Antibody formulation

The present invention is a human interleukin I beta (IL-ββ), in particular antibodies, described in WO

2002/016436.

[0002] Antibodies, like other protein therapeutic agents, are complex molecules, and in general. Liquid and non-invasive therapeutic agents. Protein degradation pathways may be associated with chemical or physical instability.

Immediately before the administration of the lyophilization of the medicinal product. However, it is required to obtain a safe preparation with effective therapeutic results.

Conveniently, liquid pharmaceutical preparations of medicinal compounds.

Daugherty and others (Advanced Drug Delivery Reviews 58, 2006 pp. 686-706) describe the obstacles to receiving stable liquid antibody preparations. WO 03/039485 refers to liquid IgG antibody preparations, e.g. Zenapax ™ (anti-CD25). WO 03/105894 relates to liquid formulations of Synagis ™ (anti-RSV protein F) of IgG1 antibodies.

A long-appreciated problem with liquid formulations, which can lead to unwanted immune reactions. In addition, the main problem was the problem of syringes or pumps, which makes them dangerous for patients.

Thus, there is a need for formulations that contain proteins, agents, and chemicals. The present invention solves the above-stated object, which is providing for new products and is also suitable for mammals, especially humans. Interleukin-ΐβ (IL-1 beta or IL-Ιβ or interleukin-ΐβ have the same meaning in this document) is a potent immune modulator that mediates a wide range of immune and inflammatory responses. Inappropriate or excessive production of diseases, such as sepsis, septic or endotoxic shock, allergies, asthma, bone loss, ischaemia, stroke, rheumatoid arthritis and other inflammatory diseases. Antibodies to IL-1 mediated disorders and diseases; see, for example, WO 95/01997 and discussion on its introduction and WO 02/16436.

A preferred prefered anti-IL-1 antibody for presentations. Id. Well. 1 and Seq Id. Well. 2, or their functional fragments. F (ab) 2, Fab, scFv, VH domain, CDR. Signal peptides in Seq. Id. Well. 1 and Seq. Id. Well. 2 is in italics according to WO 02/16436.

Is It stable It stable It stable It. Stable It stable It stable It. According to the present invention, the stability and the stability of the storage. For example, the spread of 2-5%, preferably 2-3%. The stability of the original activity is 80-125%. The biological activity of the preparation in the form of a gene assay,

Another object is to provide a stable liquid administration. Preferably, the liquid formulation is suitable for lyophilization and subsequent reconstitution. It is also a subject of mammals, especially humans.

[0003] In general, it is preferred to use small quantities of pharmaceutical formulation for subcutaneous injection (typically 1.0 ml-1.2 ml at the maximum). (Eg, 50 mg / ml-150 mg / ml or more). In the case of high-quality antimicrobial enzyme. Due to the high enzyme problem, the formation of antibody. The increased viscosity of the treatment, eg liquid preparations do not-pass, which causes discomfort for the patient; injection time; the possibility of using an auto-injector. In addition, this is a high-quality, high-quality and low-cost chemical. The term "viscosity" as used herein may be "kinematic viscosity" or "dynamic viscosity". Typically, the kinematic viscosity is expressed in centistokes (cSt). The unit of kinematic viscosity in the SI system is mm "kinematic viscosity" or "dynamic viscosity". Typically, the kinematic viscosity is expressed in centistokes (cSt). The unit of kinematic viscosity in the SI system is mm "kinematic viscosity" or "dynamic viscosity". Typically, the kinematic viscosity is expressed in centistokes (cSt). The unit of kinematic viscosity in the SI system is mm²/ s, which is 1 cSt. Dynamic viscosity is expressed in centipoise units (cP). The system of dynamic viscosity in SI system is millipaskal-second (mPa-s), with 1 cP = 1 mPa-s.

Accordingly, the present filter provides preparations of IL-ββ antibodies and has a sufficiently low viscosity.

The liquid pharmaceutical preparation of the laboratory should exhibit various predefined properties. One of the main problems in the process of soluble and insoluble aggregates during production and storage. In addition, various chemical reactions may occur in solution (deamidation, oxidation, oxidation reaction, etc.). Preferably, the liquid should have more than 24 months.

[0004] The antibody preparation, in the ACZ885, should contain an acceptable shelf life of about 24 hours. 36 months at 2-8 ° C. preferably, the lyophilized preparation of ACE885 should be at the same time. products and degradation and loss of biological activity. The formulation of the present achievements are desirable levels of stability.

In addition to the required pH and osmolality (250 to 500 mOsm / kg) for subcutaneous use. However, it has been administered in the literature that with high osmolarity (up to 1100 mOsm / kg) can be administered subcutaneously, without increasing pain perception and burning time after injection. It is also known for the high concentration of antibodies. A suitable pharmaceutical preparation, which is less than about 16 mPas, preferably 3 to 16 mPa.s, and more preferred 3-10 mPas.

[0005] In accordance with the present invention, it is necessary to maintain a successful life in the long term. The present invention in its broadest guide is a pharmaceutical preparation (formulation of the invention), stabilizing agent and a surfactant. The prepaqrat according to the invention is liquid.

The present invention relates to IL-ββ as an active ingredient and a buffer system, while the pH is 5.5 to 7.5, preferably from 5.5 to 7, more preferably from 6.2 to 6.8. More specifically, the ACZ885 is an active ingredient in the pharmaceutical industry. The pH is from 5.5 to 7.5, preferably from 5.5 to 7, more preferably from 6.2 to 6.8.

It has a pH value of 5.5 to 7.5, preferably of 5.5 to 7, preferably of 6.2 to 6.8. In a particular aspect, the pH value is any pH value; for example, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.

[0006] The concentration of a suitable buffer system for 10mM is about 50mM, or from about 10mM to about 40mM. The buffer system is histidine; and histidine is used at a concentration of 10 to 50 mM, preferably from 15 to 40 mM, more preferably from up to 30 mM.

[0007] The preparation further comprises a stabilizing agent. The stabilizing agent of the present invention is mannitol. The mannitol concentration is used in the preparation of the invention is about 300 mM, preferably from 180 to 300 mM, most preferably about 270 Mm of mannitol.

