TWI679988B - Stabilized formulations containing anti-interleukin-4 receptor(il-4r) antibodies - Google Patents

Abstract

The present invention provides a pharmaceutical formulation comprising a human antibody capable of specifically binding to human interleukin-4 receptor (hIL-4R). In addition to the anti-hIL-4R antibody, the formulation may contain at least one amino acid, at least one carbohydrate, or at least one non-ionic surfactant. The pharmaceutical formulation of the invention can exhibit a substantial degree of antibody stability after storage for several months.

Description

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

The invention relates to the field of therapeutic antibody formulations. More specifically, the invention relates to the field of pharmaceutical formulations comprising human antibodies capable of specifically binding to the human interleukin-4 receptor.

Sequence Listing

This patent specification also includes a ST.25 matching text file of the sequence listing. The article The text is incorporated herein by reference. Incorporates the same sequence listing as the text file that matches ST.25 On paper as part of this patent specification.

Therapeutic macromolecules (such as antibodies) must be formulated in a way that not only makes the molecule suitable for patient administration, but also maintains stability during storage and subsequent use. For example, a therapeutic antibody in a liquid solution is prone to degradation, aggregation, or poor chemical modification unless the solution is properly formulated. The stability of antibodies in a liquid formulation depends not only on the type of excipients used in the formulation, but also on the relative amount and ratio between the excipients. In addition, other factors must be considered in addition to stability when preparing a liquid antibody formulation. Examples of such additional considerations include the viscosity of the solution and the concentration of antibodies 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 stable, contain the appropriate antibody, and have the appropriate viscosity and other properties to facilitate administration of the formulation to a patient.

Antibodies to human interleukin-4 receptor alpha (hIL-4Rα) are an example of therapeutically relevant macromolecules that need to be properly formulated. Anti-hIL-4Rα antibodies are used clinically to treat or prevent diseases such as atopic dermatitis and allergic asthma, as well as other conditions. Range of anti-hIL-4Rα antibodies Examples have been specifically described in 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 anti-hIL-4Rα antibodies that is sufficiently stable and suitable for administration to patients.

Summary of invention

The present invention satisfies the aforementioned needs by providing a pharmaceutical formulation containing a human antibody capable of specifically binding to human interleukin-4 receptor alpha (hIL-4Rα).

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

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 includes 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) each containing a sequence identification number: 2, a sequence identification number: 3, and a sequence identification number: 4. Heavy chain variable region (HCVR); and (b) contain light chain complementarity determining regions 1, 2, and 3 (LCDR1-LCDR2-LCDR3, respectively) that contain sequence identification numbers: 6, sequence identification numbers: 7, and sequence identification numbers: 8 ), A light chain variable region (LCVR). In a specific embodiment, the antibody includes an HCVR and an LCVR each containing an amino acid sequence having a sequence identification number: 1 and a sequence identification number: 5.

In a specific embodiment, the antibody includes 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) each containing a sequence identification number: 10, a sequence identification number: 11 and a sequence identification number: 12 Heavy chain variable region (HCVR); and (b) light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3, respectively) containing sequence identification numbers: 14, sequence identification numbers: 15 and sequence identification numbers: 16 ), A light chain variable region (LCVR). In a special In a specific embodiment, the antibody includes an HCVR and an LCVR each containing an amino acid sequence having a sequence identification number: 9 and a sequence identification number: 13 respectively.

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

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 a specific embodiment, the liquid pharmaceutical buffer comprises one or more buffering agents 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 buffer solutions. 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 an acetate buffer and the second buffer is a histidine buffer. In a specific embodiment, the concentration of the 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% of "weight by 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 of polysorbate 20.

In a specific embodiment, the heat stabilizer is a sugar. In a specific embodiment In this, 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 specific 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 detackifier is L-spermine hydrochloride.

In a specific embodiment, the concentration of the detackifier is less than about 100 mM. In a specific embodiment, the concentration of the detackifier is 50 mM ± 7.5 mM. In another specific embodiment, the concentration of the detackifier 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 molar osmolality of the liquid pharmaceutical formulation is less than about 450 mOsm / kg. In a specific embodiment, the molar osmolality of the liquid pharmaceutical formulation is about 290 ± 20 mOsm / kg.

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

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

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

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 a sequence identification number: 1 and Sequence identification number: heavy chain variable region (HCVR) and light chain variable region (LCVR) of the amino acid sequence of 5; (ii) about 12.5 mg / ml ± 2 mM acetate; (iii) about 20 mM ± 3 mM (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 spermine 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 molar osmolality of the liquid pharmaceutical formulation is 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 storage at 5 ° C for six months 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 storage at 45 ° C for eight weeks when measured by size exclusion chromatography.

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

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

In one aspect, a stable low viscosity isotonic liquid pharmaceutical formulation containing at least 100 mg / ml of a stable 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 concentration of the antibody is about 150 mg / ml ± 15 mg / ml.

