EP3849513A1 - Compositions comprenant un anticorps bispécifique, un tampon et un ou plusieurs agents stabilisants - Google Patents

Compositions comprenant un anticorps bispécifique, un tampon et un ou plusieurs agents stabilisants

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Publication number
EP3849513A1
EP3849513A1 EP19778823.5A EP19778823A EP3849513A1 EP 3849513 A1 EP3849513 A1 EP 3849513A1 EP 19778823 A EP19778823 A EP 19778823A EP 3849513 A1 EP3849513 A1 EP 3849513A1
Authority
EP
European Patent Office
Prior art keywords
pharmaceutical formulation
present
concentration
beat
antibody
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19778823.5A
Other languages
German (de)
English (en)
Inventor
Sachin DUBEY
Paresh VADGAMA
Anne-Lise TERRIER
Paul Clement
Roberto Giovannini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ichnos Sciences SA
Original Assignee
Ichnos Sciences SA
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 Ichnos Sciences SA filed Critical Ichnos Sciences SA
Publication of EP3849513A1 publication Critical patent/EP3849513A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to stable formulations of a therapeutic antibody; preferably the therapeutic antibody is a monoclonal bispecific antibody.
  • the pharmaceutical formulation of a therapeutic product commonly comprises, besides an active pharmaceutical ingredient (API) with therapeutic properties, also inactive ingredients, or excipients that contribute to the stability, bioavailability and effectiveness of the API.
  • API active pharmaceutical ingredient
  • excipients include solvents, diluents, buffering agents, pH-adjusting agents, surfactants, preservatives, tonicifying agents, antioxidants etc.
  • Appropriate excipients are chosen based on the drug product presentation, on the route of administration and on the administration dosage, as well as based on their effect on the stability of the API.
  • Biomolecules like proteins, i.e. antibodies, are nowadays developed as therapeutics to treat a variety of diseases spanning from cancer to autoimmune diseases.
  • Biological molecules like proteins, i.e. antibodies, are nowadays developed as therapeutics to treat a variety of diseases spanning from cancer to autoimmune diseases.
  • To use a biological molecule for therapeutic purposes the full comprehension of its stability is necessary to elaborate proper formulation and package as well as to provide proper storage conditions and shelf life, essential for regulatory authorities.
  • analytical tests are carried out to assess the stability of the pharmaceutical formulation when subjected to different stresses (i.e. freeze-thaw cycles, shaking) and in different conditions (i.e. short term and long term storage, storage at different temperatures). Different tests are required to prove biomolecule stability, including visual inspection, sub-visible particle analysis, aggregate formation investigation etc.
  • the present invention relates to a stable pharmaceutical formulation comprising a bispecific antibody or an antibody fragment thereof, a buffer and one or more stabilizing or tonicity agents.
  • the pharmaceutical formulation is liquid, or lyophilized or reconstituted.
  • the pharmaceutical formulation comprises an antibody or antibody fragment thereof is present within said pharmaceutical formulation at a concentration between 0.05 mg/mL and 15 mg/mL.
  • the disclosed pharmaceutical formulation pharmaceutical formulation has a pH between 5.5 and 7.0.
  • the pharmaceutical formulation comprises a buffer is selected from the group comprising acetate, L-histidine, citrate and phosphate.
  • the buffer is histidine, present within said pharmaceutical formulation at a concentration comprised between 1 mM and 20 mM.
  • the pharmaceutical formulation according to the present invention comprises one or more stabilizing or tonicity agent is selected from the group comprising sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride, potassium acetate, potassium bicarbonate, potassium carbonate, potassium chloride, calcium chloride, sucrose, glutamate, mannitol, polyols, Polysorbate 20, Polysorbate 40, Polysorbate 80, Poloxamer, Poloxamer 188, Poloxamer 407, amino acids such as histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, amines, cyclodextrins, b-cyclodextrins, polyvinylpyrrolidone, polyethylene glycol 400, sorbitol, trehalose and EDTA, present within said pharmaceutical formulation at a percentage between about 0.005% and about 20%.
  • stabilizing or tonicity agent is selected from the group comprising sodium acetate, sodium bicarbonate, sodium carbon
  • the stabilizing or tonicity agent is sucrose present within said pharmaceutical formulation at a concentration comprised between about 2% and about 15%; and/or mannitol present within said pharmaceutical formulation at a concentration comprised between about 2% and about 6%; and/or glycine present within said pharmaceutical formulation at a concentration comprised between about 0.4% and about 1.2%; and/or Polysorbate 80 present within said pharmaceutical formulation at a concentration comprised between about 0.01% and about 0.11%; and/or arginine present within said pharmaceutical formulation at a concentration of about 0.4%; and/or glutamate present within said pharmaceutical formulation at a concentration of about 0.4%; and/or polyvinylpyrrolidone present within said pharmaceutical formulation at a concentration of about 0.1%; and/or b-cyclodextrin present within said pharmaceutical formulation at a concentration of about 0.1%.
