TWI690329B - Stabilized formulations containing anti-interleukin-4 receptor (il-4r) antibodies - Google Patents

Abstract

The present invention provides pharmaceutical formulations comprising a human antibody that specifically binds to human interleukin-4 receptor (hIL-4R). The formulations may contain, in addition to an anti-hIL-4R antibody, at least one amino acid, at least one sugar, or at least one non-ionic surfactant. The pharmaceutical formulations of the present invention exhibit a substantial degree of antibody stability after storage for several months.

Description

Stabilized formulation containing anti-interleukin-4 receptor (IL-4R) antibody

The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations containing human antibodies that specifically bind to human interleukin-4 receptor.

Sequence Listing

This patent specification is also accompanied by a ST.25 matching text file of the sequence listing. The text file is incorporated here by reference. Incorporate the same sequence listing paper as the ST.25 text file as part of this patent specification.

Therapeutic macromolecules (such as antibodies) must be formulated not only to make the molecule suitable for patient administration, but also to maintain stability during storage and subsequent use. For example, unless the solution is properly formulated, the therapeutic antibody in the liquid solution is susceptible to degradation, aggregation, or poor chemical modification. The stability of antibodies in liquid formulations depends not only on the type of excipients used in the formulation, but also on the relative number and ratio of the excipients. In addition, when preparing a liquid antibody formulation, in addition to stability, other factors must be considered. Examples of such additional considerations include the viscosity of the solution and the antibody concentration that can accommodate a known formulation, and the visual quality or appeal of the formulation. Therefore, when formulating a therapeutic antibody, care must be taken to maintain a formulation that is stable, contains appropriate antibodies, and has appropriate viscosity and other properties that can conveniently administer the formulation to patients.

Antibodies to human interleukin-4 receptor alpha (hIL-4Rα) are an example of therapeutically relevant macromolecules that require proper formulation. Anti-hIL-4Rα antibodies are used clinically to treat or prevent diseases such as atopic dermatitis and allergic asthma, as well as other conditions. Examples of anti-hIL-4Rα antibodies have been specifically described as U.S. Patent Nos. 7,605,237, 7,608,693, 7,465,450 and 7,186,809; and U.S. Patent Application Nos. 2010-0047254 and 2010-0021476.

Although anti-hIL-4Rα antibodies are well known, there is still a need in the art for a novel pharmaceutical formulation containing an anti-hIL-4Rα antibody that is sufficiently stable and suitable for administration to patients.

Summary of invention

The present invention meets the above needs by providing a pharmaceutical formulation containing a human antibody that can specifically bind to human interleukin-4 receptor α (hIL-4Rα).

In one aspect, a liquid pharmaceutical formulation is provided, comprising: (i) a human antibody that specifically binds to human interleukin-4 receptor alpha (hIL-4Rα); (ii) a buffer solution; ( iii) an organic co-solvent; (iv) a thermal stabilizer; and (v) a debonding agent.

In a specific embodiment, the concentration of the antibody is about 150 mg/ml ± 50 mg/ml. In another specific embodiment, the concentration of the antibody is about 150 mg/ml ± 15 mg/ml. In a specific embodiment, the concentration of the antibody is about 150 mg/ml.

In a specific embodiment, the antibody comprises any one or more amino acid sequences with sequence identification numbers: 1-8. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) containing sequence identification number: 2, sequence identification number: 3 and sequence identification number: 4 respectively The heavy chain variable region (HCVR); and (b) contain the sequence identification number: 6, sequence identification number: 7 and sequence identification number: 8 respectively, the light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3 ) Of a light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR containing amino acid sequences of sequence identification number: 1 and sequence identification number: 5, respectively.

In a specific embodiment, the antibody comprises any one or more amino acid sequences with sequence identification numbers: 9-16. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) containing sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 The heavy chain variable region (HCVR); and (b) contain the sequence identification number: 14, sequence identification number: 15 and sequence identification number: 16 respectively, the light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3 ) Of a light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR containing amino acid sequences of sequence identification number: 9 and sequence identification number: 13, respectively.

In a specific embodiment, the antibody comprises any one or more amino acid sequences with sequence identification numbers: 17-24. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) containing sequence identification number: 18, sequence identification number: 19 and sequence identification number: 20, respectively The heavy chain variable region (HCVR); and (b) the light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3) containing sequence identification number: 22, sequence identification number: 23 and sequence identification number: 24, respectively ) Of a light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR containing the amino acid sequence of sequence identification number: 17 and sequence identification number: 21, respectively.

In a specific embodiment, the pH of the liquid formulation is about 5.9±0.5. In a particular embodiment, the pH of the liquid formulation is about 5.9±0.1. In a specific embodiment, the liquid pharmaceutical buffer contains one or more buffers that can buffer from about pH 5.6 to about pH 6.2.

In a specific embodiment, the liquid pharmaceutical formulation comprises a buffer system containing at least two buffers. In a specific embodiment, the buffer system includes a first buffer solution having an effective pH range of 3.6 to 5.6 and a second buffer solution having an effective pH range of 5.5 to 7.4. In a specific embodiment, the first buffer has a pKa of about 4.8±0.3 and the second buffer has a pKa of about 6.0±0.3. In a specific embodiment, the first buffer is acetate buffer and the second buffer is histidine buffer. In a specific embodiment, the acetate concentration is 12.5±1.9 mM and the histidine concentration is 20±3 mM.

In a specific embodiment, the organic co-solvent is a nonionic polymer containing polyoxyethylene groups. In some specific embodiments, the organic co-solvent is any one or more of polysorbate 20, poloxamer 181, and polyethylene glycol 3350. In a specific embodiment, the organic co-solvent is polysorbate 20.

In a specific embodiment, the concentration of the organic co-solvent is from about 0.2%±0.03% to about 1%±0.15% "weight volume" or "w/v", where for example 0.1g/ml=10% and 0.01 g/ml=1%). In a specific embodiment, the concentration of the organic co-solvent is about 0.2% ± 0.03% w/v polysorbate 20.

In a specific embodiment, the heat stabilizer is a carbohydrate. In a specific embodiment, the sugar is selected from the group consisting of sucrose, mannose and trehalose. In a specific embodiment, the heat stabilizer is sucrose.

In a specific embodiment, the concentration of the thermal stabilizer is from about 0.9%±0.135% W/V to about 10%±1.5% w/v. In a particular embodiment, the heat stabilizer is sucrose at a concentration of about 5%±0.75% w/v.

In a specific embodiment, the detackifier is selected from the group consisting of spermine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, and sodium acetate Group of salt. In a specific embodiment, the debonding agent is L-spermine hydrochloride.

In a specific embodiment, the concentration of the debonding agent is less than about 100 mM. In a specific embodiment, the concentration of the debonding agent is 50 mM ± 7.5 mM. In another specific embodiment, the concentration of the debonding agent is 25 mM ± 3.75 mM. In a specific embodiment, the detackifier is L-spermine hydrochloride at a concentration of 25 mM ± 3.75 mM.

In a specific embodiment, the viscosity of the liquid pharmaceutical formulation is less than or equal to about 35±3.5 centipoise (cPoise). In a specific embodiment, the viscosity is about 21.5±13.5 centipoise, about 11±1.1 centipoise, or about 8.5±0.85 centipoise. In a specific embodiment, the viscosity of the liquid pharmaceutical formulation is about 8.5±0.85 centipoise.

In a specific embodiment, the liquid pharmaceutical formulation has a molar osmolarity of less than about 450 mOsm/kg. In a specific embodiment, the liquid pharmaceutical formulation has a molar osmolarity of about 290±20 mOsm/kg.

In a specific embodiment, when the liquid pharmaceutical formulation is stored at 5°C for six months when measured by size exclusion chromatography, at least 90% or at least 95% of the natural anti-hIL can be harvested from the liquid pharmaceutical formulation -4Rα antibody. In a specific embodiment, at least 98% of the natural antibody can be recovered from the liquid pharmaceutical formulation after six months of storage at 5°C when measured by size exclusion chromatography.

In a specific embodiment, at least 90% of the natural antibody can be recovered from the liquid pharmaceutical formulation after eight weeks of storage at 45°C when measured by size exclusion chromatography.

