Classifications

• • 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
• • 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
  • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • 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
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
• • 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

Definitions

• the invention relates to medicine.
• the invention is directed to stable formulations of specific anti-rabies antibodies.
• an antibody Like any protein, the biological activity of an antibody, such as its binding affinity or neutralizing activity, depends upon the conformational integrity of at least a core sequence of amino acids remaining intact while protecting the protein's multiple functional groups from degradation. Chemical and physical instability can each contribute to degradation of an antibody. Because antibodies are larger and more complex than traditional organic and inorganic drugs, the formulation of such antibodies poses special problems. Antibody stability can be affected by such factors as ionic strength, pH, temperature, repeated cycles of freeze/thaw, antibody concentration and shear forces.
• Active antibodies may be lost as a result of physical instabilities, including denaturation, aggregation (both soluble and insoluble aggregate formation), precipitation and adsorption as well as chemical instabilities, including, for example, racemization, beta-elimination or disulfide exchange, hydrolysis, deamidation, and oxidation, to name just a few. Any of these instabilities can potentially result in the formation of antibody by-products or derivatives having lowered biological activity, increased toxicity, and/or increased immunogenicity.
• physical instabilities including denaturation, aggregation (both soluble and insoluble aggregate formation), precipitation and adsorption as well as chemical instabilities, including, for example, racemization, beta-elimination or disulfide exchange, hydrolysis, deamidation, and oxidation, to name just a few. Any of these instabilities can potentially result in the formation of antibody by-products or derivatives having lowered biological activity, increased toxicity, and/or increased immunogenicity.
• Formulations that meet the requirements of the object of the invention have surprisingly been found in the form of aqueous solutions that in addition to the two different monoclonal antibodies, comprise citrate buffer, a tonicity agent and a surfactant.
• Phosphate buffer was surprisingly found to lead to instability of the specific antibodies, which instability was even increased by addition of surfactant.
• the invention thus provides formulations for the specific anti-rabies antibodies CR57 and CR4098, or functional variants thereof.
• the invention also pertains to antibody formulations comprising both CR57 and CR4098, or functional variants thereof.
• the formulations contain, besides the active ingredient (the antibody or antibodies), a citrate buffer, a tonicity agent and a surfactant.
• the formulations of the invention are stable for at least 1 year at -70 0 C and at 5°C.
• FIG. 1 The stability of anti-rabies virus antibody CR57 (Fig. 1), CR4098 (Fig. 2) and a cocktail of CR57 and CR4098 (Fig. 3) after storage for 0 (white columns), 2 (black columns) and 4 weeks (shaded columns) at 40 ⁇ 2°C/75 ⁇ 5% relative humidity as measured by HP-SEC is shown. From left to right the following buffer systems were tested: citrate (20 mM, pH 6.0); citrate (20 mM, pH 6.5); phosphate (20 mM, pH 7.0); phosphate (20 mM, 0.01% w/v polysorbate 80, pH 7.0).
• the formulations of the invention comprise at least one of, and preferably both of, antibody CR57 (heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2) and antibody CR4098 (heavy chain SEQ ID NO: 3 and light chain SEQ ID NO: 4).
• Identification, isolation, preparation and characterization of the anti-rabies virus monoclonal antibodies CR57 and CR4098 has been described in detail in WO 2005/118644 which is incorporated herein by5 reference. Functional variants of these antibodies may have similar physicochemical properties based on their high similarity and therefore are also included within the scope of the invention.
• Functional variants are defined for the present invention as antibodies with an amino acid sequence that is at least 95%, preferably at least 97%, for instance at least 98% or 99% homologous to CR59 or CR4098, and capable of competing for binding to the target o recognized by the parent molecule (the parent molecule being CR59 or CR4098, respectively) and having rabies virus neutralizing activity.
• a target for an antibody is an antigen (for the present antibodies this is rabies virus, in particular G protein thereof), and may be further defined as an epitope.