  • the pharmaceutical formulation comprises a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration selected from the group comprising about 0.1 mg/mL, about 0.3 mg/mL , about 1 mg/mL and about 10 ⁇ 2 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3.
  • the bispecific antibody or fragment thereof is present within said pharmaceutical formulation at a concentration of about 10 ⁇ 2 mg/mL and the pharmaceutical formulation is liquid.
  • the bispecific antibody or fragment thereof is present within said pharmaceutical formulation at a concentration of about 0.1 mg/mL and the pharmaceutical formulation is lyophilized or reconstituted.
  • the pharmaceutical formulation is stable at about 25°C for at least 1 month; and/or at about 5°C for at least 1 month; and/or at about -20°C for at least 48 months; at about -60°C for at least 48 months; and/or at about -80°C for at least 36 months.
  • the pharmaceutical formulation is stable at about 40°C for at least 3 months; and/or at about 25°C for at least 24 months; and/or at about 5°C for at least 36 months.
  • the disclosed pharmaceutical formulation comprises a bispecific antibody or an antibody fragment thereof that binds CD3 and HER2.
  • the bispecific antibody or an antibody fragment comprises the amino acid sequences of SEQ ID NOs: 2, 4 and 6.
  • the present invention also relates a method of manufacturing the pharmaceutical formulation of any one of the preceding claims. Disclosed by the present invention is also a method to test the pharmaceutical formulation of any one of the preceding claims to any assay for stability determination.
  • the present invention also discloses an article of manufacture comprising the pharmaceutical formulation of any one of the preceding claims.
  • the present invention relates to a stable pharmaceutical formulation comprising a bispecific antibody or an antibody fragment thereof, a buffer and one or more stabilizing or tonicity agents.
  • Temperatures above 0°C are expressed in the present invention either by preceding the temperature value by "+” or not, e.g. 25°C and +25°C can be used interchangeably according to the present invention.
  • the disclosed pharmaceutical formulation may be liquid, or lyophilized or reconstituted.
  • a "liquid” formulation is one that has been prepared in a liquid format. Such a formulation may be suitable for direct administration to a subject or, alternatively, can be packaged for storage either in a liquid form, in a frozen state or in a dried form (e.g. lyophilized) for later reconstitution into a liquid form or other forms suitable for administration to a subject.
  • a "lyophilized" formulation is one that has been prepared by freeze-drying a liquid or pre- lyophilization formulation. Freeze-drying is performed by freezing the formulation and then subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition the product temperature is below the collapse temperature of the formulation. A secondary drying stage may then be carried out, which produces a suitable lyophilized cake.
  • a "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation.
  • the reconstituted formulation should be suitable for administration (e.g. parenteral administration) to a subject to be treated with the protein of interest.
  • Suitable "reconstitution media" useful for the preparation of a reconstituted formulation include ones which are pharmaceutically acceptable (safe and non-toxic for administration to a human).
  • suitable diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI). Water for injection (WFI), a pH buffered solution e.g. phosphate-buffered saline (PBS), sterile saline solution, Ringer's solution or dextrose solution and the buffer used to prepare the pharmaceutical formulation.
  • lyophilization is carried out by at least one lyophilization cycle, comprising the steps of precooling, freezing/annealing, primary drying and secondary drying.
  • precooling is performed at a temperature comprised between about 0°C and about 10°C, preferably at a temperature of about 5°C, for a ramp time, namely the time require to change temperature from one step to other, comprised between about 5 min and about 60 min, preferably between about 10 min and about 40 min, more preferably for a ramp time of about 20 min, and for an hold time, namely the time in which the sample is kept at the defined temperature, comprised between about 5 hours and about 12 hours, preferably between about 7 hours and about 10.30 hours, more preferably for an hold time of about 7 hours.
  • Freezing/annealing is performed by a first freezing step at a temperature comprised between about -5°C and about 5°C, preferably at a temperature of about 0°C for a ramp time comprised between about 1 min and about 55 min, preferably between about 2 min and about 10 min, more preferably for a ramp time of about 5 min, and for an hold time comprised between about 20 min and about 40 min, preferably for an hold time of about 30 min; followed by a second freezing step at a temperature comprised between about -40°C and about -50°C, preferably at a temperature of about -45°C, for a ramp time comprised between about 15 min and about 2 hours, preferably between about 20 min and about 1.40 hours, more preferably for a ramp time of about 45 min, and for an hold time comprised between about 1 hour and about 3 hours, preferably for an hold time of about 2 hours; followed by a third freezing step at a temperature comprised between about -5°C and about -25°C, preferably at a temperature of about
  • the present invention also comprises lyophilization processes at any value of the temperature, the ramp time and the hold time between the above cite values.
  • buffer refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
  • a buffer of this invention has a pH in the range from about 5.0 to about 7.0; preferably the buffer has a pH selected from the group comprising pH 6, pH 6.3 and pH 6.5.
  • buffers that can control the pH in this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, amino acids, such as histidine (e.g. histidine-HCI), citrate, phosphate, other organic acid buffer, their salts and combinations of buffers.