In a specific embodiment, less than 45% of the acid form antibody can be recovered from the liquid pharmaceutical formulation after eight weeks of storage at 45°C when measured by cation exchange chromatography.

In a specific embodiment, the antibody that can be recovered from the liquid pharmaceutical formulation after being stored at 25°C for six months when measured by size exclusion exchange chromatography is less than about 4% aggregated.

In one aspect, a liquid pharmaceutical formulation is provided, comprising: (i) about 150 mg/ml ± 50 mg/ml of a human antibody that specifically binds hIL-4Rα, wherein the antibody contains sequence identification numbers: 1 and Sequence identification number: 5 amino acid sequence heavy chain variable region (HCVR) and light chain variable region (LCVR); (ii) about 12.5mg/ml ± 2mM acetate; (iii) about 20mM ± 3mM Histidine; (iv) about 5% ± 0.75% (w/v) sucrose; (v) about 0.2% ± 0.03% (w/v) polysorbate 20; and (vi) about Approximately 25 mM ± 3.75 mM arginine at a pH of 5.9 ± 0.5.

In a specific embodiment, the liquid pharmaceutical formulation has a viscosity from about 8.5±0.85 centipoise to about 11±1.1 centipoise. In a specific embodiment, the viscosity of the liquid pharmaceutical formulation is about 8.5±0.85 centipoise.

In a specific embodiment, the liquid pharmaceutical formulation is physiologically isotonic. In a specific embodiment, the liquid pharmaceutical formulation has a molar osmolarity of about 290±20 mOsm/kg.

In a specific embodiment, at least 98% of the natural anti-hIL-4Rα antibody can be recovered from the liquid pharmaceutical formulation after six months of storage at 5°C when measured by size exclusion chromatography.

In a specific embodiment, at least 90% of the natural anti-hIL-4Rα antibody can be recovered from the liquid pharmaceutical formulation after eight weeks of storage at 45°C when measured by size exclusion chromatography.

In a specific embodiment, less than 45% of the antibody in acid form can be recovered from the liquid pharmaceutical formulation after eight weeks of storage at 45°C when measured by cation exchange chromatography.

In a specific embodiment, the antibody that can be recovered from the liquid pharmaceutical formulation aggregates less than about 4% after being stored at 25°C for six months when measured by size exclusion exchange chromatography.

In one aspect, a stable low viscosity isotonic liquid pharmaceutical formulation containing at least 100 mg/ml diazepam anti-hIL-4Rα antibody is provided. In a specific embodiment, the concentration of the antibody is about 150 mg/ml ± 50 mg/ml. In a specific embodiment, the antibody concentration is about 150 mg/ml ± 15 mg/ml.

In a specific embodiment, the antibody comprises any one or more amino acid sequences with sequence identification numbers: 1-8. In a specific embodiment, the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR/LCVR combination includes sequence identification numbers: 2-3-4 and sequence identification, respectively Number: 6-7-8 amino acid sequence heavy chain and light chain complementarity determining region (HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3). In a specific embodiment, an HCVR and an LCVR of the anti-hIL-4Rα antibody.

In some embodiments, the viscosity of the formulation is less than 35±3.5 centipoise, less than 20±2 centipoise, less than 15±1.5 centipoise, or less than 10±1 centipoise. In a specific embodiment, the liquid formulation has a viscosity of about 8.5±2.5 centipoise.

In a specific embodiment, the formulation has a physiologically compatible molar osmolarity. In a specific embodiment, the formulation contains a molar osmolarity of 290±20 mOsm/kg.

In a specific embodiment, the antibody has a stability of at least about six months at about 5°C. In a specific embodiment, at least about 98% of the antibody retains its natural configuration when stored at 5°C for about six months when measured by size exclusion chromatography.

In a specific embodiment, the antibody has a storage stability of at least about eight weeks at about 45°C. In a specific embodiment, at least about 90% of the antibody retains its natural configuration when stored at 45°C for about eight weeks when measured by size exclusion chromatography. In a specific embodiment, less than about 45% of the antibody is in acid form when stored at 45°C for about eight weeks when measured by cation exchange chromatography.

In a specific embodiment, the antibody has a storage stability of at least about six months at about 25°C. In a particular embodiment, the antibody is less than about 4% aggregated when stored at 25°C for about six months when measured by size exclusion chromatography.

In a specific embodiment, the formulation includes a buffer having a pH of about 5.9±0.5. In a specific embodiment, the buffer solution includes an acetate buffer solution and a group of amino acid buffer solutions. In a specific embodiment, the concentration of the acetate is 12.5 mM ± 1.9 mM and the concentration of the histidine is 20 mM ± 3 mM.

In a specific embodiment, the formulation includes an organic co-solvent at a concentration of from about 0.2%±0.03% to about 1%±0.15% w/v. In a specific embodiment, the organic co-solvent is a non-ionic polymer containing polyoxyethylene groups. In some embodiments, the organic co-solvent is any one or more of polysorbate 20, poloxamer 181, and polyethylene glycol 3350. In a specific embodiment, the organic co-solvent is polysorbate 20 at a concentration of about 0.2%±0.03% w/v.

In a specific embodiment, the formulation contains a heat stabilizer at a concentration of from about .0.9% ± 0.135% w/v to about 10% ± 1.5% w/v. In a specific embodiment, the heat stabilizer is sucrose. In a specific embodiment, the sugar system is selected from the group consisting of sucrose, mannose and trehalose. In a specific embodiment, the heat stabilizer is sucrose at a concentration of about 5%±0.75% w/v.

In a specific embodiment, the formulation includes a debonding agent at a concentration not exceeding about 100 mM. In a specific embodiment, the debonding agent is arginine. In a specific embodiment, the detackifier is L-spermine hydrochloride at a concentration of 25 mM ± 3.75 mM.

In a specific embodiment, the stable low viscosity isotonic liquid pharmaceutical formulation has a viscosity of about 8.5 ± 2.5 centipoise and a molar osmolarity of about 290 ± 20 mOsm/kg, and includes: (i) 150 mg/ml ±15mg/ml anti-hIL-4Rα antibody, wherein the antibody contains one HCVR and one LCVR containing the amino acid sequence of sequence identification number: 1 and sequence identification number: 5 respectively; (ii) 12.5mM±1.9mM acetate Salt; (iii) 20 mg/ml ± 3 mM histidine; (iv) 0.2% ± 0.03% w/v polysorbate 20; (v) 5% ± 0.75% w/v sucrose; and ( vi) 25 mM ± 3.75 mM L-spermine hydrochloride. According to this specific embodiment, (i) when measured by size exclusion chromatography, stored at 5°C for at least about six months, at least about 98% of the antibody retains its natural configuration; (ii) when by size exclusion When stored at 45°C for at least about eight weeks when measured by chromatography, at least about 90% of the antibody retains its natural configuration; (iii) when measured by cation exchange chromatography at 45°C for eight weeks, less than about 45% Acid form antibody; and (iv) When stored at 25°C for about six months when tested by size exclusion chromatography, the antibody aggregated less than about 4%.

In one aspect, a liquid pharmaceutical formulation of any of the above aspects in a container is provided. In a specific embodiment, the container is a glass bottle. In another specific embodiment, the container is a microsyringe. In another specific embodiment, the container is a syringe. In a particular embodiment, the syringe contains a fluorocarbon coated piston. In a specific embodiment, the syringe is a low-tungsten syringe.

Other specific embodiments of the present invention can be better understood from the summary detailed later.

Detailed description of the invention

Before describing the present invention, it should be understood that the present invention is not limited to the specific specific embodiments and experimental conditions, and therefore different methods and conditions are possible.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "approximately" when used in connection with a particular stated value or range of values means that the value does not exceed 1% of the stated value. For example, the expression "about 100" described here includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any similar or identical methods and materials described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described in detail below. The entire disclosure mentioned herein is hereby incorporated by reference to fully describe the present invention.