The formulation of the invention may be used as an alternative to the group. The bulking agent is a lyophilized form. Suitable mannitol, glycine, polyethylene glycol and sorbitol. The concentration of 20-90 mM is the invention of a bulking agent.

The use of a surfactant may reduce protein aggregation in the reconstituted formulation. The amount of added surfactant after the reconstruction of the solidarity after the reconstitution. Suitable surfactants of the present invention include polysorbates (eg, polysorbates 20 or 80); poloxamers (eg poloxamer 188); triton; sodium dodecyl sulphate (SDS); sodium lauryl sulfate; octyl sodium glycoside; lauryl, myristyl, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetylbetaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- isostearamidopropyl betaine (eg, lauramidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl dimethylamine; methylcocoyl sodium or disodium methyloleoyltaurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and ethylene and propylene glycol copolymers (eg Pluronics, PF68 etc.). In a preferred embodiment, the surfactant may be selected from the group of polysorbates 20 and polysorbates 80. The concentration of the surfactant in the formulation of the invention is 0.001-0.5%, or from about 0.005-0.10%, preferably 0.01 to 0.10%, most preferably from 0.04 to 0.06% w / v. In the preparation. palmidopropyl- or isostearamidopropyl dimethylamine; methylcocoyl sodium or disodium methyloleoyltaurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and ethylene and propylene glycol copolymers (eg Pluronics, PF68 etc.). In a preferred embodiment, the surfactant may be selected from the group of polysorbates 20 and polysorbates 80. The concentration of the surfactant in the formulation of the invention is 0.001-0.5%, or from about 0.005-0.10%, preferably 0.01 to 0.10%, most preferably from 0.04 to 0.06% w / v. In the preparation. palmidopropyl- or isostearamidopropyl dimethylamine; methylcocoyl sodium or disodium methyloleoyltaurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and ethylene and propylene glycol copolymers (eg Pluronics, PF68 etc.). In a preferred embodiment, the surfactant may be selected from the group of polysorbates 20 and polysorbates 80. The concentration of the surfactant in the formulation is 0.001-0.10%, preferably 0.01 to 0.10%, most preferably from 0.04 to 0.06% w / v. In the preparation. polypropylene glycol; and ethylene and propylene glycol copolymers (eg Pluronics, PF68 etc.). In a preferred embodiment, the surfactant may be selected from the group of polysorbates 20 and polysorbates 80. The concentration of the surfactant in the formulation of the invention is 0.001-0.5%, or from about 0.005-0.10%, preferably 0.01 to 0.10%, most preferably from 0.04 to 0.06% w / v. In the preparation. polypropylene glycol; and ethylene and propylene glycol copolymers (eg Pluronics, PF68 etc.). In a preferred embodiment, the surfactant may be selected from the group of polysorbates 20 and polysorbates 80. The concentration of the surfactant in the formulation is 0.001-0.10%, preferably 0.01 to 0.10%, most preferably from 0.04 to 0.06% w / v. In the preparation. and most preferably from about 0.04 to 0.06% w / v. in the preparation. and most preferably from about 0.04 to 0.06% w / v. in the preparation.

Optionally, preservatives can be used in the formulations of the invention. Benz benz oct benz and benz, benz benz and benz, benz benz and benz, benz benz and benz. Benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. Other pharmaceutically acceptable carriers, excipients or stabilizers, as described in Remington's Science and Practice of Pharmacy 21 edition. (2005) or Art., Science and Technology of Pharamceutical Compounding, 3rd edition (2008) may be included in the formula. Acceptable carriers, excipients, or stabilizers are nontoxic to be added to the dosages, and include additional buffering agents; preservatives;

Co-Solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (eg Zn-protein complexes); biodegradable polymers such as polyesters; and / or salt-forming counterions, such as sodium.

[0009] The present filter provides, in one aspect, an ACE885 IL-ββ and a buffer system in which the pH of the liquid formulation is from 5.5 to 7.5, preferably from 5.5 to 7, more preferably from 6.2 to 6.8 in order to obtain a minimum stability, minimal aggregation and acceptable low viscosity. The translation further comprises mannitol as a stabilizing agent and one or more.

[0010] The present invention provides a pharmaceutical formulation comprising:

a) an ante to a ^ 1, an ACZ885 antibody; used at a concentration of about 10 to 150 mg / ml; and

b) a buffer system that is histidine at a concentration of about 10 to 50 mM; and while the pH of the buffer system is any pH in the range of 5.5 to 7.5, preferably 6.2 to 6.8; and

c) a stabilizing agent that is mannitol at a concentration of about 50 to 300 mM; and eventually

d) additional excipients selected from the group of bulking agent, salt, surfactant and preservative.

[0011] In certain embodiments of the invention, a bulking agent (eg mannitol or glycine) is used to prepare a pre-lyophilization formulation. The filling agent can be a homogeneous lyophilized lump substance.

[0012] A preferred liquid formulation of the present provides a concentration of 10-150 mg / ml, 270 mM mannitol, 20 mM histidine and 0.04% polysorbate 80, the pH of the formulation being 6.5.

[0013] In one embodiment, the preparation may be administered subcutaneously. The formulations of IL-1 mediated diseases or conditions, eg, inflammatory conditions, allergies and conditions, hypersensitivity reactions, autoimmune diseases, and infections of the organ or tissue transplants.

[0014] The object of the present invention is IL-1. For example, the formulations of the invention may be used to treat cardiac, pulmonary, heart and lung, liver, kidney, pancreatic, skin or corneal recipients, including rejection of homologous transplantation or rejection of a xenograft, and for the prevention of graft- versus-host disease, eg bone marrow transplantation and arteriosclerosis associated with organ transplantation.

[0015] The formulation of an autoimmune component, such as arthritis (eg rheumatoid arthritis, chronic progressive arthritis) and diseases; . rheumatic, including inflammation and rheumatic diseases with bone loss, inflammatory pain, hypersensitivity (including both respiratory hypersensitivity and skin hypersensitivity) and allergies. Specific autoimmune diseases, including, for example, haemolytic anemia, aplastic anemia, blood red cell anemia and idiopathic thrombocytopenia,

[0016] The formulations of asthma, bronchitis, pneumoconiosis, emphysema and other obstructive or inflammatory airways diseases.