  • the buffer is present within the pharmaceutical formulation at concentration between about 1 mM and about 20 mM; preferably the buffer is present within the pharmaceutical formulation at concentration selected from the group comprising 1 mM, 3 mM, 5 mM, 7 mM, 10 mM, 13mM, 15 mM, 17 mM and 20 mM.
  • the present invention also includes a buffer with a concentration at any intermediate value of the above said values.
  • the buffer is Histidine, present within the pharmaceutical formulation at a concentration of about 5 mM.
  • One or more stabilizing or tonicity agent may be added to the formulation to stabilize the protein in the lyophilized form.
  • Said stabilizing or tonicity agent is selected from the group comprising sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride (NaCI), potassium acetate, potassium bicarbonate, potassium carbonate, potassium chloride, calcium chloride (CaCI2), glutamate, sugars such as sucrose, glucose and trehalose, polyols such as mannitol, maltitol, sorbitol, xylitol, erythritol, and isomalt, polyethylene glycol, such as PEG400, Ethylenediaminetetraacetic acid (EDTA), amino acids such as histidine (e.g.
  • Non limiting examples of a typical surfactant include: non- ionic surfactants (HLB 6 to 18) such as sorbitan fatty acid esters (e.g. sorbitol), glutamic acid, glutamine, cysteine, amines, glutathione, cyclodextrin, such as such as Hydroxypropyl b-cyclodextrin (HPBCD), Hydroxypropyl-sulfobutyl b-cyclodextrin (HPSBCD), Sulfobutylether b-cyclodextrin (SBECD), b-cyclodextrin (BetaCD), a-cyclodextrin (Alpha CD) and y- cyclodextrin (Gamm CD) and surfactants.
  • Non limiting examples of a typical surfactant include: non- ionic surfactants (HLB 6 to 18) such as sorbitan fatty acid esters (e.g. sorbitol
  • glycerine fatty acid esters e.g. glycerine monocaprylate, glycerine monomyristate, glycerine monostearate
  • poly glycerine fatty acid esters e.g. decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate
  • polyoxyethylene sorbitan fatty acid esters e.g.
  • polyoxyethylene lauryl ether polyoxyethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g.
  • polyoxyethylene lanolin polyoxyethylene lanolin
  • polyoxyethylene fatty acid amides e.g. polyoxyethylene stearyl amide
  • anionic surfactants such as Cio-Cis alkyl sulfates salts (e.g. sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene Cio-Cis alkyl ether sulfates salts with an average of 2 - 4 moles of ethylene oxide (e.g. sodium polyoxyethylene lauryl sulfate), and Cs-Cis alkyl sulfosuccmate ester salts (e.g.
  • the surfactant is selected from polyoxyethylene sorbitan fatty acid esters.
  • the surfactant is selected from polyoxyethylene sorbitan fatty acid esters.
  • the surfactant is selected from polyoxyethylene sorbitan fatty acid esters.
  • the surfactant is Polysorbate 20, 21 , 40, 60, 65, 80, 81 and 85, most preferably Polysorbate 80.
  • Polysorbate 80 is also known by the brand name Tween 80TM (ICI Americas, Inc.).
  • the stabilizing or tonicity agent is selected from the group comprising sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride, potassium acetate, potassium bicarbonate, potassium carbonate, potassium chloride, calcium chloride, sucrose, glutamate, mannitol, polyols, Polysorbate 20, Polysorbate 40, Polysorbate 80, Poloxamer, Poloxamer 188, Poloxamer 407, amino acids such as histidine, arginine, glycine, methionine, proline, lysine, glutamic acid,
  • the stabilizing or tonicity agent is present within the pharmaceutical formulation at a percentage between about 0.005% and about 20%. More in particular, sucrose is present within said pharmaceutical formulation at a concentration comprised between about 0.1% and about 20%, preferably between about 2% and about 15%, more preferably at a concentration selected from the group comprising 2%, 4%, 6%, 8%, 10%, 12%, 14% and 15%; and/or mannitol is present within said pharmaceutical formulation at a concentration comprised between about 0.5% and about 10%, preferably between about 2% and about 6%, more preferably at a concentration selected from the group comprising 2%, 2.5%, 4%, 5% and 6%; and/or glycine is present within said pharmaceutical formulation at a concentration comprised between about 0.1% and about 2%, preferably between about 0.4% and about 1.2%, more preferably at a concentration selected from the group comprising 0.4%, 0.5%, 0.8%, 1%, 1.2% and 2%; and/or Polysorbate 80 is present within said pharmaceutical formulation
  • the pharmaceutical formulation according to the present invention also comprises a bispecific antibody or an antibody fragment thereof.
  • antibody or “immunoglobulin” are herein used interchangeably and referred to whole antibodies and any antigen binding fragments or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as 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
  • VL 8 region
  • the light chain constant region is comprised of one domain, CL.
  • CL complementarity determining regions
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR or FW).