Pharmaceutical formulations

The statement of "pharmaceutical formulation" herein means a combination of at least one active ingredient (eg, small molecules, macromolecules, compounds, etc. that can exhibit biological effects in human or non-human animals) and at least one inactive ingredient, which is When the active ingredient is mixed with one or more additional inactive ingredients, it can be administered to a human or non-human animal therapeutically. Unless otherwise stated, the term "formulation" here means "pharmaceutical formulation". The present invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to some specific embodiments of the present invention, the therapeutic polypeptide can specifically bind to an antibody or antigen-binding fragment thereof of human interleukin-4 antibody α (hIL-4Rα). More specifically, the invention includes (i) a human antibody that specifically binds hIL-4Rα; (ii) an acetate/histidine buffer system; (iii) an organic aid of a nonionic surfactant Solvent; (iv) a heat stabilizer for carbohydrates; and (v) a pharmaceutical formulation of a detackifier. The specific exemplary ingredients and formulations contained in the present invention are described in detail below.

Antibodies that specifically bind to hIL-4R

The pharmaceutical formulation of the present invention comprises a human antibody or antigen-binding fragment thereof that specifically binds to hIL-4Rα. The term "hIL-4Rα" as used herein means a human cytokinin receptor that specifically binds to interleukin-4 (IL-4). In certain embodiments, the antibody contained in the pharmaceutical formulation of the present invention specifically binds to the extracellular region of hIL-4Rα. An exemplary human IL-4 receptor alpha (hIL-4Rα) amino acid sequence is described in sequence identification number: 25. Antibodies to hIL-4Rα are described in US Patent Nos. 7,605,237 and 7,608,693. The extracellular region of hIL-4Rα is represented by the amino acid sequence of SEQ ID NO: 26.

The term "antibody" as used herein generally means an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide chains, and multimers thereof (Eg IgM); however, immunoglobulin molecules composed only of heavy chains (ie without light chains) also fall within the definition of "antibody". Each heavy chain includes a heavy chain variable region (herein referred to simply as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V _L) and a light chain constant region. The light chain constant region contains a domain (CL1). The V _H and V _L can be further subdivided into more hyper-conserved regions called scaffold regions (FR), known as complementarity-determining regions (CDRs). FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 configuration consisting of: each of V _H and V _L, is composed of three CDRs and four FRs in the following order from the amino terminal to carboxy-terminus.

Unless expressly stated otherwise, the term "antibody" as used herein should be considered to include complete antibody molecules as well as antigen-binding fragments thereof. The "antigen-binding region" or "antigen-binding fragment" (or simply "antibody region" or "antibody fragment") of the antibody herein means the ability to retain specific binding to hIL-4Rα or its epitope One or more fragments of the antibody.

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that can specifically bind to hIL-4Rα, which has substantially no specific binding except for hIL-4Rα Antibody to the antigen).

The terms "specifically bind" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with a antigen that is relatively stable under physiological conditions. The specific binding is characterized by having a dissociation constant of at least about 1×10 ⁻⁶ M or higher. Methods for determining whether two molecules specifically bind are known in the art and include, for example, dialysis equilibrium method, surface plasmon resonance method, and the like. However, an isolated antibody that specifically binds hIL-4Rα is cross-reactive with other antigens, such as IL-4R (homologous genes) from other strains. In the context of the present invention, a multispecific (bispecific) antibody that binds hIL-4Rα and one or more additional antigens are considered to “specifically bind” hIL-4Rα. In addition, an isolated antibody may be substantially free of other cellular materials or chemicals.

Exemplary anti-hIL-4Rα antibodies incorporated into the pharmaceutical formulations of the present invention are described in US 7,605,237 and US 7,608,693, which are incorporated herein by reference.

According to certain embodiments of the present invention, the anti-hIL-4Rα anti-system comprises a heavy chain variable region of one of IGHV 3-9 subtypes and a human IgG1 of a light chain variable region of one of IGKV 2-28 subtypes (please See Barbie and Lefranc, Human Immunoglobulin κ Variable Region (IGKV) Gene and Junction (IGKJ) Fragments, Exp. Clin. Immunogenet. 1998, 15: 171-183; and Scavner, D. et al., Human Immunoglobulin Protein presentation of heavy chain, kappa and lambda variable regions and junction regions, Exp. Clin. Immunogenet. 1999, 16:234-240).

In some embodiments, the anti-hIL-4Rα contains at least one amino acid substitution that causes a change in the charge of the antibody relative to the exposed surface of the germline IGHV 3-9 sequence or germline IGKV 2-28 sequence. The germline IGHV 3~9 and germline IGKV 2~28 sequences shown here, as well as the amino acid position designation number and the International Immunogenetics (IMGT) information system have been described in Lefranc, M.-P. et al. , IMGT ^® , International Immunogenetics Information System ^® , Nucl. Acids Res. , 37, D1006~D1012 (2009). In some embodiments, the exposed surface includes a complementarity determining region (CDR). In some embodiments, one or more substitutions of the amino acid are selected from (a) a basic amino acid to replace a natural amino acid in CDR2 (eg, at position 58) of IGHV 3-9; b) A natural amino acid substitutes a single amino acid in CDR3 of IGHV 3~9 (such as at position 107); and (c) A natural amino acid substitutes a CDR1 of IGKV 2~28 (such as in position 33). Combination of basic amino acids. The special arrangement of an antibody, especially in the charge distribution of the environmental interface (for example, in the CDR), will cause the stability of the antibody in the solution to produce unpredictable conditions.

In some embodiments, the anti-hIL-4Rα antibody comprises at least one amino acid substitution, which is produced within the framework region of the antibody variable region relative to germline IGHV 3-9 sequence or germline IGKV 2-28 sequence Changes in torsional strain. In some embodiments, one or more substitutions of the amino acid are selected from a non-proline in the framework region 3 (FR3) (eg at position 96) substituted by (a) a proline acid in IGHV 3-9 Amine acid; and (b) a combination of a non-proline amino acid substituted for a proline acid in the framework region 2 (FR2) (eg, position 46) of IGKV 2~28. Altering the peptide chain particularly refers to the torsional ability of a framework region that affects the interface between CDR and solvent, which will cause the stability of the antibody in the solution to produce unpredictable conditions.

According to some embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) of sequence identification number: 2, a HCDR2 of sequence identification number: 3, and a sequence identification number : 4 a HCDR3. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a HCVD with sequence identification number: 1.

According to some specific embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a light chain (kappa) complementarity determining region (LCDR) 1 with a sequence identification number of 6; an LCDR2 with a sequence identification number of 7; And the serial identification number: 8 a LCDR3. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an LCVD with sequence identification number: 5.

According to some other specific embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCDR1 with sequence identification number: 10, an HCDR2 with sequence identification number: 11, an HCDR3 with sequence identification number: 12, and a sequence Identification number: an LCDR1 of 14, serial identification number: an LCDR2 of 15, and serial identification number: an LCDR3 of 16. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD with sequence identification number: 9 and an LCVD with sequence identification number: 13.

According to some other specific embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCDR1 with a sequence identification number: 18, an HCDR2 with a sequence identification number: 19, an HCDR3 with a sequence identification number: 20, a sequence Identification number: an LCDR1 of 22, serial identification number: an LCDR2 of 23, and serial identification number: an LCDR3 of 24. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD with sequence identification number: 17 and an LCVD with sequence identification number: 21.

The non-limiting example antibody in the examples here is called "mAb1". This antibody is also known as H4H098P in US 7,608,693. mAb1 (H4H098 P) contains a HCVR/LCVR amino acid sequence pair with sequence identification number: 1/5, and HCDR1-HCDR2 representing the sequence identification number: 2-3-4/sequence identification number: 6-7-8 -HCDR3/LCDR1-LCDR2-LCDR3 domain.

Another non-limiting exemplary antibody used in the practice of the invention is called "mAb2". This antibody is also known as H4H083P in US 7,608,693. mAb2 (H4H083P) contains an HCVR/LCVR amino acid sequence pair with sequence identification number: 9/13, and HCDR1-HCDR2- which represents the sequence identification number: 10-11-12/sequence identification number: 14-15-16 HCDR3/LCDR1-LCDR2-LCDR3 domain.