In a specific embodiment, the antibody includes a sequence identification number: any of 1 to 8. Or more amino acid sequences. In a specific embodiment, the antibody includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR / LCVR combination includes a sequence identification number: 2-3-4 and sequence identification, respectively. The heavy and light chain complementarity determining regions (HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3) of the amino acid sequence numbered 6-7-8. 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 centistokes, 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 Moire osmolality. In a specific embodiment, the formulation comprises a molar osmolarity of 290 ± 20 mOsm / kg.

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

In a specific embodiment, the antibody has storage stability at about 45 ° C for at least about eight weeks. 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 in acid form is stored at 45 ° C. for about eight weeks when measured by cation exchange chromatography.

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

In a specific embodiment, the formulation comprises 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 amine 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 comprises an organic co-solvent at a concentration 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 a polysorbate 20 having a concentration of about 0.2% ± 0.03% w / v.

In a specific embodiment, the formulation comprises a thermal stabilizer at a concentration 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 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 detackifier at a concentration of no more than about 100 mM. In a specific embodiment, the detackifier 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 osmolality of about 290 ± 20 mOsm / kg, and comprises: (i) 150 mg / ml ± 15mg / ml anti-hIL-4Rα antibody, wherein the antibody contains an HCVR and an LCVR each containing an amino acid sequence of sequence identification number: 1 and sequence identification number: 5; (ii) 12.5mM ± 1.9mM acetic acid 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 example, (i) at least about 98% of the antibody retains its natural configuration when stored at 5 ° C for at least about six months when measured by size exclusion chromatography; (ii) when determined by size exclusion At least about 90% of the antibody retains its natural configuration when stored at 45 ° C for at least about eight weeks when measured by chromatography; (iii) when measured at 45 ° C for eight weeks when measured by cation exchange chromatography, Acid form antibodies; and (iv) less than about 4% of antibody aggregates when stored at 25 ° C for about six months when measured by size exclusion chromatography.

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

Other specific embodiments of the present invention will be better understood from the review detailed later.

Detailed description of the invention

Before explaining the present invention, it should be understood that the present invention is not limited to the specific specific examples and experimental conditions, and therefore, there may be different methods and conditions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "about" 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 (such as 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 disclosures herein are incorporated by reference in their entirety to fully describe the present invention.

Pharmaceutical formulations

The statement of "pharmaceutical formulation" herein means a combination of at least one active ingredient (such as small molecules, macromolecules, compounds, etc. that can exhibit biological effects in human or non-human animals), and The active ingredient is admixed with one or more additional inactive ingredients to be therapeutically administered to a human or non-human animal. Unless expressly stated otherwise, the term "formulation" herein means "pharmaceutical formulation". The invention provides a pharmaceutical formulation comprising at least one therapeutic polypeptide. According to some specific embodiments of the present invention, the therapeutic polypeptide is an antibody or an antigen-binding fragment thereof that specifically binds human interleukin-4 antibody α (hIL-4Rα). More specifically, the present invention includes organic antibodies containing (i) a human antibody that specifically binds hIL-4Rα; (ii) an acetate / histamine buffer system; (iii) a non-ionic surfactant Solvents; (iv) a carbohydrate stabilizer; and (v) a demuxing pharmaceutical formulation. Specific exemplary ingredients and formulations contained in 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 an 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 certain embodiments, the pharmaceutical formulations contained in the present invention Antibodies in the body specifically bind the extracellular region of hIL-4Rα. An exemplary human IL-4 receptor alpha (hIL-4Rα) amino acid sequence is described in SEQ ID NO: 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 shown in the amino acid sequence of SEQ ID NO: 26.

The term "antibody" as used herein generally refers to an immunoglobulin molecule and its multimers comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by a disulfide chain. (Such as IgM); however, immunoglobulin molecules composed only of heavy chains (ie, no light chains) fall within the definition of "antibody". Each heavy chain includes a heavy chain variable region (herein referred to as HCVR or V _H ) 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 hypervariable regions interspersed with more conserved regions called framework regions (FR) called complementarity determining regions (CDRs). Each V _H and V _{L is} composed of three CDRs and four FRs from the amine end to the carboxy end 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 the complete antibody molecule as well as its antigen-binding fragments. The "antigen-binding region" or "antigen-binding fragment" (or simply "antibody region" or "antibody fragment") of an antibody described herein means that it retains the ability to specifically bind hIL-4Rα or its epitope One or more fragments of an antibody.

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

The terms "specifically bind" and the like mean that an antibody or an antigen-binding fragment thereof forms an complex with an antigen that is relatively stable under physiological conditions. 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 plasma 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 material or chemicals.

Exemplary anti-hIL-4Rα antibodies incorporated into the pharmaceutical formulations of the 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 antibody 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 Kappa Variable Region (IGKV) Gene and Link (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 linker regions, Exp. Clin. Immunogenet. 1999, 16: 234 ~ 240).

