TW201924718A - Stabilized formulations containing anti-interleukin-4 receptor (IL-4R) antibodies - Google Patents

Abstract

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

Description

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

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

 Sequence table  

This patent specification also includes an ST.25 conforming text file of the sequence listing. This text file is hereby incorporated by reference. The sequence book paper incorporating the same content as the ST.25 conforming text file is included as part of this patent specification.

Therapeutic macromolecules (e.g., antibodies) must be formulated in a manner that not only enables the molecule to be suitable for administration to a patient, but also maintains stability during storage and subsequent use. For example, therapeutic antibodies in liquid solutions are susceptible to degradation, aggregation, or poor chemical modification unless the solution is properly formulated. The stability of the antibodies in the liquid formulation depends not only on the type of excipients used in the formulation, but also on the relative amounts and ratios between the excipients. In addition, other factors must be considered in addition to stability in the preparation of a liquid antibody formulation. Examples of such additional considerations include the viscosity of the solution and the concentration of antibody that can be adapted to a known formulation, as well as the visual quality or appeal of the formulation. Thus, when formulating a therapeutic antibody, care must be taken to maintain a formulation stable, contain appropriate antibodies, and have suitable viscosity and other properties that can be conveniently administered to the patient to the formulation.

An antibody to human interleukin-4 receptor alpha (hIL-4R[alpha]) is an example of a therapeutically relevant macromolecule that needs to be suitably formulated. Anti-hIL-4Rα antibodies are clinically used to treat or prevent diseases such as atopic dermatitis and allergic asthma, as well as other conditions. Examples of anti-hIL-4Rα antibodies have been described in particular 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 a need in the art for a novel pharmaceutical formulation containing an anti-hIL-4Rα antibody that is sufficiently stable and suitable for administration to a patient.

 Summary of invention  

The present invention satisfies the above needs by providing a pharmaceutical formulation containing a human antibody that specifically binds to human interleukin-4 receptor alpha (hIL-4Rα).

In one aspect, a liquid pharmaceutical formulation comprising: (i) a human antibody that specifically binds to human interleukin-4 receptor alpha (hIL-4Rα); (ii) a buffer; Iii) an organic cosolvent; (iv) a thermal stabilizer; and (v) a detackifier.

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 sequence identification number: 1-8. In a specific embodiment, the antibody comprises (a) a heavy chain complementarity determining region 1, 2 and 3 (HCDR1-HCDR2-HCDR3) comprising sequence identification number: 2. sequence identification number: 3 and sequence identification number: 4, respectively. One heavy chain variable region (HCVR); and (b) each containing a sequence identification number: 6, sequence identification number: 7 and sequence identification number: 8 light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3 A light chain variable region (LCVR). In a specific embodiment, the antibody comprises an HCVR and an LCVR comprising an amino acid sequence of sequence identification number: 1 and sequence identification number: 5, respectively.

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

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

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

In a specific embodiment, the liquid pharmaceutical formulation comprises a buffer system comprising at least two buffers. In one embodiment, the buffer system comprises a first buffer having an effective pH range of 3.6 to 5.6 and a second buffer 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 acetate has a concentration of 12.5 ± 1.9 mM and a histidine concentration of 20 ± 3 mM.

In one embodiment, the organic cosolvent is a nonionic polymer containing a polyoxyethylene group. In some embodiments, the organic co-solvent is any one or more of polysorbate 20, poloxamer 181, and polyethylene glycol 3350. In a particular 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 by volume" or "w/v", wherein, for example, 0.1 g/ml = 10% and 0.01 g/ml=1%). In a particular embodiment, the concentration of the organic co-solvent is about 0.2% ± 0.03% w/v polysorbate 20.

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

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

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

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

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

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

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

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

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

In one aspect, a liquid pharmaceutical formulation comprising: (i) about 150 mg/ml ± 50 mg/ml of a human antibody that specifically binds hIL-4Rα, wherein the antibody comprises 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) acetate of about 12.5 mg/ml ± 2 mM; (iii) about 20 mM ± 3 mM Histamine; (iv) about 5% ± 0.75% (w/v) sucrose; (v) about 0.2% ± 0.03% (w/v) polysorbate 20; and (vi) about Approximately 25 mM ± 3.75 mM arginine at 5.9 ± 0.5 pH.

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

In a specific embodiment, the liquid pharmaceutical formulation is physiologically isometric. In a specific embodiment, the liquid pharmaceutical formulation has an osmolality of about 290 ± 20 mOsm/kg.

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

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

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

In one aspect, a stable 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 concentration of the antibody is about 150 mg/ml ± 15 mg/ml.

In a specific embodiment, the antibody comprises any one or more amino acid sequences of sequence number: 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 a sequence identification number: 2-3-4 and sequence identification, respectively. ID: The heavy and light chain complementarity determining regions of the amino acid sequence of 6-7-8 (HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3). In a specific embodiment, an HCVR and an LCVR of the anti-hIL-4Rα antibody.