Yet another non-limiting exemplary antibody used in the practice of the present invention is called "mAb3". This antibody is also known as H4H095P in US 7,608,693. mAb3 (H4H095P) contains an HCVR/LCVR amino acid sequence pair with sequence identification number: 17/21, and HCDR1-HCDR2- with representative sequence identification number: 18-19-20/sequence identification number: 22-23-24 HCDR3/LCDR1-LCDR2-LCDR3 domain.

The content of the antibody or antigen-binding fragment in the pharmaceutical formulation of the present invention depends on the particular properties desired by the formulation and the specific circumstances and uses of the formulation to be used. In some specific embodiments, the pharmaceutical formulation contains about 100±10 to about 200±20 mg/ml, about 110±11 to about 190±19 mg/ml, about 120±12 to about 180±18 mg/ml, Liquid formulations of antibodies of about 130±13 to about 170±17 mg/ml, about 140±14 to about 160±16 mg/ml, or about 150±15 mg/ml. For example, the formulation of the present invention contains about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, about 120 mg/ml, about 125 mg/ml, about 130 mg/ ml, about 131mg/ml, about 132mg/ml, about 133mg/ml, about 134mg/ml, about 135mg/ml, about 140mg/ml, about 145mg/ml, about 150mg/ml, about 155mg/ml, about 160mg/ ml, about 165mg/ml, about 170mg/ml, about 175mg/ml, about 180mg/ml, about 185mg/ml, about 190mg/ml, about 195mg/ml, or about 200mg/ml to specifically bind hIL-4Rα Antibody or antigen-binding fragment thereof.

Excipients and pH

The pharmaceutical formulation of the present invention contains one or more excipients. The term "excipient" as used herein means any non-therapeutic substance added to the formulation to provide the desired consistency, viscosity or stabilizing effect.

In certain embodiments, the pharmaceutical formulation of the present invention contains at least one organic co-solvent that can stabilize the type and amount of the hIL-4Rα antibody under vigorous operation such as shaking. In some specific embodiments, the term "stable" means that the formation of aggregated antibodies that can prevent the total amount of antibodies (on a molar basis) during the violent operation exceeds 2%. In some embodiments, vigorous operation involves shaking the solution containing the antibody and organic co-solvent for about 120 minutes.

In certain embodiments, the organic co-solvent is a nonionic surfactant, such as alkyl poly(oxyethylene). Specific nonionic surfactants that can be incorporated into the formulations of the present invention include, for example, polysorbate esters such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and poly-sorbate 85; poloxamer such as poloxamer 181, poloxamer 188, polo-xamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitol monolaurate and polyoxyethylene sorbitol monolaurate. poloxamer 181 is also known as PLURONIC F68.

The content of the organic co-solvent in the pharmaceutical formulation of the present invention depends on the desired special properties of the formulation and the specific circumstances and uses of the formulation to be used. In certain embodiments, the surfactant content in the formulation is from about 0.1±0.01% to about 2±0.2%. For example, the formulation of the present invention may contain about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, or about 0.30% of polysorbate 20 Or poloxamer 181. For example, the formulation of the present invention may contain about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0% PEG 3350.

Exemplary organic co-solvents that can stabilize the hIL-4Rα antibody contain 0.2%±0.02% polysorbate 20, 0.2%±0.02% polyxamer 181, or 1%±0.1% PEG 3350.

The pharmaceutical formulation of the present invention also includes one or more heat stabilizers that can stabilize the type and amount of the hIL-4Rα antibody under heat stress. In some embodiments, the term "stable" means that when the solution containing the antibody and the heat stabilizer is stored at about 45°C up to about 28 days, it can maintain more than about 92% of the natural configuration of the antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and the heat stabilizer is stored at about 45°C up to about 28 days, the aggregated antibody can be maintained below about 5%.

In certain embodiments, the heat stabilizer is a sugar or sugar alcohol selected from sucrose, trehalose and mannose, or any combination thereof, and its content in the formulation depends on the specific situation and the formulation is intended to be used Depending on the purpose. In certain embodiments, the formulation contains about 2.5% to about 10%, about 3% to about 9.5%, about 3.5% to about 9%, about 4% to about 8.5%, about 4.5% to about 8 %, about 5% to about 7.5%, about 5.5% to about 7%, or about 6.0% to about 6.5% sugar or sugar alcohol. For example, the pharmaceutical formulation of the present invention may contain about 2.5%±0.375%, about 3%±0.45%, about 3.5%±0.825%, about 4.0%±0.6%, about 4.5%±0.675%, about 5.0%±0.75 %, about 5.5%±0.825%, about 6.0%±0.9%, about 6.5%±0.975%, about 7.0%±1.05%, about 7.5%±1.125%, about 8.0%±1.2%, about 8.5%±1.275% , About 9.0%±1.35%, or about 10.0%±1.5% of sugar or sugar alcohol (such as sucrose, trehalose or mannose).

The pharmaceutical formulation of the present invention may also contain a buffer or buffer system that maintains a stable pH and helps stabilize the hIL-4Rα antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and the buffer is stored at about 45°C up to about 14 days, the aggregated antibody can be maintained below 3.0%±0.5%. In some specific embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C up to about 6 months, it can maintain an aggregated antibody of less than 3.7%±0.5% . In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 45°C up to about 14 days, it can be maintained at least 95% as determined by size exclusion chromatography ±0.5% natural configuration antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C up to about 6 months, it can be maintained for at least 96 by size exclusion chromatography. %±0.5% natural configuration antibody. In some specific embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 45°C up to about 14 days, it can be maintained at least 62% by cation exchange chromatography. 0.5% neutral configuration antibody. In some specific embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C up to about 6 months, it can be maintained at least 54% as measured by cation exchange chromatography ±0.5% neutral configuration antibody. "Neutral configuration" means the portion of the antibody in the ion exchange resin that is dialyzed from the main peak, which tends to be more "alkaline" on one side and more "acidic" on the other side.

The pharmaceutical formulation of the present invention has a pH from about 5.2 to about 6.4. For example, the pharmaceutical formulations of the invention can have from about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, or about 6.4 PH. In some specific embodiments, the pH is about 5.3±0.2, about 5.9±0.2, or about 6.0±0.2.

In some embodiments, the buffering agent or buffering system comprises at least one buffering agent that completely overlaps or is in the partial range of pH 5.2 to 6.4. In a specific embodiment, the buffer or buffer system contains two buffers, the first of which is in the effective pH range of 3.6 to 5.6 and the second is in the effective pH range of 5.5 to 7.4. In a specific embodiment, the first buffer has a pKa of about 4.8±0.3 and the second buffer has a pKa of about 6.0±0.3. In certain embodiments, the buffer system includes acetate buffer and histidine buffer. In some embodiments, each mole of acetate contains about 1.3 to 1.9 parts of histidine. In certain embodiments, each mole of acetate contains about 1.6±0.25 parts of histidine. In certain specific embodiments, the concentration of the acetate is about 2.5 mM to about 22.5 mM, about 3.0 mM to about 22 mM, about 3.5 mM to about 21.5 mM, about 4.0 mM to about 21.0 mM, about 4.5 mM to about 20.5mM, about 5.0mM to about 20mM, about 5.5mM to about 19.5mM, about 6.0mM to about 19.0mM, about 6.5mM to about 18.5mM, about 7.0mM to about 18.0mM, about 7.5mM to about 17.5mM, About 8.0 mM to about 17.0 mM, about 8.5 mM to about 16.5 mM, about 9.0 mM to about 16.0 mM, about 9.5 mM to about 15.5 mM, about 10.0 mM to about 15.0 mM, about 10.5 mM to about 14.5 mM, about 12.5 mM to about 1.875 mM, about 11.0 mM to about 14.0 mM, about 11.5 mM to about 13.5 Mm, or about 12.0 mM to about 13.0 mM. In certain specific embodiments, the concentration of the histidine is about 10 mM to about 30 mM, about 11 mM to about 29 mM, about 12 mM to about 28 mM, about 13 mM to about 27 mM, about 14 mM to about 26 mM, about 15 mM to about 25 mM , About 16 mM to about 24 mM, about 17 mM to about 23 mM, about 18 mM to about 22 mM, or about 19 mM to about 21 mM. In certain embodiments, the slowing system comprises about 12.5 mM acetate at about pH 5.9 and about 20 mM histidine.

