anti-IgE antibodies, such as E25, E26, E27 (described in W099/01556 as rhuMAbE-25, rhuMAbE-26, and rhuMAbE-27, respectively) or their fragments and derivatives.
• anti-IgE antibody is a humanized murine antibody or a fully human antibody.
• the anti-IgE antibody is Omalizumab, which is also named “E25”.
• Another preferred anti-IgE antibody is named “E26” as further defined hereinbelow.
• anti-IgE antibodies are described in the prior art, and in greater detail in the International applications WO 93/04173 and WO 99/01556.
• WO 99/01556 specifically describes Omalizumab, also named E25, in FIG. 12, and in the sequences ID-No. 13-14.
• Antibody molecules comprising a E26 sequence are described in WO 99/01556 and are selected from the group of F(ab) fragment (Sequence ID Nos. 19-20), sFv fragment (Sequence ID No. 22) and F(ab)′ 2 fragment (Sequence Nos. 24-25), in accordance to FIGS. 12-15.
• the terms E25 and E26 shall be construed accordingly.
• the IgE antibodies of the instant invention do not result in histamine release from mast cells or basophils.
• U.S. Pat. No. 5,449,760 generally describes anti-IgE antibodies that bind soluble IgE but not IgE on the surface of B cells or basophils. Antibodies such as these bind to soluble IgE and inhibit IgE activity by, for example, blocking the IgE receptor binding site, by blocking the antigen binding site and/or by simply removing the IgE from circulation. Additional anti-IgE antibodies and IgE-binding fragments derived from the anti-IgE antibodies are described in U.S. Pat. No. 5,656,273. U.S. Pat. No. 5,543,144 describes further anti-IgE antibodies that are suitable for this invention, in particular anti-IgE antibodies that bind soluble IgE and membrane-bound IgE on IgE-expressing B cells but not to IgE bound to basophils.
• the antibody acid solution can for example be mixed with sugars, detergents and/or other additives. Accordingly the present invention also describes methods suitable for the preparation of long-term stable liquid formulations of antibodies including such additives. Also provided are the aqueous solutions including such additives themselves.
• liquid solvents e.g. an alcohol, e.g. isopropanol,
• sugars or a sugar alcohols e.g. mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose or dextrans,
• c) detergents e.g. Tween 20, 60 or 80 (polysorbate 20, 60 or 80)
• buffering agents e.g. acetate buffer
• preservatives e.g. benzalkonium chloride, benzethonium chloride, tertiary ammonium salts and chlorhexidine diacetate.
• g) carriers e.g. polyethylene glycol (PEG), recombinant human serum albumin
• antioxidants e.g. ascorbic acid and methionine
• biodegradable polymers e.g. polyesters
• salt-forming counterions e.g. sodium
• a “preservative” within the meaning of the invention is a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example.
• preservatives may advantageously be included in solutions suitable for nasal administration or in solutions for use with multiple pen injectors.
• Preferred compounds to be added as further additives are detergents such as Tween 20, sugars such as sucrose, fructose, mannitol and preservatives.
• detergents such as Tween 20
• sugars such as sucrose, fructose, mannitol and preservatives.
• additives derived from animal origin such as gelatine or serum albumin (e.g. BSA) are excluded from formulations of the invention.
• acceptable additives are nontoxic to recipients at the dosages and concentrations employed.
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, alternatively, sterility of the entire mixture may be accomplished by autoclaving the ingredients, except for protein, at about 120° C. for about 30 minutes, for example.
• the percentage of the acid solution and the amount of additives used can vary and depends on the intended use. For example during different manufacturing steps the concentration of the acid solution can differ from the concentration of the final product.
• additives such as ethanol, phosphate buffer saline (PBS), or citrate buffer, may induce gelation, increased viscosity and/or aggregation of the antibody in question under certain pH conditions. If the problems cannot be avoided by routine changes in pH, such additives should preferably not be used for preparing compositions of this invention.
• a liquid formulation may, for example, be made by adding the additives to an aqueous solution of the antibody and then stirring to dissolve.
• Any suitable stirrer may be used, e.g. a vortex mixer. It is preferred to dissolve the antibody in an aqueous solution of the acid and then to add an aqueous solution of the additives.
• the stirring may preferably be carried out under an inert gas atmosphere, such as nitrogen or argon, and the resulting solution may preferably be degassed under vacuum.