In some embodiments, the formulation has a viscosity of 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 particular embodiment, the liquid formulation has a viscosity of about 8.5 ± 2.5 centipoise.

In a specific embodiment, the formulation has a physiologically compatible molar osmolality. In a particular embodiment, the formulation comprises an osmolality of 290 ± 20 mOsm/kg.

In a specific embodiment, the antibody has a stability of at least about six months at about 5 °C. In a specific embodiment, at least about 98% of the antibody is stored for about six months at 5 ° C as determined by size exclusion chromatography to retain its natural conformation.

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

In a specific embodiment, the antibody has a storage stability of at least about six months at about 25 °C. In a specific embodiment, the antibody is stored for less than about 4% aggregation at about 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 comprises an acetate buffer and a set of amine acid buffers. In a specific embodiment, the acetate concentration is 12.5 mM ± 1.9 mM and the histamine concentration is 20 mM ± 3 mM.

In a particular embodiment, the formulation comprises an organic cosolvent having a concentration of from about 0.2% ± 0.03% to about 1% ± 0.15% w/v. In a specific embodiment, the organic cosolvent is a nonionic polymer containing a polyoxyethylene group. In some embodiments, the organic co-solvent is any one or more of polysorbate 20, poloxamer 181, and polyethylene glycol 3350. In a particular 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 of from about 0.9% ± 0.135% w/v to about 10% ± 1.5% w/v. In a specific embodiment, the thermal stabilizer is sucrose. In a particular embodiment, the sugar system is selected from the group consisting of sucrose, mannose, and trehalose. In a particular embodiment, the thermal stabilizer is sucrose at a concentration of about 5% ± 0.75% w/v.

In a specific embodiment, the formulation comprises a detackifying agent at a concentration of no more than about 100 mM. In a specific embodiment, the detackifying agent is arginine. In a specific embodiment, the detackifying agent is a L-spermine hydrochloride salt having a concentration of 25 mM ± 3.75 mM.

In a specific embodiment, the stabilized low viscosity isotonic liquid pharmaceutical formulation has a viscosity of about 8.5 ± 2.5 centipoise and an osmolality of about 290 ± 20 mOsm/kg, and comprises: (i) 150 mg/ml ±15 mg/ml of anti-hIL-4Rα antibody, wherein the antibody comprises an HCVR and an LCVR comprising sequence identification number: 1 and amino acid sequence of sequence identification number: 5; (ii) 12.5 mM ± 1.9 mM acetic acid a salt; (iii) 20 mg/ml ± 3 mM histidine; (iv) 0.2% ± 0.03% w/v polysorbate 20; (v) 5% ± 0.75% w/v sucrose; Vi) 25 mM ± 3.75 mM L-spermine hydrochloride. According to this embodiment, (i) at least about 98% of the antibody retains its natural configuration when stored at 5 ° C for at least about six months as determined by size exclusion chromatography; (ii) 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 as determined by chromatography; (iii) stored at 45 ° C for less than about 45% at 45 ° C when measured by cation exchange chromatography The acid form of the antibody; and (iv) less than about 4% aggregation of the antibody 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 of the container is provided. In a specific 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 particular embodiment, the syringe comprises a fluorocarbon coated piston. In a particular embodiment, the syringe is a low tungsten syringe.

Further embodiments of the invention will be apparent from the following detailed description.

Detailed description of the invention

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

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "about" as used herein with respect to 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 herein includes 99 and 101 and all values therebetween (eg, 99.1, 99.2, 99.3, 99.4, etc.).

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

Pharmaceutical preparation

The statement "pharmaceutical formulation" herein means at least one active ingredient (for example, a small molecule, a macromolecule, a compound, etc., which exhibits a biological effect in a human or non-human animal) and a combination of at least one inactive ingredient, when The active ingredient can be therapeutically administered to a human or non-human animal when the active ingredient is combined with one or more additional inactive ingredients. The term "formulation" as used herein means "medical formulation" unless otherwise stated. The invention provides a pharmaceutical formulation comprising at least one therapeutic polypeptide. According to some embodiments of the invention, the therapeutic polypeptide specifically binds to an antibody or antigen-binding fragment thereof of human interleukin-4 antibody alpha (hIL-4Rα). More specifically, the invention encompasses human antibodies comprising (i) a specific binding to hIL-4Rα; (ii) a monoacetate/histidine buffer system; (iii) an organic helper of a nonionic surfactant a solvent; (iv) a thermal stabilizer for a carbohydrate; and (v) a pharmaceutical formulation of a detackifier. The specific exemplary ingredients and formulations contained in the present invention are described in detail below.

