TWI782325B - 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 formulations containing anti-interleukin-4 receptor (IL-4R) antibodies

The present invention is in the field of therapeutic antibody formulations. More specifically, the invention is in the field of pharmaceutical formulations comprising human antibodies that specifically bind the human interleukin-4 receptor.

sequence listing

This patent specification is accompanied by a ST.25 conforming text file of the ordered list. The contents of that text file are hereby incorporated by reference. The paper sequence listing with the same content as the ST.25 conforming text file is incorporated as a part of this patent specification.

Therapeutic macromolecules, such as antibodies, must be formulated in a manner that not only renders the molecule suitable for patient administration, but also maintains stability upon storage and subsequent use. For example, therapeutic antibodies in liquid solutions are prone to degradation, aggregation, or undesirable chemical modification unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation, but also on the relative amounts and ratios of the excipients. In addition, other factors besides stability must be considered when preparing a liquid antibody formulation. Examples of such additional considerations include solution viscosity and antibody concentration that can be adapted to a known formulation, as well as 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 the appropriate antibody, and has the appropriate viscosity and other properties to enable convenient administration of the formulation to a patient.

Antibodies to human interleukin-4 receptor alpha (hIL-4Rα) are an example of a therapeutically relevant macromolecule that requires appropriate formulation. Anti-hIL-4Rα antibodies are used clinically to treat or prevent diseases such as atopic dermatitis and allergic asthma, among other conditions. Range of anti-hIL-4Rα antibodies Examples are described, inter alia, in US Patent Nos. 7,605,237, 7,608,693, 7,465,450, and 7,186,809; and US Patent Application Nos. 2010-0047254 and 2010-0021476.

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

Summary of Invention

The present invention meets the above needs by providing pharmaceutical formulations containing human antibodies that specifically bind human interleukin-4 receptor alpha (hIL-4Rα).

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

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 of SEQ ID NO: 1-8. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) comprising SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively a heavy chain variable region (HCVR); and (b) light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3) containing SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively ) of a light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5, respectively.

In a specific embodiment, the antibody comprises any one or more amino acid sequences of SEQ ID NO: 9-16. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) comprising SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively a heavy chain variable region (HCVR); and (b) light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively ) of a light chain variable region (LCVR). In a particular embodiment, The antibody comprises an HCVR and an LCVR comprising the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 13, respectively.

In a specific embodiment, the antibody comprises any one or more amino acid sequences of SEQ ID NO: 17-24. In a specific embodiment, the antibody comprises (a) heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3) comprising SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively a heavy chain variable region (HCVR); and (b) light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively ) of a light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR comprising the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 21, respectively.

In a specific embodiment, the pH of the liquid formulation is about 5.9±0.5. In a specific embodiment, the pH of the liquid formulation is about 5.9±0.1. In one embodiment, the liquid pharmaceutical buffer comprises one or more buffering agents that buffer from about pH 5.6 to about pH 6.2.

In one embodiment, the liquid pharmaceutical formulation comprises a buffer system comprising at least two buffers. In one embodiment, the buffer system includes a first buffer with an effective pH range of 3.6-5.6 and a second buffer with an effective pH range of 5.5-7.4. 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 concentration of acetate is 12.5±1.9 mM and the concentration of histidine is 20±3 mM.

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.

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", wherein 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 one embodiment, the heat stabilizer is a sugar. In a specific embodiment In the present invention, 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 heat stabilizer is from about 0.9%±0.135% w/v to about 10%±1.5% w/v. In a specific embodiment, the heat stabilizer is sucrose at a concentration of about 5% ± 0.75% w/v.

In a specific embodiment, the viscosity reducer is a type selected from the group consisting of arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride and sodium acetate. Set of salt. In a specific embodiment, the viscosity reducing agent is L-arginine hydrochloride.

In one embodiment, the concentration of the viscosity reducing agent is less than about 100 mM. In a specific embodiment, the concentration of the viscosity reducing agent is 50mM±7.5mM. In another specific embodiment, the concentration of the viscosity reducing agent is 25mM±3.75mM. In a specific embodiment, the viscosity reducing agent is L-arginine hydrochloride at a concentration of 25 mM±3.75 mM.

In one 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 liquid pharmaceutical formulation has a viscosity of about 8.5 ± 0.85 centipoise.

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

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

In a specific embodiment, at least 90% of the antibody in native form can be recovered from the liquid pharmaceutical formulation after storage at 45°C for eight weeks as determined by size exclusion chromatography.

In one embodiment, less than 45% of the antibody in the acidic form can be recovered from the liquid pharmaceutical formulation after storage at 45° C. for eight weeks as determined by cation exchange chromatography.

In a specific embodiment, when determined by size exclusion exchange chromatography at Less than about 4% of antibodies recovered from the liquid pharmaceutical formulation after six months of storage at 25°C were 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 to hIL-4Rα, wherein the antibody comprises SEQ ID NO: 1 and The heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the amino acid sequence of SEQ ID NO: 5; (ii) about 12.5mg/ml ± 2mM acetate; (iii) about 20mM ± 3mM (iv) about 5%±0.75% (w/v) of sucrose; (v) about 0.2%±0.03% (w/v) of polysorbate 20; and (vi) at about Approximately 25 mM ± 3.75 mM arginine at pH 5.9 ± 0.5.

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

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

In a specific embodiment, at least 98% of the native form of the anti-hIL-4Rα antibody is recovered from the liquid pharmaceutical formulation after storage at 5° C. for six months as determined by size exclusion chromatography.

In a specific embodiment, at least 90% of the native form of the anti-hIL-4Rα antibody is recovered from the liquid pharmaceutical formulation after storage at 45° C. for eight weeks as determined by size exclusion chromatography.

In one embodiment, less than 45% of the antibody in the acidic form can be recovered from the liquid pharmaceutical formulation after storage at 45° C. for eight weeks as determined by cation exchange chromatography.

In a specific embodiment, the antibody recoverable from the liquid pharmaceutical formulation after six months storage at 25° C. is less than about 4% aggregated as determined by size exclusion exchange chromatography.

In one aspect, a diazepam low viscosity isotonic liquid pharmaceutical formulation comprising 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 is present at a concentration of about 150 mg/ml ± 15 mg/ml.