[0017] The formulations of acute and hyperactive IL-1, in particular, IL-1, in particular IL-1, or the promotion of TNF release by IL-1, eg acute infections, for example, septic shock (eg endotoxic shock and adult respiratory distress syndrome), meningitis, pneumonia and severe burns, and for the treatment of cachexia and cachexia associated with the morbid release of TNF, subsequent infection, cancer or organ dysfunction, especially wasting associated with or associated with HIV infection.

[0018] The formulations of bone fragments, and osteoarthritis, osteoarthritis and other arthritis and, in general, bone loss, including age related bone loss, and more particularly, periodontal disease.

[0019] The formulations of the present syndrome in patients such as mammals, and especially humans. The auto-inflammatory diseases, a group of inherited disorders characterized by recurrent episodes of autoantibodies or antigen-specific T-cells are lacking in contrast to autoimmune diseases. In addition, the autoinflammatory syndromes of the inventions show an increased secretion of IL-1beta (activation of NFkB and apoptosis of non-healed leukocytes). The auto-inflammatory syndromes of the invention are Muckle-Wells (MWS) syndromes, LADA (latent autoimmune diabetes in adults), auto-inflammatory paroxysmal cold (FCAS), congenital cryopyrinopathy (CAPS), neonatal inflammation syndrome (NOMID), chronic infant neurosurgeon syndrome (CINCA), familial Mediterranean fever (FMF) and / or some form of juvenile arthritis, such as systemic juvenile idiopathic arthritis (SJIA), and form of juvenile rheumatoid arthritis such as systemic juvenile idiopathic rheumatoid arthritis and / or some form of adult rheumatoid arthritis. Preferably, the Muckle Wells syndrome. Juvenile rheumatoid arthritis and rheumatoid arthritis in adults and / or Muckle Wells syndrome. Chronic infant neurodegenerative syndrome (CINCA), familial Mediterranean fever (FMF), and forms of juvenile rheumatoid arthritis, a form of juvenile rheumatoid arthritis, such as systemic juvenile idiopathic rheumatoid arthritis and / or some form of adult rheumatoid arthritis. Preferably, the Muckle Wells syndrome. Juvenile rheumatoid arthritis and rheumatoid arthritis in adults and / or Muckle Wells syndrome. Chronic infant neurodegenerative syndrome (CINCA), familial Mediterranean fever (FMF) and / or some form of juvenile arthritis, a form of juvenile rheumatoid arthritis, such as systemic juvenile idiopathic rheumatoid arthritis and / or some form of adult rheumatoid arthritis. Preferably, the Muckle Wells syndrome. Juvenile rheumatoid arthritis and rheumatoid arthritis in adults and / or Muckle Wells syndrome. such as systemic form of juvenile idiopathic arthritis (SJIA), a form of juvenile rheumatoid arthritis such as systemic juvenile idiopathic rheumatoid arthritis and / or some form of adult rheumatoid arthritis. Preferably, the Muckle Wells syndrome. Juvenile rheumatoid arthritis and rheumatoid arthritis in adults and / or Muckle Wells syndrome. such as systemic form of juvenile idiopathic arthritis (SJIA), a form of juvenile rheumatoid arthritis such as systemic juvenile idiopathic rheumatoid arthritis and / or some form of adult rheumatoid arthritis. Preferably, the Muckle Wells syndrome. Juvenile rheumatoid arthritis and rheumatoid arthritis in adults and / or Muckle Wells syndrome.

The IL-1 blockade. The pathologicals of the invention are also useful in the treatment of chronic pathologies associated with diabetes mellitus, as well as retinopathy, wound healing, vascular disease (including restenosis of angioplasty), renal dysfunction, chronic renal failure and metabolic syndrome, and obesity. The formulations of the invention may also be useful in the treatment of migraine, synovitis, gout, arthrosis / gouty arthritis or chondrocalcinosis, chronic obstructive pulmonary disease (COPD), ventilation due to lung injury, various pain states as as morphine-resistant pain, neuropathic pain, pain before the date of birth, discogenic pain, inflammatory pain, headache or migraine. IL-1 beta is involved in the development of neurogenic signals. In addition, the formulations of the invention of atherosclerosis, acute renal colic, biliary colic and pain associated with these disorders. The syndrome of familial Mediterranean fever (FMF) associated with these disorders. Fever syndromes: familial Mediterranean fever (FMF), periodic syndrome associated with tumor necrosis factor receptor, hyperimmunoglobulinemia-D syndrome (HIDS), also referred to as recurrent syndrome biliary colic and pain associated with these disorders. The syndrome of familial Mediterranean fever (FMF), periodic syndrome associated with tumor necrosis factor receptor, hyperimmunoglobulinemia-D syndrome (HIDS), also referred to as recurrent syndromefever associated with mevalonate kinase , familial auto-inflammatory disease and recurrent fever, aphthous stomatitis, pharyngitis, lymphadenitis syndrome (PFAPA), where IL-1 beta is the dominant cytokine. Other diseases in which IL-1beta is the predominant cytokine, which is the invention of anti-synthetase syndrome, MAS macrophage activation syndrome, Behcet's disease, Blau syndrome, PAPA syndrome, Schnizler's syndrome, Sweet's syndrome. IL-1beta-ligand-receptor inhibition and IL-1beta compounds of the invention can also be used in the treatment of vasculitis; giant cell arteritis (GCA), Henoch-Schoenlein purpura, primary systemic vasculitis, Kawasaki disease (mucosal lymph node syndrome), Takayasu arteritis, nodular arteritis, basal cryoglobulinaemia vasculitis, microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS), urticar vasculitis. In addition, the formulations of autoimmune diseases such as sarcoidosis, pemphigus, ankylosing spondylitis, Alzheimer's disease, amyloidosis, secondary amyloidosis and adult onset. Still's disease (AOSD). The formulations of the invention can be used to treat HLA-B27 related to arthritis, ankylosing spondylitis, Morbus Reiter arthritis and enteropathic arthritis. The IL-1beta compounds of the invention can be used to treat rheumatic fever, polymyalgia, and giant cell arteritis.