An antibody that specifically binds hIL-4R

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

The term "antibody" as used herein generally refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) linked to each other by a disulfide chain, and multimers thereof. (eg IgM); however, immunoglobulin molecules consisting only of heavy chains (ie no light chains) are also within the definition of "antibody". Each heavy chain comprises a heavy chain variable region (referred to herein simply 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 comprises a light chain variable region (abbreviated herein as LCVR or V _L) and a light chain constant region. The light chain constant region comprises a domain (CL1). The V _H and V _L, may then be further subdivided into more interspersed conserved region called the framework region (FR) of the hypervariable regions called complementarity determining regions (CDRs). FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 configuration consisting of: each of V _H and V _L, is composed of three CDRs and four FRs in the following order from the amino terminal to carboxy-terminus.

Unless otherwise stated, the term "antibody" as used herein shall be taken to include the full antibody molecule as well as its antigen-binding fragment. An "antigen-binding region" or "antigen-binding fragment" (or simply "antibody region" or "antibody fragment") of an antibody as used herein means retaining the ability to specifically bind to hIL-4Rα or its epitope. One or more fragments of an antibody.

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody capable of specifically binding to hIL-4R[alpha], which has substantially no specific binding except for hIL-4R[alpha]. Antibody to the antigen).

The term "specifically binds" and the like means that an antibody or antigen-binding fragment thereof forms a 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 x 10 ^-6 M or higher. Assay methods for whether two molecules specifically bind are known in the art and include, for example, dialysis balance methods, surface plasma resonance methods, and the like. However, an isolated antibody that specifically binds hIL-4Rα is cross-reactive with other antigens, such as IL-4R (homologous gene) from other lines. 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. Furthermore, an isolated antibody can be substantially free of other cellular material or chemicals.

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

According to some embodiments of the invention, the anti-hIL-4Rα anti-system comprises a heavy chain variable region of one of the IGHV 3-9 subtypes and a human IgG1 of the light chain variable region of one of the IGKV 2-28 subtypes (please See Barbie and Lefranc, Human Immunoglobulin κ Variable Region (IGKV) Gene and Linker (IGKJ) Fragments, Exp. Clin. Immunogenet. 1998, 15: 171~183; and Scaviner, D. et al., Human Immunoglobulin Protein expression in the 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 charge change 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 the amino acid position assignment number and the International Immunogenetics (IMGT) information system, have been described by Lefranc, M.-P. et al. , IMGT ^® , International Immunogenetics Information ^System® , Nucl. Acids Res. , 37, D1006~D1012 (2009). In some embodiments, the exposed surface comprises a complementarity determining region (CDR). In some embodiments, the one or more substitutions of the amino acid are selected from the group consisting of (a) a basic amino acid substituted for a native amino acid of CDR2 of IGHV 3-9 (eg, at position 58); b) a native amino acid substituted for the amino acid of IGHV 3~9 (as in position 107); and (c) a native amino acid substituted for CDR1 of IGKV 2~28 (as in position 33) A combination of basic amino acids. A particular arrangement of an antibody, particularly within the charge distribution of the environmental interface (e.g., CDRs), will result in an unpredictable condition for the stability of the antibody within the solution.

In some embodiments, the anti-hIL-4Rα antibody comprises at least one amino acid substitution which is produced in the framework region of the antibody variable region relative to the germline IGHV 3-9 sequence or the germline IGKV 2-28 sequence Torsional strain changes. In some embodiments, the one or more substitutions of the amino acid are selected from the group consisting of (a) a proline to the framework region 3 (FR3) of IGHV 3-9 (eg, at position 96). And (b) a combination of a non-proline amino acid substituted for a proline in the framework region 2 (FR2) of IGKV 2-28 (as in position 46). Altering the peptide chain, particularly the ability to reverse the CDR and solvent interface in a framework region, will result in an unpredictable condition for the stability of the antibody in solution.

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

According to some embodiments of the 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: And an LCD number of serial identification number: 8. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an LCVD of sequence 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 HCDR2 of sequence identification number: 11 and an HCDR3 of sequence identification number: 12. Identification number: an LCDR1 of 14, an LCDR2 of sequence identification number: 15, and an LCDR3 of sequence identification number: 16. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD of sequence number: 9 and an LCVD of sequence 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 HCDR2 of sequence identification number: 19, an HCDR3 of sequence identification number: 20, a sequence Identification number: an LCDR1 of 22, an LCDR2 of sequence identification number: 23, and an LCDR3 of sequence identification number: 24. In certain embodiments, the anti-hIL-4Rα antibody or antigen-binding fragment thereof comprises an HCVD of sequence number: 17 and an LCVD of sequence number: 21.

A non-limiting example 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 with sequence identification number: 1/5, and HCDR1-HCDR2 with sequence identification number: 2-3-4/SEQ ID NO: 6-7-8 -HCDR3/LCDR1-LCDR2-LCDR3 domain.