In a specific embodiment, the antibody comprises any one or more amino acid sequences of SEQ ID NO: 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 comprises the complementary heavy and light chains of the amino acid sequences of SEQ ID NO: 2-3-4 and SEQ ID NO: 6-7-8, respectively Determining region (HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3). In a specific embodiment, the anti-hIL-4Rα antibody has an HCVR and an LCVR.

In some embodiments, the formulation has a viscosity of 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 osmolarity. In a specific embodiment, the formulation comprises an osmolarity of 290 ± 20 mOsm/kg.

In a specific embodiment, the antibody is stable at about 5°C for at least about six months. In a specific embodiment, at least about 98% of the antibody retains its native conformation when stored at 5°C for about six months as determined 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 native conformation when stored at 45°C for about eight weeks as determined by size exclusion chromatography. In a specific embodiment, less than about 45% of the antibody is in the acidic form when stored at 45°C for about eight weeks, as determined 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 specific embodiment, less than about 4% of the antibody comprises aggregated forms when stored at 25°C for about six months as determined by size exclusion chromatography.

In a specific embodiment, the formulation comprises a buffer having a pH of about 5.9±0.5. In one embodiment, the buffer comprises an acetate buffer and a histamine buffer. 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 comprises 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 In a specific embodiment, the organic co-solvent is polysorbate 20 with a concentration of about 0.2%±0.03% w/v.

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

In a specific embodiment, the formulation comprises a viscosity reducing agent at a concentration of no more than about 100 mM. In a specific embodiment, the viscosity reducer is arginine. In a specific embodiment, the viscosity reducing agent is L-arginine hydrochloride at a concentration of 25 mM±3.75 mM.

In a specific embodiment, the diazepam low viscosity isotonic liquid pharmaceutical formulation has a viscosity of about 8.5±2.5 centipoise and a molar osmolality of about 290±20 mOsm/kg, and comprises: (i) 150 mg/ml The anti-hIL-4Rα antibody of ± 15mg/ml, wherein the antibody comprises a HCVR and a LCVR respectively containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5; (ii) acetic acid of 12.5mM ± 1.9mM salt; (iii) histidine at 20 mg/ml±3 mM; (iv) polysorbate 20 at 0.2%±0.03% w/v; (v) sucrose at 5%±0.75% w/v; and ( vi) 25 mM ± 3.75 mM L-arginine hydrochloride. According to this embodiment, (i) at least about 98% of the antibody stored at 5°C for at least about six months retains its native conformation as determined by size exclusion chromatography; (ii) when determined by size exclusion chromatography At least about 90% of the antibody retains its native conformation when stored at 45°C for at least about eight weeks when assayed by cation exchange chromatography; (iii) less than about 45% is in the acidic form when assayed by cation exchange chromatography when stored at 45°C for eight weeks the antibody; and (iv) less than about 4% of the antibody comprises aggregated forms when stored at 25°C for about six months as determined by size exclusion chromatography.

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

Other specific embodiments of the invention will become apparent from the review of the detailed description that follows.

Detailed Description of the Invention

Before the present invention is described, it is to be understood that this invention is not limited to the specific embodiments and experimental conditions described, as various methods and conditions may be varied.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Herein the term "about" when applied to a particular stated value or range of values means that the value is no more than 1% of the stated value. For example, the expression "about 100" mentioned herein includes 99 and 101 and all values therebetween (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those 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. All publications mentioned herein are hereby incorporated by reference to fully describe the present invention.

pharmaceutical preparations

The statement "pharmaceutical formulation" here means a combination of at least one active ingredient (such as a small molecule, macromolecule, compound, etc. that can exhibit biological effects in humans or non-human animals) and at least one inactive ingredient, which when The active ingredient may be administered therapeutically to a human or non-human animal in admixture with one or more additional inactive ingredients. The term "formulation" herein means "pharmaceutical formulation" unless expressly stated otherwise. The invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to some embodiments of the present invention, the therapeutic polypeptide is an antibody or an antigen-binding fragment thereof that can specifically bind to human interleukin-4 antibody alpha (hIL-4Rα). More specifically, the present invention includes organic co-stimulators comprising (i) a human antibody that specifically binds hIL-4Rα; (ii) an acetate/histidine buffer system; (iii) a nonionic surfactant. solvents; (iv) a heat stabilizer of carbohydrates; and (v) a pharmaceutical formulation of a viscosity reducer. Specific exemplary ingredients and formulations encompassed by the invention are described in detail below.

Antibodies that specifically bind hIL-4R

The pharmaceutical formulation of the present invention comprises a human antibody or antigen-binding fragment thereof that specifically binds hIL-4Rα. The term "hIL-4Rα" as used herein means a human cytokine receptor that specifically binds interleukin-4 (IL-4). In some specific embodiments, the compound contained in the pharmaceutical preparation of the present invention The antibody in the product specifically binds to the extracellular region of hIL-4Rα. An exemplary human IL-4 receptor alpha (hIL-4Rα) amino acid sequence is set forth in SEQ ID NO:25. Antibodies to hIL-4Ra are described in US Pat. 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 refers to immunoglobulin molecules and multimers thereof comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide chains (such as IgM); however, immunoglobulin molecules composed only of heavy chains (ie, no light chains) also fall within the definition of "antibody". Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or _VL ) and a light chain constant region. The light chain constant region comprises one domain (CL1). The _VH and _VL can be further subdivided into interspersed with more conserved regions called framework regions (FR) and hypervariable regions called complementarity determining regions (CDRs). Each _VH and _VL is composed of three CDRs and four FRs from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Unless expressly stated otherwise, the term "antibody" as used herein should be considered to include whole antibody molecules as well as antigen-binding fragments thereof. The "antigen-binding region" or "antigen-binding fragment" (or simply referred to as "antibody region" or "antibody fragment") of the antibody herein refers to an antibody that retains the ability to specifically bind hIL-4Rα or its epitope (epitope). One or more fragments of an antibody.

The term "isolated antibody" as used herein means an antibody that is substantially free of other antibodies having different antigenic specificities (for example, an isolated antibody capable of specifically binding to hIL-4Rα, which has substantially no specific binding to hIL-4Rα except hIL-4Rα. Antibodies to the antigen).