• the inert gas atmosphere and degassing both may help to prolong the stability of the solution.
• After preparation the solution may be stored in glass or plastics containers.
• the aqueous solution or liquid formulation of the invention further includes CaCl 2 and/or MgCl 2 .
• concentration of CaCl 2 is within the range of 50-200 mM, more preferably 50-130 mM, preferably 100-130 mM, most preferably about 100 mM.
• concentration of MgCl 2 is within the range of 50-200 mM, more preferably 50-130 mM, preferably 100-130 mM, most preferably about 100 mM.
• the aqueous solution or liquid formulation of the invention further includes a detergent, such as for example Tween 20, Tween 60 or Tween 80.
• the aqueous solution or liquid formulation of the invention further includes at least one sugar.
• the aqueous solution or liquid formulation of the invention further includes at least one sugar selected from the group comprising trehalose, sucrose, mannitol, sorbitol, fructose, maltose, lactose or a dextran.
• the aqueous solution or liquid formulation of the invention does not include maltose.
• aqueous solution or liquid formulation of the invention further includes at least one buffering agent.
• One desirable anti-IgE antibody aqueous solution discovered herein includes an anti-IgE antibody in amount between 100 and 200 mg/ml, preferably of about 190 mg/ml or of about 220 mg/ml, and CaCl 2 or MgCl 2 in an amount between 50 and 200 mM, preferably of about 50 mM or of about 100 mM, optionally a buffer and optionally a detergent, such as a Tween 20, e.g. at a concentration of about 0.02%.
• this anti-IgE formulation is stable at 8° C. for at least 1 year.
• the aqueous solution or liquid formulation of the invention may, for example, be used with standard ampoules, vials, pre-filled syringes or multiple administration systems.
• the aqueous solution may be administered to the patient by subcutaneous administration.
• the formulation may be injected using a syringe.
• other injection devices for administration of the formulation are available such as injector pens, and subcutaneous patch delivery systems such as, for example, chip devices.
• the aqueous solution may also be administered to the patient by inhalation devices.
• Conventional systems for delivery of molecules through the nasal passages and the lung include metered dose inhalers, and liquid jet and ultrasonic nebulizers.
• the present invention also provides a delivery system which contains the aqueous solution selected from the group of single use injection syringes or inhalation devices.
• the delivery system comprises a container.
• Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes.
• the container may be formed from a variety of materials such as glass or plastic.
• the container holds the aqueous solution and the label on, or associated with, the container may indicate directions for use.
• the label may for example indicate that the aqueous solution is useful or intended for subcutaneous administration.
• the container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the aqueous solution.
• aqueous solution or liquid formulation according to the invention for the production of a delivery system for the use treatment of a disease.
• an article of manufacture which contains the aqueous solution of the present invention and provides instructions for its use.
• an article of manufacture is provided herein which comprises:
• the article of manufacture may further comprise a second container which holds a diluent (eg. bacteriostatic water for injection comprising an aromatic alcohol).
• a diluent eg. bacteriostatic water for injection comprising an aromatic alcohol
• 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.
• a slow release formulation comprising the aqueous solution or liquid formulation of the invention.
• a slow release formulation selected from the group of polymeric nano or microparticles, or from gels.
• the slow release formulation is a gel such as a hyaluronic acid gel.
• slow release formulations offer other advantages for delivery of protein drugs including protecting the protein over an extended period from degradation or elimination, and the ability to deliver the protein locally to a particular site or body compartment thereby lowering overall systemic exposure.
• the present invention also contemplates injectable depot formulations in which the protein is embedded in a biodegradable polymeric matrix.
• Polymers that may be used are the homo- and co-polymers of lactic and glycolic acid (PLGA).
• PLGA degrades by hydrolysis to ultimately give the acid monomers and is chemically unreactive under the conditions used to prepare, for example, microspheres and thus does not modify the protein.
• the protein is released by a combination of diffusion and polymer degradation.
• the hydrolysis rate can be varied thereby allowing release to last from days to months.
• the present invention provides a nasal spray comprising the aqueous solution or liquid formulation of the present invention.
• an acidic component for the preparation of an aqueous solution comprising an antibody having a concentration of at least 50 mg/ml is provided.