Another non-limiting exemplary antibody for use 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 with sequence identification number: 9/13, and HCDR1-HCDR2- with sequence identification number: 10-11-12/SEQ ID NO: 14-15-16 HCDR3/LCDR1-LCDR2-LCDR3 domain.

Yet another non-limiting exemplary antibody for use 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 with sequence ID: 17/21, and HCDR1-HCDR2- representing sequence identification number: 18-19-20/SEQ ID NO: 22-23-24 HCDR3/LCDR1-LCDR2-LCDR3 domain.

The amount of antibody or antigen-binding fragment thereof in a pharmaceutical formulation of the invention will depend on the particular nature of the formulation and the particular circumstances and uses in which the formulation is intended to be employed. In certain embodiments, the pharmaceutical formulation comprises from about 100 ± 10 to about 200 ± 20 mg/ml, from about 110 ± 11 to about 190 ± 19 mg/ml, from about 120 ± 12 to about 180 ± 18 mg/ml, A liquid formulation of the antibody of from about 130 ± 13 to about 170 ± 17 mg/ml, from about 140 ± 14 to about 160 ± 16 mg/ml, or about 150 ± 15 mg/ml. For example, a formulation of the invention contains about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, about 120 mg/ml, about 125 mg/ml, about 130 mg/ Ml, about 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/ Specific binding of hIL-4Rα to 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. Antibody or antigen-binding fragment 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 that is added to the formulation to provide the 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 in a vigorous manner, such as in an oscillating state. In some embodiments, the term "stable" means that the total amount of antibody that can be prevented (on a molar basis) is more than 2% formed during vigorous manipulation. In some embodiments, the intense operation is to oscillate a solution containing the antibody and the organic co-solvent for about 120 minutes.

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

The amount of organic co-solvent in the pharmaceutical formulations of the present invention will depend on the particular nature of the formulation and the particular circumstances and uses for which the formulation is intended to be employed. In certain embodiments, the surfactant is present in the formulation at a level of from about 0.1 ± 0.01% to about 2 ± 0.2%. For example, a formulation of the 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, a formulation of the 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.

An exemplary organic co-solvent that stabilizes the hIL-4Rα antibody contains 0.2% ± 0.02% polysorbate 20, 0.2% ± 0.02% polyxamer 181, or 1% ± 0.1% PEG 3350.

The pharmaceutical formulations of the present invention also comprise one or more heat stabilizers which are capable of stabilizing the type and amount of the hIL-4Rα antibody under heat stress. In some embodiments, the term "stable" means that a solution containing the antibody and the heat stabilizer is maintained at about 45 ° C for up to about 28 days to maintain greater than about 92% of the native conformational antibody. In some embodiments, the term "stable" means that less than about 5% of the aggregated antibody can be maintained when the solution containing the antibody and the heat stabilizer is stored at about 45 ° C for up to about 28 days.

In certain embodiments, the thermal stabilizer is selected from the group consisting of sucrose, trehalose, and mannose, or any combination thereof, of a sugar or sugar alcohol, the amount of which is within the formulation, as the case may be, and the formulation is intended to be used. Depending on the purpose. In certain embodiments, the formulation contains from about 2.5% to about 10%, from about 3% to about 9.5%, from about 3.5% to about 9%, from about 4% to about 8.5%, from about 4.5% to about 8 %, from about 5% to about 7.5%, from about 5.5% to about 7%, or from about 6.0% to about 6.5% of a sugar or sugar alcohol. For example, a pharmaceutical formulation of the 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 as a means of maintaining a stable pH and helping to stabilize the hIL-4Rα antibody. In some embodiments, the term "stable" means that the solution containing the antibody and buffer can maintain less than 3.0% ± 0.5% of aggregated antibody when stored at about 45 ° C for up to about 14 days. In some embodiments, the term "stable" means that a concentrated antibody that maintains less than 3.7% ± 0.5% when the solution containing the antibody and buffer is stored at about 25 ° C for up to about 6 months. . 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 be maintained at least 95% by size exclusion chromatography. ±0.5% of natural conformational antibodies. 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 be maintained at least 96 by size exclusion chromatography. %±0.5% of natural conformational antibodies. 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 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 for up to about 6 months, it can be maintained at least 54% by cation exchange chromatography. ±0.5% neutral conformational antibody. "Neutral conformation" means the portion of the antibody that is dialyzed from the main peak in the ion exchange resin, which tends to be more "alkaline" on one side and more "acidic" on the other side.

