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
  • A61K39/39591—Stabilisation, fragmentation
• • 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
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • 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
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present disclosure relates to a formulation of an anti-IL13R antibody and uses thereof, a method of treatment using the formulation, in particular for treating a condition disclosed herein, and a process of making the formulation,
• IL- 13 has been associated with various conditions, Including but not limited to various respiratory and allergy-mediated disorders, fibrosis, scleroderma, atopic dermatitis, Inflammatory bowel disease and certain cancers; see, e.g., Wynn, TA, 2003 Annu. Rev, Immunol. 21:425-456; Terabe st al, 2000 Nat. Immunol. 1 (61: 515 -520; Fuss st al, 2004 J. Ciin. Invest 113 (10): 1490-1497; Simms st al, 2002 Curr. Opin. Rheumatol. 14 (6) :717-722; and Hasegawa et al, 1997 J. Rheumatol. 24 (2): 328-332.
• IL-13 is an attractive target for the treatment, of such diseases.
• One possible way to inhibit the activity of IL-13 would be to interfere with the binding of IL-13 to its receptor IL-13R, for example by using an antibody specific to IL-13R, such as an antibody specific to lL-13Rai.
• An effective antibody antagonist to 1L-13R «1 may also interfere with the binding of IL-13 and prevent heterodimerization of IL-4Ra and IL-13Ral.
• Such an antibody coukl inhibit signaling of both IL-13 and IL-4 through the type II receptor while sparing IL-4 signalling through the type I receptor, Signalling through the type I receptor is essential in the induction phase of the immune response during which Th2 cells differentiate. T cells do notexpress IL-13RaI so the type II receptor plays no role in Th2 differentiation.
• an IL- 13 Rai antibody should not affect the overall Thi/Th2 balance, Signalling through the type II IL-4/1L-13 receptor is critical during the effector- A- stage of the immune response during established allergic inflammation.
• blockade of the type II receptor should have a beneficial effect on many of the sy mptoms of asthma and other lL-13R-mediated conditions and should, therefore, be an effective disease modifying agent
• Antibodies against IL- 13Rai have been described in the art; see, eg, WO 97/15663, WO 03/80675; WO 03/46009; WO 06/072564; Gauchat et al, 1998 Ear. J. Immunol. 28:4286-4298; Gauchat et al, 2000 Eur. J, Immunol. 30:3157-3164; Clement et a/, 1997 Cytokine 9(11) :959 (Meeting Abstract); Ogata et al, 1998 J. Biol. Chem, 273:9864-9871; Graber et al, 1998 Eur. J. Immunol.
• CSL334 now known as ASLANOO-4/ebiasakimab], described in W02008/060813 as antibody 10G5-6.
• Ebiasakimab has been shown to bind to human lL-13Rcd with a high affinity (for example Kd may be 500pM).
• Ebiasakimab was shown to effectively antagonise IL- 13 function through inhibiting the binding of IL-13 to its receptor IL-13Ral and to inhibit IL-13 and IL-4 induced eotaxin release in NHDF cells, IL-13 and IL-4 induced STAT6 phosphorylation in NHDF ceils and IL-13 stimulated release of TARC in blood or peripheral blood mononuclear cells.
• optimised formulation for ebiasakimab is required because it can be difficult to handle and manufacture.
• Creating high concentration antibody formulations, for example with an antibody/fragment concentration of greater than 150mg/ml is beneficial for patients because it allows smaller injection volumes but is not easy to achieve. Formation of aggregates and particles in parenteral formulations can be extremely dangerous to patients and must be avoided.
• optimised formulations are needed to address these problems and/or also to maximise the shelf life,, delivery, potency and efficacy of the same.
• a high concentration antibody formulation for thereof, said formulation comprising:
• an anti-IL- 13R antibody or antigen binding fragment thereof for example 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 mg/rnl, in particular 150 mg/rnl, 175 mg/ml or 200 mg/ml;
• 170 to 250 mM of arginine (such as Arg-HCl or Arg-Glu), for example 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245 or 250 mM, in particular 150 mM, 175 mM or 250 mM;
• 20 to 50 mM histidine buffer for example 20, 25, 30, 35, 40, 45 or 50 mM, such as 20 mM or 50 mM histidine buffer;
• a non-ionic surfactant for example 0.01-0.03% w/w, such as 0.02% w/w; wherein the pH of the formulation is in the range 6.0 to 7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0, in particular 6.5; and wherein the anti lL 13R antibody or antigen binding fragment thereof comprises a VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3; and comprises a VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4, a VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5, and a VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6.
• the formulation comprises 150 to 200 mg/ml, such as 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 mg/ml of the anti- IL13R antibody or a binding fragment thereof.
• osmolarity of the formulation is in the range 350 to 550 mOsmo/kg for example 350, 355, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, ,465, 470, 475, 480, 485, 490, 495, 500, 505, 515, 520, 525, 530, 535, 540, 545, 550, such as 405 to 435 mOsmo/kg.
• the sugar is selected from mannitol, sorbitol, dextrose, galactose, fructose, lactose, trehalose and sucrose.
• the formulation according to paragraph 17, wherein the sugar is sucrose.
• the formulation according to any one of paragraphs 1 to 18, comprising 0.02% w/w of a nonionic surfactant comprising 0.02% w/w of a nonionic surfactant
• the formulation according to paragraph 20, wherein the non-ionic surfactant is polysorbate 20.
• the anti-lL13R antibody comprises a VII domain comprising an amino acid sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising an amino acid sequence shown in SEQ ID NO: 8 or a sequence at least 95% identical thereto.
• an anti-IL-13R antibody or antigen binding fragment thereof wherein the anti- IL13R antibody comprises a VL domain comprising an amino acid sequence shown in SEQ ID NO: 8 or a sequence at least 95% identical thereto, and a VH comprising an amino acid sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto; 250 mM of Arg- HCl; 20 mM histidine buffer; 0.02% polysorbate 20; 75 mM phenylalanine and wherein the pH of the formulation is 6.5.