  • CDR complementarity determining regions
  • FR or FW framework regions
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino- terminus to carboxy- terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3 and FW4.
  • Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region.
  • Human constant light chains are classified as kappa (CK) and lambda (CX) light chains.
  • Heavy chains are classified as mu (m), delta (d), gamma (y), alpha (a), or epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • isotype as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgGl (IGHG1), lgG2 (IGHG2), lgG3 (IGHG3), lgG4 (IGHG4), IgAl (IGHA1), lgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE).
  • the IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, lgG2, lgG3 and lgG4.
  • Antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward ES et al., (1989) Nature, 341 : 544-546) which consists of a single variable, (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vi) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird RE et al, (1988) Science 242: 423-426; Huston JS et al, (1988) Proc.
  • the disclosed antibody is monoclonal.
  • monoclonal antibody refers to antibodies that are produced by clone cells all deriving from the same single cell, and that specifically bind the same epitope of the target antigen. When therapeutic antibodies are produced, the generation of monoclonal antibodies is preferred over polyclonal antibodies. In fact, while monoclonal antibodies are produced by cells originating from a single clone and bind all the same epitope, polyclonal antibodies are produced by different immune cells and recognize multiple epitopes of a certain antigen. Monoclonal antibodies assure batch to batch homogeneity, reduced cross-reactivity and high specificity toward the target. Monoclonal antibodies can be expressed, for instance in host cells, using recombinant DNA, giving rise to a recombinant antibody.
  • the antibody is a recombinant antibody.
  • recombinant antibody refers to an antibody that has been produced by any process involving the use of recombinant DNA.
  • a recombinant antibody can be engineered in such a way to improve characteristics such as immunogenicity, binding affinity, molecular size, specificity, half-life, and format.
  • recombinant antibodies include, but are not limited to engineered antibodies, chimeric antibodies, CDRs grafted antibodies (such as humanized antibodies), fully human antibodies, antibody fragments, Fc-engineered antibodies, multispecific antibody (such as bispecific, trispecific, tetraspecific antibody), monomeric and multimeric antibodies (such as homo-dimeric and hetero-dimeric antibodies).
  • the antibody of the present invention is a hetero-dimeric antibody.
  • hetero-dimeric antibody or “hetero-dimeric fragment” or “hetero-dimer” as used herein includes an immunoglobulin molecule or part of comprising at least a first and a second polypeptide, like a first and a second domain, wherein the second polypeptide differs in amino acid sequence from the first polypeptide.
  • a hetero-dimeric immunoglobulin comprises two polypeptide chains, wherein the first chain has at least one non identical domain to the second chain, and wherein both chains assemble, i.e. interact through their non-identical domains.
  • a hetero-dimeric immunoglobulin comprises at least two domains, wherein the first domain is non identical to the second domain, and wherein both domains assemble, i.e. interact through their protein-protein interfaces. More preferably the hetero-dimeric immunoglobulin, has binding specificity for at least two different ligands, antigens or binding sites, i.e. is bispecific.
  • bispecific antibody refers to any antibody having two binding sites that can bind two different epitopes of the same antigen, or two different antigens.
  • the antibody or antibody fragment thereof is present within said pharmaceutical formulation at a concentration between 0.05 mg/mL and 15 mg/mL, more in particular the concentration selected from the group comprising about 0.1 mg/mL, about 0.3 mg/mL , about 1 mg/mL and about 10 ⁇ 2 mg/mL.
  • the present invention also includes antibody or antibody fragment thereof present within the disclosed pharmaceutical formulation at a concentration at any intermediate value of the above said values.
  • the pharmaceutical formulation comprises a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration selected from the group comprising about 0.1 mg/mL, about 0.3 mg/mL, about 1 mg/mL and about 10 ⁇ 2 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3.
  • the pharmaceutical formulation comprises a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 10 ⁇ 2 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3, and said pharmaceutical formulation is liquid.
  • the pharmaceutical formulation comprises a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 0.1 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3, and said pharmaceutical formulation is lyophilized or reconstituted.
  • the hetero-dimeric bispecific antibody may be generated by BEAT ® technology (WO2012131555).
  • the bispecific antibody referred to as BEAT ® 1
  • BEAT ® 1 comprises the amino acid sequences of SEQ ID NOs: 1 to 6.
  • the pharmaceutical formulation according to the present invention is stable.
  • a “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed for example in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones A (1993) Adv Drug Delivery Rev, 10: 29- 90. Stability can be measured at a selected temperature for a selected time period.
  • analytical tests useful to determine said stability include but are not limited to: monitoring of the visual appearance as a significant change in the appearance of sample may indicate product degradation and/or microbial contamination leading to safety risk for the patients; sub-visible particles analysis, as the presence of higher sub-visible particles in parental solutions may lead to immunogenic responses; protein content measurement (e.g. by measuring absorbance at 280 nm wavelength (A280) by UV-VIS Spectroscopy or by SoloVPE) as any significant variation from its target concentration would not provide effective dose to patients; pH measurement as changes in pH may be indicative of degradation of buffering agents and lead to protein instability; size variants monitoring (e.g.