• the targets of the parent molecules have been disclosed in WO 2005/118644, and determining competition for binding to the target can be done by routine 5 methods known to the skilled person.
• the functional variants are human antibodies, and preferably are IgGl molecules.
• a functional variant is at least 95%, 97%, 98%, or 99% identical in amino acid sequence with the parent antibody.
• the term "functional variant”, as used herein, thus refers to a monoclonal antibody that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid0 sequences of the parental monoclonal antibody.
• the functional variant may have conservative sequence modifications including amino acid substitutions, additions and deletions.
• Amino acid modifications can be introduced by standard techniques known in the art, such as site- directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis and random PCR-mediated mutagenesis in the nucleic acid encoding the antibodies.
• Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
• nonpolar side chains e.g., glycine, alanine, valine, leucine, iso leucine, proline, phenylalanine, methionine
• beta-branched side chains e.g., threonine, valine, iso leucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
• a variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both.
• Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art. Computer algorithms such as inter alia Gap or Bestfit known to a person skilled in the art can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues.
• the formulation according to the invention comprises a first anti-rabies virus monoclonal antibody that has a kappa light chain and a second anti-rabies virus monoclonal antibody that has a lambda light chain. This allows easy determination of the antibody concentration for each antibody, as specific ELISAs can be performed for each of the kappa and the lambda light chain.
• monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
• a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.
• the monoclonal antibodies of the invention (CR57 and CR4098 and functional variants thereof) for the formulations of the present invention are human antibodies and are in the IgG class of antibodies, preferably IgGl.
• the term "specifically binding” means immunospecifically binding to an antigen or a fragment thereof and not immunospecifically binding to other antigens.
• a monoclonal o antibody that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., radioimmunoassays (RIA), enzyme- linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art.
• Monoclonal antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens.
• monoclonal antibodies or fragments5 thereof that immunospecifically bind to an antigen do not cross-react with other antigens.
• pharmaceutically acceptable excipient any inert substance that is combined with an active molecule such as monoclonal antibody for preparing an agreeable or convenient dosage form.
• pharmaceutically acceptable excipient is an excipient that is o non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation comprising the monoclonal antibody.
• by-product includes undesired products, which detract or diminish the proportion of therapeutic/prophylactic antibody in a given formulation.
• Typical by-products 5 include aggregates of the antibody, fragments of the antibody, e.g. produced by degradation of the antibody by deamidation or hydrolysis, or mixtures thereof.
• aggregates are complexes that have a molecular weight greater than the monomer antibody.
• Antibody degradation products may include, for example, fragments of the antibody, for example, brought about by deamidation or hydrolysis.
• degradation products are complexes0 that have a molecular weight less than the monomer antibody. In the case of an IgG antibody, such degradation products are less than about 150 kD.
• a “stable/stabilized” formulation as used herein is one in which the antibody therein essentially retains its physical stability/identity/integrity and/or chemical stability/identity/integrity and/or biological activity 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. Adv. Drug Delivery Rev. 10:29-90 (1993), for example. Stability can be measured at a selected temperature and other storage conditions for a selected time period.
• the stability may be determined by at least one of the methods selected from the group consisting of visual inspection, SDS-PAGE, IEF, HPSEC, RFFIT, and kappa/lambda ELISA.
• a monoclonal antibody "retains its physical stability" in a pharmaceutical formulation, if it shows no signs of aggregation, precipitation and/or denaturation upon visual examination of colour and/or clarity, or as measured by UV light scattering, SDS-PAGE or by (high pressure) size exclusion chromatography (HPSEC).
• the CR57 and CR4098 antibodies are stable in the formulations of the invention upon storage at 5 ⁇ 3°C for at least 18 months, i.e.
• the monomer peak in the HPSEC chromatogram comprises an area of >95% of the total area of all peaks (in Table 9 it can be seen that the main peak area is even >99%).
• Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification ⁇ e.g. clipping) which can be evaluated using (HP)SEC, 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) which can be evaluated by ion-exchange chromatography, for example.
• An antibody "retains its biological activity" in a pharmaceutical formulation at a given time, if the biological activity of the antibody at a given time is at least about 90% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared as determined in an antigen binding assay or virus neutralizing assay, for example.
• the invention encompasses a pharmaceutical formulation comprising at least an active ingredient, preferably in a therapeutically effective amount, and at least a pharmaceutically acceptable excipient.
• the pharmaceutical formulation comprises a citrate buffer, a tonicity agent, a surfactant and two anti-rabies virus monoclonal antibodies, wherein the antibodies are different from one another.
• the formulation may be solid, e.g. o frozen or lyophilised, but is preferably liquid, e.g. aqueous.
• the formulation may comprise at least two distinct anti-rabies virus monoclonal antibodies, in particular (i) CR57 (antibody with amino acid sequence of heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2) or a functional variant thereof and (ii) CR4098 (antibody with amino acid sequence of heavy chain SEQ ID NO: 3 and light chain SEQ ID NO: 4) or a functional variant thereof.
• the formulation according to the invention has a rabies virus neutralizing potency ranging from about 250 IU/ml to about 1500 IU/ml, e.g. from about 300 IU/ml to about 1400 IU/ml, typically from about 380 IU/ml to about 1350 IU/ml.
• a suitable and known assay for neutralizing activity is a RFFIT assay.
• the rabies virus neutralizing potency of the formulations of the invention after 12 months of storage at 5 ⁇ 3°C is at least 80%, preferably at least 90%, more 5 preferably at least 95%, more preferably at least 98%, and in particular 100% of the rabies virus neutralizing potency of the formulations of the invention before storage.
• the rabies virus neutralizing potency of the formulations of the invention after 3 months of storage at 25 ⁇ 2°C is at least 90%, preferably at least 95%, more preferably at least 98%, and in particular 100% of the rabies virus neutralizing potency of the formulations of0 the invention before storage.
• the formulations according to the invention comprise a surfactant, also known as stabilizer.
• Surfactants may include, but are not limited to, polysorbates.
• surfactants e.g. non-ionic or ionic detergents
• the invention provides a formulation according to the invention, wherein the surfactant is a polysorbate such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 or polysorbate 80, with polysorbate 80 being preferred.
• polysorbate 80 is present in the formulations in an amount from about 0.0005% w/v to about 0.05% w/v, preferably from about 0.005% w/v to about 0.03% w/v, more preferably o from about 0.008% w/v to about 0.015% w/v. In a preferred embodiment polysorbate 80 is present in an amount of about 0.01% w/v.
• the invention provides formulations according to the invention, wherein the citrate buffer, e.g. sodium citrate dehydrate (2.5 mg/ml)/citric acid monohydrate (0.3 mg/ml) buffer, is present at a concentration from about 5 mM to about 255 mM, preferably from about 7 mM to about 20 mM, more preferably from about 8 mM to about 15 mM, and particularly from about 9 mM to about 12 mM. In a preferred embodiment the citrate buffer is present at a concentration of about 10 mM.
• the citrate buffer e.g. sodium citrate dehydrate (2.5 mg/ml)/citric acid monohydrate (0.3 mg/ml) buffer
• the invention is concerned with formulations according to the invention, wherein the pH ranges from about 5.2 to about 6.8, typically from about 5.5 to o about 6.5, preferably from about 5.7 to about 6.3, more preferably from about 5.8 to about 6.2 and particularly from about 5.9 to about 6.1. In a preferred embodiment the pH is about 6.0.
• the tonicity agent is sodium chloride.
• Other salts can for instance also be used as tonicity agents, or for instance sugars, and the like, as 5 long as they are pharmaceutically acceptable, as is known to the skilled person.