• the formulation according to paragraph 37, for use m the treatment of inflammation or an autoimmune disease, for example chronic inflammation.
• fibrosis including pulmonary fibrosis, such as cystic fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis; liver fibrosis, such as cirrhosis; heart disease, such as atrial fibrosis, endomyocardial fibrosis, old myocardial infarction; arthrofibrosis; Dupuytren's contracture; keloid fibrosis; mediastinal fibrosis; myelofibrosis; nephrogenic systemic fibrosis; retroperitoneal fibrosis; and scleroderma] Hodgkin's disease, ulcerative colitis, Chron’s disease, atopic dermatitis, eosinophilic esophagitis, allergic rhinitis, asthma and chronic pulmonary disease (including chronic obstructive pulmonary disease], in particular asthma. 40.
• fibrosis including pulmonary fibrosis, such as cystic fibrosis, id
• fibrosis including pulmonary fibrosis, such as cystic fibrosis, iodiopathic pulmonary fibrosis, progressive massive fibrosis; liver fibrosis, such as cirrhosis; heart disease, such as atrial fibrosis, endomyocardial fibrosis, old myocardial infarction; artbrofibrosis; Dupuytren's contracture; keloid fibrosis; mediastinal fibrosis; myelofibrosis; nephrogenic systemic fibrosis; retroperitoneal fibrosis; and scleroderma) Hodgkin's disease, ulcerative colitis, Chron’s disease, atopic dermatitis, eosinophilic esophagitis, allergic rhinitis, asthma and chronic pulmonary disease (including chronic obstructive pulmonary disease), Sez
• a method of treatment comprising administering an effective amount of a formulation according to any one of paragraphs 1 to 36.
• a method of treating inflammation comprising administering an effective amount of a formulation according to any one of paragraphs 1 to 36.
• fibrosis including pulmonary fibrosis, such as cystic fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis; liver fibrosis, such as cirrhosis; heart disease, such as atriai fibrosis, endomyocardial fibrosis, old myocardial infarction; arthrofibrosis; Dupuytren’s contracture; keloid fibrosis; mediastinal fibrosis; myelofibrosis; nephrogenic systemic fibrosis; retroperitoneal fibrosis; and scleroderma] Hodgkin’s disease, ulcerative colitis, Chron’s disease, atopic dermatitis, eosinophilic esophagitis, allergic rhinitis, asthma and chronic pulmonary disease (including chronic obstructive pulmonary disease), in
• Antibodies such as eblasakimab need to be formulated to high concentration to allow the desired dose in man to be administered in the smallest possible volume.
• High concentration formulations pose unique challenges as phenomena like phase separation can be observed. Aggregation is also a common feature at high antibody concentration.
• the formulation needs to contain very high levels of antibody molecules as "monomer”, for example 99% monomer or more.
• the formulation needs to be stable when stored.
• Eblasakimab seems to have a hydrophobic portion in the protein, which for example interacts with hydrophobic interaction columns in foe absence of high salt concentrations. This hypothesised hydrophobic portion adds additional complexity when formulating the antibody and preventing aggregation.
• the antibodies of the present disclosure are particularly difficult to formulate.
• a difficult challenge presented by higher concentration antibody formulations is the tendency for such formulations to be overly viscous.
• optimise formulations in order to keep viscosity at an acceptably low level, i.e. close to a target viscosity range of 20-25 cP, such as 20cP.
• the present inventors have optimised the formulation of the present disclosure and established that the IL-13R antibodies, such as eblasakimab, are most suitable for formulation within a narrow set of parameters.
• the formulations of the present disclosure are highly monomeric, for example at least 98% monomeric (such as 98 to 99.5% monomeric) even when formulated with high antibody concentration.
• the formulations of the present disclosure also have good viscosity at high antibody concentrations.
• the formulation is suitably stable, for example in some embodiments no change in monomer or less than a 0.5% reduction in monomer was observed when stored at 4°C or 25° for 90 days. Accelerated ‘stress test' studies at 40°C also show the formulations of the present disclosure to be stable over a period of 60 days, for example using potency m easurem e n ts.
• the formulations of the present disclosure have a viscosity in the range of 10 to 30, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 cP (centipoise), such as 20 cP, for exampie at ambient temperature.
• cP centipoise
• the viscosity is measured using a viscometer, such as rotational viscometer, an electroraagneticaHy spinning-sphere (EMS) viscometer, or a Stabinger viscometer.
• EMS electroraagneticaHy spinning-sphere
• the viscosity is measured using a rheometer, such as shear rheometer, dynamic shear rheometer, an extensional rheometer, a capillary rheometer.
• die viscosity is measured using a Kinexus-ultra+ rheometer (Netzsch).
• the osmolarity of the formulation is in the range 350 to 450 mOsmo/kg, such as 390 to 430 mOsmo/kg, in particular 410 +/-5m0smo/kg.
• the formulation comprises 150 to 210 mg/ml or an ant.i-JL13R antibody, for example 150 to 175mg/mi, such as 150, 155, 160, 165, 170, 175 mg/ml. In embodiment, the formulation comprises 175 mg/ml to 210 mg/ml, such as 175, 180, 185, 190, 195, 200, 205 or 210 mg/ml. In one embodiment the formulation comprises 150 mg/ml of ant.i-IL13R antibody. In another embodiment, the formulation comprises 175 mg/ml of anti-IL13R antibody. In one embodiment, the formulation comprises 200 mg/ml.
• the formulation comprises 170 to 260 mM of arginine, for example 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 or 260 mM,
• the formulation comprises 150 mM, 175 mM, 200 mM or 250 mM arginine.
• the formulation comprises 150 mM arginine.
• the formulation comprises 175 mM arginine.
• the formulation comprises 200 mM arginine.
• the formulation comprises 250 mM arginine.