The terms "specifically bind" and the like mean that an antibody or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding is characterized by a dissociation constant of at least about 1 x 10 ^-6 M or higher. Methods for determining whether two molecules specifically bind are known in the art and include, for example, dialysis equilibration, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds hIL-4Rα has cross-reactivity 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 is considered to "specifically bind" hIL-4Rα. Furthermore, an isolated antibody can be substantially free of other cellular material or chemicals.

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

According to some specific embodiments of the present invention, the anti-hIL-4Rα antibody system comprises a human IgG1 of one of the heavy chain variable regions of IGHV 3-9 subtype and one of the light chain variable region of IGKV 2-28 subtype (please See Barbie and Lefranc, Human immunoglobulin kappa variable (IGKV) gene and junction (IGKJ) fragments, Exp. Clin. Immunogenet. 1998, 15: 171-183; and Scaviner, D. et al., Human immunoglobulin Protein display 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 comprises at least one amino acid substitution that results in a change in the charge of the exposed surface of the antibody relative to the germline IGHV 3-9 sequence or the germline IGKV 2-28 sequence. The germline IGHV 3-9 and germline IGKV 2-28 sequences shown here, together with the amino acid position designation numbers and the International Immunogenetics (IMGT) Information System, have been described in Lefranc, M.-P. et al. , IMGT ^® , International Immunogenomics Information System ^® , Nucl.Acids Res. , 37, D1006~D1012 (2009). In some embodiments, the exposed surface comprises a complementarity determining region (CDR). In some embodiments, one or more substitutions of the amino acid are selected from (a) a basic amino acid replacing a natural amino acid in CDR2 (such as at position 58) of IGHV 3-9; ( b) a natural amino acid substitution of an amino acid in CDR3 (such as at position 107) of IGHV 3-9; and (c) a natural amino acid substitution of an amino acid in CDR1 of IGKV 2-28 (such as at position 33) A combination of basic amino acids. The particular arrangement of an antibody, particularly within the charge distribution at the environmental interface (eg at the CDRs), can lead to unpredictable situations in the stability of the antibody in solution.

In some embodiments, the anti-hIL-4Rα antibody comprises at least one amino acid substitution within the framework region of the antibody variable region relative to the germline IGHV 3-9 sequence or the germline IGKV 2-28 sequence Change in torsional strain. In some embodiments, the one or more substitutions of the amino acid are selected from the group consisting of (a) a proline substitution in framework region 3 (FR3) (eg at position 96) of IGHV 3-9 to a non-proline and (b) a non-proline amino acid substituting a proline in framework region 2 (FR2) (eg, position 46) of IGKV 2-28. Changing the peptide chain, especially the twisting ability in a framework region that affects the interface between the CDR and the solvent, will lead to unpredictable conditions in the stability of the antibody in solution.

According to some specific embodiments of the present invention, the anti-hIL-4Rα antibody or its antigen conjugate The composite fragment comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, a HCDR2 of SEQ ID NO:3, and a HCDR3 of SEQ ID NO:4. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a HCVD of SEQ ID NO:1.

According to certain 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 of SEQ ID NO:6, an LCDR2 of SEQ ID NO:7, and an LCDR3 of SEQ ID NO:8. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a LCVD of SEQ ID NO:5.

According to some other specific embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a HCDR1 of SEQ ID NO: 10, a HCDR2 of SEQ ID NO: 11, a HCDR3 of SEQ ID NO: 12, a An LCDR1 of ID NO:14, an LCDR2 of SEQ ID NO:15, and an LCDR3 of SEQ ID NO:16. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a HCVD of SEQ ID NO:9 and an LCVD of SEQ ID NO:13.

According to some other specific embodiments of the present invention, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCDR1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, an HCDR3 of SEQ ID NO: 20, SEQ ID NO: 20, An LCDR1 of ID NO:22, an LCDR2 of SEQ ID NO:23, and an LCDR3 of SEQ ID NO:24. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises a HCVD of SEQ ID NO: 17 and a LCVD of SEQ ID NO: 21.

A non-limiting exemplary antibody in the examples herein is referred to as "mAbl". This antibody is also known as H4H098P in US 7,608,693. mAb1(H4H098 P) comprises a HCVR/LCVR amino acid sequence pair having SEQ ID NO: 1/5, and HCDR1-HCDR2 representing SEQ ID NO: 2-3-4/SEQ ID NO: 6-7-8 - HCDR3/LCDR1-LCDR2-LCDR3 domain.

Another non-limiting exemplary antibody useful in the practice of the invention is referred to as "mAb2". This antibody is also known as H4H083P in US 7,608,693. mAb2(H4H083P) comprises a HCVR/LCVR amino acid sequence pair having SEQ ID NO:9/13, and HCDR1-HCDR2- representing SEQ ID NO:10-11-12/SEQ ID NO:14-15-16 HCDR3/LCDR1-LCDR2-LCDR3 domain.

Yet another non-limiting exemplary antibody useful in the practice of the invention is referred to as "mAb3". This antibody is also known as H4H095P in US 7,608,693. mAb3 (H4H095P) comprises a HCVR/LCVR amino acid sequence pair having SEQ ID NO: 17/21, and HCDR1-HCDR2- representing SEQ ID NO: 18-19-20/SEQ ID NO: 22-23-24 HCDR3/LCDR1-LCDR2-LCDR3 domain.

The amount of antibody or antigen-binding fragment thereof in the pharmaceutical formulation of the present invention depends on the desired specific properties of the formulation and the particular circumstances and uses for which the formulation is intended to be used. In certain 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, A liquid formulation of the antibody at 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 formulations of the present invention contain 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 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 ml, about 165 mg/ml, about 170 mg/ml, about 175 mg/ml, about 180 mg/ml, about 185 mg/ml, about 190 mg/ml, about 195 mg/ml, or about 200 mg/ml of hIL-4Rα Antibodies or antigen-binding fragments thereof.