  • FDS Forced Degradation Studies
  • the liquid pharmaceutical formulation comprising a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 10 ⁇ 2 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3, is stable at about 25°C for at least 1 month; and/or at about 5°C for at least 1 month; and/or at about -20°C for at least 48 months; at about -60°C for at least 48 months; and/or at about -80°C for at least 36 months.
  • the lyophilized or reconstituted pharmaceutical formulation comprising a bispecific antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 0.1 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 5 mM, sucrose present within said pharmaceutical formulation at a percentage of about 5% and Polysorbate 80 present within said pharmaceutical formulation at a percentage of about 0.02%, and wherein said pharmaceutical formulation has pH of about 6.3, is stable at about 40°C for at least 3 months; and/or at about 25°C for at least 24 months; and/or at about 5°C for at least 36 months.
  • the present invention relates to a pharmaceutical formulation for use in the treatment of cancer characterized by the overexpression of HER2 and in particular selected from the group comprising breast, ovarian, bladder, salivary gland, endometrial, pancreatic and non-small-cell lung cancer (NSCLC).
  • NSCLC non-small-cell lung cancer
  • the present invention also relates to a pharmaceutical formulation for use in the treatment of a patient in need thereof using combinations of the disclosed bispecific antibody and a second immuno-oncology agent to the patient either sequentially or simultaneously.
  • the term “subject” includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
  • the subject is human.
  • a “patient” for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans.
  • the antibodies of the present invention have both human therapy and veterinary applications.
  • treatment or “treating” in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder.
  • successful administration of an antibody prior to onset of the disease results in treatment of the disease.
  • successful administration of an antibody after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.
  • Treatment and “treating” also encompasses administration of an antibody after the appearance of the disease in order to eradicate the disease.
  • Those "in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
  • the antibody or of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. More preferred routes of administration are intravenous or subcutaneous.
  • parenteral administration 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, epidural and intrasternal injection and infusion.
  • an antibody of the invention can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the antibody of the present invention can be administered at a single or multiple doses.
  • dose or “dosage” as used in the present invention are interchangeable and indicates an amount of drug substance administered per body weight of a subject or a total dose administered to a subject irrespective to their body weight.
  • the pharmaceutical formulations provided may be administered to individuals. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a subject. Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of medical doctors. Appropriate doses of antibody are well known in the art (Ledermann JA et al., (1999) Int J Cancer 47: 659-664; Bagshawe KD et a/., (1991) Antibody, Immunoconjugates and Radiopharmaceuticals, 4: 915-922).
  • the precise dose will depend upon a number of factors, including the size and location of the area to be treated, body weight of the subject, the precise nature of the antibody (e.g. whole antibody or fragment) and any additional therapeutic agents administered before, at the time of or after administration of the antibody.
  • Figure 1 Graphics related to physical-chemical attributes (concentration by UV detection, pH by SevenExcellence pH meter, A) and to size variants (SE-HPLC, B) for BEAT ® 1 control and stress temperature samples.
  • Figure 2 Graphics related to purity determination by cGE (NR) for BEAT ® 1 BDS and FBDS temperature control and stress samples.
  • Figure 3 Graphics related to purity determination by cGE (R) for BEAT ® 1 BDS and FBDS temperature control and stress samples.
  • Figure 4 Graphics related to charge variants determination by CEX for BEAT ® 1 FBDS and BDS temperature control samples.
  • Figure 5 Graphics related to size variants determination by SE-HPLC for BEAT ® 1 FBDS and BDS samples treated with oxidizing agents.
  • Figure 6 Overlays (left) and Graphs (right) related to charge variants determination by CEX for BEAT ® 1 BDS and FBDS samples treated with oxidizing agents.
  • Figure 7 Fit Least Squares reports for SE-HPLC analysis of BEAT ® 1 BDS (A) and FBDS (B) showing plots of the percentage of aggregates, main and fragments.
  • Figure 8 Profiler plot with maximized desirability after SE-HPLC analysis (maximum aggregate and fragments levels, minimum main levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 BDS.
  • Figure 9 Profiler plot with maximized desirability after SE-HPLC analysis (maximum aggregate and fragments levels, minimum main levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 FBDS.
  • Figure 11 Fit Least Squares reports for cGE (NR) analysis of BEAT ® 1 BDS (A) and FBDS (B) showing plots of the percentage of BEAT and total fragments.
  • Figure 12 Overlays related to purity determination by cGE (NR) for BEAT ® 1 BDS under pH 4 (A) and pH 9 (B) incubation at 40°C, 10 days.
  • Figure 13 Profiler plot with maximized desirability after cGE (NR) analysis (maximum total fragments levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 BDS.
  • Figure 14 Profiler plot with maximized desirability after cGE (NR) analysis (maximum total fragments levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 FBDS.
  • NR cGE
  • Figure 15 Fit Least Squares reports for CEX analysis of BEAT ® 1 BDS (A) and FBDS (B) showing plots of the percentage of acidics, main and basics.