• the arginine is Arg-HCl. In another embodiment, the arginine is Arg-GIu. in one embodiment arginine is L-arginine.
• the formulation comprises 150 mM Arg-HCl. In one embodiment the formulation comprises 175 mM Arg-HCl. In one embodiment the formulation comprises 200 mM Arg-HCl. In one embodiment the formulation comprises 250 rnM Arg-HCl,
• the formulation comprises 20 to 50 mM histidine buffer, for example 20, 25, 30, 35, 40, 45 or 50 mM, such as 20 mM or 50 mM histidine buffer. In one embodiment the formulation comprises 20 mM histidine buffer. In another embodiment the formulation comprises 50 mM histidine buffer.
• the formulation comprises 0.01-0,03% of a non-ionic surfactant, such as 0.01, 0.015, 0,02, 0.025 or 0,030 %, in particular 0.02%, in one embodiment, the formulation comprises 0.01-0.03%, such as 0.01, 0.015, 0.02, 0.025 or 0.030 %, in particular 0.02% volume per volume (v/v) of a non-ionic surfactant, In one embodiment the formulation comprises 0.01-0.03%, such as 0.01, 0.015, 0.02, 0.025 or 0.030 %, in particular 0.02% weight per volume (w/v) of a non-ionic surfactant In one embodiment the formulation comprises 0.01-0.03%, such as 0.01, 0.015, 0.02, 0.025 or 0.030 %, in particular 0.02% weight per weight (w/w) of a non-ionic surfactant. In one embodiment the formulation comprises 0.02% w/w of a non-ionic surfactant.
• the non-ionic surfactant is polysorbate, such as polysorbate 20, 40, 60, or 80. in one embodiment the non-ionic surfactant is polysorbate 20.
• the formulation comprises 0.01-0.03%, such as 0.02% polysorbate 20 (for example as %w/w, %w/v, %v/w or %v/v). In one embodiment the formulation comprises 0.02% w/w polysorbate 20.
• the pH of the formulation is in the range 6.0 to 7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9 or 7.0
• the pH is 6.0, 6.5 or 7.0.
• the pH is 6.5.
• the formulation further comprises phenylalanine, such as 45 to 90 mM phenylalanine, for example 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 mM, In one embodiment the formulation comprises 50, 75 or 80 mM phenylalanine. Thus, in one embodiment the formulation comprises 50 mM phenylalanine. In one embodiment the formulation comprises 75 mM phenylalanine. In one embodiment the formulation comprises 80 mM phenylalanine.
• phenylalanine such as 45 to 90 mM phenylalanine, for example 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 mM
• the formulation comprises 50, 75 or 80 mM phenylalanine.
• the formulation comprises 50 mM phenylalanine.
• the formulation comprises 75 mM phenylalanine.
• the formulation comprises 80 mM phenylalanine.
• the formulation further comprises CaCH, for example 10, 20, 30, 40, 50 or 60 mM CaCIj. In one embodiment the formulation comprises 50 mM CaCfo.
• the formulation further comprises 50 to 200mM of a sugar, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM of a sugar.
• the formulation comprises 180mM of a sugar.
• the sugar is selected from mannitol, sorbitol, dextrose, galactose, fructose, lactose, trehalose and sucrose.
• the sugar is sucrose.
• the formulation comprises 180 mM sucrose.
• certain formulations of the present disclosure have 1% or less protein aggregation, for example when stored for 90 days at temperature in the range 2 to 25°C.
• the presently disclosed anti-IL13R antibody formulation is particularly suitable for stable longterm storage of the anti-IL13R antibody.
• the formulation is stored at a temperature in the range 2 to 8°C, such as 2, 3, 4, 5, 6, 7 or 8 °C, such as 4 °C.
• a parenteral formulation for example for infusion or injection.
• liquid parenteral formulation as a concentrate for dilution with a liquid for injection, such as glucose, saline or water for injection.
• liquid parenteral formulation is provided in a final concentration for administration without dilution, for example for injection or for infusion.
• the formulation comprises: 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof; 250 mM of arginine; 20 mM histidine buffer; 0.02% of a non-ionic surfactant; and wherein the pH of the formulation is 6.5.
• the formulation comprises: 175 mg/ml of an anti-lL-13R antibody or antigen binding fragment thereof; 250 mM of Arg-HCI; 20 mM histidine buffer; 0,02% of polysorbate 20; and wherein the pH of the formulation is 6.5.
• the formulation comprises; 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof; 250 mM of arginine: 20 mM histidine buffer: 0.02% of a non-ionic surfactant; phenylalanine, such as 45 to 85 mM phenylalanine; and wherein the pH of the formulation is 6.5.
• the formulation comprises: 175 mg/ml of an anti-lL-13R antibody or antigen binding fragment thereof; 250 mM of arginine; 20 mM histidine buffer; 0.02% of a non-ionic surfactant; 75 mM phenylalanine: and wherein the pH of the formulation is 6.5.
• the formulation comprises; 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof; 250 mM of Arg-HCl; 20 mM histidine buffer; 0.02% of polysorbate 20;
• the antibody or binding fragment employed in the formulation of the present disclosure is monoclonal.
• the antibody or binding fragment employed in the formulation of the present disclosure is human. In one embodiment the antibody or binding fragment employed in the formulation of the present, disclosure is chimeric or humanised.
• the antibody or binding fragment thereof comprises a variable heavy region comprising a CDRH1 with a sequence shown in SEQ ID NO: 1, a CDRH2 with a sequence shown in SEQ ID NO: 2, and a CDRH3 with a sequence shown in SEQ ID NO: 3; and a variable light region comprising CDRL1 with a sequence shown in SEQ ID NO: 4, a CDRL2 with a sequence shown in SEQ ID NO: 5, and a CDRL3 with a sequence shown in SEQ ID NO: 6.
• the antibody or binding fragment thereof comprises a VH domain comprising a sequence shown in SEQ. ID NO: 7 or a sequence at feast 95% identical thereto.