Excipients and pH

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

In certain embodiments, the pharmaceutical formulations of the present invention comprise at least one organic co-solvent capable of stabilizing the type and amount of the hIL-4Rα antibody under severe handling such as shaking. In some embodiments, the term "stable" means that it can prevent the formation of aggregated antibodies of more than 2% of the total amount of antibodies (on a molar basis) during intense manipulation. In some embodiments, the vigorous operation involves shaking the solution containing the antibody and the organic co-solvent for about 120 minutes.

In certain embodiments, the organic co-solvent is a nonionic surfactant, such as an alkyl poly(oxyethylene). Specific nonionic surfactants that can be incorporated into the formulations of the invention include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and poly-sorbate 85; poloxamers such as poloxamer 181, poloxamer 188, polo-xamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, Sorbitan Monolaurate and Polyoxyethylene Sorbitan 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 specific properties of the formulation and the specific circumstances and uses for which the formulation is intended to be used. In certain embodiments, the amount of surfactant in the formulation is from about 0.1 ± 0.01% to about 2 ± 0.2%. For example, formulations 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% polysorbate 20 or poloxamer 181. For example, formulations 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 capable of stabilizing the type and amount of the hIL-4Rα antibody under heat stress. In some embodiments, the term "stable" means that greater than about 92% of the antibody in its natural configuration can be maintained when the solution containing the antibody and a heat stabilizer is stored at about 45°C for up to about 28 days. In some embodiments, the term "stable" means that when the solution containing the antibody and a heat stabilizer is stored at about 45° C. for up to about 28 days, less than about 5% of the aggregated antibody can be maintained.

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 circumstances and the intended use of the formulation depends 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, pharmaceutical formulations 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% sugar or sugar alcohol (such as sucrose, trehalose or mannose).

The pharmaceutical formulations of the invention may also contain a buffer or buffer system to maintain a stable pH and help stabilize the hIL-4Rα antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 45° C. for up to about 14 days, it can maintain less than 3.0% ± 0.5% of the aggregated antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C for up to about 6 months, it can maintain less than 3.7% ± 0.5% of the aggregated antibody . In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 45°C for up to about 14 days, it can maintain at least 95% ± 0.5% natural form antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C for up to about 6 months, it can maintain at least 96% as determined by size exclusion chromatography. ±0.5% of antibodies in their native form. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 45°C for up to about 14 days, it can maintain at least 62% ± 0.5% neutral conformational antibody. In some embodiments, the term "stable" means that when the solution containing the antibody and buffer is stored at about 25°C for up to about 6 months, it can maintain at least 54% as determined by cation exchange chromatography. ±0.5% of neutral conformational antibodies. By "neutral conformation" is meant the portion of the antibody dialyzed from the main peak in the ion exchange resin, which typically has one of the two sides with a more "basic" peak and the other with a more "acidic" peak.

The pharmaceutical formulations of the invention have a pH of from about 5.2 to about 6.4. For example, the pharmaceutical formulations of the present invention may have a pH of 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 embodiments, the pH is about 5.3±0.2, about 5.9±0.2, or about 6.0±0.2.

In some embodiments, the buffer or buffer system comprises at least one buffer that completely overlaps or is within a partial range of pH 5.2-6.4. In one embodiment, the buffer or buffer system comprises two buffers, the first of which is effective in the pH range of 3.6-5.6 and the second of which is effective in the pH range of 5.5-7.4. In a specific embodiment, the first buffer has about A pKa of 4.8±0.3 and the second buffer has a pKa of about 6.0±0.3. In certain embodiments, the buffer system comprises acetate buffer and histidine buffer. In certain embodiments, the molar acetate contains about 1.3-1.9 parts of histidine per 1 part of molar acetate. In certain embodiments, the molar acetate contains about 1.6 ± 0.25 parts histidine per 1 part molar acetate. In certain embodiments, the acetate is present at a concentration of 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 8.0mM to about 17.0mM, about 8.5mM to about 16.5mM, about 9.0mM to about 16.0mM, about 9.5mM to about 15.5mM, about 10.0mM to about 15.0mM, about 10.5mM to about 14.5mM, 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 embodiments, the histidine is present at a concentration of 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 supplement system comprises about 12.5 mM acetate and about 20 mM histidine at about pH 5.9.

The pharmaceutical formulations of the invention also comprise 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, below At about 14 cPoise, below about 13 cPoise, below about 12 cPoise, below about 10 cPoise, or below about 9 cPoise.

In certain embodiments, the concentration of arginine, preferably L-arginine hydrochloride, contained in the pharmaceutical formulation of the present invention is about 25 mM±3.75 mM, about 50 mM±7.5 mM, or about 100 mM±15 mM . In certain embodiments, the arginine is present at a concentration of 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 26mM.

Representative formulation

According to one aspect of the present invention, the pharmaceutical formulation is a low-viscosity, generally physiologically isotonic liquid formulation comprising: (i) a protein that specifically binds to hIL-4Rα (such as mAb1, mAb2 or mAb3 [as described above] ]) a human antibody at a concentration of about 100 mg/ml or higher; (ii) a buffer system sufficient to buffer at about pH 5.9±0.6; (iii) a sugar specifically used as a heat stabilizer; (iv) protecting the internal structure of the antibody an organic co-solvent for integrity; and (v) an amino acid that maintains a viscosity ready for subcutaneous injection.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binding to hIL-4Rα and comprising a substituted IGHV 3~9 type heavy chain variable region and a substituted IGLV 2~28 type light chain variable region ( Human IgG1 antibody such as mAb1) at a concentration of about 100 mg/ml to about 200 mg/ml; (ii) a buffer system comprising acetate and histidine sufficient to buffer at about pH 5.9±0.6; (iii) as a heat stabilizer sucrose; (iv) polysorbate as organic cosolvent; and (v) arginine as viscosity reducer.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binding hIL-4Rα and comprising a HCDR1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3, a HCDR3 of SEQ ID NO: 4 , an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7, and a human IgG1 antibody at a concentration of about 150 mg/ml ± 25 mg/ml of an LCDR3 of SEQ ID NO: 8; (ii) sufficient to buffer at about Acetate at about 12.5 mM±1.9 mM and histidine at about 20 mM±3 mM at pH 5.9±0.3; (iii) sucrose at about 5%±0.75% w/v; (iv) at about 0.2%±0.03% polysorbate 20 w/v; and (v) arginine as L-arginine hydrochloride at about 25 mM±3.75 mM.