The pharmaceutical formulations of the present invention also include one or more excipients for maintaining low viscosity or reducing the viscosity of formulations containing high concentrations of protein (eg, typically >100 mg/ml protein). In some embodiments, the arginine content of the formulation is sufficient to maintain the viscosity of the liquid formulation below about 35 cPoise, below about 30 cPoise, below about 25 cPoise, below about 20 cPoise, below about 15 cPoise, low At about 14cPoise, below about 13cPoise, below about 12cPoise, below about 10cPoise, or below about 9cPoise.

In certain embodiments, the pharmaceutical formulation of the present invention preferably contains L-spermine hydrochloride with an arginine concentration of about 25 mM ± 3.75 mM, about 50 mM ± 7.5 mM, or about 100 mM ± 15 mM . In certain embodiments, the concentration of arginine is about 20 mM to about 30 mM, about 21 mM to about 29 mM, about 21.25 mM to about 28.75 mM, about 22 mM to about 28 mM, about 23 mM to about 27 mM, or about 24 mM To about 26 mM.

Representative formulation

According to one aspect of the invention, the pharmaceutical formulation is a low-viscosity, usually physiologically isotonic liquid formulation comprising: (i) specific binding to hIL-4Rα (eg mAb1, mAb2 or mAb3 [as described above ]) Human antibodies with a concentration of about 100 mg/ml or higher; (ii) a buffer system sufficient to buffer at about pH 5.9±0.6; (iii) sugars especially used as heat stabilizers; (iv) protection of antibody internal structures Complete organic co-solvent; and (v) monoamino acid that maintains viscosity that is easy to use for subcutaneous injection.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binds hIL-4Rα and comprises a substituted IGHV 3-9 heavy chain variable region and a substituted IGLV 2 28 light chain variable region ( Human mAb1 antibody with a concentration of about 100 mg/ml to about 200 mg/ml, such as mAb1); (ii) a buffer system containing acetate and histidine sufficient to buffer at about pH 5.9±0.6; (iii) as a heat stabilizer Sucrose; (iv) polysorbate as an organic co-solvent; and (v) arginine as a debonding agent.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specific binding to hIL-4Rα and a HCDR1 comprising sequence identification number: 2; a HCDR2 sequence identification number: 3; a HCDR3 sequence identification number: 4 1. The sequence identification number: 6-LCDR1, the sequence identification number: 7-LCDR2, and the sequence identification number: 8-LCDR3 human IgG1 antibody with a concentration of about 150mg/ml±25mg/ml; (ii) sufficient to buffer at about about 12.5mM±1.9mM acetate and about 20mM±3mM histidine at pH 5.9±0.3; (iii) sucrose at about 5%±0.75% w/v; (iv) at about 0.2%±0.03% w/v polysorbate 20; and (v) arginine as L-spermine hydrochloride at about 25 mM ± 3.75 mM.

Other non-limiting examples of pharmaceutical formulations of the present invention have been described elsewhere herein, including the examples described below.

Stability and viscosity of pharmaceutical formulations

The pharmaceutical formulations of the present invention generally exhibit a high degree of stability. The term "safety" with respect to the pharmaceutical formulation here means that the antibody in the pharmaceutical formulation maintains an acceptable degree of chemical structure or biological function under defined storage conditions. The antibody contained in a formulation may not maintain 100% of its chemical structure or biological function after being stored for a period of time, but it is still in a stable state. In some cases, maintaining the antibody structure or function at about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage for a period of time may be considered "stable."

Its stability can be determined by measuring the percentage of natural antibody retained in the formulation after storage at a defined temperature for a period of time. The percentage of natural antibodies can be determined in particular by size exclusion chromatography (eg size exclusion high performance liquid chromatography [SE-HPLC]). The phrase "an acceptable degree of stability" as described herein means that at least 90% of natural antibodies can be detected in the formulation after storage for a period of time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 can be detected in the formulation after storing for a period of time at a defined temperature %, 99%, or 100% natural antibodies. The defined period of time determined to be stable is at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The defined temperature measured as a stable storable pharmaceutical formulation may be any temperature from about -80°C to about 45°C, for example, stored at about -30°C, about -20°C, about 0°C, about 4°C~8 °C, about 5 °C, about 25 °C, or about 45 °C. For example, if a pharmaceutical formulation can be measured by SE-HPLC after being stored at 5°C for 3 months, a natural antibody greater than about 90%, 95%, 96%, 97%, or 98% can be considered stable. If a pharmaceutical formulation is stored at 5°C for 6 months and can be measured by SE-HPLC with greater than about 90%, 95%, 96%, 97% or 98% natural antibody, it can also be considered stable. If a pharmaceutical formulation is stored at 5°C for 9 months, SE-HPLC can detect greater than about 90%, 95%, 96%, 97% or 98% of natural antibodies, it can also be considered stable. If a pharmaceutical formulation is stored at 25°C for 3 months, it can be considered stable if it can be measured by SE-HPLC with a natural antibody greater than about 90%, 95%, 96%, or 97%. If a pharmaceutical formulation is stored at 25°C for 6 months after being tested by SE-HPLC, a natural antibody greater than about 90%, 95%, 96% or 97% can also be considered stable. If a pharmaceutical formulation is stored at 25°C for 9 months, it can be considered stable if it can be measured by SE-HPLC with a natural antibody greater than about 90%, 95%, 96%, or 97%.

The stability can be determined after storage for a period of time at a defined temperature by in particular measuring the percentage of aggregated antibody in the formulation, where the stability is inversely proportional to the percentage of aggregated antibody formed. The percentage of aggregated antibodies can be determined in particular by size exclusion chromatography (eg size exclusion high performance liquid chromatography [SE-HPLC]). The phrase "an acceptable degree of stability" as described herein means that up to 5% of aggregated antibodies can be detected in the formulation after storage at a given temperature for a period of time. In certain embodiments, an acceptable degree of stability means that up to about 5%, 4%, 3%, 2%, 1%, 0.5 can be detected in the formulation after storage at a given temperature for a period of time % Or 0.1% aggregated antibody. The defined period of time determined to be stable is at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The defined temperature measured as a stable storable pharmaceutical formulation may be any temperature from about -80°C to about 45°C, for example, stored at about -30°C, about -20°C, about 0°C, about 4°C~8 °C, about 5 °C, about 25 °C, or about 45 °C. For example, if a pharmaceutical formulation is stored at 5°C for 3 months, if it is measured below about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody, it can be considered stable . A medicinal formulation stored at 5°C for 6 months can also be considered stable if it has measured less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of aggregated antibody. If a pharmaceutical formulation is stored at 5°C for 9 months, it can also be considered stable if it measures less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies. If a pharmaceutical formulation is stored at 25°C for 3 months, it can be regarded as stable if the measured aggregated antibody is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. If a pharmaceutical formulation is stored at 25°C for 6 months, if it is measured below about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of aggregated antibody, it can also be considered stable. A medicinal preparation stored at 25°C for 9 months can be considered stable if it has measured less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of aggregated antibody.

The stability of the antibody can be determined in particular by measuring the ratio of the antibody migrating to a more acidic part ("acid form") to the main part of the antibody ("neutral configuration") during ion exchange, wherein the stability Inversely proportional. Without being bound by theory, deamidation of antibodies can lead to antibodies with more negative charges and are therefore more acidic than non-deamidated antibodies (see for example Robinson, N., Deamidation of Proteins, PNAS , April 16, 2002, 99(8):5283~5288). The "acidified" or "deamidated" antibodies can be determined in particular by ion exchange chromatography (eg cation exchange high performance liquid chromatography [CEX-HPLC]). The phrase "an acceptable degree of stability" as described herein means that up to 45% of the antibody in acid form can be detected in the formulation after a period of storage at a defined temperature. In certain embodiments, an acceptable degree of stability means that up to about 45%, 40%, 35%, 30%, 25%, 20 can be detected in the formulation after storage at a given temperature for a period of time %, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% acid form antibodies. The defined period of time determined to be stable is at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The defined temperature measured as a stable storable pharmaceutical formulation may be any temperature from about -80°C to about 45°C, for example, stored at about -30°C, about -20°C, about 0°C, about 4°C~8 °C, about 5 °C, about 25 °C, or about 45 °C. For example, if a pharmaceutical formulation is stored at 5°C for 3 months, if it is measured below about 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% acid form antibodies can be considered stable. If a pharmaceutical formulation is stored at 25°C for 3 months, if it is measured below about 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7% , 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% antibody in acid form can also be considered stable. If a pharmaceutical formulation is stored at 45°C for 8 weeks, if it is measured below about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% acid form antibodies can also be considered stable. If a pharmaceutical formulation is stored at 40°C for 2 weeks, if it is measured below about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% acid form antibodies can also be considered stable.