  • Figure 16 Profiler plot with maximized desirability after CEX analysis (maximum acidic and basic levels, minimum main levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 BDS.
  • Figure 17 Profiler plot with maximized desirability after CEX analysis (maximum acidic and basic levels, minimum main levels) at pH 4 (left) and pH 9 (right) under 40°C temperature incubation for BEAT ® 1 FBDS.
  • Figure 18 Graphics related to size variants determination by SE-HPLC for BEAT ® 1 FBDS and BDS samples under light stress.
  • Figure 19 Graphics related to purity determination by cGE (NR) for BEAT ® 1 FBDS and BDS samples under light stress.
  • Figure 20 Overlays (left) and graphics (right) related to charge variants by CEX for BEAT ® 1 FBDS and BDS samples under light stress.
  • Figure 24 Overlays related to purity determination by cGE (NR) for BEAT ® 1 BDS samples under forced glycation (lyophilized condition).
  • one of the preferred composition for lyophilized presentation comprises of 5 mM histidine, 5% sucrose, 0.02% polysorbate 80 at pH 6.3 have BEAT ® 1 at a concentration of 0.1 mg/mL.
  • BEAT ® 1 BDS and FBDS were formulated by diluting the BDS to the desired concentration (0.1 mg/mL) with its formulation buffer: 5 mM L-Histidine, 5% Sucrose, 0.02% Polysorbate 80, pH 6.3, and then filled in 500 mL Freeze-PakTM STS Charter medical bags (FP50ML44) followed by
  • Oxidation stress condition consisted in oxidizing BEAT ® 1 BDS and FBDS with AAPH (2,2'-Azobis(2- amidinopropane) dihydrochloride) or H2O2 (hydrogen peroxide) which generally oxidizes Tryptophan (Trp) and Methionine (Met) residues respectively.
  • Oxidation by AAPH Sigma Aldrich was carried out by mixing protein with AAPH in the final molar concentration of 1:42 (protei AAPH).
  • a stock solution of AAPH, diluted in highly purified water (HPW) was prepared prior to mixing with BEAT ® 1 BDS and FBDS.
  • Oxidation by H2O2 was carried out by mixing protein with H2O2 in order to achieve a final concentration of 0.01% H2O2 .
  • a stock solution was prepared just as AAPH oxidizing agent.
  • the mixed samples with AAPH and H2O2 were kept at 25°C and removed at the specific time points (Table 1). d) Acidic and basic pH
  • Light stress consisted in incubating BEAT ® 1 BDS and FBDS in a binder light and climatic chamber (Binder). Following conditions have been used for light exposure experiment: 50 mL Freeze-PakTM STS Charter medical bags (10 mL filling volume), open vessel (50 mL falcon caps with 4 mL filling volume). The selected bag type is the same as the one used for the storage of the BDS material. Bags were lay down in the light exposure chamber. Vials were kept horizontally and caps were placed in a corning plate. Bags/vials/caps were kept on the first shelf of the light chamber, positioned directly below the light source and close together such that the distance from the light source remains similar.
  • a 1:1 mass ratio of glucose and a buffer exchange in Phosphate Buffer Saline (PBS) pH 7.0 - 7.5 was used to induce glycation at 37°C.
  • PBS Phosphate Buffer Saline
  • Two different glycations conditions were employed, one in liquid and the other one after lyophilization.
  • the buffer exchange step was done by putting sufficient material (BEAT ® 1 BDS) into a 15 mL amicon and by fulfilling with PBS pH 7.4. After centrifuging the amicon 15 min at 4000g, the flowthrough was discarded and the washing steps were repeated twice. BEAT ® 1 BDS was then collected by pipetting.
  • Glycation induction was assessed by adding a 1:1 mass ratio of Alpha-D(+)-Glucose, 99% + %anhydrous (Sigma Aldrich) in the falcon containing the BDS after buffer exchange. The solution was then filtered by using a syringe (Millipore) and a centrifugal filter (Becton Dickenson). Control samples were prepared by adding PBS instead of glucose. For glycation in lyophilized conditions, same steps than liquid glycation induction were followed but after filtration aliquots were prepared in 2 mL Fiolax glass vials (SCHOTT TopLyo, Adelphi Healthcare), stoppered and with a flip-off cap on top.
  • SCHOTT TopLyo Adelphi Healthcare
  • Table 8 shows a summary of the different stresses and enzymatic digestions applied to BEAT ® 1 BDS and FBDS with their respective time points.