Other non-limiting examples of pharmaceutical formulations of the invention are described elsewhere herein, including the Examples described below.

Stability and viscosity of pharmaceutical formulations

The pharmaceutical formulations of the invention generally exhibit a high degree of stability. The term "safety" in relation to the pharmaceutical formulation herein means that the antibody in the pharmaceutical formulation maintains an acceptable level of chemical structure or biological function under defined storage conditions. The antibody contained in a formulation is still in a stable state even if its chemical structure or biological function cannot maintain 100% after storage for a period of time. In certain instances, the antibody may be considered "stable" when it retains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after storage for a period of time.

Stability can be determined by, inter alia, measuring the percentage of native antibody remaining in the formulation after storage at a defined temperature for a period of time. The percentage of native antibodies can be determined, inter alia, by size exclusion chromatography (eg size exclusion high performance liquid chromatography [SE-HPLC]). The phrase "an acceptable degree of stability" as used herein means that at least 90% of the native antibody is detectable in the formulation after storage at a given temperature for a period of time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% are detectable in the formulation after storage at a defined temperature for a period of time. %, 99%, or 100% natural antibodies. The defined period of time measured as 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 of a storable pharmaceutical formulation determined to be stable 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 to 8°C °C, about 5 °C, about 25 °C, or about 45 °C. For example, a pharmaceutical formulation may be considered stable if greater than about 90%, 95%, 96%, 97%, or 98% of native antibodies can be detected by SE-HPLC after storage at 5°C for 3 months. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 97%, or 98% of native antibodies can be detected by SE-HPLC after storage at 5°C for 6 months. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 97%, or 98% of native antibodies can be detected by SE-HPLC after storage at 5°C for 9 months. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, or 97% of native antibodies can be detected by SE-HPLC after storage at 25°C for 3 months. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, or 97% of native antibodies can be detected by SE-HPLC after storage at 25°C for 6 months. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, or 97% of native antibodies can be detected by SE-HPLC after storage at 25°C for 9 months.

Stability can be determined after storage at a defined temperature for a period of time by, inter alia, measuring the percentage of aggregated antibody within the formulation, wherein stability is inversely proportional to the percentage of aggregated antibody formed. In particular, by size exclusion chromatography (such as size exclusion high performance liquid chromatography [SE-HPLC]) to determine the percentage of aggregated antibody. The phrase "an acceptable level of stability" as used herein means that up to 5% of aggregated antibody is detectable in the formulation after storage at a given temperature for a period of time. In certain embodiments, an acceptable level of stability means that up to about 5%, 4%, 3%, 2%, 1%, 0.5% is detectable in the formulation after storage at a given temperature for a period of time % or 0.1% aggregated antibody. The defined period of time measured as 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 of a storable pharmaceutical formulation determined to be stable 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 to 8°C °C, about 5 °C, about 25 °C, or about 45 °C. For example, a pharmaceutical formulation may be considered stable if less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies are measured after storage at 5°C for 3 months . A pharmaceutical formulation may also be considered as having less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of antibodies in aggregated form after storage at 5°C for 6 months. stable. A pharmaceutical formulation may also be considered stable if less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in an aggregated form is measured after storage at 5°C for 9 months . A pharmaceutical formulation may also be considered stable if less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in an aggregated form is measured after storage at 25°C for 3 months . A pharmaceutical formulation may also be considered stable if less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in an aggregated form is measured after storage at 25°C for 6 months . A pharmaceutical formulation may also be considered stable if less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in an aggregated form is measured after storage at 25°C for 9 months .

Stability can be determined, inter alia, by measuring the ratio of the antibody's migration to the more acidic part (the "acid form") to the main part of the antibody (the "neutral conformation") during ion exchange, wherein stability is related to the acidic part of the antibody Inversely proportional. Without being bound by theory, deamidation of antibodies may result in antibodies with a more negative charge and thus more acidic relative to non-deamidated antibodies (see e.g. Robinson, N., Protein Deamidation, pp. PNAS , April 16, 2002, 99(8):5283~5288). "Acidified" or "deamidated" antibodies may be determined, inter alia, by ion exchange chromatography (eg cation exchange high performance liquid chromatography [CEX-HPLC]). The phrase "an acceptable degree of stability" as used herein means that up to 45% of the acidic form of the antibody can be detected in the formulation after storage at a defined temperature for a period of time. In certain embodiments, an acceptable level of stability means that up to about 45%, 40%, 35%, 30%, 25%, 20 %, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the acidic form of the antibody. The defined period of time measured as 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 of a storable pharmaceutical formulation determined to be stable 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 to 8°C °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 less than about 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, Antibodies in the acidic form at 4%, 3%, 2%, 1%, 0.5%, or 0.1% are considered stable. If a pharmaceutical preparation is stored at 25°C for 3 months and is less than about 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7% , 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the acidic form of the antibody may also be considered stable. When a pharmaceutical formulation is stored at 45°C for 8 weeks, if it measures less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, Antibodies in the acidic form at 2%, 1%, 0.5%, or 0.1% can also be considered diazepam. When a pharmaceutical preparation is stored at 40°C for 2 weeks, if it is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, Antibodies in the acidic form at 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% can also be considered stable.

Other methods may be used to determine the stability of the formulations of the invention, such as thermal stability by differential scanning calorimetry (DSC), mechanical stability by controlled agitation, and absorbance at about 350 or about 405 nm for solutions. 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 OD ₄₀₅ of the formulation changes from time zero to less _than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.02, 0.01, or lower), it was considered stable.

Stability can also be assessed by measuring an antibody's biological activity or binding affinity to its target. For example, after a formulation of the present invention is stored at 5°C, 25°C, 45°C, etc. for a period of time (eg, 1 to 12 months), the anti-IL-4Rα antibody contained in the formulation has a binding affinity of at least 90% before storage. %, 95% or higher binding to IL-4Rα, it can be considered stable. by Binding affinity is determined eg by ELISA or plasmon resonance. Biological activity is determined by an IL-4Rα activity assay, eg, by contacting an IL-4Rα expressing cell with a formulation comprising the anti-IL-4Rα antibody. Binding of such cells to the antibody can be assayed directly, eg, via FACS analysis. Alternatively, the downstream activity of the IL-4Rα system is determined in the presence of the antibody and an IL-4Rα agonist, and then compared to the activity of the IL-4Rα system without antibody. In some embodiments, the IL-4Rα can be endogenous in the cell. In other embodiments, the IL-4Rα can be ectopically expressed in the cell.