Other methods can be used to determine the stability of the formulations of the present invention, such as thermal stability by differential scanning calorimetry (DSC), mechanical stability by controlled agitation, and absorbance measurement solutions at about 350 or about 405 nm Turbidity. For example, if a pharmaceutical formulation of the present invention is stored at about 5°C to about 25°C for 6 or more months, if the formulation changes from zero time point OD _{405 to} less than OD _{405 by} about 0.05 (such as 0.04, 0.03, 0.02, 0.01, or lower) is considered stable.

The stability of an antibody can also be assessed by measuring the biological activity of the antibody or its affinity to its target. For example, after a formulation of the present invention is stored at, for example, 5°C, 25°C, and 45°C for a period of time (eg, 1 to 12 months), the anti-IL-4Rα antibody contained in the formulation has an antibody binding affinity of at least 90 before storage When %, 95% or higher is bound to IL-4Rα, it can be regarded as stable. The binding affinity is determined by, for example, ELISA or plasma resonance. The biological activity is measured by the IL-4Rα activity detection method, for example, a cell expressing IL-4Rα is contacted with the formulation containing the anti-IL-4Rα antibody. The binding effect of such cells to the antibody can be directly measured, for example via FACS analysis. Alternatively, the downstream activity of the IL-4Rα system is measured in the presence of the antibody and an IL-4Rα agonist, and then the activity of the IL-4Rα system without antibody is compared. In some specific embodiments, the IL-4Rα can be endogenously endogenous to the cell. In other specific embodiments, the IL-4Rα may be ectopically expressed in the cell.

Other methods for determining the stability of antibodies in the formulation are illustrated in the examples below.

In certain embodiments, the pharmaceutical formulations of the present invention exhibit low to moderate viscosity. The "viscosity" mentioned here may be "dynamic viscosity" or "absolute viscosity". "Dynamic viscosity" is the measured flow resistance of a liquid under the influence of gravity. When two equal amounts of liquid are placed in the same capillary viscometer and flow under gravity, a viscous liquid requires a lower viscosity liquid to flow through the capillary for a longer time. For example, if a liquid takes 200 seconds and another liquid takes 400 seconds to complete its flow, the second liquid has twice the viscosity of the first on a dynamic viscosity scale. "Absolute viscosity" refers to the product of dynamic viscosity and liquid density (absolute viscosity = dynamic viscosity x density), which is sometimes referred to as dynamic or simple viscosity. The size of the dynamic viscosity is L ² /T, the length of the L system and the time of the T system. Generally, the dynamic viscosity is expressed as centipoise (cSt). The SI unit of dynamic viscosity is mm ² /s, which refers to 1cSt. The unit of absolute viscosity is centipoise (cP). The SI unit of absolute viscosity is centipa-second (mPa-s), and its 1cP=1mPa-s.

As described herein, a low viscosity liquid formulation of the present invention will exhibit an absolute viscosity below about 15 centipoise (cP). For example, if a liquid formulation of the present invention is considered to have "low viscosity" when measured by a standard viscosity measurement method, the formulation will exhibit about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9cP, about 8cP, or lower absolute viscosity. As described herein, a medium viscosity liquid formulation of the present invention will exhibit an absolute viscosity ranging from about 35 cP to about 15 cP. For example, if a liquid formulation of the present invention is considered to have "medium viscosity", the formulation will exhibit about 34cP, about 33cP, about 32cP, about 31cP, about 30cP, about 29cP, about 28cP, about 27cP, about 26cP , About 25cP, about 24cP, about 23cP, about 22cP, about 21cP, about 20cP, about 19cP, about 18cP, about 17cP, about 16cP, or about 15.1cP absolute viscosity.

As described in the examples below, the inventors have unexpectedly discovered that by formulating the antibody from about 25 mM to about 100 mM arginine, antibodies containing high concentrations of anti-hIL-4Rα antibodies (eg, from about 100 mg/ml up to at least 200 mg/ml) can be obtained ) Of low to medium viscosity liquid formulations. In addition, it has been further found that by adjusting the sucrose content to less than about 10%, the viscosity of the formulation can be reduced to a greater extent.

Container for the pharmaceutical preparation and method of administration

The pharmaceutical formulation of the present invention can be placed in any container suitable for storing drugs and other therapeutic compositions. For example, the pharmaceutical formulation can be placed in a sealed and sterilized plastic or glass container with a defined volume such as a vial, ampoule, syringe, cartridge, or vial. The formulation of the present invention can be placed in different types of small glass bottles such as transparent and opaque (such as amber) glass or plastic bottles. Likewise, any type of syringe can be used to contain or administer the pharmaceutical formulation of the present invention.

The pharmaceutical formulation of the present invention can be placed in a "normal tungsten" syringe or a "low tungsten" syringe. As is understood by those of ordinary skill in the art, the process of manufacturing a glass syringe usually involves the use of a hot tungsten rod that penetrates the glass to create a small hole that can draw liquid from the syringe. This process causes a trace amount of tungsten to deposit on the inner surface of the barrel. Then washing and other process steps can be used to reduce the amount of tungsten in the syringe. The term "ordinary tungsten" as described herein means that the barrel contains more than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the barrel contains less than 500 ppb of tungsten. For example, a low tungsten syringe according to the present invention may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80 , 70, 60, 50, 40, 30, 20, 10 or less ppb tungsten.

The rubber plunger used for the syringe and the rubber stopper used to close the opening of the glass bottle can be coated to avoid contamination of the syringe or the medicine in the bottle, or to preserve its stability. Therefore, the pharmaceutical formulation of the present invention according to some specific embodiments can be placed in a syringe containing a coated plunger, or in a glass bottle sealed with a coated rubber stopper. For example, the plunger or rubber plug may be coated with a fluorocarbon film. Examples of coated plungers or rubber stoppers suitable for glass bottles and syringes containing the pharmaceutical formulations of the present invention have been described in US Patent Nos. 4,997,423, 5,908,686, 6,286,699, 6,645,635 and 7,226,554, the contents of which are fully incorporated by reference this. Coating rubber for the particular context of the invention the plunger and stoppers are available from West Pharmaceutical Services Company (Lionville, PA city) market product "FluroTec ^®".

According to certain embodiments of the present invention, the pharmaceutical formulation can be placed in a low tungsten syringe containing a fluorocarbon coated piston.

The pharmaceutical formulation can be administered to patients via parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal, pulmonary, or oral administration. Many portable injection pens or automatic injection infusion devices can be used for subcutaneous infusion of the pharmaceutical formulation of the present invention. Examples include, but are not limited to AUTOPEN ^TM (Owen Mumford, Woodstock, UK), DISETRONIC ^TM pen (Disetronic Medical, Bergdorf, Switzerland), HUMALOG MIX 75/25 ^TM pen, HUMALOG ^TM pen, HUMALIN 70/30 ^TM pen (Indian Eli Lilly, Indianapolis, N.A.), NOVOPEN ^TM I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk, Copenhagen, Denmark), BD ^TM Pen (Becton Dickinson, Franklin, New Jersey) , OPTIPEN ^™ , OPTIPEN PRO ^™ , OPTIPEN STARLET ^™ , and OPTICLIK ^™ (Sanofi-Aventis, Frankfurt, Germany). Examples of portable pens or automatic infusion devices that have been used for subcutaneous infusion of the pharmaceutical composition of the present invention include, but are not limited to SOLOSTAR ^TM pens (Sanofi-Aventis), FLEXPEN ^TM (Novo Nordisk), and KWIKPEN ^TM (Eli Lilly) ; SURECLICK ^TM autoinjector (Amgen, Thousand Oaks, California), PENLET ^TM (Hasselmeier, Stuttgart, Germany), EPIPEN ^TM (Dey, LP), and HUMIRA ^TM pen (Abbott Laboratories, Abbott Park, Illinois).