In some embodiments, the anti-hIL-4Rα comprises at least one amino acid substitution, which results in a change in the charge of the antibody relative to the exposed surface of 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, as well as amino acid position designation numbers and the International Immunological Genetics (IMGT) information system have been described in Lefranc, M.-P. et al. , IMGT ^® , Immune Genetics Information System ^® , Nucl. Acids Res. , 37, D1006 ~ D1012 (2009). In some embodiments, the exposed surface includes a complementary determining region (CDR). In some specific embodiments, the one or more substitutions of the amino acid are selected from (a) replacing a natural amino acid in CDR2 (as in position 58) of IGHV 3-9 with a basic amino acid; ( b) a natural amino acid replaces an amino acid in CDR3 of IGHV 3-9 (as in position 107); and (c) a natural amino acid replaces CDR1 of IGKV 2-28 in (as in position 33) a A combination of basic amino acids. The special arrangement of an antibody, especially within the charge distribution of the environmental interface (such as the CDR), will cause an unexpected situation in the stability of the antibody in the solution.

In some embodiments, the anti-hIL-4Rα antibody comprises at least one amino acid substitution, which is generated in the framework region of the variable region of the antibody relative to the germline IGHV 3-9 sequence or the germline IGKV 2-28 sequence. Changes in torsional strain. In some specific embodiments, the one or more substitutions of the amino acid are selected from the group consisting of (a) a proline acid and a non-proline group in the framework region 3 (FR3) (eg, position 96) of IGHV 3-9 Amino acid; and (b) a combination of a non-proline amino acid substituted with a proline in framework region 2 (FR2) (as in position 46) of IGKV 2-28. Alteration of the peptide chain, in particular, refers to the ability of the backbone region to affect the torsion ability of the CDR and the solvent interface, which will cause the stability of the antibody in the solution to produce unexpected conditions.

According to some specific embodiments of the present invention, the anti-hIL-4Rα antibody or its antigen binding The combined fragment includes a heavy chain complementary determining region (HCDR) with a sequence identification number: 2, an HCDR2 with a sequence identification number: 3, and an HCDR3 with a sequence identification number: 4. In certain 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) of sequence identification number 6 and an LCDR2 of sequence identification number: 7, And an LCDR3 with a sequence identification number: 8. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an LCVD of 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 of sequence identification number: 10, an HCDR of sequence identification number: 11 of HCDR2, an sequence of HCDR3 of sequence identification number: 12, An identification number: an LCDR of 14; a serial identification number: an LCDR2 of 15; and an LCDR3 of a serial identification number: 16. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD having a sequence identification number: 9 and an LCVD having a 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 of sequence identification number: 18, an HCDR of sequence identification number: 19 of HCDR2, an sequence of HCDR3 of sequence identification number: 20, An LCDR1 with an identification number: 22, an LCDR2 with a serial identification number: 23, and an LCDR3 with a serial identification number: 24. In some specific embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD having a sequence identification number: 17 and an LCVD having a sequence identification number: 21.

A non-limiting exemplary antibody in the examples herein is referred to as "mAb1". This antibody is also known as H4H098P in US 7,608,693. mAb1 (H4H098P) contains an HCVR / LCVR amino acid sequence pair with a 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 useful in the practice of the present 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 a sequence identification number: 9/13, and HCDR1-HCDR2- representing the sequence identification number: 10-11-12 / sequence identification number: 14-15-16 HCDR3 / LCDR1-LCDR2-LCDR3 domain.

Yet another non-limiting exemplary antibody useful in the practice of the present invention is referred to as "mAb3". This antibody is also known as H4H095P in US 7,608,693. mAb3 (H4H095P) contains an HCVR / LCVR amino acid sequence pair with a sequence identification number: 17/21, and HCDR1-HCDR2- representing the 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 thereof in the pharmaceutical formulation of the present invention depends on the particular properties desired for the formulation and the particular situation and application in 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, 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, and about 130 mg / ml. ml, about 131 mg / ml, about 132 mg / ml, about 133 mg / ml, about 134 mg / ml, about 135 mg / ml, about 140 mg / ml, about 145 mg / ml, about 150 mg / ml, about 155 mg / ml, about 160 mg / 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 specifically binds hIL-4Rα Antibodies or antigen-binding fragments thereof.