[0020] For the above indications, on the basis of IL-6, the host, the mode of administration. Frequently Asked Questions (FAQs) The preparation of the patient is in the patient's mind; it may be administered as above.rding to the invention once a month to every 2 to 3 months, or less often. The ACZ885 is preferably administered intravenously, but also via subcutaneous or intramuscular injection. For the purpose, the preparation can be injected using a syringe. For example, a formulation containing ACZ885 is administered by an autovalent injector, a normal syringe that can be pre-filled, optionally in a sterile package, optionally syringes with safety devices. The micro-needle and coated patches with reservoirs are also suitable for administration. [0021] According to known methods, IL-1 antibody, ie ACZ885 is preferably administered to mammals. such as intravenous bolus administration, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. The formulations must be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes, before or after, lyophilization and reconstitution. Alternatively, the sterility can be achieved by autoclaving the ingredients, with 120 ° C for about 30 minutes. This is easily achieved by filtration through sterile filtration membranes, before or after, lyophilization and reconstitution. Alternatively, the sterility of the entire mixture can be achieved by autoclaving the ingredients, with the exception of the antibodies, eg at 120 ° C for about 30 minutes. This is easily achieved by filtration through sterile filtration membranes, before or after, lyophilization and reconstitution. Alternatively, the sterility can be achieved by autoclaving the ingredients, with 120 ° C for about 30 minutes.

The treatment of obstructive pulmonary disease (COPD), CAPS, Muckle-Wells syndrome (RA), juvenile rheumatoid arthritis MWS), osteoarthritis (OA) and promote type 2 diabetes and gout.

[0023] Treatment refers to both treatment and prophylactic or preventive measures. The term 'squeeze' is the term 'animal' for animals, animals, pets, cats, cows, etc. Preferably, the mammal is a human.

A disorder is an IL-1. This includes chronic or acute diseases or diseases, including the pathological conditions. Non-limiting examples of diseases and disorders. The therapeutically effective amount is an induction and improvement of the given disorder.

Legend to figures:

[0024]

Figure 1 provides an overview of the results obtained by RP-HPLC (top) and SEC (bottom) for the ACZ885 preparation after storage for 4 weeks at 40 ° C.

Figure 2 shows the results of ACZ885 formulation after storage for 4 weeks at 40 ° C

EXAMPLES

Preparation of the liquid preparation and its lyophilizate [0025] The ACZ885 preparation is developed, which allows it to be administered intravenously after reconstitution and subsequent dilution as well as subcutaneously after reconstitution. Four different buffer systems (citrate, histidine, sodium succinate and sodium / potassium phosphate buffer, 40 mM each) were selected to check their suitability for ACZ885 preparations.

Shaking, freezing and thawing in cycles have been used as stress tests to evaluate buffer systems with respect to protein aggregation. Protein aggregation can be most effectively avoided by using a histidine or citrate buffer in the pH range 5.0 - 7, as shown in Table 1.

Table 1. Analytical results of a buffer system screening test

Total pH of aggregates

Buffer salt pKa values ​​pH (measured) (planned) measured SEC [%]

<td rowspan="2">histidine</td><td>pKa = 6.1, group</td><td rowspan="2">6.0 6.5</td><td rowspan="2">5.8 6.2</td><td rowspan="2">0.37 0.21</td>

<td>imidazoles</td>

<td></td><td></td><td>7.0</td><td>6.6</td><td>0.38</td>

<td></td><td rowspan="2">pK a1 = 4.19,</td><td>5.0</td><td>5.2</td><td>0.39</td>

<td rowspan="2">succinate</td><td rowspan="2">5.5</td><td rowspan="2">5.6</td><td rowspan="2">0.39</td>

<td rowspan="2">pKa2 = 5.57</td>

<td></td><td>6.0</td><td>6.0</td><td>0.42</td>

<td></td><td></td><td>6.0</td><td>6.2</td><td>0.47</td>

<td>phosphate</td><td>pKa2 = 7.21</td><td>6.5</td><td>6.7</td><td>0.74</td>

<td></td><td></td><td>7.0</td><td>7.0</td><td>3.94</td>

<td>Citrate</td><td rowspan="2">pK a2 = 4.76, pKa3 = 6.40</td><td>5.0 5.5</td><td>5.3 5.7</td><td>0.38 0.40</td>

<td></td><td>6.0</td><td>6.2</td><td>0.39</td>

[0026] The pH was tested in the range from 3.5 to 8.0 with an increase of 0.5 unit. After 4 weeks of storage at 40 ° C various optimal pH from 6.2 to 6.8 were chosen based on the results from the various analytical techniques used, as shown in Fig. 1. Table 2 Analytical results for freeze drying

<td>Code</td><td>Buffer (20mM)</td><td>Addition sucrose</td><td>Viscosity [MPas]</td><td>osmolarity [MOsm]</td><td>Sum aggregates measured SEC [%]</td>

<td>F1</td><td>histidine pH 6.2</td><td>-</td><td>14.8</td><td>382</td><td>5.4</td>

<td>F2</td><td>histidine pH 6.2</td><td>90 mM</td><td>11.0</td><td>392</td><td>0.6</td>

<td>F3</td><td>Citrate pH 6.0</td><td>-</td><td>13.2</td><td>386</td><td>3.9</td>

<td>F4</td><td>Citrate pH 6.0</td><td>90 mM</td><td>11.0</td><td>445</td><td>0.6</td>

[0027] The results for the histidine-citrate-buffered samples were similar. Citrate buffer may be critical in subcutaneous formulations due to increased perception of pain after application; Histidine may therefore be more beneficial than citrate for subcutaneous use. After selection of appropriate buffer systems, the effect of stabilizing agents on protein aggregation was examined. Formulations containing sucrose, mannitol, glycine, sorbitol or trehalose were analyzed after 6 and 16 weeks storage at 5 ° C and 40 ° C. The yellow color was observed in preparations containing sucrose stored at 40 ° C, probably as a result of the Maillard reaction between amino groups of the protein or histidine and reducing sugar. A preparation containing sucrose or mannitol, was chosen for the preparation after lyophilization and reconstituted. Mannitol containing formulations were selected for the liquid preparation. Further studies were conducted to evaluate the effect of surfactant concentration on the physicochemical stability of the formulation of the invention. The following particulate data indicated the highest values ​​for the formulation without surfactant, which means that Tween is beneficial for the physical stability of the samples. At a concentration of 0.10% Tween, the particulate data appear to be higher compared to lower concentrations. The following particulate data indicated the highest values ​​for the formulation without surfactant, which means that Tween is beneficial for the physical stability of the samples. At a concentration of 0.10% Tween, the particulate data appear to be higher compared to lower concentrations. The following particulate data indicated the highest values ​​for the formulation without surfactant, which means that Tween is beneficial for the physical stability of the samples. At a concentration of 0.10% Tween, the particulate data appear to be higher compared to lower concentrations.