The pharmaceutical formulations of the invention have a pH of from about 5.2 to about 6.4. For example, a pharmaceutical formulation of the invention can have from about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, or about 6.4. pH. In some 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 the range of pH 5.2 to 6.4. In a specific embodiment, the buffer or buffer system comprises two buffers, the first of which is in the effective pH range of 3.6 to 5.6 and the second 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 certain embodiments, from about 1.3 to about 1.9 parts of histidine is present per 1 part molar ratio of acetate. In certain embodiments, about 1.6 ± 0.25 parts of histidine is present per 1 part molar ratio of acetate. In certain embodiments, the acetate concentration is from about 2.5 mM to about 22.5 mM, from about 3.0 mM to about 22 mM, from about 3.5 mM to about 21.5 mM, from about 4.0 mM to about 21.0 mM, from about 4.5 mM to about 20.5 mM, from about 5.0 mM to about 20 mM, from about 5.5 mM to about 19.5 mM, from about 6.0 mM to about 19.0 mM, from about 6.5 mM to about 18.5 mM, from about 7.0 mM to about 18.0 mM, from about 7.5 mM to about 17.5 mM, From about 8.0 mM to about 17.0 mM, from about 8.5 mM to about 16.5 mM, from about 9.0 mM to about 16.0 mM, from about 9.5 mM to about 15.5 mM, from about 10.0 mM to about 15.0 mM, from about 10.5 mM to about 14.5 mM, about 12.5 mM to about 1.875 mM, from about 11.0 mM to about 14.0 mM, from about 11.5 mM to about 13.5 Mm, or from about 12.0 mM to about 13.0 mM. In certain embodiments, the concentration of the histamine is from about 10 mM to about 30 mM, from about 11 mM to about 29 mM, from about 12 mM to about 28 mM, from about 13 mM to about 27 mM, from about 14 mM to about 26 mM, from about 15 mM to about 25 mM. From about 16 mM to about 24 mM, from about 17 mM to about 23 mM, from about 18 mM to about 22 mM, or from 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 formulations of the present invention also comprise one or more excipients for maintaining a low viscosity or reducing the viscosity of a formulation containing a high concentration of protein, such as typically >100 mg/ml protein. In some embodiments, the formulation has a arginine acid content sufficient to maintain the viscosity of the liquid formulation below about 35 c Poise, below about 30 c Poise, below about 25 c Poise, below about 20 c Poise, below about 15 c Poise, low. At about 14 c Poise, below about 13 c Poise, below about 12 c Poise, below about 10 c Poise, or below about 9 c Poise.

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

Representative formulation

According to one aspect of the invention, the pharmaceutical formulation is a low viscosity, generally physiologically isotonic liquid formulation comprising: (i) specifically binding to hIL-4R[alpha] (eg, 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 for use as a thermal stabilizer; (iv) protecting the internal structure of the antibody An intact organic co-solvent; and (v) an amino acid that maintains the viscosity of the subcutaneous injection.

According to a specific embodiment, the pharmaceutical formulation comprises: (i) specifically binding to hIL-4Rα and comprising a substituted IGHV 3-9 heavy chain variable region and a substituted IGLV 2-28 light chain variable region ( a human IgG1 antibody having a concentration of from about 100 mg/ml to about 200 mg/ml as in mAb 1); (ii) a buffer system comprising acetate and histidine sufficient to buffer at about pH 5.9 ± 0.6; (iii) as a thermal stabilizer Sucrose; (iv) a polysorbate as an organic co-solvent; and (v) arginine as a debonding agent.

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

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

Stability and viscosity of pharmaceutical formulations

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

The stability can be determined by, for example, measuring the percentage of natural antibody retained in the formulation after a defined temperature storage period. The percentage of native antibodies can be determined, inter alia, by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). A phrase "an acceptable level of stability" as used herein means that at least 90% of the native antibody is detectable in the formulation after storage for a given period of time. In some embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 can be detected in the formulation after a defined temperature storage period. %, 99%, or 100% natural antibodies. The defined period of time that is 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 at which the stable susceptable medicinal formulation is determined can be any temperature from about -80 ° C to about 45 ° C, for example, stored at about -30 ° C, about -20 ° C, about 0 ° C, about 4 ° C ~ 8 °C, about 5 ° C, about 25 ° C, or about 45 ° C. For example, a pharmaceutical formulation can be considered stable if it is greater than about 90%, 95%, 96%, 97%, or 98% by SE-HPLC after storage for 3 months at 5 °C. A pharmaceutical formulation can also be considered stable if it is greater than about 90%, 95%, 96%, 97%, or 98% by SE-HPLC after 6 months of storage at 5 °C. A pharmaceutical formulation that is greater than about 90%, 95%, 96%, 97%, or 98% by SE-HPLC after storage for 9 months at 5 °C can also be considered stable. A natural antibody greater than about 90%, 95%, 96%, or 97% can be detected by SE-HPLC after storage of a pharmaceutical formulation at 25 ° C for 3 months, and can also be considered as stability. A natural antibody greater than about 90%, 95%, 96%, or 97% can be detected by SE-HPLC after storage of a pharmaceutical formulation at 25 ° C for 6 months, and can also be considered as stability. A natural antibody greater than about 90%, 95%, 96%, or 97% can be detected by SE-HPLC after storage of a pharmaceutical formulation at 25 ° C for 9 months, and can also be considered as stability.