Excipients and pH

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

In certain embodiments, the pharmaceutical formulation of the present invention comprises at least one organic co-solvent capable of stabilizing the type and amount of the hIL-4Rα antibody under severe manipulations, such as shaking conditions. In some embodiments, the term "stable" means that the formation of aggregated antibodies, which can prevent the total amount of antibodies (on a mole basis) during the intense operation, exceeds 2%. In some embodiments, the intense 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 surface active Agents, such as alkyl poly (oxyethylene). Specific non-ionic surfactants that can be incorporated into the formulations of the present 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, 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 particular properties desired for the formulation and the particular situation and application in which the formulation is intended 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 0.18%, 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 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 cosolvents that 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 contains 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 when the solution containing the antibody and heat stabilizer is stored at about 45 ° C up to about 28 days, it can maintain greater than about 92% of the natural conformational 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 the group consisting of sucrose, trehalose, and mannose, or any combination thereof. The content of the heat stabilizer 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%, and about 4.5% Sugar or sugar alcohol to about 8%, about 5% to about 7.5%, about 5.5% to about 7%, or about 6.0% to about 6.5%. 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%, and 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 formulation of the present invention may also contain a buffer or a 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 buffer is stored at about 45 ° C for 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 the buffer is stored at about 25 ° C for about 6 months, the aggregated antibody can maintain less than 3.7% ± 0.5% . In some embodiments, the term "stable" means that at least 95% can be maintained by size exclusion chromatography when the solution containing the antibody and buffer is stored at about 45 ° C up to about 14 days. ± 0.5% natural conformation 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 by size exclusion chromatography for at least 96 % ± 0.5% natural conformation antibody. 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 62% by cation exchange chromatography. 0.5% neutral conformation 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 by at least 54% as determined by cation exchange chromatography. ± 0.5% neutral conformation antibody. The "neutral configuration" means the portion of the antibody in the ion exchange resin dialyzed from the main peak, which generally tends to be more "basic" on one side and more "acidic" on the other.

The pharmaceutical formulation of the present invention has a pH from about 5.2 to about 6.4. For example, the pharmaceutical formulation of the present invention may 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 The 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 in a range of pH 5.2 to 6.4. In a specific embodiment, the buffering agent Or the buffer system contains two buffering agents, 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 comprises an acetate buffer and a histidine buffer. In some specific embodiments, each part of the molar ratio 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 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 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 slow system comprises about 12.5 mM acetate at about pH 5.9 and about 20 mM histidine.

The pharmaceutical formulation of the present invention also includes one or more excipients for maintaining a low viscosity or reducing the viscosity of a formulation containing a high concentration 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 35 cPoise, below 30 cPoise, below 25 cPoise, below 20 cPoise, below 15 cPoise, low At about 14 cPoise, below about 13 cPoise, below about 12 cPoise, below about 10 cPoise, or below about 9 cPoise.

In some embodiments, the concentration of arginine, preferably L-spermine hydrochloride, in the pharmaceutical formulation of the present invention is about 25mM ± 3.75mM, about 50mM ± 7.5mM, or about 100mM ± 15mM . In some specific embodiments, the concentration of the spermine 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 present invention, the pharmaceutical formulation is a low-viscosity, usually physiologically isotonic liquid formulation, comprising: (i) specifically binding to hIL-4Rα (eg, mAb1, mAb2, or mAb3 [as described above) ]) 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 especially used as a heat stabilizer; (iv) protecting the internal structure of the antibody Integral organic co-solvents; and (v) monoamino acids that maintain viscosity that is easy to use for subcutaneous injection.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binding hIL-4Rα and comprising a substituted IGHV type 3-9 heavy chain variable region and a substituted IGLV type 2-28 light chain variable region ( Such as mAb1) human IgG1 antibody at a concentration of about 100 mg / ml to about 200 mg / ml; (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) a polysorbate as an organic co-solvent; and (v) arginine as a tackifier.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binding hIL-4Rα and an HCDR1 comprising a sequence identification number: 2; an HCDR2 having a sequence identification number: 3; an HCDR3 having a sequence identification number: 4 1. An LCDR with a sequence identification number: 6; an LCDR2 with a sequence identification number: 7; an LCDR3 with a sequence identification number: 8; a human IgG1 antibody at a concentration of about 150 mg / ml ± 25 mg / ml; (ii) sufficient to buffer at about about 12.5 mM ± 1.9 mM acetate and about 20 mM ± 3 mM histamine 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 invention have been 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" as used herein with respect to the pharmaceutical formulation means that the antibodies within the pharmaceutical formulation retain an acceptable degree of chemical structure or biological function under defined storage conditions. Store antibodies in a formulation for a period of time After time, even though its chemical structure or biological function cannot be maintained at 100%, it is still stable. In some cases, the structure or function of the antibody maintained at about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage for a period of time can be considered "stable."

Stability can be determined after storage at a defined temperature for a period of time, in particular by measuring the percentage of natural antibodies retained in the formulation. The percentage of natural antibodies can be determined in particular by size exclusion chromatography, such as size exclusion high performance liquid chromatography [SE-HPLC]. As used herein, the phrase "an acceptable level of stability" means that at least 90% of the natural antibodies are 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 can be detected 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 determined to be a stable storable pharmaceutical formulation can be any temperature from about -80 ° C to about 45 ° C, such as 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 capable of measuring greater than about 90%, 95%, 96%, 97%, or 98% of natural antibodies by SE-HPLC after storage at 5 ° C for 3 months, it can be considered stable. If a pharmaceutical formulation has a natural antibody greater than about 90%, 95%, 96%, 97%, or 98% measured by SE-HPLC after storage at 5 ° C for 6 months, it can also be considered stable. If a pharmaceutical formulation has a natural antibody greater than about 90%, 95%, 96%, 97%, or 98% measured by SE-HPLC after storage at 5 ° C for 9 months, it can also be considered stable. If a pharmaceutical formulation is capable of measuring more than about 90%, 95%, 96%, or 97% of natural antibodies by SE-HPLC after storage at 25 ° C for 3 months, it can also be considered stable. If a pharmaceutical formulation has a natural antibody greater than about 90%, 95%, 96%, or 97% measured by SE-HPLC after storage at 25 ° C for 6 months, it can also be considered stable. If a pharmaceutical formulation is capable of measuring greater than about 90%, 95%, 96%, or 97% of natural antibodies by SE-HPLC after storage at 25 ° C for 9 months, it can also be considered stable.