  • Table 8 Stability time points for BEAT ® 1 BDS and FBDS stress conditions. X: Selected time point. N/A: Not Applicable at this time point
  • Table 9 Summary of the analytical methods performed for BEAT ® 1 study.* For general appearance, the degree of opalescence (clear, opalescent), the coloration (colorless, Brownish, Yellowish) and the particulates were observed. a) Total protein determination
  • Protein concentration enables to determine the quantity of proteins in a solution which absorb ultraviolet light at 280 nm wavelength (A280), due to the presence of aromatic amino acids in the protein structure,
  • the concentration of the sample must be 0.1-10.0 mg/mL. Otherwise, if sample concentration is >10.0 mg/mL, which is our case with BEAT ® 1 BDS, a dilution with the corresponding sample buffer is required. For all BDS samples, the same dilution (2X) was used. Protein concentration by UV-Visible spectrophotometer is determined using Nanodrop 2000 equipment (Thermo scientific). b) pH measurement
  • the pH is characterized in order to control the stability of our protein in solution. A change in the pH value could lead to degradation in proteins. With the time, the pH value could change due to the evaporation of the water and the concentration of the excipients of the buffer. Before taking a measurement, calibration of the pH meter is necessary. Minimum 300 pi of solution is required to measure the pH with the nano probe. pH measurements are determined using SevenExcellence pH meter (Mettler Toledo). c) Size Exclusion-High Performance Liquid Chromatography (SE-HPLC)
  • Size Exclusion-HPLC allows to determine purity of BEAT ® 1 in purified samples.
  • the SE-HPLC permits to obtain a percentage value of monomer, aggregate and fragment but is mainly interesting for the percentage of aggregates. A decrease in the percentage value of monomer could mean that the main species is degraded or form either aggregates or fragments.
  • a column with eluent A (0.1M sodium phosphate, 0.15M NaCI, pH 6.8) was used for chromatographic separation (protein separated according to their molecular weight) and at a flow rate of 1.0 mL/min. Samples were not diluted for BEAT ® 1 FBDS and diluted to 0.3 mg/mL in eluant A for BDS. 50 uL of sample was injected for the chromatographic system.
  • CEX-HPLC is used to investigate the identity of BEAT ® 1.
  • the method allows the determination of the charge profile (positive and negative charges) of BEAT ® 1 purified samples. If the product is degraded, chemical changes in the molecule could appear and lead to changes in percentages of peak area.
  • a step gradient using eluant A (20 mM MES, pH 6.9) and eluant B (20 mM MES, 150 mM NaCI, pH 7.0) at 1.0 mg/mL was applied.
  • the different forms of antibodies in solution are separated according to their interactions with a cation-exchange column and analyzed after passing through a UV-detector (absorbance at 280 nm).
  • CEX was performed to monitor BEAT ® 1 charge variants using a ProPac WCX-10 column (4.0 x 250 mm; Dionex).
  • cGE is used to determine the purity of monoclonal antibodies in terms of fragments and main species.
  • Non-reduced conditions allows to detect fragments and main species (BEAT) of BEAT ® 1.
  • Reduced conditions allows to detect LC, HC and scFv-Fc species of BEAT ® 1.
  • the percentage value of main species could decrease if the main species is degraded.
  • Samples were separated by electrophoresis through a sieving gel matrix (Beckman Coulter Kit) in a 50 um I.D. bare-fused silica capillary with 57 cm effective length using a Beckman Coulter PA 800 system with DAD/PDA detector (Diode Array/Photodiode Array Detector).
  • 40°C temperature is a stress factor as we can suppose that after 1 day reversible aggregates are decreased followed by an increase in the percentage of aggregates when incubation is longer. Moreover, when incubation at high temperature is extended just as a stress factor, this induces aggregates. BDS and FBDS have similar trends, and compared to TO unstressed samples for all temperatures at either time point, the level of aggregates decrease. The FBDS with a lower concentration, shows a lower level of aggregates. To conclude, this study highlighted that high temperatures have a positive effect on the formation of aggregates.
  • Oxidation of methionine and tryptophan are known degradation pathways for monoclonal antibodies.
  • the induced chemical modifications may impact the biological activity of antibodies and may have biological consequences (Andrea Bertolotti-Ciarlat, & al, 2009).
  • These amino acids are also accompanied by other degradants such as aggregates.
  • BEAT ® 1 BDS and FBDS samples were induced with FI O or AAPH.
  • AAPFI and FI O oxidizing agents did not impact the physical (appearance) attributes of the BDS and FBDS.
  • AAPFI let to more degradation compared to F ⁇ Ch for BDS in terms of size variants. Aggregates increased mostly and significantly between day 3 and day 10 (1.2% to 33.8% compared to TO) while a slight variation was observed at 25°C (control). As a consequence, this increasing trend could be attributed to oxidation by AAPFI and not to the temperature of incubation. Under FI O oxidation, aggregates decreased from day 3 and increased at day 10 (reversible aggregates). The same trend was observed for the control 25°C with slightly lower change, hence this variation could be attributed to the temperature of incubation. Fragmentation also slightly increased from day 10 for both oxidizing agents
  • Oxidation is known to influence potency/binding activity of monoclonal antibodies (Xuan Gao & al, 2014), thus it is important to analyze the bioactivity of the molecule after oxidation for BEAT ® 1.
  • MS mass spectrometry
  • pH treatment at pH 4 enables to investigate the aggregation and fragmentation mechanisms and the stability characteristics of proteins in acidic conditions.