• the antibody or binding fragment thereof comprises a VL domain comprising a sequence shown in SEQ ID NO; 8 or a sequence at least 95% identical thereto.
• the antibody or binding fragment thereof comprises a VH domain comprising a sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown In SEQ ID NO: 8 or a sequence at least 95% identical thereto.
• the antibody or binding fragment thereof comprises a VH domain comprising a sequence shown in SEQ ID NO: 7 and a VL domain comprising a sequence shown in SEQ ID NO: 8.
• the antibody or binding fragment thereof is eblasakimab.
• the formulation comprises: 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof comprising a VH domain comprising a sequence shown in SEQ ID NO; 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ ID NO: 8;
• the formulation comprises: 175 mg/ml of an antl-IL-13R antibody or antigen binding fragment thereof comprising a VH domain comprising a sequence shown in SEQ ID NO; 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ ID NO: 8; 250 mM of Arg-HCI; 20 mM histidine buffer; 0.02% of polysorbate 20; and wherein the pH of the formulation is 6.5, in one embodiment the formulation comprises: 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof comprising a VH domain comprising a sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ ID NO: 8; 250 mM of arginine; 20 mM histidine buffer; 0.02% of a non-ionic surfactant; phenylalanine, such as 45 to 85 mM phenylalanine: and
• the formulation comprises: 175 mg/ml of an anti-lL-13R antibody or antigen binding fragment thereof comprising a VH domain comprising a sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ ID NO: 8; 250 mM of arginine: 20 mM histidine buffer; 0.02% of a non-ionic surfactant: 75 mM phenylalanine; and wherein the pH of the formulation is 6.5.
• the formulation comprises: 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof comprising a VH domain comprising a sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ. ID NO: 8; 250 mM of Arg-HCI; 20 mM histidine buffer; 0.02% of polysorbate 20; 75 mM phenylalanine; and wherein the pH of the formulation is 6.5.
• the formulation comprises: 175 mg/ml of an anti-IL-13R antibody or antigen binding fragment thereof comprising a VH domain with a sequence shown in SEQ ID NO: 7 or a sequence at least 95% identical thereto and a VL domain comprising a sequence shown in SEQ ID NO; 8: 250 mM of Arg-HCI; 20 mM histidine buffer; 0.02% of polysorbate 20; 75 mM phenylalanine; and wherein the pH of the formulation is 6.5.
• Long term as used herein refers to a period of at least 6 months, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 months.
• the disclosed formulation storage for at least 12 months, such as 12 months, 18 months and 24 months.
• Non-ionic surfactant refers to surfactants that have covalently bonded oxygencontaining hydrophilic groups which are bonded to hydrophobic parent structures.
• non-ionic surfactants include ethoxylates, such as fatty alcohol ethoxylates (such as narrow-range ethoxylate, octaethylene glycol monododecyl ether and pentaethylene glycol monododecyl ether], alkylphenol ethoxylates (such as nonoxynols and Triton X-100), fatty acid ethoxylates, ethoxylated amines and/or fatty acid amides (such as polyethoxylated tallow amine, cocamide monoehtnolamine and cocamide diethanolamine), terminally blocked ethoxylates (such as poloxamers); fatty acid esters of polyhydroxycompounds; fatty acid esters of glycerol (such as glycerol monosteacetates
• the non-ionic surfactant is selected from the group comprising ethoxylates: fatty acid esters of polyhydroxy compounds; fatty acid esters of glycerol; tatty acid esters of sorbitol; Tweens; fatty acid esters of sucrose; alkyl polyglucosides; and polysorbates.
• Parenteral formulation as employed herein refers to a formulation designed not to be delivered through the G1 tract. Typical parenteral delivery routes include injection (including bolus injection), implantation or Infusion. In one embodiment the formulation is provided in a form for bolus delivery.
• parenteral formulation is administered intravenously. In one embodiment the parenteral formulation is administered subcutaneously.
• Injection refers to the administration of a liquid formulation into the body via a syringe or syringe driver.
• Injection includes intravenous, subcutaneous, intra-tumoral or intramuscular administration.
• the injection is generally over a short period of time, such as 5 minutes or less.
• injection can be administered slowly or continuously, for example using a syringe driver.
• Injections generally involve administration of smaller volumes than infusions.
• the injection is administered as a slow injection, for example over a period of 1.5 to 30 minutes.
• Siow injection as employed herein is manual injection with syringe.
• one dose of the formulation less than lOOmls, for example 30mls, such as administered by a syringe driver.
• Infusion as employed herein means the administration of fluids by drip, infusion pump, or equivalent device.
• the infusion is administered over a period in the range of 1 to 120 minutes (for example 1 to 5 minutes), such as about 1, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110, 115 or 120 minutes.
• Interleukin-13 receptor as used herein is a type I cytokine receptor, which binds to interleukin- 13. It consists of two subunits, encoded by ILlSRal and IL4R, respectively. These two genes encode the proteins IL-13Ral and lL-4Ra. These form a dimer with IL-13 binding to the IL- 13Ral chain and 1L-4R « stabilises this interaction. Due to the presence of the IL4R subunit, 1L13R can also instigate IL-4 signalling.
• tins occurs via activation of the Janus kinase (jAK'j/Signal Transducer and Activator of Transcription (STAT) pathway, resulting in phosphorylation of STAT6.
• STAT Janus kinase
• Human lL- 13Ral has the Uniprot number P3597.
• IL-13Ra2 previously called IL-13R and lL-13Rct, is another receptor which is able to bind to IL-13.
• this protein binds IL-13 with high affinity, but it does not bind IL-4.
• Human IL-13R «2 has the Uniprot number Q14627.
• Anti-IL13R antibody as used herein refers to an antibody hospice has specificity for IL13R, for example IL13Rctl or IL13Rct2.