• the tonicity agent is present at a concentration from about 50 mM to about 250 mM, typically from about 75 mM to about 225 mM, preferably from about 100 mM to about 200 mM, and more preferably from about 125 mM to about 175 mM. In a preferred embodiment the tonicity agent is present at a concentration of about 150 mM.
• the osmolality of the formulations according to the invention ranges from about 250 m ⁇ sm/kg to about 350 m ⁇ sm/kg, preferably from about 270 m ⁇ sm/kg to about 330 mOsm/kg, more preferably from about 280 mOsm/kg to about 320 mOsm/kg, and particularly from about 290 mOsm/kg to about 310 mOsm/kg.
• the osmolality is about 300 mOsm/kg.
• the formulations are preferably substantially isotonic, i.e. having substantially the same osmotic pressure as human blood. Isotonicity can be measured using vapour pressure or ice-freezing type osmometers, for example.
• the osmolality of the formulations of the invention can for instance be regulated by one or more tonicity agents.
• each antibody in the formulations of the invention preferably is between about 0.1 and 2.0 mg/ml, typically between about 0.1 and 1 mg/ml. In certain non- limiting embodiments, the concentration of each antibody is 0.15 ( ⁇ 20%) mg/ml. In other non-limiting embodiments each antibody is present in a concentration of 0.3 ( ⁇ 20%) mg/ml (i.e. total 0.6 mg/ml for two antibodies).
• the (protein) ratio of the two antibodies is between 5:1 and 1 :5, preferably between 2:1 and 1 :2 and particularly about 1 :1.
• the formulation according to the invention may comprise other excipients including, but not limited to, amino acids and salts thereof, sugars, proteins, diluents, solubilizing agents, pH-modif ⁇ ers, soothing agents, additional buffers, other inorganic or organic salts, antioxidants, or the like.
• the formulations of the present invention comprise no other excipients next to a citrate buffer, a tonicity agent and a surfactant.
• the anti-rabies virus monoclonal antibodies CR57 and CR4098 are stable at about 2°C to about 8°C for at least about 1 year, typically at least about 18 months. Preferably they may be stable at about 2-8°C for at least about 2 years, more preferably 3 years.
• the anti-rabies virus monoclonal antibodies are stable in the formulations according to the invention at about 25 ⁇ 2°C for at least about 2 months. Besides that, the anti-rabies virus monoclonal antibodies are stable in the formulations according to the invention at about 40 ⁇ 2°C for at least 2 weeks.
• the formulations are suitable for administering intramuscularly, intradermally, subcutaneous Iy, injected locally into a wound, or a combination thereof. Therefore, the formulations are preferably sterile. Methods for making formulations sterile are well known in the art and include filtration through sterile filtration membranes or autoclaving the ingredients of the formulation, with the exception of the antibodies, at about 120 0 C for about 30 minutes, for example.
• the formulations are substantially free of endotoxin.
• Endotoxins are low molecular weight complexes of about 10 kDa that are associated with the outer cell wall of gram-negative bacteria that can produce pyrogenic reactions upon parenteral administration to a patient. Accordingly, the FDA has set an upper limit of 5 EU per dose per kilogram body weight in a single one-hour period for intravenous drug applications (see, e.g., The United States Pharmacopeial Convention (USP), Pharmacopeial Forum 26 (1):223 (2000)).
• the formulation has a concentration of endotoxin of less than about 5.0 endotoxin units per milliliter (EU/ml) (a concentration of less than about 5.0 EU/ml is referred to herein as substantially free of endotoxin), preferably less than about 2.5 EU/ml, more preferably less than about 1.0 EU/ml, even more preferably less than about 0.5 EU/ml and particularly less than about 0.30 EU/ml.
• the formulation has a concentration of endotoxin that ranges from about 0.001 EU/ml to about 5.0 EU/ml.
• Methods for measuring endotoxins are known to a person skilled in the art and include, but are not limited to, gel-clot assays, turbidimetric (spectrophotometric) assays and chromogenic assays.