Stability can be determined after storage at a defined temperature for a period of time, in particular by measuring the percentage of aggregated antibodies in the formulation, where stability and the percentage of aggregated antibodies formed The fraction is inversely proportional. The percentage of aggregated antibodies can be determined in particular by size exclusion chromatography, such as size exclusion high performance liquid chromatography [SE-HPLC]. As used herein, the phrase "an acceptable level of stability" 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 level 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% of aggregated 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 determined to be a stable storable pharmaceutical formulation can be any temperature from about -80 ° C to about 45 ° C, such as 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, a pharmaceutical formulation can be considered stable if it detects less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies after storage at 5 ° C for 3 months. . A pharmaceutical formulation can also be considered stable if it detects aggregated antibodies below about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after storage at 5 ° C for 6 months. A pharmaceutical formulation that has been stored at 5 ° C for 9 months can also be considered stable if it detects less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies. A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies after storage at 25 ° C for 3 months can also be considered stable. A medicinal formulation that has been stored for 6 months at 25 ° C can also be considered stable if it detects less than about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of aggregated antibodies. A pharmaceutical formulation that has been stored at 25 ° C for 9 months and if it detects less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies can also be considered stable.

Its stability can be determined, in particular, by measuring the ratio of antibody migration to the more acidic part ("acid form") to the main part of the antibody ("neutral configuration") during ion exchange, where the stability is compared to the acid form part of the antibody Inversely proportional. Without being bound by theory, deamidation of antibodies can result in antibodies with more negative charges and therefore are more acidic than non-deamidated antibodies (see, for example, Robinson, N., Protein deamidation, PNAS , April 16, 2002, 99 (8): 5283 ~ 5288). An "acidified" or "deamidated" antibody can be measured, in particular, by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]). As used herein, the phrase "an acceptable level of stability" means that up to 45% of the acid form 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% 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% of the acid form 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 determined to be a stable storable pharmaceutical formulation can be any temperature from about -80 ° C to about 45 ° C, such as 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% of the acid form antibody can be considered stable. If a pharmaceutical formulation is stored at 25 ° C for 3 months, if it 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 acid form antibody can also be considered stable. If a pharmaceutical formulation is stored at 45 ° C for 8 weeks, if it is less than 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 less than 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 using micro-scanning calorimetry (DSC), mechanical stability using controlled agitation, and absorbance measurement solutions at about 350 or about 405 nm. Turbidity. For example, a pharmaceutical formulation of the present invention is stored at about 25 deg.] C to about 5 ℃ after 6 months or more if the formulation is less than about 0.05 OD ₄₀₅ (e.g., 0.04,0.03,0.02 zero time OD ₄₀₅ changes from, 0.01, or lower) is considered stable.

The stability of an antibody can also be assessed by measuring its biological activity or its affinity for binding to its target. For example, after a formulation of the present invention is stored at, for example, 5 ° C, 25 ° C, 45 ° C for a period of time (for example, 1-12 months), the anti-IL-4Rα antibody contained in the formulation has an antibody binding affinity of at least 90 before storage %, 95% or higher when bound to IL-4Rα can be considered stable. By Binding affinity is determined, for example, by ELISA or plasma resonance. Biological activity is measured by an IL-4Rα activity assay, such as contacting a cell expressing IL-4Rα with a formulation containing the anti-IL-4Rα antibody. The binding effect of such cells to the antibody can be determined directly, 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, but the activity of the IL-4Rα system without antibodies is compared. In some embodiments, the IL-4Rα may be endogenous to the cell. In other embodiments, the IL-4Rα may be ectopically expressed in the cell.

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

In certain embodiments, the pharmaceutical formulations of the invention exhibit low to moderate viscosity. As used herein, "viscosity" can be "dynamic viscosity" or "absolute viscosity". "Dynamic viscosity" is the measured flow resistance of a liquid under the influence of gravity. When two equal volumes of liquid are placed in the same capillary viscometer and flow under gravity, a viscous liquid needs a lower viscosity liquid to flow through the capillary for a longer time. For example, if one liquid takes 200 seconds and another liquid takes 400 seconds to complete its flow, a dynamic viscosity scale marks the second liquid to be twice the viscosity of the first. "Absolute viscosity" refers to a product with 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 L series length and the T series time. In general, dynamic viscosity is expressed in centiseconds (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 below about 15 centipoise (cP). For example, if a liquid formulation of the present invention is considered to have "low viscosity" when measured by standard viscosity measurement methods, 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 between about 35 cP and about 15 cP. For example, if a liquid formulation of the 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, and about 26 cP Absolute viscosity of 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.