• the anti-IL13R antibody of the present disclosure- is specific for ILlSRocl. In one embodiment, the anti-IL13R antibody binds to an epitope comprising the amino acid sequence FFYQ.
• the anti-IL13R antibodies of the present disclosure may comprise a complete antibody molecule having full length heavy and light chains or a binding fragment thereof Binding fragments include but are not limited to Fab, modified Fab, Fab', F(ab')?, Fv, single domain antibodies (such as VH, VL, VHH, IgNAR V domains), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example HoIIiger and Hudson, 2005, Nature Biotech. 23(9):1126-H36; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3], 209-217).
• antibody fragments for use in the present invention include the Fab and Fab' fragments described in W02005/003169, W02005/003170 and W02005/003171.
• Other antibody fragments for use in the present invention include Fab-Fv and Fab-dsFv fragments described in W02010/035012 and antibody fragments comprising those fragments.
• Multi-valent, antibodies may comprise multiple specificities or may be monospecific (see for example V/0 92/22853 and V/005/113605).
• the antibody and fragments thereof, for use in the present disclosure may be from any species including for example mouse, rat, shark, rabbit, pig, hamster, camel, llama, goat or human.
• Chimeric antibodies have a non-human variable regions and human constant regions.
• An antibody or binding fragment for use in the present invention can be derived from any ciass (e.g. IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule.
• the antibody employed in the present disclosure is IgG4 or lgG4 with a 241P mutation.
• the antibody or binding fragment employed in the formulation of the present disclosure has affinity of 5nM or higher (higher affinity is a lower numerical value), for example 500pM, such as 250pM or higher, in particular 125pM or less.
• GYSFTSYW1G (SEQ ID NO: 1 )
• V1YPGDSYTR SEQ ID NO: 2
• MPNWGSLDH (SEQ ID NO: 3)
• the anti-IL13R antibody or binding fragment employed in the present disclosure comprises a CDRH l comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 3.
• the anti-IL13R antibody or binding fragment employed in the present disclosure comprises a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 4, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 5, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 6.
• the VH sequence comprises SEQ ID NO: 7 or a sequence at least 95% identical thereto.
• the VL sequence comprises SEQ ID NO: 8 or a sequence at least 95% identical thereto.
• the VH sequence comprises SEQ ID NO: 7 or a sequence at least 95% identical thereto and the VL sequence comprises SEQ ID NO: 8 or a sequence at least 95% identical thereto, in one embodiment the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 8.
• Variable region as employed herein refers to the region in an antibody chain comprising the CDRs and a suitable framework.
• the heavy chain comprises a sequence independently selected from the group comprising: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and a sequence at. least 95% identical to any one of the same.
• the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 14 or a sequence at least 95% identical thereto.
• the heavy chain comprises SEQ ID NO; 9 or a sequence at least 95% identical thereto and the light chain comprises SEQ ID NO: 14 or a sequence at least 95% identical thereto.
• the heavy chain comprises SEQ ID NO: 10 or a sequence at least 95% identical thereto and the light chain comprises SEQ ID NO: 14 or a sequence at least 95% identical thereto.
• the heavy chain comprises SEQ ID NO: 11 or a sequence at least 95% identical thereto and the light chain comprises SEQ ID NO: 14 or a sequence at least 95% identical thereto.
• the heavy chain comprises SEQ ID NO: 12 or a sequence at least 95% identical thereto and the light chain comprises SEQ ID NO: 14 or a sequence at least. 95% identical thereto.
• the heavy chain comprises SEQ ID NO: 13 or a sequence at least 95% identical thereto and the light chain comprises SEQ ID NO: 14 or a sequence at least 95% identical thereto.
• the heavy chain is SEQ ID NO: 9 and the light chain is SEQ ID NO: 14. In one embodiment the heavy chain is SEQ ID NO: 10 and the light, chain is SEQ ID NO: 14. In one embodiment the heavy chain is SEQ ID NO: 11 and the light chain is SEQ ID NO: 14. In one embodiment the heavy chain is SEQ ID NO: 12 and the light chain is SEQ ID NO; 14. In one embodiment the heavy chain is SEQ ID NO: 13 and the light chain is SEQ ID NO: 14.
• Derived from as employed herein refers to the fact that the sequence employed or a sequence highly similar to the sequence employed was obtained from the original genetic material, such as the light or heavy chain of an antibody.
• At least 95% identical as employed herein is intended to refer to an amino acid sequence which over its full length is 95% identical or more to a reference sequence, such as 96, 97, 98 or 99% identical.
• Software programmes can be employed to calculate percentage identity'.
• an antibody or binding fragment thereof, employed in a formulation of the present disclosure is humanised.
• Humanised which include CDR-grafted antibodies] as employed herein refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, for example US 5,585,089; WO91/09967). it will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived. For a review, see Vaughan etal, Nature Biotechnology', 16, .535-539, 1998.
• any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
• human frameworks which can be used in the present invention are KOI , NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al, supra).
• KOL and NEWM can be used for the heavy chain
• REI can be used for the light chain and EU
• LAY and POM can be used for both the heavy chain and the light chain.
• human germline sequences may be used: these are available at: http: //vbase.mrc-cpe.cam. ac.uk/
• the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
• the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et M.. 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions which may need to be changed is set. forth in WO91/09967.
• anti-IL13R antibodies of the present disclosure are fully human, in particular one or more of the variable domains are fully human.
• Fully human molecules are those in which the variable regions and the constant regions (where present] of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, not necessarily from the same antibody.
• Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts e.g. as described in general terms in EP0546Q73 Bl, US 5,545,806, US 5,569,825, US 5,62.5,126, US 5,633,425, US 5,661,016, US5, 770,429, EP 0438474 and EP0463151.
• Constant region as employed herein is intended to refer to the constant region portion located between two variable domains, for example non- cognate variable domains, in the heavy chain.
• the presently disclosed anti-lL13R antibody may comprise one or more constant regions, such as a naturally occurring constant domain or a derivate of a naturally occurring domain.