Here, a microsyringe is also used to infuse the pharmaceutical formulation of the present invention. As described herein, "microsyringe" means that a large amount (eg, up to about 2.5 ml or more) of the therapeutic formulation is slowly administered over a long period (eg, about 10, 15, 20, 25, 30 minutes, or longer). Hypodermic infusion set. Please see, for example, US 6,629,949, US 6,659,982; and Meehan et al., J. Controlled Release 46: 107~116 (1996). The micro-infusion set is most suitable for infusion of high-dose therapeutic proteins containing high concentrations (such as about 100, 125, 150, 175, 200 mg/ml, or higher), or viscous solutions.

In a specific embodiment, the liquid pharmaceutical formulation containing about 150±15 mg/ml anti-IL-4Rα antibody is administered subcutaneously in a volume of about 1±0.15 ml from a pre-filled syringe in the auto-injector. In another specific embodiment, the formulation is administered from a microsyringe in a volume between about 1 ml and 2.5 ml. The formulation can be pre-filled into a storage bag or cartridge in a micro-syringe.

Therapeutic use of pharmaceutical preparations

The pharmaceutical formulations of the present invention are particularly useful for treating, preventing or alleviating any disease or disorder related to IL-4 activity, including diseases or disorders mediated by IL-4Rα activation. Non-limiting exemplary diseases and disorders that can be treated or prevented by administering the pharmaceutical formulation of the present invention include various allergic diseases such as atopic dermatitis, allergic conjunctivitis, allergic rhinitis, asthma, and other IgE/Th2 Leading diseases.

Accordingly, the present invention includes methods of treating, preventing or alleviating any disease or disorder related to IL-4 activity or IL-4Rα activation (including any of the above-mentioned exemplary diseases, disorders and conditions). The treatment method of the present invention comprises administering the above formulation containing anti-hIL-4Rα antibody to an organism. The organism to which the pharmaceutical formulation is administered can be, for example, any human or non-human animal that needs such treatment, prevention, or remission, or can benefit from inhibiting or relieving IL-4 or IL-4Rα-mediated activity. For example, the organism may be an individual who is diagnosed or believed to be at risk of suffering from the above-mentioned diseases or disorders. The present invention further includes any of the pharmaceutical formulations disclosed herein for the manufacture, use in the treatment, prevention or alleviation of any disease or disorder related to IL-4 activity or IL-4Rα activation (including any of the above exemplary diseases, disorders and conditions) the use of.

The following examples provide a complete disclosure and description of how those skilled in the art make and use the methods and compositions of the present invention, and are not intended to limit the scope of the invention deemed by the inventor to be their invention. Although efforts have been made to ensure the accuracy of the numbers used (eg, quantity, temperature, etc.), some experiments may still cause certain errors and deviations. Unless otherwise stated, the parts are mole parts, the molecular weight is the average molecular weight, the temperature is in degrees Celsius, and the pressure is at or near atmospheric pressure.

The activity of forming a preliminary formulation involves screening organic co-solvents, heat stabilizers and buffers in the liquid formulation of mAb1 (anti-IL-4Rα antibody of the invention) to confirm that the excipients are compatible with the protein and contribute to its Stability, while maintaining the molar osmolarity and viscosity for subcutaneous injection. The buffer conditions were also checked to determine the optimal pH for the highest protein stability.

Example 1. Organic cosolvent

It has been found that mAb1 is unstable when oscillating tightly. The analysis of reverse phase high performance liquid chromatography (RP-HPLC) and size exclusion high performance liquid chromatography (SE-HPLC) proved that when mAb1 was shaken at room temperature, the protein loss and the increase of agglutination protein were confirmed ( Table 1, please see the "no cosolvent" data). Adding an organic co-solvent to the mAb1 solution can prevent protein degradation measured by SE-HPLC and RP-HPLC (Table 1). However, it has been found that the addition of some organic co-solvents will reduce the thermal stability of mAb1 (Table 2). It has been found that formulations containing PEG 3350 (3%) and PEG 300 (10% and 20%) will lose recovered protein measured by RP-HPLC after heat stress (Table 2). In addition, formulations containing PLURONIC F68 (poloxamer 181) (0.2%), PEG 300 (10% and 20%) and propylene glycol (20%) formed more aggregates as measured by SE-HPLC than formulations without co-solvents Thing. Polysorbate 20 (0.2%) and polysorbate 80 (0.2%) have considerable stability against vibration and thermal stress.

According to Table 1, 0.3 ml of mAb1 in a 2 ml glass bottle at 15 mg/ml in 10 mM phosphate pH 6.0 and various organic co-solvents were shaken for about 120 minutes. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation without shaking.

According to Table 2, 0.3 ml of mAb1 in a 2 ml glass bottle at 15 mg/ml in 10 mM phosphate pH 6.0 and various organic co-solvents were stored at about 45°C for about 28 days. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. The percentage of total recovered mAb1 was determined by reverse-phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not subjected to heat stress.

Example 2. Thermal stabilizer

Check the ability of various heat stabilizers, such as sugar, amino acids and inorganic salts, to inhibit the degradation of mAb1 when stored at about 45°C. A summary of the research on heat stabilizers is shown in Table 3. Formulations containing sucrose or trehalose have a higher mAb1 stabilizing effect (determined by SE-HPLC) when cultured in a high-temperature solution. Because sucrose has been used for a long time in the safety of monoclonal antibody formulations, it was chosen as a stabilizer.

According to Table 3, 0.3 ml of mAb1 in a 2 ml glass bottle at 25 mg/ml in 10 mM acetate pH 5.3 and various heat stabilizers were stored at about 45°C for about 28 days. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. All samples can ignore the difference in turbidity. The percentage of total recovered mAb1 was determined by reverse-phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak residence time, respectively. The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not subjected to heat stress.

Example 3. Buffer and pH

The effect of pH and buffer type on the stability of mAb1 was also examined. 15 mg/ml of mAb1 was incubated with different buffers at different pH values ranging from pH 4.5 to 7.0. Protein stability was monitored by SE-HPLC and cation exchange HPLC (CEX-HPLC). When mAb1 was formulated in pH 6.0 histidine buffer or pH 5.3 acetate buffer, it had the maximum protein stability determined by SE-HPLC and CEX-HPLC (Table 4 and Table 5). The acetate buffer system has a wider pH stability range and a lower rate of variable charge formation compared to formulations containing histidine buffer (Table 5). Therefore, acetate buffer at pH 5.3 was selected for the formulation of mAb1 drugs.

According to Table 4, 10 mM of various buffers were mixed with 0.3 ml of 15 mg/ml mAb1 and 0.2% polysorbate 20 in 2 ml glass bottles and stored at about 45°C for about 14 days. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. All samples can ignore the difference in turbidity. The percentage of total recovered mAb1 was determined by reverse-phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak residence time, respectively. The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not subjected to heat stress.

According to Table 5, 10 ml of various buffers were mixed with 0.3 ml of 15 mg/ml mAb1 and 0.2% polysorbate 20 in 2 ml glass bottles and stored at about 45°C for about 14 days. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. All samples can ignore the difference in turbidity. The percentage of total recovered mAb1 was determined by reverse-phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak residence time, respectively. The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not subjected to heat stress.

6.5)下溶液內mAb1可被去醯胺。反之，低於pH 5.0時已發現可增加形成變異分子量之mAb1的速率。根據這些數據，該mAb1配製物的pH被維持在約pH 5.6和pH 6.2之間。已發現mAb1可穩定存在於此pH範圍。 Formulation test is shown in alkaline conditions (pH

6.5) mAb1 can be deamidated in the lower solution. Conversely, below pH 5.0 it has been found to increase the rate of formation of mAb1 of varying molecular weight. Based on these data, the pH of the mAb1 formulation is maintained between about pH 5.6 and pH 6.2. It has been found that mAb1 can be stably present in this pH range.