• Post exposure prophylaxis is indicated for persons possibly exposed to a rabid animal. Possible exposures include bite exposure (i.e. any penetration of the skin by teeth) including animal bites, and non-bite exposure.
• the formulations according to the invention can be administered to a subject in need thereof for use in prevention and/or treatment, e.g. post exposure prophylaxis, of a rabies virus infection.
• the formulations of the invention may be employed in conjunction with other molecules useful in diagnosis, prophylaxis and/or treatment of rabies virus. For instance, they can be co-administered with a vaccine against rabies virus. Alternatively, the vaccine may also be administered before or after administration of the formulations of the invention.
• Rabies vaccines include, but are not limited to, purified chick embryo cell (PCEC) vaccine (RabAvert, Rabipur), human diploid cell vaccine (HDCV; Imovax vaccine) or rabies vaccine adsorbed (RVA). 5
• PCEC purified chick embryo cell
• HDCV human diploid cell vaccine
• RVA rabies vaccine adsorbed
• a single bolus of the formulations of the invention are administered.
• the dosing regimen of post exposure prophylaxis is administration of five doses of rabies vaccine intramuscularly in the deltoid muscle on days 0, 3, 7, 14 and 28 after exposure in individuals not previously immunized against rabies virus.
• the formulations according to the invention should be administered into and around the wounds on day 0 or otherwise as soon as possible o after exposure, with the remaining volume given intramuscularly at a site distant from the vaccine.
• Non-vaccinated individuals are advised to be administered anti-rabies virus antibodies.
• a therapeutically effective amount of antibody or antibodies is administered, which amount is effective or at least partially effective for PEP of rabies, i.e. rabies virus is neutralized.
• the invention provides a pharmaceutical unit dosage form comprising an effective amount of a formulation according to the invention for post exposure prophylaxis treatment of a subject through administration of the dosage form to the subject.
• the subject is a human.
• the human may be an adult or may be an infant.
• pharmaceutical unit dosage form refers to a physically o discrete unit suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic/prophylactic effect in association with the required pharmaceutical carrier, diluent, or excipient.
• the unit dosage form may be a container comprising the formulation.
• Suitable 5 containers include, but are not limited to, sealed ampoules, vials, bottles, syringes, and test tubes.
• the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example the container may be a vial having a stopper pierceable by a hypodermic injection needle).
• the container is a vial.
• the vial preferably comprise a volume from about 0.3 ml to about 3 ml.
• the0 vial contains anti-rabies virus antibodies in an amount from about 0.1 mg to about 2.0 mg.
• the vial contains a total of 750-2000 IU of rabies virus neutralizing monoclonal antibodies per vial.
• This type of vial can suitably be used for administration to an adult, while a vial containing a total of 250-750 IU of rabies virus neutralizing monoclonal antibodies per vial can suitable be used for administration to an infant.
• the antibodies are typically formulated in the formulations of the invention in a therapeutically effective amount. Dosage regimens can be adjusted to provide the optimum desired response (e.g. a therapeutic response).
• a suitable dosage range may for instance be 10-30 IU/kg body weight, such as about 20 IU/kg body weight.
• the pharmaceutical unit dosage form may be present in a kit, further comprising a instructions for use.
• the kit may further comprise more containers comprising pharmaceutically acceptable excipients and include other materials desirable from a commercial and user standpoint, including filters, needles, syringes.
• Associated with the kits can be instructions customarily included in commercial packages of therapeutic, prophylactic or diagnostic products, that contain information about for example the indications, usage, dosage, manufacture, administration, contra-indications and/or warnings concerning the use of such therapeutic, prophylactic or diagnostic products.
• the kit comprises instructions to use the appropriate volume necessary to achieve a dose of about 5 IU/kg to about 40 IU/kg, e.g. 20 IU/kg.