• a derivative of a naturally occurring domain as employed herein is intended to refer to where one, two, three, four or five amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is/are retained.
• an antibody for use in the present invention may be conjugated to one or more effector molecule(s). It will be appreciated that the effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies of the present invention.
• an antibody fragment linked to an effector molecule this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule, Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et at, 1982 , Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).
• effector molecule includes, for example, biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
• biologically active proteins for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
• effector molecules may include detectable substances useful for example in diagnosis.
• detectable substances include various enzymes, prosthetic groups, fluorescent, materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography], and nonradioactive paramagnetic metal ions. See 054,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics.
• Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent, materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 1251, 1311, Ulin and 99Tc.
• the effector molecule may increase the half-life of the antibody in vivo, and/or reduce immunogenicity of the antibody and/or enhance the delivery of an antibody across an epithelial barrier to the immune system
• suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in W005/117984.
• the effector molecule is a polymer it may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.
• synthetic polymers include optionally substituted straight or branched chain poly (ethyleneglycol), poiy(propyleneglycol) polyfvinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleriegiycoi) or derivatives thereof.
• synthetic polymers include lactose, amylose, dextran, glycogen or ci e r i vati ve s th e re o f.
• Derivatives as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like.
• the reactive group may be linked directly or through a linker segment to fee polymer. It will be appreciated feat the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.
• Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoiy(ethyleneglycol) or a derivative thereof and especially with a molecular weight in the range from about ISOOODa to about 40000Da.
• antibodies for use in the present invention are attached to poly(ethyieneglycol) (PEG) moieties.
• the antibody is an antibody fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group.
• Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example US 5,219,996; US 5,667,425; WO98/25971, W02008/038024).
• die antibody molecule of the present invention is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy' chain one or more amino acids to allow the attachment of an effector molecule.
• the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.
• the formulation herein is administered in combination with another therapy.
• “In combination” as employed herein is intended to encompass where the anti-lL13K antibody is administered before, concurrently with, or after another therapy.
• Therapeutic dose as employed herein refers to the amount of the anti-lL13R antibody, such as eblasakimab that is suitable for achieving the intended therapeutic effect when employed in a suitable treatment regimen, for example ameliorates symptoms or conditions of a disease, in particular without eliciting dose limiting side effects.
• Suitable therapeutic doses are generally a balance between therapeutic effect and tolerable toxicity, for example where the side-effect and toxicity are tolerable given the benefit achieved by the therapy.
• a formulation according to the present disclosure (including a formulation comprising same) is administered monthly, for example in a treatment cycle or as maintenancetherapy.
• the background section may be employed as basis to make amendments.
• Embodiments may be combined when technically appropriate.
• Figure 1A shows a schematic diagram of the Kinexus ultra+ rheometer used for the viscosity measurements
• Figure IB shows a graph of the viscosity of the eblasakimab bulk drug product ( BDP)
• Figure 2 show’s a series of graphs indicating the viscosity of Formulations 1 to 15
• Figure 3 shows a graph comparing the viscosity of formulations comprising different Arg-
• Figure 4 shows a graph showing the viscosity of formulations comprising 150 mM Arg-HCl + add i ti onal excip ients
• Figure 5 shows graph comparing die viscosity of Formulations 1 to 15
• Figure s show’s a comparison of viscosity measurements for eblasakimab BDP from the 1 st and 2 nd step screens
• Figure 7 shows a series of graphs comparing the viscosity of Formulation 3 and 6
• Figure 8 shows a graph of a repeat viscosity measurement for Formulation 3 to check reproducibility of viscosity 7 measurements
• Figure 9 shows a series of graphs indicating the viscosity of Formulations 16 to 30
• Figure 10 shows a graph of the viscosity of Arg-Glu containing formulations vs Arg-HCl formulations
• Figure 11 show's a graph showing the viscosity of formulations comprising 150 mM Arg-HCl o additional excipients
• Figure 12 shows a graph comparing the viscosity of 150 mM Arg-HCl formulations at different pH
• Figure 13 shows a graph comparing the viscosity of 20 mM vs 50 mM His buffer formulations
• Figure 14 show’s a graph comparing the viscosity of Formulations 16 to 30
• Figure 15 show's a series of graphs of viscosity measurements for Formulations 31 and 32 in comparison with Formulations 3 and 16
• Figure 17 show's a comparison of viscosity measurements for Formulations 33 to 45
• Figure 18 shows a graph of viscosity measurements for 175 mg/ml eblasakimab formulations
• Figure 19 show’s a graph of viscosity measurements for 150 mg/ml eblasakimab formulations
• Figure 20 show's a graph of the stability test 3-month results for viscosity'
• Figure 21 shows a graph of the stability test 3-month results for osmolality
• Figure 22 shows a graph of the stability test 3-month results for protein concentration (soloVPE). Order from left to right for each formulation: CO; tlm, 050; tlm, 250; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 23 shows a graph of the stability test 3-month results for pH. Order from left to right for each formulation: W; tlm. 050; tlm, 250; tlm, 40C; t3m, 050; t.3m, 250; and t3m, 400
• Figure 24 shows a table of the stability test 3-month results based on visual inspection of the formulations
• Figure 25 shows a graph of the stability test 3-month results for turbidity. Order from left to right for each formulation: tO; tlm, 05C; tlm, 250: tlm, 400; t3m, 050; t3m, 250; and t3m, 400