• a process for the preparation of a therapeutical liquid formulation comprising an antibody, wherein in a first step an aqueous solution including an antibody at a concentration of at least 50 mg/ml and at least one acidic component is prepared, and, in a second step, at least one pharmaceutically acceptable additive is added to said aqueous solution.
• a process for the preparation of a therapeutical formulation including an antibody which process comprises adding an acidic component on the last purification step of the preparation of said antibody.
• Such last step may, for example, be an elution step, a buffer exchange step or a step comprising continuous diafiltration.
• a process for the preparation of a therapeutical liquid formulation comprising an antibody at a concentration of more than 50 mg/ml, wherein in a first step an antibody solution in a suitable buffer is concentrated to a concentration between about 10 mg/ml and about 50 mg/ml; in a second step, the concentrated solution obtained in the first step is diafiltered with an aqueous solution of at least one acidic component, optionally containing MgCl 2 and/or CaCl 2 and/or further suitable additives; and, in a third step, the solution obtained in the second step is further concentrated to a concentration of more than 50 mg/ml.
• the aqueous solution of at least one acidic component may be a solution of acetic acid, such as a solution of between about 0.01% and about 0.1% acetic acid.
• MgCl 2 and/or CaCl 2 may be present at a concentration within the range of 50-200 mM, preferably 50-130 mM, more preferably 100-130 mM, most preferably about 100 mM.
• these aqueous solutions further include a detergent and/or a sugar.
• an antibody solution in a suitable buffer is concentrated to a concentration of between about 10 mg/ml and about 50 mg/ml;
• the concentrated solution obtained in the first step is diafiltered with an aqueous solution of at least one acidic component
• the solution obtained in the second step is further concentrated to an intermediate concentration of between about 100 and 200 mg/ml, preferably between about 100 and 150 mg/ml;
• the intermediate concentrated solution obtained in the third step is diafiltered with an aqueous solution of at least one acidic component containing MgCl 2 and/or CaCl 2 and/or further suitable additives; and,
• the solution obtained in the fourth step is further concentrated to a concentration of more than 150 mg/ml.
• the diafiltration is generally carried out at constant retentate volume, with at least 5 volumes, or preferably 8 volumes, of diafiltration buffer.
• a solution of MgCl 2 and/or CaCl 2 and/or further suitable additives may directly be added to the intermediate concentrated solution obtained in the third step of the above 5-step process. If MgCl 2 and/or CaCl 2 and/or further suitable additives are directly added, the fourth step (i.e. diafiltration with an aqueous solution of at least one acidic component containing MgCl 2 and/or CaCl 2 and/or further suitable additives) thereafter may be omitted if no further adjustment of the respective concentrations of the salts and/or additives is required.
• the 5-step process of the invention which adds the salts and/or additives only to an intermediate concentrated solution of antibody avoids the appearance of aggregates and/or turbidity in solutions of the process.
• a concentrated aqueous solution of MgCl 2 (or CaCl 2 ), for example at concentration 1 M, is added directly into an ultrafiltration system, to give approximately the desired resulting concentration (for example 50 mM or 100 mM).
• carboxylic acids such as acetic acid
• carboxylic acids such as acetic acid
• no salt of a carboxylic is added in the process.
• the present invention also provides the aqueous solution of the invention for use in medicine.
• the use of the aqueous solution for the manufacture of a medicament for the treatment of disease, such as for example an allergic disease, is provided.
• the appropriate dosage 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 antibody used, and the discretion of the attending physician.
• the antibody 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 antibody may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
• the uses for a formulation including an anti-IgE antibody include the treatment or prophylaxis of IgE-mediated allergic diseases, parasitic infections, interstitial cystitis and asthma, in particular allergic athma, allergic rhinitis and atopic dermatitis, for example.
• a therapeutically effective amount of the anti-IgE antibody may be administered to the patient.
• CZE capillary zone electrophoresis
• the buffer of a solution of 40 mg/ml E25 in the production buffer (10 mM histidine buffer 10 % sucrose) was exchanged in a diafiltration equipment to 0.1% acetic acid. After that the E25 solution was concentrated by ultrafiltration to 161 mg/ml. The solution was fluid, no aggregation or opalescence was observed. The recovery was very good, about 95%. This solution of 161 mg/ml was further concentrated by filtration through centrifugation using Centricone tubes. Fluid, clear solutions of E25 in 0.1% acetic acid with concentrations of 214 mg/ml and also 297mg/ml were obtained. The solutions can be easily handled through syringe needles and permit the development of a single use prefilled syringe with small volume (e.g., 0.5 ml to 1 ml).