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

Container for the pharmaceutical preparation and administration method

The pharmaceutical formulations of the present invention can be placed in any container suitable for storing drugs and other therapeutic compositions. For example, the pharmaceutical formulation may be placed in a sealed and sterilized plastic or glass container having a defined capacity, such as a vial, ampoule, syringe, pill box, or vial. The formulations of the invention can be placed in different types of small glass bottles such as clear and opaque (such as amber) glass or plastic bottles. Likewise, any type of syringe can be used to contain or administer the pharmaceutical formulations 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 understood by those of ordinary skill in the art, the process of manufacturing glass syringes typically involves the use of a hot tungsten strip as a penetrating glass to create small holes that can draw liquid from the syringe. This process results in a trace amount of tungsten being deposited on the inner surface of the syringe. Washing and other process steps can then be used to reduce the amount of tungsten in the syringe. The term "ordinary tungsten" as used herein means that the syringe contains more than 500 billion parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe 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 of tungsten.

The rubber plunger for the syringe and the rubber stopper for closing 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 certain embodiments may 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 stopper may be coated with a fluorocarbon film. Examples of coating plungers or rubber stoppers suitable for use in glass bottles and syringes containing the pharmaceutical formulation of the present invention have been described in U.S. Patent Nos. 4,997,423, 5,908,686, 6,286,699, 6,645,635, and 7,226,554, the contents of which are incorporated by reference in their entirety 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 invention, the pharmaceutical formulation may be placed in a low-tungsten syringe containing a fluorocarbon-coated piston.

The pharmaceutical formulation may be administered to a patient via, for example, injection (e.g., subcutaneously, intravenously, intramuscularly, intraperitoneally, etc.) or parenteral route for transdermal, mucosal, nasal, pulmonary or oral administration. Many portable pens or automatic infusion devices can be used for subcutaneous infusion of a pharmaceutical formulation of the invention. Examples include, but are not limited to AUTOPEN ^TM (Owen Mumford, Woodstock, UK), DISETRONIC ^TM (Disetronic Medical Company, Bergdorf, Switzerland), HUMALOG MIX 75/25 ^TM , HUMALOG ^TM , HUMALIN 70/30 ^TM (Indian Eli Lilly, Indianapolis), NOVOPEN ^TM I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk, Copenhagen, Denmark), BD ^TM (Becton Dickinson, Franklin, NJ) OPTIPEN ^TM , OPTIPEN PRO ^TM , OPTIPEN STARLET ^TM , and OPTICLIK ^TM (Sanofi-Aventis Company, Frankfurt, Germany). Examples of portable pens or automatic injection infusion devices that have been used for subcutaneous infusion of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR ^TM pen (Sanofi-Aventis), FLEXPEN ^TM (Novo Nordisk), and KWIPKEN ^TM (Eli Lilly) SURECLICK ^™ autoinjector (Amgen, Thousand Oaks, California), PENLET ^™ (Haselmeier, Stuttgart, Germany), EPIPEN ^™ (Dey, LP), and HUMIRA ^™ pen (Abbott Lab, Abbott Park, Ill.).

A microsyringe is also used here for infusion of the pharmaceutical formulations of the invention. As used herein, "micro-syringe" means slowly administering a large amount (e.g., up to about 2.5 ml or more) of a therapeutic formulation over a long period of time (e.g., about 10, 15, 20, 25, 30 minutes or more) Subcutaneous infusion set. See, for example, 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 high-dose therapeutic proteins, or viscous solutions containing high concentrations (eg, about 100, 125, 150, 175, 200 mg / ml, or higher).

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

Therapeutic uses of pharmaceutical formulations

The pharmaceutical formulations of the present invention are particularly useful for 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 example diseases and disorders that can be treated or prevented by administration of the pharmaceutical formulations of the present invention include various allergic diseases such as atopic dermatitis, allergic conjunctivitis, allergic rhinitis, asthma, and other IgE / Th2 mediators Disease.

Accordingly, the invention includes methods of treating, preventing, or alleviating any disease or disorder associated with 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-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 remission, or who can benefit from inhibiting or relieving IL-4 or IL-4Rα-mediated activity. For example, the organism may be an individual diagnosed or considered to be at risk of suffering from the above-mentioned diseases or disorders. The invention further includes any of the pharmaceutical formulations disclosed herein for use in the treatment, prevention, or alleviation of any disease or disorder (including any of the exemplary diseases, disorders, and conditions described above) associated with IL-4 activity or IL-4Rα activation. the use of.

The following examples are provided to provide a complete disclosure and illustration of how those skilled in the art can make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Although efforts have been made to ensure the accuracy of the numbers used (e.g., quantity, temperature, etc.), some errors and deviations may still occur in some experiments. Unless otherwise stated, parts are moles, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

The formation of preliminary formulations involves the screening of organic cosolvents, heat stabilizers and buffers in liquid formulations of mAb1 (anti-IL-4Rα antibodies of the invention) to confirm that the excipients are compatible with the protein and help it. Stability, while maintaining Molar osmolarity and viscosity for subcutaneous injection. Buffer conditions were also checked to determine the optimal pH for highest protein stability.