The effect of the pH and buffer type of 20 mM histidine pH 6, 12.5 mM acetate pH 5.3 or a mixture of 20 mM histidine and 12.5 acetate pH 5.9 on the stability of mAb1 was further evaluated (Table 6). Comparing individual buffer systems, mAb1 containing histidine and about pH 5.9 acetate is the most stable. When mAbl was formulated in this mixed buffer system (SE-HPLC), it had the slowest aggregation rate (Table 6).

According to Table 6, 0.4 ml of 150 mg/ml mAb1, 10% sucrose, 0.2% polysorbate 20 mixed with various buffers in a 2 ml glass bottle was stored at about 45°C for about 14 days. The optical density (OD) at 405 nm was used to measure the turbidity and to compare the relative change of the OD of the starting material at 405 nm. All samples can ignore the difference in turbidity. The percentage of total recovered mAb1 was determined by reverse-phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC). Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak residence time, respectively. The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not subjected to heat stress.

Example 4. Control of viscosity and tension

The viscosity and tonicity (expressed as molar osmolarity) of various excipients mixed with high concentrations of mAb1 (ie 150 mg/ml, 175 mg/ml, and 200 mg/ml) were evaluated. The contents of sucrose, sodium chloride and L-arginine hydrochloride were adjusted to form a low viscosity and physiological isotonic formulation containing high concentration of mAb1 that is easy to comfortably and quickly infuse mAb1 in high volume (Table 7). Liquid formulation of pH 5.9 containing 25mM arginine, 20mM histidine, 12.5mM acetate, 5% (w/v) sucrose, 0.2% (w/v) polysorbate 20 and 150mg/ml mAb1 (Formulation A ) Is the best formulation with low viscosity (about 8.5cPoise) and physiological isotonicity (about 293mOsm/kg) while still maintaining the stability of mAb1.

Example 5. Characterization of Formulation A

The main degradation pathways in the process of forming the mab1 liquid formulation are the production of aggregates, fracture products and charge variants. The formation of these degradation products can be reduced by formulating mAb1 in a pH 5.9 formulation containing 20 mM histidine, 12.5 mM acetate, 0.2% polysorbate 20, 5% sucrose, and 25 mM L-arginine hydrochloride. It has been found that these formulated 150 mg/ml mAb1 are clear to slightly opalescent liquid solutions that are substantially free of particles visible to the naked eye.

The formulated mAb1 has physical and chemical stability under various stress (incubation at 25°C and 45°C) and immediate storage conditions (5°C) (Table 8). The mAb1 does not affect its appearance when stored at 25°C (3 months) or 5°C for 6 months. In addition, it has been found that it does not affect the pH, turbidity of the solution or the recovery of mAb1. After the formulated mAb1 was stored at 25°C for 3 months, the antibody measured by SE-HPLC was not significantly degraded and the degradation measured by CEX-HPLC was only 3.3% higher. It has been found that increasing the amount of degradation measured by SE-HPLC and CEX-HPLC after 8 weeks of storage at 45°C shows that the formation of aggregation and charge variation is the main degradation pathway of this mAb1 antibody molecule. The formulated mAb1 antibody was not degraded when stored at 5°C for 6 months.

According to Table 8, OD=optical density; RP-HPLC=reverse phase high performance liquid chromatography; SE-HPLC=size exclusion high performance liquid chromatography; and CEX-HPLC=cation exchange high performance liquid chromatography. Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak residence time, respectively.

Example 6. Container

The filter-sterilized formulation containing mAb1 has been determined to be stable. The manufacturing system of clinical supply uses a microporous MILLIPAK filter device, while the laboratory uses a filter of the same composition (microporous Millex DURAPORE).

With a minimum of 2.5 ml of 150 mg/ml mAb1, 5% (w/v) sucrose at pH 5.9, 25 mM L-arginine hydrochloride, 0.2% (w/v) polysorbate 20, 12.5 mM acetate, 20 mM histamine Fill the acid into a 5ml glass bottle. Use 0.5ml of excess formulation in a 5ml bottle to ensure that 2.0ml of formulation can be withdrawn. This excess is not used to compensate for losses during the manufacture of mAb1 or formulations containing mAb1, degradation during manufacture, degradation during storage (shelf life), or to extend the expiration date.

Compared with storage in glass bottles, the stability of the formulated mAb1 (Formulation A) is not affected when stored in polypropylene glycol tubes, polystyrene tubes, polycarbonate tubes, or in glass bottles containing stainless steel blocks (Table 9).

According to Table 9, 150 mg/ml mAb1 at pH 5.9, 5% sucrose, 25 mM spermine hydrochloride, 0.2% PS-20, 20 mM histidine, and 12.5 mM acetate were stored in various materials at 40°C for 14 days . OD=optical density; RP-HPLC=reverse phase high performance liquid chromatography; SE-HPLC=size exclusion high performance liquid chromatography; and CEX-HPLC=cation exchange high performance liquid chromatography. Turbidity is recorded as the difference in turbidity at an OD of 405 nm compared to the starting material. Acidic or basic substances are defined as the total amount of mAb1 peaks dialyzed from the CEX-HPLC column earlier or later than the main peak residence time, respectively.

<110> Regeneron Pharmaceuticals, Inc.

<120> Stabilized formulation containing anti-interleukin-4 receptor (IL-4R) antibody

<130> 6032

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Claims (13)

A stable liquid pharmaceutical formulation comprising: (i) a human antibody at a concentration from 15 mg/ml to less than 100 mg/ml, wherein the antibody specifically binds to human interleukin-4 receptor alpha (hIL-4Rα ) And contains a heavy chain variable region (HCVR), which contains the amino acid sequence of SEQ ID NO: 1; and a light chain variable region (LCVR), which contains the amino acid sequence of SEQ ID NO: 5; (ii ) 10 mM to 15 mM acetate; (iii) 15 mM to 25 mM histidine; (iv) 2.5% w/v to 10% w/v sucrose; (v) 0.1% w/v to 0.3% w/v Polysorbate; and (vi) 25 mM to 100 mM arginine; wherein the liquid pharmaceutical formulation has a pH of 5.6 to 6.2. For example, a liquid pharmaceutical formulation according to item 1 of the patent application, in which acetate is present at a concentration of 12.5 mM ± 1.85 mM and histidine is present at a concentration of 20 mM ± 0.3 mM. For example, the liquid pharmaceutical formulation of claim 1 of the patent application, wherein the polysorbate is polysorbate 20 or polysorbate 80. For example, the liquid pharmaceutical formulation of claim 1 of the patent application, wherein the polysorbate is polysorbate 20 and its concentration is 0.2%±0.03% weight/volume. For example, the liquid pharmaceutical formulation of claim 1 of the patent application, wherein the polysorbate is polysorbate 80 and its concentration is 0.2%±0.03% weight/volume. For example, the liquid pharmaceutical formulation of the first patent application, in which sucrose is present at a concentration of 5% ± 0.75% weight/volume. For example, the liquid pharmaceutical formulation of the first patent application, in which arginine is present at a concentration of 25 mM ± 3.75 mM. For example, the liquid pharmaceutical formulation of claim 1 of the patent application, in which arginine is present at a concentration of 50 mM ± 7.5 mM. For example, the liquid pharmaceutical formulation of the first patent application scope, in which the pharmaceutical formulation is contained in a glass bottle. For example, the liquid pharmaceutical formulation of the first patent application scope, in which the pharmaceutical formulation is contained in the syringe. For example, the liquid pharmaceutical formulation of the first patent application scope, in which the formulation is contained in a micro-infusion set. For example, the liquid pharmaceutical formulation of claim 10, wherein the syringe contains a fluorocarbon-coated plunger. For example, the liquid pharmaceutical formulation of the patent application scope item 10 or 12, wherein the syringe is a low tungsten syringe.