  • the aggregation propensity increases with low pH at a fixed temperature or with increasing temperature at a fixed pH (Erinc Sahin & al, 2011).
  • pH 9 has been tested in order to also follow aggregation and fragmentation mechanisms depending on the temperature applied and to determine the deamidation susceptibility of asparagine residues located at different sites.
  • DoE experiment was proposed and executed.
  • the prediction of the model shows a R 2 ranging from 0.70 (% acidics) to 0.59 (% basics) for BDS and 0.78 to 0.75 for FBDS (Figure 15).
  • the levels of variant species are significantly influenced by pH incubation and less by time and temperature incubation.
  • Figure 16 we can observe a low level of acidics (29.05%) which rises significantly at pH 9 (62.61%) and 40°C incubation for BDS (FBDS show slightly lower changes, Figure 17). Inversely, the level
  • BEAT ® 1 BDS has been filled in BDS bags and in open vessels (50 mL Falcon caps) and FBDS in stoppered glass vials. BEAT ® 1 formulations have been exposed to light as described in ICH guidelines. Light stress did not impact the physical (appearance) and physico-chemical attributes (protein concentration and pH) of the BDS and FBDS.
  • Glycation is an important protein modification that could potentially affect bioactivity and molecular stability.
  • the HC peak is strongly affected by glycation in lyophilized condition as compared to liquid condition because all along the glycation kinetic a mass shift to the right can be observed and finally from week 2 both HC and ScFv-Fc peaks could't be differentiated and are completely overlapping.
  • glycated HC and ScFv-Fc show the same molecular weight which is observed on the overlays by one wide peak instead of two sharp peaks.
  • Results for cGE (R) analysis are shown in appendix 2, Figure 23 and Figure 24.
  • BEAT ® 1 BDS target BEAT ® 1 concentration 10 ⁇ 2 mg/mL 5 mM Histidine, 5% w/v Sucrose, and 0.02% w/v Polysorbate 80 at solution pH of 6.3, filled into 50 mL bags (Freeze-PakTM STS 50 mL, Product code: FP50ML44, Charter Medical) with a fill volume of 15 mL per bag, was studied in different conditions and on including: storage for up to 48 months (48 M) at ⁇ -60 °C (Table 10); storage for up to 48 months (48 M) at -20 ⁇ 5°C (Table 11); storage for up to 1 month at + 5 ⁇ 3°C (Table 12 and Table 19); storage for up to 1 month at 25 ⁇ 3°C (Table 13 and Table 20); 3 cycles of FT from -60°C or -20 ⁇ 5°C to 5°C (Table 14); temperature excursions (Table 15); 3 cycles of Freeze-Thaw at both -80 ⁇ 20°C and -20
  • BEAT ® 1 BDS stability was assessed by comparing the obtained results to reference standards.
  • the measured parameters included pH measurement, physical appearance of the liquid, A280 measurement, SDS-PAGE, 5 SE-HPLC, cGE, CEX-HPLC, HIC-HPLC, clEF (iCE3), Binding ELISA, functional assay, SDS-PAGE and bioburden analysis.
  • BEAT ® 1 BDS is stable: - after 3 cycles of freeze-thaw at both -80 ⁇ 20°C and -20 ⁇ 5°C in Charter Medical bag (Freeze-PakTM
  • the drug product (DP) is formulated in a 5 mM (0.776 mg/mL) L-Histidine buffer containing 5% w/v (50 mg/mL) Sucrose and 0.02% w/v (0.2 mg/mL) Polysorbate 80 (Tween 80). It is presented as a sterile lyophilized powder for injection; lyophilization was carried out as described in Table 2. Post reconstitution with WFI, the solution contains GBR 1302 at a concentration of 0.1 mg/mL at a pH of 6.3. Required no. of BEAT ® 1 DP vials were stored under various temperature conditions of +5 ⁇ 3 °C (Table 25), +25 ⁇ 2 °C (Table 26) and +40 ⁇ 2 °C (Table 27) in order to assess its stability.
  • BEAT ® 1 DP is found to be within specification for all the analytical assays for 36 months when stored at +5 ⁇ 3 °C and 24 months when stored at +25 ⁇ 2 °C.
  • the DP conformed to criteria of purity (based on SE-HPLC, SDS-PAGE and cGE non-reduced and reduced), charge profile (based on iCE3 and CEX-HPLC), protein content (A280), sub-visible particles, moisture content (Karl Fisher) and binding to target (Binding ELISA and Functional Assay).
  • No significant increase in the reconstitution time of lyophilized cake was observed during stability period.
  • the physical appearance and pH values of reconstituted solution confirmed to be within the predefined specifications.

Abstract

La présente invention concerne des formulations stables d'un anticorps thérapeutique ; de préférence, l'anticorps thérapeutique est un anticorps bispécifique monoclonal.
EP19778823.5A 2018-09-11 2019-09-11 Compositions comprenant un anticorps bispécifique, un tampon et un ou plusieurs agents stabilisants Withdrawn EP3849513A1 (fr)

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