Example 1. Organic Cosolvents

MAb1 has been found to be unstable under oscillating stress. Analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) and size-exclusion high-performance liquid chromatography (SE-HPLC) demonstrated the loss of protein and the increase of agglutinating proteins when mAb1 was shaken at room temperature ( Table 1, please see the "no-solvent" data). Adding an organic co-solvent to the mAb1 solution prevented protein degradation as 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 the recovered protein as 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 the help of solvents Thing. Polysorbate 20 (0.2%) and polysorbate 80 (0.2%) are fairly stable against oscillations and thermal stress.

According to Table 1, 0.3 ml of mAb 1 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 turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. The percentage of natural and aggregated mAb1 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 without shaking.

According to Table 2, 0.3 ml of mAb 1 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 turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. The percentage of total mAb1 recovered was determined by a reverse phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAb1 was determined by size exclusion HPLC (SE-HPLC) method. The SE-HPLC results shown in the "Starting Materials" table are the averages of the formulations without heat stress.

Example 2. Heat stabilizer

Various heat stabilizers, such as sugars, amino acids, and inorganic salts, were examined for their ability to inhibit degradation of mAb1 when stored at about 45 ° C. A summary of a study of heat stabilizers is shown in Table 3. Formulations containing sucrose or trehalose have a higher mAb1 stabilization effect when cultured in high temperature solutions (determined by SE-HPLC). Sucrose was selected as a stabilizer because of its long history of safety in monoclonal antibody formulations.

According to Table 3, 0.3 ml of mAb 1 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 turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. All samples can ignore the difference in turbidity. The percentage of total mAb1 recovered was determined by a reverse phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAb1 was determined by size exclusion HPLC (SE-HPLC) method. Acidic or basic substances are defined as the ratios from the cation exchange (CEX-HPLC) column respectively. The total amount of mAb1 peaks with the main peak having a dwell time earlier or later. The SE-HPLC results shown in the "Starting Materials" table are the averages of the formulations without heat stress.

Example 3. Buffers and pH

The effects of pH and buffer species on mAb1 stability were also examined. 15 mg / ml mAb1 was cultured 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 a pH 6.0 histidine buffer or pH 5.3 acetate buffer, it had the maximum protein stability as determined by SE-HPLC and CEX-HPLC (Tables 4 and 5). The acetate buffer system has a broader pH stability range and a lower rate of variably charge formation compared to formulations containing histidine buffers (Table 5). Therefore, an acetate buffer at pH 5.3 was selected for the formulation of the mAb1 drug.

According to Table 4, a 2 ml glass bottle of 10 ml of various buffers mixed with 0.3 ml of 15 mg / ml mAb1 and 0.2% of polysorbate 20 was stored at about 45 ° C for about 14 days. The turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. All samples can ignore the difference in turbidity. The percentage of total mAb1 recovered was determined by a reverse phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAb1 was determined by size exclusion HPLC (SE-HPLC) method. Acidic or basic substances are defined as the total amount of mAb1 peaks that were dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak, respectively. The SE-HPLC results shown in the "Starting Materials" table are the averages of the formulations without heat stress.

According to Table 5, a 2 ml glass bottle of 10 mM of various buffers mixed with 0.3 ml of 15 mg / ml mAb1 and 0.2% of polysorbate 20 was stored at about 45 ° C for about 14 days. The turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. All samples can ignore the difference in turbidity. The percentage of total mAb1 recovered was determined by a reverse phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAb1 was determined by size exclusion HPLC (SE-HPLC) method. Acidic or basic substances are defined as the total amount of mAb1 peaks that were dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak, respectively. The SE-HPLC results shown in the "Starting Materials" table are the averages of the formulations without 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 formation tests are shown under alkaline conditions (pH

6.5) mAb1 in solution can be desmamine. In contrast, below pH 5.0 has been found to increase the rate at which mAb1 of varying molecular weight is formed. Based on these data, the pH of the mAb1 formulation was maintained between about pH 5.6 and pH 6.2. MAb1 has been found to be stable in this pH range.

The effects of pH and buffer species on the stability of mAb1 were 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 individual buffer systems, the formulation of mAb1 with histidine and acetate at pH 5.9 is the most stable. MAb1 has the slowest aggregation rate when formulated in this mixed buffer system (SE-HPLC) (Table 6).

According to Table 6, 0.4 ml of 150 mg / ml mAb1, 10% sucrose, and 0.2% polysorbate 20 mixed with various buffers in a 2 ml glass bottle were stored at about 45 ° C. for about 14 days. The turbidity was measured at an optical density (OD) at 405 nm and the relative changes in OD of the starting materials at 405 nm were compared. All samples can ignore the difference in turbidity. The percentage of total mAb1 recovered was determined by a reverse phase HPLC (RP-HPLC) method. The percentage of natural and aggregated mAb1 was determined by size exclusion HPLC (SE-HPLC) method. Acidic or basic substances are defined as the total amount of mAb1 peaks that were dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak, respectively. The SE-HPLC results shown in the "Starting Materials" table are the averages of the formulations without heat stress.