Additional methods for determining the stability of antibodies in formulations are described in the Examples below.

In certain embodiments, pharmaceutical formulations of the invention exhibit low to moderate viscosity. The "viscosity" described here may be "kinetic viscosity" or "absolute viscosity". "Dynamic viscosity" is the measured resistance of a liquid to flow under the influence of gravity. When two liquids of equal volume are placed in the same capillary viscometer and flow under gravity, it takes a longer time for a viscous liquid than the less viscous liquid to flow through the capillary. For example, if one liquid takes 200 seconds and another liquid takes 400 seconds to complete its flow, the second liquid is twice as viscous as the first on a dynamic viscosity scale. "Absolute viscosity" refers to the product of kinetic viscosity and liquid density (absolute viscosity = kinetic viscosity x density), which is sometimes called kinetic or simple viscosity. The dimension of dynamic viscosity is L ² /T, where L is length and T is time. Typically, dynamic viscosity is expressed in centistokes (cSt). The SI unit of dynamic viscosity is mm ² /s, which refers to 1 cSt. The unit of absolute viscosity is centipoise (cP). The SI unit of absolute viscosity is centiPascal-second (mPa-s), where 1cP=1mPa-s.

As described herein, a low viscosity liquid formulation of the present invention will exhibit an absolute viscosity of less than about 15 centipoise (cP). For example, a liquid formulation of the invention that is deemed to have "low viscosity" as determined by standard viscosity measurements 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 of between 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 34 cP, about 33 cP, about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP , about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, about 20 cP, about 19 cP, about 18 cP, about 17 cP, about 16 cP, or about 15.1 cP absolute viscosity.

As described in the Examples below, the inventors have unexpectedly discovered that by formulating the antibody with arginine from about 25 mM to about 100 mM, it is possible to obtain antibodies containing high concentrations of anti-hIL-4Rα antibodies (e.g., from about 100 mg/ml up to at least 200 mg/ml). ) of low to medium viscosity liquid formulations. In addition, it has further been found that by adjusting the sucrose content below about 10%, the viscosity of the formulation can be reduced even more.

Container and method of administration for the pharmaceutical preparation

The pharmaceutical formulations of this invention may be placed in any container suitable for the storage of pharmaceutical and other therapeutic compositions. For example, the pharmaceutical formulation may be placed in a sealed and sterilized plastic or glass container of defined volume, such as a vial, ampoule, syringe, cartridge or vial. The formulations of the invention can be placed in different types of glass vials such as clear and opaque (eg amber) glass or plastic bottles. Likewise, any type of syringe may be used to contain or administer the pharmaceutical formulations of the 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 making a glass barrel typically involves the use of a hot tungsten rod that penetrates the glass to create a small hole through which liquid can be drawn from the barrel. This process results in the deposition of trace amounts of tungsten on the inner surface of the barrel. Washing and other process steps can then be used to reduce the amount of tungsten in the barrel. The term "ordinary tungsten" as used herein means that the barrel contains greater than 500 parts per billion (ppb) tungsten. The term "low tungsten" means that the syringe contains less than 500ppb 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 of tungsten.

Rubber plungers used in syringes and rubber stoppers used to close openings in glass vials can be coated to avoid contamination of the syringes or vials, or to preserve their stability. Thus, pharmaceutical formulations of the present invention according to certain embodiments may be placed in a syringe containing a coated plunger, or in a glass vial sealed with a coated rubber stopper. For example, the plunger or rubber stopper can be coated with a fluorocarbon film. Examples of coated plungers or rubber stoppers suitable for use in glass vials and syringes containing pharmaceutical formulations of the present invention are described in U.S. Pat. this. Specially coated rubber plungers and stoppers for use in the context of the present invention are commercially available as "FluroTec®" from West Pharmaceutical Services, Inc. (Lionville, PA).

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

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

Here too, a microsyringe is used to infuse the pharmaceutical formulations of the invention. As used herein, "microsyringe" means the slow administration of large quantities (e.g., up to about 2.5 ml or more) of a therapeutic formulation over an extended period of time (e.g., about 10, 15, 20, 25, 30 minutes or longer) hypodermic infusion set. See eg US 6,629,949, US 6,659,982; and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinfusion sets are most suitable for infusion of large doses of therapeutic proteins containing high concentrations (eg, about 100, 125, 150, 175, 200 mg/ml, or higher), or viscous solutions.

In one embodiment, the liquid pharmaceutical formulation containing about 150±15 mg/ml anti-IL-4Rα antibody is administered subcutaneously from a prefilled syringe in an autoinjector in a volume of about 1±0.15 ml. In another specific embodiment, the formulation is administered from a microsyringe in a volume of between about 1 ml and 2.5 ml. The formulation can be prefilled into a pouch or cartridge of a microsyringe.

Therapeutic use of pharmaceutical preparations

The pharmaceutical formulations of the present invention are particularly useful in the treatment, prevention or alleviation of any disease or disorder associated with IL-4 activity, including diseases or disorders mediated by activation of IL-4Rα. Non-limiting examples of diseases and disorders that can be treated or prevented by administering the pharmaceutical formulations of the invention include various allergic diseases such as atopic dermatitis, allergic conjunctivitis, allergic rhinitis, asthma, and other IgE/Th2 mediated leading diseases.

Accordingly, the present invention includes methods of treating, preventing or ameliorating any disease or disorder associated with IL-4 activity or IL-4Ra activation, including any of the above exemplary diseases, disorders and conditions. The treatment method of the present invention comprises administering the above-mentioned anti-hIL-4Rα antibody-containing formulation to an organism. The organism to which the pharmaceutical formulation is administered can be, for example, any human or non-human animal in need of such treatment, prevention or alleviation, or that would benefit from inhibition or alleviation of IL-4 or IL-4Ra mediated activity. For example, the organism can be an individual who has been diagnosed or thought to be at risk of developing a disease or disorder as described above. The present invention further includes any such pharmaceutical formulations disclosed herein in the manufacture for use in the treatment, prevention or alleviation of any disease or disorder associated with IL-4 activity or IL-4Rα activation (including any of the exemplary diseases, disorders and conditions described above) the use of.