• Figure 26 shows a table of the stability test 3-month results for colour. Order from left to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 40C; t3m, 050; t3m, 250; and t3m, 400
• Figure 27A shows a graph of die stability test 3-month results for subvisible particle content using micro-flow imaging (MFI), > 2 pm. Order from left to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 40C; t :3m, 050; t3m, 25C; and t3m, 400
• Figure 278 shows a graph of the stability test 3-month results for subvisible particle content (MFI), > 10 pm. Order from left to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 400; t3m, 050; t3m, 25C; and t3m, 40C
• Figure 27C show’s a graph of the stability test 3-month results for subvisible particle content (MFI), > 25 pm. Order from left to right for each formulation: t.0; tlm, 050; tlm, 250; tlm, 40C; t3m, 05C; t3m, 250; and t3m, 400
• Figure 28 shows a table of the stability test 3-month results for protein mass recovery (HP- SEC). Order from left, to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 40C; t3m, 050; t3rn, 250; and t3rn, 40 C
• Figure 29A show's a graph of the stability test 3-month results for aggregate % content (HP- SEO). Order from left to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 400; L3m, 050; t3m, 250; and t3m, 400
• Figure 298 shows a graph of the stability test 3-month results for monomer % content f HP- SEC). Order from left to right for each formulation: tO; tlm, 050; tlm, 25C; tlm, 40C; t3m, 050; t3m, 250; and t3m, 400
• Figure 29C shows a graph of the stability test 3-month results for fragment % content (HP- SEC). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40C; 13m, 050; f.3m, 25C; and t.3m, 40C
• Figure 29D shows a summary table a comparison of aggregate, monomer and fragment % content with the 100 mg/ml eblasakimab formulation (HP-SEC)
• Figure 30A shows a graph of the stability test 3-month results for non-reducing aggregate % content (cGE). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 3 OB show's a graph of the stability 7 test 3-month results for non-reducing monomer % content (cGE). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 30C shows a graph of the stability test 3-month results for non-reducing intact protein % content (cGE). Order from left to right for each formulation: tO; tlm, 050; tlm, 25C; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 30D show’s a summary table a comparison of intact protein % ⁇ content with the 100 mg/ ml eblasakimab formulation
• Figure 31A shows a graph of the stability test 3-month results for reducing non-glycosylated heavy chain (NGHC) % ⁇ content (cGE). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40 C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 3 IB show’s a graph of the stability test 3-month results for reducing heavy chain (HC) % content (cGE). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40C; 13m, 05C; t3m, 25C; and t3m, 40C
• Figure 31C shows a graph of the stability test 3-month results for reducing light chain (LC) % content (cGE). Order from left to right for each formulation: tO; tlm, 05C; tlm, 25C; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 3 ID shows a graph of the stability test 3-month results for reducing LC HC % content (cGE). Order from left to right for each formulation: tO; tlm, 050; tlm, 250; tlm, 40C; t3m, 05C; t3m, 25C; and t3m, 40C
• Figure 3 IE show's a graph of the stability test 3-month results for reducing impurity' % content (cGE). Order from left to right for each formulation: tO; tlm, 05C: tlm, 25C; tlm, 40C; t3m, 050; t3m, 25C; and t3m, 40C
• Figure 3 IF shows a summary table a comparison of reducing LC +HC % and NGHC % content wan the 100 mg/rol eblasakimab formulation (cGE)
• Figure 32 A show’s a graph of the stability test 3-month results for acidic peak % content (IEX)
• Figure 32B show's a graph of the stability test 3-month results for natural peak % content (IEX)
• Figure 32C shows a graph of the stability test. 3-month results for basic peak % content (IEX)
• Figure 32D shows a summary table a comparison of acidic, neutral and basic peak % content w’ith the 100 mg/ml ASLAN004 formulation (IEX)
• Example 1 - Step 1 (Primary) Screen Formulations 1 to 15 15 different formulations were initially produced:
• Formulations 8-13 Ser, Thr, Giy, Pro screening
• Eblasakimab BDP samples were concentrated 2 times to ⁇ 7.5 ml and diluted with 7,5 ml formulation buffer.
• each formulation was concentrated to yield the target concentration of 150 mg/mi, 175 mg/mi or 200 mg/ml.
• Protein concentration [Solo-VPE], osmolarity and pH was measured for each formulation.
• the average dynamic viscosity values were calculated from the reading between 540 to 600 sec, The eblasakimab samples are likely to behave as non-Newtonian fluids and the shear rate applied is constant. Thus, in the context of the present disclosure, the dynamic viscosity and shear viscosity are taken to be the same and these terms are used interchangeably in the Examples.
• Figure IB shows the viscosity measurements for the eblasakimab BDP with polysorbate 20 added. The results of the viscosity measurements for Formulations 1 to 15 are shown in Figure 2.
• Table 2 shows the formulations ranked by viscosity at different target concentration ranges.
• Figure 3 shows the relationship between Arg-HCl concentration and viscosity The results suggest that minimal viscosity is reached with 150 mM Arg-HCl and that higher Arg-HCl concentration does not further reduce viscosity. However, this was investigated further below,
• Figure 4 shows the impact of the different excipients on viscosity. The results indicate that. Arg-Glu reduces viscosity similarly to Arg-HCl and that adding an additional excipient to 150 mM Arg-HCl did not further reduce viscosity. This was investigated further below.
• Figure 5 shows the correlation between viscosity and ASLAN 004 concentration.
• each formulation was concentrated to yield the target concentration of 150 mg/ml, 175 mg/ml or 200 mg/ml.
• Poly-sorbate-20 was added to achieve a target concentration of 0.02% (w/w).
• Figure 7 shows a comparison between Formulation 3 and Formulation 16.
• Note Figure 8 indicates that, die viscosity reading for Formulation 3 was reproducible.
• Table 4 shows the formulations ranked by viscosity at different target concentration ranges.
• Figure 10 shows a comparison between the viscosities of Arg-HCI and Arg- Gin formulations at various concentrations. The results suggest that there was a more pronounced reduction in viscosity for the Arg-HCI compared to the Arg-Glu formulations, in particular, Increasing the Arg- HCI from 175 mM to 250 mM seemed to reduce the viscosity substantially.
• FIG 11 shows the impact of the different excipients on viscosity. The results indicate that the addition of Phenylalanine and CaCH helps to reduce viscosity.