Table 3 Analytical results for Tween: subvitoid particles by weakening radiation (solid particles) after 10 months

<td colspan="5">5 ° C</td><td colspan="2">25 ° C</td>

<td>Concentration</td><td>Cząstki /</td><td>Cząstki /</td><td>Cząstki /</td><td>Cząstki /</td><td>Cząstki /</td><td>Cząstki /</td>

<td>Tween</td><td>ml</td><td>ml</td><td>ml</td><td>ml</td><td>ml</td><td>ml</td>

<td></td><td>&gt; 1.0 μm</td><td>&gt; 1.0 μm</td><td>&gt; 10.9 μm</td><td>&gt; 25.7 μm</td><td>&gt; 10.9 μm</td><td>&gt; 25.7 μm</td>

<td colspan="7">Lack</td>

<td>Tween</td><td>6353</td><td>11644</td><td>14</td><td>0</td><td>3</td><td>0</td>

<td>0.01% Tween 80</td><td>862</td><td>2044</td><td>0</td><td>0</td><td>2</td><td>0</td>

<td>0.04% Tween 80</td><td>1217</td><td>2077</td><td>2</td><td>0</td><td>0</td><td>0</td>

<td>0.10% Tween 80</td><td>2180</td><td>2424</td><td>0</td><td>0</td><td>3</td><td>0</td>

<td>0.01% Tween 20</td><td>1349</td><td>2105</td><td>0</td><td>0</td><td>0</td><td>0</td>

<td>0.04% Tween 20</td><td>1410</td><td>1077</td><td>1</td><td>0</td><td>2</td><td>0</td>

<td>0.10% Tween 20</td><td>1071</td><td>2382</td><td>2</td><td>0</td><td>2</td><td>0</td>

[0028] Samples of liquid formulations containing 150 mg / ml ACZ885, 20 mM histidine, 270 mM mannitol, 0.04% (m / v) Tween 80 pH 6.5 were stored at 5 ° C, 25 ° C and 40 ° C. ° C for up to 24 months. At 5 ° C, no larger amounts of both soluble and insoluble aggregates could be detected. Biological activity, determined using the reporter gene assay, as described below, was in the range of 70 - 125%. These data (see tables 4 and 6) showed that the test formulation was stable during storage for 24 months.

Table 4 Analytical data of the screening test, 5 ° C

Periods 16.5

<td>storage and</td><td>Beginning</td><td>2 months</td><td>4 months</td><td>10 months</td><td>month and</td><td>24 months</td>

<td>Measured rating SEC [mg / ml]</td><td>152.2</td><td>155.0</td><td>151.9</td><td>153.0</td><td>153.0</td><td>151.9</td>

<td>Gene evaluation</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>reporter</td><td>121</td><td>nd</td><td>97</td><td>99</td><td>110</td><td>99 (n = 4)</td>

<td>[%]</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Appearance in the solution: color</td><td>colorless</td><td>colorless</td><td>Slightly brown (B8-B9)</td><td>Slightly brown (B8-B9)</td><td>Slightly brown (B8)</td><td>Brown (B5-B6) No visible</td>

particles

turbidity

LLS [Da]

PH value

By-products or degradation measured by SEC [%]

Impurities measured with SDSPAGE

nd

155'750

6.7

AP1: 0.3

0.8

nd

151'800

6.7

AP1: 0.4

0.8

nd

147'400

6.6

AP1: 0.4 DPx: 1.2

0.8

Impurities measured

Bioanalyzer

nd

nd

nd

CEX [%]

nd

nd

nd

nd

142'300

6.6

AP1: 0.6 DPx: not decomposed

Strongly opalescent e (23

NTU)

147'800

6.6

AP1: 0.6 DPx: not decomposed

Iridescent (17 NTU)

155'700

6.6

AP1: 0.8 DPx: 0.2 DP3: 0.2

0.8 0.9 1.1

nd

nd

H1: 0.7

M1: 3.1

M2: 0.3 M3: 0.7

L1: 0.0

L2: 0.3 Total: 5.2

0K: 48.7 1K: 19.6 2K: 15,8

H1: 0.7

M1: 2.1

M2: 0.0 M3: 0.6

L1: 0.0

L2: 0.3 Sum: 3.8

0K: 43.4

1K: 19.1

2K: 10.5

Sum

The sum of others:

other: 27.0

15.9

APx to DP3: from the highest to the lowest molecular weight; AP1: dimer, DPx: P100, DP3: P50

Table 5 Analytical data of the screening test, 25 ° C

periods

<td>storage and</td><td>Beginning</td><td>2 months</td>

<td>Measured rating</td><td rowspan="2">152.2</td><td rowspan="2">146.7</td>

<td>SEC [mg / ml]</td>

16.5

<td>4 months</td><td>10 months</td><td>month and</td><td>24 months</td>

<td>145.8</td><td>149.8</td><td>141.8</td><td>134.2</td>

Gene evaluation

<td>reporter</td><td>121</td><td>nd</td><td>100</td><td>85</td><td>74</td><td>70 (n = 4)</td>

<td colspan="7">[%]</td>

<td>Appearance in the solution: color</td><td>colorless</td><td>colorless</td><td>Slightly brown (B8-B9)</td><td>Slightly brown (B8-B9)</td><td>Slightly brown (B6) Firmly iridescent</td><td>Brown (B5-B6) None visible particles Firmly</td>

<td>turbidity</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>e (22 NTU)</td><td>iridescent (24 NTU)</td>

<td>LLS [Da]</td><td>155'750</td><td>150'250</td><td>152'250</td><td>148'700</td><td>172'250</td><td>189'800</td>