The following examples provide a complete disclosure and illustration of how one skilled in the art can make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. While every effort has been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.), some errors and deviations may have occurred in some experiments. Unless indicated otherwise, parts are parts in moles, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

The activity of forming the primary formulation involved screening organic co-solvents, heat stabilizers and buffers in the liquid formulation of mAbl (anti-IL-4Rα antibody of the invention) to confirm that the excipients are compatible with the protein and facilitate its Stability, while maintaining the molar osmolality and viscosity for subcutaneous injection. Buffer conditions were also checked to determine the optimum pH for highest protein stability.

Example 1. Organic co-solvents

mAb1 has been found to be unstable under oscillatory stress. RP-HPLC Analytical (RP-HPLC) and size-exclusion high-performance liquid chromatography (SE-HPLC) analysis demonstrated protein loss and increase in aggregated protein when mAb1 was shaken at room temperature (Table 1, see "No cosolvent "data). Addition of organic co-solvents to mAbl solutions prevented protein degradation measured by SE-HPLC and RP-HPLC (Table 1). However, the addition of some organic co-solvents was found to reduce the thermal stability of mAbl (Table 2). Formulations containing PEG 3350 (3%) and PEG 300 (10% and 20%) were found to lose recovered protein as measured by RP-HPLC after heat stress (Table 2). Furthermore, formulations containing PLURONIC F68 (poloxamer 181) (0.2%), PEG 300 (10% and 20%) and propylene glycol (20%) formed more aggregation as measured by SE-HPLC than formulations without cosolvent things. Polysorbate 20 (0.2%) and polysorbate 80 (0.2%) are fairly stable against oscillations and heat stress.

According to Table 1, 0.3ml of 15mg/ml mAb1 in 10mM phosphate pH 6.0 in a 2ml glass bottle 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 compare the relative change in OD at 405 nm of the starting materials. The percentage of native and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC) method. The SE-HPLC results shown in the "Starting Materials" table are the average value of each formulation without shaking.

According to Table 2, 0.3 ml of 15 mg/ml mAb1 in 10 mM phosphate pH 6.0 in a 2 ml glass bottle 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 compare the relative change in OD at 405 nm of the starting materials. The percentage of total recovered mAbl was determined by reverse phase HPLC (RP-HPLC) method. The percentage of native and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC) method. The SE-HPLC results shown in the "Starting Materials" table are the average of each formulation that was not heat stressed.

Example 2. Heat stabilizer

Various heat stabilizers, such as sugars, amino acids and inorganic salts were examined for their ability to inhibit the degradation of mAbl upon storage at about 45°C. A summary of studies on heat stabilizers is shown in Table 3. Formulations containing sucrose or trehalose had a higher mAbl stabilizing effect (determined by SE-HPLC) when incubated in high temperature solution. Sucrose was chosen as the stabilizer because of its long history of safety in monoclonal antibody formulations.

According to Table 3, 0.3 ml of mAbl at 25 mg/ml in 10 mM acetate pH 5.3 in 2 ml glass vials 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 compare the relative change in OD at 405 nm of the starting materials. All samples had negligible differences in turbidity. The percentage of total recovered mAbl was determined by reverse phase HPLC (RP-HPLC) method. The percentage of native and aggregated mAbl was determined by size exclusion HPLC (SE-HPLC) method. Acidic or basic species were defined as the total amount of the mAb1 peak dialyzed from the cation exchange (CEX-HPLC) column at earlier or later retention times than the main peak, respectively. shown in the "Starting Materials" table SE-HPLC results are the average of formulations that were not heat stressed.

Example 3. Buffers and pH

The effect of pH and buffer type on mAbl stability was also examined. 15 mg/ml of mAbl 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). The greatest protein stability was determined by SE-HPLC and CEX-HPLC when mAbl was formulated in pH 6.0 histidine buffer or pH 5.3 acetate buffer (Table 4 and Table 5). The acetate buffer system has a wider range of pH stability and a lower variable charge formation rate than the formulation containing histidine buffer (Table 5). Therefore, acetate buffer at pH 5.3 was chosen for the formulation of mAbl drug.

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

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

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。 The formulation formation test was shown in alkaline conditions (pH

6.5) mAb1 in the lower solution can be deamidated. Conversely, pH below 5.0 has been found to increase the rate of formation of mAbl of variable molecular weight. According to these data, the pH of the mAbl formulation was maintained between about pH 5.6 and pH 6.2. mAb1 was found to be stable in this pH range.

The effect of pH and buffer type on the stability of mAbl was further evaluated in formulations containing 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 (Table 6). Comparing the individual buffer systems, mAb 1 was most stable in its formulation with histidine and acetate at about pH 5.9. The slowest rate of aggregation was when mAbl was formulated in this mixed buffer system (SE-HPLC) (Table 6).

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

Example 4. Control of Viscosity and Tension

Evaluate mixing various excipients with high concentrations of mAb1 (i.e. 150 mg/ml, 175 mg/ml and 200 mg/ml) viscosity and tonicity (expressed as molar osmolarity). The contents of sucrose, sodium chloride and L-arginine hydrochloride were adjusted to form a low viscosity and physiological isotonic formulation containing high concentrations of mAb1 for comfortable and rapid high-volume subcutaneous infusion of mAb1 (Table 7). The liquid formulation (formulation A ) is the optimal formulation with low viscosity (approximately 8.5 cPoise) and physiological isotonicity (approximately 293 mOsm/kg) while still maintaining mAbl stability.

Example 5. Characterization of Formulation A

The main degradation pathways during the formation of mab1 liquid formulations were the generation of aggregates, fragmentation products and charge variants. The formation of these degradation products can be reduced by formulating mAbl 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. These mAb1 formulated at 150 mg/ml were found to be clear to slightly opalescent liquid solutions substantially free of macroscopic particles.