Example 4. Control of viscosity and tension

The viscosity and tension (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 a high concentration of mAb1, which was easy and comfortable for high-volume subcutaneous infusion of mAb1 (Table 7). Liquid formulation with pH 5.9 containing 25 mM arginine, 20 mM histidine, 12.5 mM acetate, 5% (w / v) sucrose, 0.2% (w / v) polysorbate 20, and 150 mg / ml mAb1 (Formulation A ) Is the best formulation with low viscosity (about 8.5 cPoise) and physiological isotonicity (about 293mOsm / kg) while still maintaining the stability of mAb1.

Example 5. Characterization of Formulation A

The main degradation pathway in the formation of the mab1 liquid formulation is the generation of aggregates, fracture products and charge variants. Formation of these degradation products can be reduced by formulating mAb1 in a formulation containing 20 mM histidine, 12.5 mM acetate, 0.2% polysorbate 20, 5% sucrose, and 25 mM L-arginine hydrochloride, pH 5.9. These 150 mg / ml mAbl have been found to be a transparent to micro-emulsion liquid solution that is substantially free of particles visible to the naked eye.

This formulated mAb1 is physically and chemically stable under various stresses (25 ° C and 45 ° C culture) and immediate storage conditions (5 ° C) (Table 8). The mAb1 did 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 solution pH, turbidity, or mAb1 recovery. After the formulated mAb1 was stored at 25 ° C for 3 months, the antibody determined by SE-HPLC was not significantly degraded and the degradation determined by CEX-HPLC was only 3.3% higher. Found on After storage at 45 ° C for 8 weeks, the amount of degradation determined by SE-HPLC and CEX-HPLC was increased to show that the formation of aggregation and charge variation were 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 substances are defined as the total amount of mAb1 peaks that were dialyzed from a cation exchange (CEX-HPLC) column earlier or later than the main peak, respectively.

Example 6. Container

Filter-sterilized mAb1-containing formulations have been tested for stability. The clinical supply manufacturing system uses a microporous Millipak filter device, while the research room uses a filter of the same composition (microporous Millex DURAPORE).

150mg / ml mAb1, 5% (w / v) sugarcane with a minimum of 2.5ml at pH 5.9 A 5 ml glass bottle was filled with sugar, 25 mM L-arginine hydrochloride, 0.2% (w / v) polysorbate 20, 12.5 mM acetate, and 20 mM histidine. Use 0.5 ml of the overdose in a 5 ml bottle to ensure that 2.0 ml of the formulation can be drawn. This excess is not intended to compensate for losses during manufacture of mAb1 or mAb1-containing formulations, degradation during manufacture, degradation during storage (shelf life), or extended shelf life.

Compared to 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 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, 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. Haze was recorded as the difference in turbidity at an OD of 405 nm compared to the starting material. Acidic or basic materials are defined as the total amount of mAb1 peaks that were dialyzed from the CEX-HPLC column earlier or later than the main peak, respectively.

<110> Regeneron Pharmaceuticals, Inc.

<120> Stabilizing formulations containing anti-interleukin-4 receptor (IL-4R) antibodies

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Claims (8)

A stable liquid pharmaceutical formulation comprising: (i) a human antibody having a concentration from 15 mg / ml to less than 100 mg / ml, wherein the antibody specifically binds human interleukin-4 receptor alpha (hIL-4Rα ) And comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1; and a light chain variable region (LCVR) comprising 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 spermine; wherein the liquid pharmaceutical formulation has a pH of 5.6 to 6.2. For example, the liquid pharmaceutical preparation according to item 1 of the application, wherein the acetate is present at a concentration of 12.5 mM ± 1.85 mM and the histidine is present at a concentration of 20 mM ± 0.3 mM. For example, the liquid pharmaceutical formulation of item 1 of the patent application scope, wherein the polysorbate is polysorbate 20 or polysorbate 80. For example, the liquid pharmaceutical formulation of item 1 of the patent application scope, wherein the polysorbate is polysorbate 20 and its concentration is 0.2% ± 0.03% weight / volume. For example, the liquid pharmaceutical formulation according to item 1 of the application, wherein the polysorbate is polysorbate 80 and its concentration is 0.2% ± 0.03% weight / volume. For example, the liquid pharmaceutical formulation of item 1 of the patent application scope, wherein sucrose is present at a concentration of 5% ± 0.75% weight / volume. For example, the liquid pharmaceutical formulation of item 1 of the patent application scope, wherein the arginine is present at a concentration of 25mM ± 3.75mM. For example, the liquid pharmaceutical formulation of item 1 of the patent application scope, wherein the arginine is present at a concentration of 50 mM ± 7.5 mM.