• the present invention is concerned with a method for improving the storage of two anti-rabies virus monoclonal antibodies in one, e.g. a single, formulation by formulating the antibodies (CR57 and CR4098 or functional variants) in a liquid pharmaceutical formulation according to the invention.
• the formulation may be stored at a temperature from about 2°C to about 40 0 C, e.g. between about 2-8°C.
• the formulations may also be stored at temperatures below 2°C, e.g. at about -20 0 C, -70 0 C, etc.
• the invention also pertains to liquid pharmaceutical formulations comprising a single anti-rabies virus monoclonal antibody, i.e. either CR57 or CR4098 or a functional variant of one of these.
• these formulations comprise all features and excipients as described hereinabove.
• they contain citrate buffer (5-25 mM) and have pH 5.5-6.5, e.g.
• a tonicity agent e.g. sodium chloride, 50-250 mM, e.g. about 150 mM
• a surfactant e.g. polysorbate 80 (0.0005%-0.05%, e.g. about 0.01%)
• formulations according to the invention may comprising a single anti-rabies virus monoclonal antibody in an amount from about 0.1 mg/ml to about 6.0 mg/ml, typically from about 1.0 mg/ml to about 4.0 mg/ml, e.g. from about 2.0 mg/ml to about 3.0 mg/ml.
• the formulation has a rabies virus neutralizing potency ranging from about 300 IU/mg to about 1600 IU/mg, e.g. from about 500 IU/mg to about 1250 IU/mg.
• Formulations comprising a single antibody as described above may be combined/mixed with one another to obtain the formulations of the invention comprising two antibodies, i.e. an antibody cocktail.
• the bioreactor was first operated in batch mode followed by fed-batch mode. Medium containing the respective antibody was harvested and clarified by centrifugation and filtrated before further downstream processing.
• the downstream purification process consisted of standard chromatographic and filtration steps followed by a buffer exchange to formulation buffer lacking polysorbate 80 and concentration to obtain the desired antibody concentration.
• the obtained drug substance either antibody CR57 or antibody CR4098 was stored at -80 0 C until further use.
• the drug substances were diluted with formulation buffer, antibody concentrations were measured, and both antibody dilutions were mixed and filtered before final filling.
• HPSEC HPSEC was used in part to assess the presence of degradation products of the antibodies due to aggregation or proteolysis.
• SDS-PAGE was used in part to assess the integrity of the intact antibody and the presence of impurities and potential degradation products.
• Protein concentration was measured to assess the maintenance of the formulation's protein concentration within an acceptable range.
• IEF was used to assess the presence and integrity of antibody iso forms that may be present in the formulations and monitor them over time to assess changes that may occur due to deamidation or loss of sialic acid. Appearance of the formulations was conducted based on visual inspection for clarity, colour and the presence of particulates.
• pH was measured to assess the maintenance of the formulation's pH within an acceptable range of about 5.5 to about 6.5.
• Osmolality was measured to assess the maintenance of the formulation's osmolality within an acceptable range of about 250 mOsm/kg to about 350 mOsm/kg.
• anti-rabies virus antibody formulations formulated in citrate buffers were compared to anti-rabies virus antibody formulations formulated in phosphate buffers.
• Formulations comprising either CR57 (0.1 mg/ml), CR4098 (0.15 mg/ml) or a mixture/cocktail (1 :1.5 mixture) of CR57 (0.1 mg/ml) and CR4098 (0.15 mg/ml) in a 20 mM citrate buffer (pH 6.0) or a 20 mM citrate buffer (pH 6.5) were stable upon storage up to 4 weeks at 5 ⁇ 3°C/ambient relative humidity,
• citrate-based formulations comprising different polysorbate 80 concentrations were analysed (see Table 1).
• the formulations were prepared as follows.
• the antibodies CR57 and CR4098 drug substances
• the concentration of CR57 was 2.5 mg/ml and the 5 concentration of CR4098 was 1.0 mg/ml.