<td>PH value</td><td>6.7</td><td>6.7</td><td>6.6</td><td>6.6</td><td>6.6</td><td>6.6</td>

<td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>APx: 0.4</td>

<td></td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>AP2: 0.2</td>

<td colspan="7">Side products</td>

<td></td><td>AP1: 0.3</td><td>AP1: 0.7</td><td>AP1: 0.9</td><td>AP1: 1.8</td><td>AP1: 2.6</td><td>AP1: 3.7</td>

<td colspan="7">or degradation</td>

<td></td><td>-</td><td>-</td><td>-</td><td>AP: 0.2</td><td>AP: 0.2</td><td>AP: 0.5</td>

<td colspan="7">measured SEC [%]</td>

<td></td><td>-</td><td>-</td><td>DPx: 1.4</td><td>DPx: 3.7</td><td>DPx: 1.7</td><td>DPx: 2.6</td>

<td></td><td>-</td><td>DP3: 0.1</td><td>DP3: 0.2</td><td>DP3: 0.6</td><td>DP3: 1.1</td><td>DP3: 1.5</td>

<td colspan="7">pollution</td>

<td>measured by SDS-</td><td>0.8</td><td>1.5</td><td>1.7</td><td>3.6</td><td>4.2</td><td>6.7</td>

<td colspan="7">PAGE</td>

<td>pollution</td><td></td><td></td><td></td><td></td><td>H1: 1.7 H2: 0.0 H3: 0.0 M1: 4.1 M2: 1.0</td><td>H1: 2.1 H2: 0.4 H3: 0.3 M1: 3.7 M2: 1.1</td>

<td>measured Bioanalyzer</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>M3: 0.7 M4: 0.3 L1: 0.4 L2: 0.8 Sum: 8.9 0K: 36.2</td><td>M3: 0.7 M4: 0.0 L1: 0.6 L2: 0.7 Sum: 9.7 0K: 12.6</td>

<td>CEX [%]</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>1K: 16.6 2K: 11.0</td><td>1K: 6.7 2K: 3.3</td>

Sum The sum of the others: 77.4

36.2

APx to DP3: from the highest to the lowest molecular weight; AP1: dimer, DPx: P100, DP3: P50

Table 6 Analytical data of the screening test, 40 ° C

<td>periods</td><td colspan="2">2</td><td>4</td><td>10</td><td>16.5</td>

<td>storage and</td><td>Beginning</td><td>months</td><td>months</td><td>months</td><td>month and</td>

<td>Measured rating SEC [mg / ml]</td><td>152.2</td><td>142.9</td><td>128.1</td><td>114.1</td><td>94.5</td>

<td>Gene test</td><td></td><td></td><td></td><td></td><td></td>

<td>reporter</td><td>121</td><td>78</td><td>53</td><td>nd</td><td>nd</td>

<td>[%]</td><td></td><td></td><td></td><td></td><td></td>

<td>Appearance in the solution: color</td><td>colorless</td><td>Slightly brown (B7)</td><td>Slightly brown (B6-B7)</td><td>Yellow (G4-G5)</td><td>Brown (B4)</td>

<td></td><td></td><td></td><td></td><td></td><td>Pose</td>

<td>turbidity</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>scale (58</td>

<td></td><td></td><td></td><td></td><td></td><td>NTU)</td>

<td>LLS [Da]</td><td>155'750</td><td>162'950</td><td>192'500</td><td>259'650</td><td>403'350</td>

<td>PH value</td><td>6.7</td><td>6.6</td><td>6.6</td><td>6.6</td><td>6.6</td>

<td></td><td>-</td><td>APx: 0.4</td><td>APx: 1.9</td><td>APx: 4.4</td><td>APx: 6.1</td>

<td>By-products or degradation measured by SEC [%]</td><td>AP1: 0.3</td><td>AP2: 0.1 AP1: 2.8 AP: 0.2</td><td>AP2: 0.3 AP1: 5.0 AP: 0.8 DPx: 5.5</td><td>AP2: 1.4 AP1: 10.2 AP: 2.2 DPx: 11.7</td><td>AP2: 2.1 AP1: 15.0 AP: 3.3 DPx: 12.2</td>

<td></td><td>-</td><td>DP3: 0.9</td><td>DP3: 1.9</td><td>DP3: 4.4</td><td>DP3: 8.6</td>

<td>pollution</td><td></td><td></td><td></td><td></td><td></td>

<td>measured by SDS-</td><td>0.8</td><td>5.8</td><td>6.7</td><td>12.5</td><td>13.3</td>

<td>PAGE</td><td></td><td></td><td></td><td></td><td>Look</td>

<td>CEX [%]</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>chromatogram</td>

<td></td><td></td><td></td><td></td><td></td><td>gram</td>

APx to DP3: from the highest to the lowest molecular weight; AP1: dimer, DPx: P100, DP3: P50

Reconstituted lyophilised preparation [0029] Liquid formulations for ACZ885 according to the invention are suitable for lyophilization. Lyophilization can be carried out under normal conditions well known in the pharmaceutical field. The bulking agent may be comprising adding weight and visibility to a lyophilisate, such as glycine. After the antibody, buffer (from 10 to 40 mM), stabilizing agent and surfactant are mixed together, the formulation is lyophilised.

Table 7 Preparations for technical stability (before freeze-drying)

<td>Code</td><td>Saccharose [MM]</td><td>histidine pH 6.0-6.2 [MM]</td><td>Tween 80 [%]</td>

<td>F1</td><td>60</td><td>10</td><td>0.02</td>

<td>F3</td><td>90</td><td>10</td><td>0.02</td>

[0030] The reproduction is usually carried out at a temperature of 15-25 ° C to ensure complete hydration. The lyophilisate is reconstituted in sterile water.

[0031] The target concentration after reconstitution is 150 mg / ml, each formulation being lyophilized and reconstituted in water.

Stability [0032] Various formulations are stored for three months at 2-8 ° C, 25 ° C and 40 ° C.

Aggregations after lyophilization and storage are used as an indicator of protein stability.