The stability can be determined after a defined period of temperature storage by measuring, in particular, the percentage of aggregated antibody in the formulation, wherein stability is inversely proportional to the percentage of aggregated antibody formed. The percentage of aggregated antibodies can be determined, inter alia, by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). A phrase "an acceptable level of stability" as used herein means that up to 5% of aggregated antibodies are detectable in the formulation after storage for a given temperature. In some 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 for a given temperature for a period of time. % or 0.1% of aggregated antibodies. The defined period of time that is 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 at which the stable susceptable medicinal formulation is determined can be any temperature from about -80 ° C to about 45 ° C, for example, stored at about -30 ° C, about -20 ° C, about 0 ° C, about 4 ° C ~ 8 °C, about 5 ° C, about 25 ° C, or about 45 ° C. For example, a pharmaceutical formulation that is measured to be less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after storage for 3 months at 5 ° C can be considered as stable. . A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after 6 months of storage at 5 °C can also be considered stable. A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after 9 months of storage at 5 °C can also be considered as stable. A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after storage for 3 months at 25 ° C can also be considered stable. A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after 6 months storage at 25 °C can also be considered stable. A pharmaceutical formulation that is less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibody after storage for 9 months at 25 °C can also be considered as stable.

Stability can be determined, inter alia, by measuring the ratio of antibody migration to a more acidic moiety ("acid form") to the major portion of the antibody ("neutral configuration") during ion exchange, wherein the stability and acid form portion of the antibody In inverse proportion. Without being immersed in theory, deamidation of antibodies can result in antibodies with more negative charges and therefore more acidic than non-deaminamide antibodies (see, for example, Robinson, N., protein deamidation, PNAS , April 16, 2002, 99(8): 5283~5288). The "acidified" or "deamidated" antibodies can be determined, inter alia, by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]). The phrase "an acceptable level of stability" as used herein means that up to 45% of the acid form of the antibody can be detected in the formulation after a defined temperature storage period. In some 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 for a given temperature for a period of time. %, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the acid form of the antibody. The defined period of time that is 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 at which the stable susceptable medicinal formulation is determined can be any temperature from about -80 ° C to about 45 ° C, for example, stored at about -30 ° C, about -20 ° C, about 0 ° C, about 4 ° C ~ 8 °C, about 5 ° C, about 25 ° C, or about 45 ° C. For example, if a pharmaceutical formulation is stored at 5 ° C for 3 months, if it is less than about 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the acid form of the antibody can be considered as stable. A pharmaceutical formulation is less than about 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7% after storage for 3 months at 25 °C. 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the acid form of the antibody may also be considered as stable. A pharmaceutical formulation is less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, after storage for 8 weeks at 45 °C. 2%, 1%, 0.5% or 0.1% of the acid form of the antibody can also be considered as stable. A pharmaceutical formulation is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, if measured after storage for 2 weeks at 40 °C, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the acid form of the antibody may also be considered as stable.

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

The stability can also be assessed by determining the biological activity of the antibody or the affinity attached to its target. For example, an anti-IL-4Rα antibody contained in the formulation of the present invention after storage for a period of time (e.g., 1 to 12 months), for example, at 5 ° C, 25 ° C, 45 ° C, etc., has an antibody binding affinity of at least 90 prior to storage. When %, 95% or higher is bound to IL-4Rα, it can be considered as stability. Binding affinities are determined by, for example, ELISA or plasma resonance. The biological activity is determined by the IL-4Rα activity assay, for example, by expressing a IL-4Rα cell in contact with a formulation containing the anti-IL-4Rα antibody. The binding of such cells to the antibody can be directly determined, for example, 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, while the activity of the IL-4Rα system without antibodies is compared. In some embodiments, the IL-4Rα can be endogenous to the cell. In other specific embodiments, the IL-4Rα can be ectopically expressed within the cell.

Other methods for determining antibody stability in a formulation are illustrated in the examples below.

In certain embodiments, the pharmaceutical formulations of the invention exhibit low to moderate viscosity. The "stickiness" described herein may be "dynamic viscosity" or "absolute viscosity". "Dynamic viscosity" is the flow resistance measured by 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 requires a lower viscosity liquid to flow through the capillary for a longer period of time. For example, if one liquid takes 200 seconds and the other liquid takes 400 seconds to complete its flow, the second liquid is the first two-fold viscosity on a dynamic viscosity scale. "Absolute viscosity" refers to a product of dynamic viscosity and liquid density (absolute viscosity = dynamic viscosity x density), which is sometimes referred to as power or simple viscosity. The size of the dynamic viscosity is L ² /T, the length of the L system and the T system time. Typically, the dynamic viscosity is expressed as a centist (cSt). The SI unit of dynamic viscosity is mm ² /s, which means 1 cSt. The unit of absolute viscosity is centipoise (cP). The SI unit of absolute viscosity is centiples per second (mPa-s), which is 1 cP = 1 mPa-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, if a liquid formulation of the present invention is considered to have "low viscosity" as determined by standard viscosity measurements, 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 of from about 35 cP to 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, about 26 cP. An 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 by formulating the antibody from about 25 mM to about 100 mM arginine, it is possible to obtain high concentrations of anti-hIL-4Rα antibodies (eg, from about 100 mg/ml up to at least 200 mg/ml). Low to medium viscosity liquid formulation. Furthermore, it has been further discovered that by adjusting the sucrose content to less than about 10%, the viscosity of the formulation can be reduced to a greater extent.