• a buffer containing 10 mM citrate (pH 6.0) and 50 mM sodium chloride and a buffer containing 10 mM citrate (pH 6.0), 50 mM sodium chloride and 5% polysorbate 80 were prepared.
• the formulations were prepared as described in Table 2. The final volume was reached with a buffer containing 10 mM citrate (pH 6.0) and 50 mM sodium chloride. The osmolality of all formulations was determined and sodium o chloride was added to bring the formulations to an osmolality of about 300 mOsm/kg (iso- osmotic), i.e. the final concentration of sodium chloride in the formulations was 150 mM.
• protein concentration was the same before and after o filtration as measured by A280 protein concentration determination.
• the results of the HPSEC analysis are shown in Table 4.
• the protein components were separated through HPSEC using an isocratic elution method, which allows rapid analysis and high resolution of protein components and also has an improved reproducibility.
• formulations with 0.01% (w/v) polysorbate 80 had a higher purity than similar formulations with 0.03% (w/v) polysorbate 80 (compare purity of formulation 1 with 2, formulation 3 with 4, and formulation 5 with 6). The same impurities were observed for both polysorbate 0 concentrations.
• IEF o illustrates the pi of the antibodies and is also helpful in indicating the conformational microheterogenicity of the antibodies.
• LAL Limulus Amoebocyte Lysate
• the formulation comprising CR57 contained ⁇ 0.30 EU/ml
• the formulation comprising CR4098 contained ⁇ 0.30 EU/ml
• the formulation containing the cocktail of both antibodies contained ⁇ 0.24 EU/ml.
• CR57 and CR4098 are considered to be stable for at least 6 months at the real time storage condition of-70 ⁇ 10°C as well as at least 6 months at the accelerated condition of 5 ⁇ 3°C in formulations comprising citrate (10 niM, pH 6.0), sodium chloride (150 mM) and 0.01% (w/v) polysorbate 80.
• CR57 and CR4098 are stable for at least 18 months at the storage condition of-70 ⁇ 10°C as well as at for least 12 months (similar results were found after 9 months, data not shown) at the accelerated condition of 5 ⁇ 3°C in formulations comprising citrate (10 mM, pH 6.0), sodium chloride (150 mM) and 0.01% (w/v) polysorbate 80.
• the antibody cocktail showed stable potency up to 6 months at 5 ⁇ 3°C and 25 ⁇ 2°C (see Table 9). A slightly lower potency value was obtained after 3 months at5 40 ⁇ 2°C. All data were within the target specification of about 380 to about 1350 IU/ml.
• the amount of total protein present in the antibody cocktail as determined by OD280 was stable over the tested time period of 3 months at 40 ⁇ 2°C, and 6 months at 5 ⁇ 3°C and 25 ⁇ 2°C.
• the IgG kappa and lambda ELISA results i.e. presented as the ratio of both o antibodies in the antibody cocktail
• the HPSEC patterns of the antibody cocktail stored at 5 ⁇ 3°C and at 25 ⁇ 2°C for up to 6 months compare well with the pattern at t 0 months.
• the target specification of "main peak area > 95%” was met in all cases (see Table 9).
• the dimer peak remained ⁇ 1% (surface area) and no degradation peaks were detected in any of the samples tested.
• Antibody cocktail stored5 at 40 ⁇ 2°C showed minor degradation after 3 months as well as an slightly increased dimer peak (2.6% (surface area)) compared to antibody cocktails stored at the two lower temperatures. However, even at 40 ⁇ 2°C the target specification of "main peak area > 95%” was met at all storage time periods tested (see Table 9).
• the antibody cocktail is stable for at least 6 months at the storage condition of 5 ⁇ 3°C and 25 ⁇ 2°C and for at least 3 months at the storage 5 condition of 40 ⁇ 2°C.
• Table 1 Composition of the antibody formulations.

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