Reporter Gene Assay [0033] The biological activity of ACZ885 was measured using a receptor gene assay using a genetically modified cell line. This cell line was obtained from human embryonic kidney cells and stably transfected with a reporter construct in which the NF-kappa b promoter (responsive IL-1 promoter) was linked upstream of the luciferase gene. Transfection was performed by the simultaneous introduction of the neomycin resistance gene. In this cell line, exposure to IL-β stimulated the expression of luciferase in a dose-dependent manner. Adding gradual amounts of ACZ885 to a fixed submaximal dose

IL-β caused a decrease in luciferase expression during the incubation period of up to 18 hours. At the end of the incubation period, the amount of luciferase was quantified based on its enzymatic activity in the cell lysate. Luciferase catalyzed the transformation of luciferin substrate into oxylucyferin, a chemiluminescent product. The chemiluminescence type obtained was then determined using a suitable luminometer.

The biological potency of the ACZ885 test sample was determined by comparing its ability to inhibit IL-1 dependent induction of luciferase activity with the ACZ885 reference standard.

Samples and standard were normalized based on the protein content. Relative potency was calculated using a parallel line test according to the European Pharmacopoeia. The final result was expressed as the relative potency (in percent) of the sample compared to the reference standard.

Reagents and buffers [0034]

- Basal cell culture medium MEM + Earle + L-glutamine;

- Fetal calf serum (FCS) heat inactivated, tested mycoplasma;

- Geneticin;

- enzyme-free cell dissociation buffer based on PBS;

- Basic medium for OptiMEM-I + GlutaMAX-I tests;

- The luciferase substrate for chemiluminescent illumination;

- Recombinant interleukin-1 beta (IL-1β).

[0035] (1) Different concentrations of reference standard and test samples were prepared by performing several 1: 2 dilutions of the initial solution of 400 ng / ml ACZ885;

(2) 2x10⁴ the cells resuspended in the assay medium were applied to a 96-well microtiter plate (3). The test was started by adding IL-1beta solution. Incubation in a humid CO2 incubator lasted up to 18 h;

(4) After incubation, the luciferase substrate solution was added to all wells. The plate was then incubated in the dark for 10 minutes and the luminescence of each well was determined in a suitable luminescence microtiter plate reader;

(6) The unweighted average of the relative potency of the sample was calculated using a parallel line estimate of EP from at least two independent experiments.

Table 8 Analytical results for technical stability

<td rowspan="3">Code</td><td colspan="2" rowspan="2">Time</td><td rowspan="3">pH</td><td rowspan="3">Test measured to^sUV [Mg / ml]</td><td rowspan="3">Protein / izoquant iso-asp [%]</td><td rowspan="3">Mass molecular kowa measured to and LLS [KDa]</td><td colspan="3">Sum</td>

<td rowspan="2">aggregate that measured to and SEC [%]</td><td rowspan="2">Test measured to y SEC [Mg / ml]</td><td rowspan="2">Biotest [%]</td>

<td>Playback ania</td><td>Opalizac I</td>

<td>F1 5 ° C (AND)</td><td>4 min 45s</td><td>lack</td><td>6.2</td><td>171.8</td><td>3.8</td><td>149.2</td><td>1.0</td><td>164.8</td><td>103</td>

<td>5 ° C (II)</td><td>4 min 30s</td><td>lack</td><td>6.2</td><td>172.5</td><td>3.9</td><td>149.4</td><td>1.0</td><td>167.7</td><td></td>

<td>25 ° C (AND)</td><td>4 min 45s</td><td>lack</td><td>6.2</td><td>171.9</td><td>5.6</td><td>152.7</td><td>2.7</td><td>163.6</td><td>99</td>

<td>25 ° C (II)</td><td>4 min 45s</td><td>lack</td><td>6.2</td><td>168.4</td><td>6.3</td><td>153.3</td><td>2.7</td><td>160.0</td><td></td>

<td>40 ° C (AND)</td><td>4 min 30s</td><td>lack</td><td>6.2</td><td>172.5</td><td>9.5</td><td>161.6</td><td>7.6</td><td>152.7</td><td>93</td>

<td>40 ° C (II) *</td><td>&gt; 12 min</td><td>lack</td><td>6.2</td><td>166.2</td><td>10.3</td><td>162.9</td><td>7.9</td><td>153.0</td><td></td>

<td>F3 5 ° C (AND)</td><td>4 min 00s</td><td>lack</td><td>6.2</td><td>167.4</td><td>3.4</td><td>149.6</td><td>0.8</td><td>163.1</td><td>101</td>

<td>5 ° C (II)</td><td>4 min 00s</td><td>lack</td><td>6.2</td><td>162.8</td><td>3.1</td><td>149.1</td><td>0.8</td><td>163.0</td><td></td>

<td>25 ° C (AND)</td><td>4 min 30s</td><td>lack</td><td>6.2</td><td>159.4</td><td>4.9</td><td>151.4</td><td>1.7</td><td>158.2</td><td>91</td>

<td>25 ° C (II)</td><td>4 min 30s</td><td>lack</td><td>6.2</td><td>155.1</td><td>4.7</td><td>152.3</td><td>1.6</td><td>161.7</td><td></td>

<td>40 ° C (AND)</td><td>4 min 45s</td><td>lack</td><td>6.2</td><td>164.4</td><td>6.6</td><td>156.6</td><td>4.5</td><td>154.9</td><td>95</td>

<td>40 ° C (II) *</td><td>4 min 45s</td><td>lack</td><td>6.2</td><td>166.01</td><td>6.1</td><td>155.7</td><td>4.5</td><td>156.8</td><td></td>

* part of the lumpy substance adhered to the bottom [0036] Preparation 3 showed the lowest level of aggregation. Formulations 1 and 3 showed biological activity after storage of about 90-105% of primary activity.

Administration of the preparation [0037] The appropriate dosage (e.g. of a therapeutically effective amount) of ACZ885 depends, for example, on the condition to be treated, the severity and course of the disease, whether ACZ885 is administered for preventive or curative purposes, prior treatment, history clinical trial and respond to ACZ885 and the attending physician.