Container for the pharmaceutical preparation and administration method

The pharmaceutical formulation of the present invention can be placed in any container suitable for storing the drug and his therapeutic composition. For example, the pharmaceutical formulation can be placed into a sealed and sterilized plastic or glass container having a defined volume such as a vial, ampoule, syringe, vial or vial. The formulations of the present invention can be placed into different types of vials such as clear and opaque (e.g., amber) glass or plastic bottles. Likewise, any type of syringe can be used to house or administer the pharmaceutical formulations of the present invention.

The pharmaceutical formulations of the present invention can be placed into "normal tungsten" syringes or "low tungsten" syringes. As is known to those of ordinary skill in the art, the process of making a glass syringe typically involves the use of a hot tungsten strip that penetrates the glass to create an aperture that can draw liquid from the barrel. This process causes a trace amount of tungsten to deposit on the inner surface of the barrel. Washing and other processing 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 parts per billion (ppb) tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe in accordance with the present invention can 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.

A rubber plunger for the syringe and a rubber stopper for closing the opening of the glass bottle can be coated to avoid contamination of the syringe or the drug in the bottle, or to preserve its stability. Thus, the pharmaceutical formulations of the present invention according to certain embodiments can be placed in a syringe containing a coated plunger or in a glass bottle sealed with a 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 bottles and syringes containing the pharmaceutical formulations of the present invention are described in U.S. Patent Nos. 4,997,423, 5,908,686, 6,286, 699, 6, 645, 635 and 7, 226, 554, the entire contents of this. The special coated rubber plungers and rubber stoppers used in the present invention are commercially available from West Pharmaceutical Services (Lionville, PA) under the market product "FluroTec ^® ".

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

The pharmaceutical formulation can be administered to a patient via an enteral route such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, mucosal, nasal, pulmonary or oral administration. Many portable injection pens or automatic injection infusion devices can be used for subcutaneous infusion of the pharmaceutical formulations of the present invention. Examples include, but are not limited AUTOPEN ^TM (UK Woodstock City Owen Mumford Company), DISETRONIC ^TM pen (Switzerland Bergdorf City Disetronic Medical), HUMALOG MIX 75/25 ^TM pen, HUMALOG ^TM pen, HUMALIN 70/30 ^TM pen (Indian Eli Lilly, Indianapolis, NV, NOVOPEN ^TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk, Copenhagen, Denmark), BD ^TM pen (Becton Dickinson, Franklin, New Jersey) ^{, OPTIPEN TM, OPTIPEN PRO TM,} OPTIPEN STARLET TM, and OPTICLIK ^TM (Sanofi-Aventis in Frankfurt, Germany company). Examples of injection pens have been used in portable subcutaneous infusion pharmaceutical composition of the present invention, an automatic injection or infusion device to include, but not limited to SOLOSTAR ^TM pen ^{(Sanofi-Aventis), FLEXPEN TM} (Novo Nordisk), and KWIKPEN ^TM (Eli Lilly) ; SURECLICK ^TM auto-injector (Amgen, Thousand Oaks, CA City), PENLET ^TM (Germany Stuttgart City Haselmeier ^{company), EPIPEN TM (Dey, LP} ), and HUMIRA ^TM pen (Abbott Park, Illinois City, Abbott laboratories).

The pharmaceutical formulations of the invention are also infused herein using a microsyringe. As used herein, "microsyringe" means that a large amount (eg, up to about 2.5 ml or more) of therapeutic formulation is administered slowly over a prolonged period of time (eg, about 10, 15, 20, 25, 30 minutes or longer). 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). Microinfusions are best suited for infusion of large doses of therapeutic proteins, or viscous solutions, at 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 of anti-IL-4Rα antibody is administered subcutaneously from a prefilled syringe in an autoinjector at 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 pre-filled into a pouch or cartridge of a microsyringe.

Therapeutic use of pharmaceutical preparations

The pharmaceutical formulations of the invention are particularly useful for the treatment, prevention or amelioration of any disease or disorder associated with IL-4 activity, including diseases or disorders mediated by IL-4Rα activation. Non-limiting exemplary diseases and disorders that can be treated or prevented by 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. Leading disease.