The formulated mAbl was physically and chemically stable under various stress (25°C and 45°C incubation) and immediate storage conditions (5°C) (Table 8). The appearance of this mAbl was not affected when stored at 25°C (3 months) or 5°C for 6 months. Furthermore, no effect was found on solution pH, turbidity or the amount of mAb1 recovered. After the formulated mAbl was stored at 25°C for 3 months, the antibody was not significantly degraded by SE-HPLC and the degradation by CEX-HPLC was only 3.3% higher. It has been found that after storage at 45°C for 8 weeks, the amount of degradation determined by SE-HPLC and CEX-HPLC is increased to show the shape Aggregation and charge variation are the main degradation pathways of the mAb1 antibody molecule. The formulated mAb1 antibody was not found to be 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 species were defined as the total amount of the mAb1 peak dialyzed from the cation exchange (CEX-HPLC) column at earlier or later retention times than the main peak, respectively.

Example 6. Container

The stability of the filter sterilized formulation containing mAbl has been determined. Millipore MILLIPAK filter units are used for manufacturing in the clinical supply, while filters of the same composition (Millipore Millex DURAPORE) are used in the laboratory.

With a minimum of 2.5ml of 150mg/ml mAb1 at pH 5.9, 5% (w/v) sucrose, 25mM L-arginine hydrochloride, 0.2% (w/v) polysorbate 20, 12.5mM acetate, 20 Fill mM histidine into a 5ml glass bottle. 0.5ml of over-formulation was used in the 5ml bottle to ensure that 2.0ml of formulation could be drawn. This excess is not intended to compensate for loss of mAbl or mAbl-containing formulations during manufacture, degradation during manufacture, degradation during storage (shelf life), or to extend shelf life.

The stability of the formulated mAbl (Formulation A) was not affected when stored in polypropylene glycol tubes, polystyrene tubes, polycarbonate tubes, or in glass vials containing stainless steel blocks compared to storage in glass vials (Table 9).

According to Table 9, 150 mg/ml mAb1 at pH 5.9, 5% sucrose, 25 mM arginine hydrochloride, 0.2% PS-20, 20 mM histidine, 12.5 mM acetate were stored in various materials at 40°C for 14 days . OD = Optical Density; RP-HPLC = Reversed Phase High Performance Liquid Chromatography; SE-HPLC = Size Exclusion High Performance Liquid Chromatography; and CEX-HPLC = Cation Exchange High Performance Liquid Chromatography. Turbidity was recorded as the difference in turbidity at OD at 405 nm compared to the starting material. Acidic or basic species were defined as the total amount of the mAb1 peak that was dialyzed from the CEX-HPLC column at an earlier or later retention time than the main peak, respectively.

<110> Regeneron Pharmaceuticals, Inc.

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

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Claims (18)

A liquid pharmaceutical preparation comprising: (i) a human antibody specifically binding to human interleukin-4 receptor alpha (hIL-4Rα) at a concentration of 100±10 mg/ml to 200±20 mg/ml, wherein the antibody comprising (a) a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3); and (b) a light chain variable region (LCVR) comprising the amino acid sequence comprising SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively Light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3); (ii) a buffer containing acetate at a concentration of 5.0mM to 20mM; (iii) containing a concentration of 0.1% w/v to 2% w/ Organic co-solvent of polysorbate in v; (iv) heat stabilizer comprising sucrose or trehalose at a concentration of 2.5% w/v to 10% w/v; and (v) spermine at a concentration of 25mM to 100mM A viscosity reducer for acids; wherein the formulation has a pH of 5.6 to 6.2. Such as the liquid pharmaceutical preparation of item 1 of the patent scope, wherein: (a) the concentration of the antibody is 100 mg/ml; or (b) the concentration of the antibody is 150 mg/ml; or (c) the concentration of the antibody is 175 mg /ml; or (d) the concentration of the antibody is 200mg/ml. Such as the liquid pharmaceutical preparation of item 1 of the scope of the patent application, wherein the concentration of the antibody is 150±15mg/ml. Such as the liquid pharmaceutical preparation of claim 1, wherein the buffer contains acetate at a concentration of 10mM to 15mM. Such as the liquid pharmaceutical preparation of item 1 of the patent scope of the application, wherein the buffer solution further comprises Histidine at a concentration of 10mM to 30mM. Such as the liquid pharmaceutical preparation according to claim 5, wherein the buffer contains histidine at a concentration of 15mM to 25mM. Such as the liquid pharmaceutical preparation of claim 1, wherein the buffer contains acetate at a concentration of 12.5mM±1.85mM and histidine at a concentration of 20mM±0.3mM. Such as the liquid pharmaceutical preparation of item 1 of the patent scope, wherein the organic co-solvent is selected from polysorbate 20 or polysorbate 80. Such as the liquid pharmaceutical preparation of item 8 of the scope of the patent application, wherein the organic co-solvent is polysorbate 80 with a concentration of 2%±0.03% w/v. Such as the liquid pharmaceutical preparation of item 8 of the scope of the patent application, wherein the organic co-solvent is polysorbate 20 with a concentration of 0.2%±0.03% w/v. For example, the liquid pharmaceutical preparation in item 1 of the scope of the patent application, wherein the heat stabilizer is sucrose with a concentration of 5%±0.75% w/v. Such as the liquid pharmaceutical preparation in item 1 of the scope of the patent application, wherein the viscosity reducing agent is arginine with a concentration of 25mM±3.75mM or 50mM±7.5mM. LCDR3. For example, the liquid pharmaceutical preparation of claim 1, wherein the antibody comprises HCVR comprising the amino acid sequence of SEQ ID NO: 1 and LCVR comprising the amino acid sequence of SEQ ID NO: 5. Such as the liquid pharmaceutical preparation of any one of items 1 to 13 in the scope of the patent application, which is placed in a glass bottle. Such as the liquid pharmaceutical preparation of any one of items 1 to 13 in the scope of the patent application, it is put into a syringe. Such as the liquid pharmaceutical preparation of any one of items 1 to 13 in the scope of the patent application, it is put into a micro-syringe. For example, the liquid pharmaceutical preparation of any one of items 1 to 13 in the scope of the patent application, which is placed in an automatic injection delivery device. Such as the liquid pharmaceutical preparation of any one of items 1 to 13 in the scope of the patent application, which is placed in a one-shot delivery device.