• a solution of 40 mg/ml E25 in the final production buffer (containing 0.02% Tween 20) was dialyzed against 0.1% acetic acid.
• the resulted E25 solution in 0.1% acetic acid (still containing Tween 20 detergent) was concentrated by filtration through centrifugation using Centricone: a concentration of 243 mg/ml E25 was reached.
• the solution fluidity was similar to the fluidity of the solutions without Tween 20, showing that the detergent is compatible with the high protein concentrated formulation.
• the starting solution is a solution of purified antibody at low concentration (lower than the high concentrations of the invention) in an aqueous buffer, for example in the buffer resulting from the preceding process step (for example in the case of E25: 25 mM TRIS buffer pH 8 containing about 200 mM NaCl).
• the pH of this solution is adjusted to a value below the isoelectric point of the antibody, for example to pH 5, with an acid, for example with 5% acetic acid.
• the resulting solution is then concentrated and diafiltered by ultrafiltration, preferably in a tangental-flow filtration system, using a membrane able to retain quantitatively the antibody, for example with a cutoff of 30 kD or 10 kD.
• the antibody solution is concentrated to an intermediate concentration, for example 40 mg/ml. Normally the retentate obtained is opalescent, due to antibody aggregation.
• the concentrated solution is diafiltered with an aqueous acetic acid solution (for example 0.01% or 0.1% acetic acid) containing MgCl 2 or CaCl 2 (for example at concentration 50 mM or 100 mM) and optionally containing other additives (for example a sugar).
• aqueous acetic acid solution for example 0.01% or 0.1% acetic acid
• MgCl 2 or CaCl 2 for example at concentration 50 mM or 100 mM
• other additives for example a sugar
• the diafiltered solution is further concentrated to a high concentration, for example higher or equal to 240 mg/ml.
• the final turbid retentate is then recovered out of the ultrafiltration system.
• the antibody solution is concentrated to an intermediate concentration, for example 40 mg/ml. Normally the retentate obtained is opalescent, due to antibody aggregation.
• the concentrated solution is diafiltered with an aqueous solution containing only acetic acid (for example 0.01% or 0.1% acetic acid).
• the diafiltration is generally carried out at constant retentate volume, with at least 5 volumes, or preferably 8 volumes, of diafiltration buffer. Normally, a decrease of the turbidity is observed during the diafiltration and the solution turns clear.
• the diafiltered solution is further concentrated to a higher intermediate concentration, preferably of about 120-130 mg/ml. Then, a concentrated aqueous solution of MgCl 2 (or CaCl 2 ), for example at concentration 1 M, is added directly into the ultrafiltration system, to give approximately the desired resulting concentration (for example 50 mM or 100 mM). After mixing by retentate recirculation, a decrease of the retentate pressure is observed, due to the resulting lower viscosity. The retentate obtained remains clear or slightly turbid.
• the solution is diafiltered with the same acetic acid solution as used for the first diafiltration (for example 0.01% or 0.1% acetic acid), but this time containing additionally MgCl 2 (or CaCl 2 ) at the desired concentration (for example 50 mM or 100 mM), in order to adjust exactly this concentration in the retentate.
• the diafiltration is generally carried out at constant retentate volume, with at least 5 volumes, or preferably 8 volumes, of diafiltration buffer.
• the diafiltered solution is further concentrated to a high concentration, for example higher or equal to 240 mg/ml.
• the final clear or slightly turbid retentate is then recovered out of the ultrafiltration system.
• the starting solution was a solution of purified E25 antibody at concentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200 mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the following steps were carried out:
• the solution was concentrated to 40 mg/ml.
• a third step the diafiltered solution was concentrated to 127 mg/ml and, after retentate recirculation during 5 minutes with the filtrate line closed, a sample was taken for viscosity measurement. Then, an aqueous solution of 1 M MgCl 2 was added directly into the ultrafiltration system, to give approximately a resulting MgCl 2 concentration of 50 mM. After reconcentration to the initial retentate volume (i.e. the volume before the addition of MgCl 2 ) and after retentate recirculation during 3 minutes with the filtrate line closed, a sample of the retentate was taken for viscosity measurement.