ACZ885 heavy chain variable region Seq. Id. No. 1

<img file="PL2376533T3_D0001.tif" />

Light chain ACZ885 variable region Seq. Id. No. 2

<img file="PL2376533T3_D0002.tif" />

DEDICATED SEQUENCE [0038] <110> Novartis AG <120> IL-1b antibody preparation <130> 53159 <160> 4 <170> PatentIn version 3.3.

<210> 1 <211> 117 <212> PRT <213> Man

<td colspan="12">&lt;400&gt; 1</td><td rowspan="2">Leu</td><td rowspan="2">Leu</td><td rowspan="2">Arg 15</td><td rowspan="2">Gly</td>

<td>Underworld 1</td><td colspan="4">Glu Phe Gly Leu 5</td><td>Cheese</td><td>Trp</td><td>val</td><td>phe</td><td>Leu 10</td><td>val</td><td>ala</td>

<td>val</td><td>Gin</td><td>Cys</td><td>Gin</td><td>val</td><td>Gin</td><td>Leu</td><td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly</td><td>V 1 *</td><td>val</td><td>Gin</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Pro</td><td>Gly</td><td>Arg</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>ala</td><td>ala</td><td>Cheese</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Cheese</td><td>V &lt;1</td><td>Tyr</td><td>Gly</td><td>Underworld</td><td>own</td><td>Trp</td><td>val</td><td>Arg</td><td>Gin</td><td>ala</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>Trp</td><td>val</td><td>ala</td><td>How much</td><td>lle</td><td>Trp</td><td>Tyr</td><td>Asp</td><td>Gly</td><td>Asp</td><td>own</td><td>Gin</td><td>Tyr</td><td>Tyr</td><td>ala</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>Asp</td><td>Cheese</td><td>val</td><td>lys</td><td>Gly</td><td>Arg</td><td>phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>own</td><td>Cheese</td><td>lys</td><td>own</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>Thr</td><td>Leu</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Gly</td><td>Pro</td><td>phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>tht</td><td>Leu</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 <210> 2 10 <211> 126 <213> PRT <213> Man <400> 2

Met Leu Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 15 10 15

Cheese Arg Gly Glu Ile Val Leu Thr Gin Cheese Pro Asp Phe Gin Cheese Val

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Thr</td><td>Pro</td><td>lys</td><td>Glu</td><td>lys</td><td>val</td><td>Thr</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>How much</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>His</td><td>Trp</td><td>lyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Asp</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>lys</td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>How much</td><td>lys</td><td>Tyr</td><td>Alu</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>phe</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td><td>Cheese</td><td>Arg</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>15</td><td></td><td></td><td></td><td></td><td>80</td>

<td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>own</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>Leu</td><td>Glu</td><td>ala</td><td>Glu</td><td>Asp</td><td>ala</td><td>ala</td><td>ala</td><td>Tyr</td><td>Tyr</td><td>Cya</td><td>His</td><td>Gin</td><td>Cheese</td><td>Cheese</td><td>set</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td>Leu</td><td>Pro</td><td>phe</td><td>Thr</td><td>phe</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Thr</td><td>lys</td><td>val</td><td>Asp</td><td>He</td><td>Lya</td><td></td><td></td>

<td></td><td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td></td><td>125</td><td></td><td></td><td></td>

<210> 3 <211> 410 <212> DNA <213> Human <400> 3

<td>atggagtttg</td><td>ggctgagctg</td><td>ggttttcctc</td><td>gttgctcttt</td><td>taagaggtgt</td><td>ccagtgtcag</td><td>60</td>

<td>gtgcagctgg</td><td>tggagtctgg</td><td>gggaggcgtg</td><td>gtccagcctg</td><td>ggaggtccct</td><td>gagactctcc</td><td>120</td>

<td>tgtgcagcgt</td><td>ctggattcac</td><td>cttcagtgtt</td><td>tatggcatga</td><td>actgggtccg</td><td>ccaggctcca</td><td>180</td>

<td>ggcaaggggc</td><td>tggagtgggt</td><td>ggcaabtatt</td><td>tggtatgatg</td><td>gagataatca</td><td>atactatgca</td><td>240</td>

<td>gactccgtga</td><td>agggccgatt</td><td>caccatctcc</td><td>agagacaatt</td><td>ccaagaacac</td><td>gctgtatctg</td><td>300</td>

<td>caaatgaaeg</td><td>gcctgagagc</td><td>cgaggacacg</td><td>gctgtgtatt</td><td>attgtgcgag</td><td>agatcttagg</td><td>360</td>

<td>actgggcctt</td><td>ttgactactg</td><td>gggccaggga</td><td>accctggtca</td><td>ccgtctcctc</td><td></td><td>410</td>

<210> 4 10 <211> 378 <212> DNA <213> Human <400> 4

<td>&gt; And gttgęc% &amp; t</td><td>[^ Agaagtgat</td><td>tgggLhtcLg</td><td>:: l gi: l.ętgg j</td><td>fc. tc '~ ag ct tt</td><td>gagęygLęaa</td><td>40</td>

<td>aitgtgctgi</td><td>cŁcagtctcc</td><td>agacŁttcag</td><td>ŁctgŁgactc</td><td>caaaggagaa</td><td>agtcaocatc</td><td>120</td>

<td>acctgccg-hh</td><td>ccagzcagag</td><td>CATT &lt;jgtfigt</td><td>agcttacact</td><td>ggtaccagca</td><td>gaaaocagat</td><td>iao</td>

<td>csgtctccM</td><td>agctcgtcat</td><td>oaegtatgct</td><td>tcccagtcct</td><td>tctcaggggz</td><td>cccctcgigg</td><td>J40</td>

<td>ttcflgtggca</td><td>gtggetctgg</td><td>gacngatttc</td><td>accctcacca</td><td>tcafttagcct</td><td>ggnłgctgafl</td><td>300</td>

<td>ęatgctgcag</td><td>cgtrtt tactg</td><td>t.-ar n &lt;gagr</td><td>ng f, ig r t. 7..1C</td><td>ęattcacttt</td><td>cggccrt.ggg</td><td>5 * 0</td>

<td>rtczr ^ .aaghgg</td><td>/ 17. Al-tZAAri</td><td></td><td></td><td></td><td></td><td>37th</td>

Novartis AG, Switzerland Plenipotentiary:

EP 2376533B1

Z-14433/16