• Figure 12 shows the impact of pH on viscosity for 150 mM Arg-HCI formulations. The results demonstrate that increasing the pH from 6,5 to 7 or reducing it to 6.0 did not improve viscosity. Thus, it would seem that any pH between 6.0 to 7.0 is suitable.
• Figure 13 shows the results of an experiment to assess the impact of increasing His buffer concentration. The results suggest that increasing the histidine concentration from 20 to 50 mM for dm 175 mg/ml and 200 mg/ml ASLAN004 formulations reduce the viscosity.
• Figure 14 shows the correlation between viscosity and ASLAN004 concentration.
• the purpose of the 3 rd screen was to attempt to gain a better understanding of the viscosity differences observed between the l 5t and 2 nd step screens and to confirm toe formulations’ viscosity range for the formulation stress testing.
• Formulations 3/16 and 29 had the best overall viscosities.
• a further 2 formulations were prepared based on these formulations: g6.5 20 mH His 250mM Arg-HO 525 F29
• the formulations were prepared as follows:
• formulations were prepared using the same method described in the secondary screen (Example 2] above.
• Formulations 41 to 45 were prepared 2 w'eeks after formulations 33 to 40.
• Figure 18 shows an overview of the viscosity measurements for the 2 nd , 3 rd and 4 th screens for the 175 mg/ml eblasakimab formulations.
• the results suggest that the formulations tested in the 4 th screen that comprised Arg-HCl and Phenylalanine had the lowest viscosities, i.e. Formulations 35, 38, 39 and 41.
• Figure 19 show’s an overview of the viscosity measurements for the 2 nd and 3 rd screens for the 150 mg/ml eblasakimab formulations. The results suggest that Formulations 21 and 29 had the lowest viscosities.
• Figure 21 shows the osmolality results for the 6 formulations. The differences observed were within the expected method variability.
• Figure 22 shows the protein concentration results for the 6 formulations. There was no distinct, change observed in the 3-mont.h samples compared to the day 0 samples.
• Figure 23 shows the pH results for the 6 formulations. The differences observed were within the expected method variability.
• Figure 24 shows results of the visual Inspection of 6 formulations, in general, the formulations appeared opalescent and with the exception of F49 at 40 °C, the particles were at the limit of being visible. This suggests thatparticie formation was minimal.
• Figure 25 shows turbidity results for the 6 formulations. The results suggest that the turbidity of the formulations was comparable between the Day 0, 1-month and 3-month samples. The lowest turbidity values were observed for the samples stored at 25 °C, Figure 26 shows the colour change results for the 6 formulations. With the exception of F49 at 40 °C, the rest of formulations remained within the brown spectrum (B5 ) after 3 months storage. No change in colouration was observed between the 1-month and 3-month samples stored at 2-8 °C or 25 °C. Conversely, there was a small change in colouration after storage for 3 months at 40 °C compared to 1 month of storage at 40 °C.
• Figures 27A to 27C show the results of the subvisible particle content experiments performed using micro-flow imaging (MFf
• FIGS 28 and 29A to 29D show the results of experiments conducted using HP-SEC. Details of the instrument and parameters used are as follows:
• Figure 28 suggest that the % protein mass recovery of all the formulations stored at 40 °C for 3 months is slightly lower compared to the formulations stored at 5 °C or 25 °C. Nonetheless, there did not appear to be a significant difference between the formulations.
• the results in Figures 29A to 29C indicate that there was a similar loss of monomer content, increase of high molecular weight and fragment content across the 6 formulations. Nonetheless, the loss or monomer was relatively low - almost no loss after 3 months at 5 °C, ⁇ 1% loss after 3 months at 25 °C, ⁇ 4% loss after 3 months at 40 °C, A higher aggregate content was observed for F49 compared to other formulations.
• Figure 29D demonstrates that the intact protein % concentration measured using SEC for Formulation F49 was very similar to that of the 100 mg/ml eblasakimab formulation.
• FIGS 30A to 30D show the results of the experiments conducted using capillary gel electrophoresis (cGEJ for the non-reduced samples. Details of the instrument and parameters used are as follows:
• Figures 30A to 30C suggest that there was almost no loss of Intact protein after 3 months at 5 °C, a loss of less than 3% after 3 months at 25 °C, and a loss of less then 15% after 3 months at 40 °C, primarily due to fragmentation.
• Figure 301) demonstrates that the intact protein % concentration measured using cGE for Formulation F49 was very similar to that of the 100 mg/ml eblasakimab formulation.
• Figures 31A to 3 IF show the results of the experiments conducted using capillary gel electrophoresis (cGE) for the reduced samples.
• Figures 31A to 31D indicate that there was no significant change in relative light chain (LC) content There was a slight decrease in heavy chain (HC) content after 5 months at 25 °C and 40 °C.
• LC and HC content was considered together [Figure 3 ID)
• Figure 31E shows the impurity levels after 1 month and 3 months storage. From the results, the inventors believe it is likely that the decrease in relative HC content after 3 months is partly clue to deglycosylation of the reduced samples, and also the increasing impurity levels with time. This trend was seen across the various formulations.
• Figure 3 IF demonstrates that the LC + HC% and NGHC% measured for Formulation F49 was very similar to that of the 100 mg/ml eblasakimab formulation.
• Figures 32A to 32D show the results of the ion-exchange chromatography (lEX) experiments. Details of the instrument and parameters used are as follows:
• Figures 32A to 32C indicate that there was a similar loss of neutral species, and a similar increase of acidic and basic species for all the formulations over the 3 months storage. There was almost no loss of neutral species after 3 months at 5 °C, less than 10% loss of neutral species after 3 months at 25 °C, and less than 45% loss of neutral species after 3 months at 40 °C.
• Figure 32D demonstrates that the acidic content, neutral content and basic content measured for Formulation F49 was very similar to that of the 100 mg/ml eblasakimab formulation.

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