• the solution was diafiltered at constant retentate volume with 8 volumes of 0.1% acetic acid containing 50 mM MgCl 2 .
• the diafiltered solution was concentrated to about 260 mg/ml. After recovery of the retentate out of the ultrafiltration system and filtration through a 0.2 ⁇ m filter, a sample was taken for viscosity measurement. An other sample was diluted to 200 mg/ml with 0.1% acetic acid containing 50 mM MgCl 2 , also for viscosity measurement.
• Example 8 The same experiment as stated in Example 8 was carried out several times, changing only the acetic acid concentration used for the diafiltration buffers, but keeping the final MgCl 2 concentration equal to 50 mM.
• the different high concentrated E25 solutions obtained were then diluted to about 200 mg/ml, using the respective diafiltration buffers (i.e. the corresponding acetic acid solutions containing 50 mM MgCl 2 ), for pH and viscosity measurements.
• this “transition concentration” of about 0.0075% acetic acid corresponds to 1.3 mM, which corresponds to the E25 molar concentration corresponding to 200 mg/ml. Accordingly, in one embodiment of the invention the concentration of the acidic component of the invention is so chosen as to be about equal or above the molar concentration of the antibody of the aqueous solution or formulation of the invention.
• the starting solution was a solution of purified E25 antibody at concentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200 mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the following steps were carried out:
• the solution was concentrated to 40 mg/ml.
• the concentrated solution was diafiltered at constant retentate volume with 8 volumes of 0.01% acetic acid containing 50 mM MgCl 2 .
• the diafiltered solution was concentrated to 230-240 mg/ml. After recovery of the retentate out of the ultrafiltration system and filtration through a 0.2 ⁇ m filter, two samples was taken for viscosity measurement (the first one as is, the second one after addition of 0.02% of Tween 20). Two other samples were diluted to about 210 mg/ml with 0.01% acetic acid containing 50 mM MgCl 2 , also for viscosity measurement (the first one as is, the second one after addition of 0.02% of Tween 20).
• the three liquid formulations have a stability of at least 6 months at5° C.
• aqueous solution [127 mg/ml E25, 0.1% acetic acid] were prepared by ultrafiltration in a tangential-flow filtration system (membrane area: 150 cm 2 , membrane cutoff: 10 kD, hold up volume of the system: 9 ml, retentate pressure: 2-3 bar), according to the three first steps of the 5-steps procedure described in Example 7:
• the starting solution was a solution of purified E25 antibody at concentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200 mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the following steps were carried out:
• the diafiltered solution was concentrated to about 120 mg/ml and a sample was taken for viscosity measurement.
• Example 12 The same experiment as stated in Example 12 was repeated using 0.1% acetic acid for the diafiltration step, but this time the diafiltered solution was concentrated to about 240 mg/ml (instead of 120 mg/ml). After recovery of the retentate out of the ultrafiltration system and filtration through a 0.2 ⁇ m filter, a sample was taken for viscosity measurement. Other samples were taken as well, for viscosity measurements after various dilution steps with 0.1% acetic acid.
• the beneficial effect of acetic acid allows to prepare antibody solutions at a concentration up to about 180 mg/ml, having a viscosity significantly lower than 50 mPa ⁇ s.
• the starting solution was a solution of purified E25 antibody at concentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200 mM NaCl. After pH adjustment to pH 4.7 with 0.5 M citric acid, corresponding to a resulting citric acid concentration of about 6.6 mM, the following steps were carried out:
• the concentrated solution was diafiltered at constant retentate volume with 8 volumes of purified water having a pH of about 4.4, preliminarily adjusted with a few droplets of 0.5 M citric acid, corresponding to a resulting citric acid concentration in the range of about 0.05 to 0.1 mM.
• the diafiltered solution was concentrated as high as possible. After recovery of the retentate out of the ultrafiltration system and filtration through a 0.2 ⁇ m filter, a sample was taken for concentration and pH measurements.
• the maximal reachable concentration was 155 mg/ml, with a resulting pH of 4.5.
• the maximal concentration obtained by using 0.1% acetic acid (without other additives) with the same ultrafiltration equipment was about 240 mg/ml.

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