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

The following examples are provided to provide a complete disclosure and illustration of the methods and compositions of the present invention, and are not intended to limit the scope of the invention. While efforts have been made to ensure the accuracy of the numbers used (eg, quantity, temperature, etc.), some errors and deviations may still occur in some experiments. Unless otherwise stated, the parts are in the form of moles, the molecular weight is the average molecular weight, the temperature is in degrees Celsius, and the pressure is at or near atmospheric pressure.

The activity of forming the preliminary formulation involves screening organic cosolvents, thermal stabilizers and buffers in the liquid formulation of mAb1 (anti-IL-4Rα antibody of the invention) to confirm that the excipient is compatible with the protein and contributes to its Stability, while maintaining the osmolality and viscosity for subcutaneous injection. Buffer conditions were also checked to determine the optimum pH for the highest protein stability.

Example 1. Organic cosolvent

It has been found that mAb1 is unstable under oscillating stress. The protein loss and the increase of agglutination protein when mAb1 was shaken at room temperature were confirmed by reversed-phase high performance liquid chromatography (RP-HPLC) and size exclusion high performance liquid chromatography (SE-HPLC) analysis. Table 1, please see the "no solvent" data). The addition of an organic cosolvent to the mAbl 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 mAb 1 (Table 2). Formulations containing PEG 3350 (3%) and PEG 300 (10% and 20%) have been found to lose the recovered protein as measured by RP-HPLC after heat compression (Table 2). In addition, formulations containing PLURONIC F68 (poloxamer 181) (0.2%), PEG 300 (10% and 20%) and propylene glycol (20%) formed more aggregation by SE-HPLC than formulations without solvent. Things. Polysorbate 20 (0.2%) and polysorbate 80 (0.2%) are quite stable to oscillations and heat stress.

According to Table 1, 0.3 ml of a 2 ml glass vial was shaken with 15 mg/ml of mAb 1 in 10 mM phosphate pH 6.0 and various organic co-solvents for about 120 minutes. The turbidity was measured at an optical density (OD) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). The SE-HPLC results shown in the "Starting Materials" table are the average of the various formulations that were not shaken.

According to Table 2, 0.3 ml of 15 mg/ml of mAb 1 in a 2 ml glass vial at 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) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The percentage of total recovered mAb1 was determined by reverse phase HPLC (RP-HPLC). The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). The SE-HPLC results shown in the "Starting Materials" table are the average of the formulations that are not subjected to heat stress.

Example 2. Thermal stabilizer

Various thermal stabilizers, such as sugars, amino acids, and inorganic salts, were examined for their ability to inhibit degradation of mAb 1 at about 45 °C. A summary of the study of thermal stabilizers is shown in Table 3. Formulations containing sucrose or trehalose have a higher mAbl stabilization effect (as determined by SE-HPLC) when cultured in a high temperature solution. Because sucrose has long been used in the safety history of monoclonal antibody formulations, it has been chosen as a stabilizer.

According to Table 3, 0.3 ml of 2 ml glass jars of 25 mg/ml of mAb 1 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) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The turbidity difference can be ignored for all samples. The percentage of total recovered mAb1 was determined by reverse phase HPLC (RP-HPLC). The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). An acidic or basic substance is defined as the total amount of mAb1 peaks that are dialyzed from the cation exchange (CEX-HPLC) column earlier than the main peak or at a later residence time. The SE-HPLC results shown in the "Starting Materials" table are the average of the formulations that are not subjected to heat stress.

Example 3. Buffer and pH

The effect of pH and buffer type on the stability of mAbl was also examined. 15 mg/ml of mAb 1 was incubated with different buffers at various pH values ranging from pH 4.5 to 7.0. Protein stability was monitored by SE-HPLC and cation exchange HPLC (CEX-HPLC). The maximum protein stability as determined by SE-HPLC and CEX-HPLC was obtained when mAb1 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 broader pH stability range and a lower rate of variation charge formation relative to formulations containing histidine buffer (Table 5). Therefore, a pH 5.3 acetate buffer was selected for the formulation of the mAbl drug.

According to Table 4, 10 ml of each buffer was mixed with 0.3 ml of a 2 ml glass bottle of 15 mg/ml mAb1, 0.2% polysorbate 20 at about 45 ° C for about 14 days. The turbidity was measured at an optical density (OD) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The turbidity difference can be ignored for all samples. The percentage of total recovered mAb1 was determined by reverse phase HPLC (RP-HPLC). The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). An acidic or basic substance is defined as the total amount of mAb1 peaks that are dialyzed from the cation exchange (CEX-HPLC) column earlier than the main peak or at a later residence time. The SE-HPLC results shown in the "Starting Materials" table are the average of the formulations that are not subjected to heat stress.

According to Table 5, 10 ml of each buffer was mixed with 0.3 ml of a 2 ml glass vial of 15 mg/ml mAb1, 0.2% polysorbate 20 at about 45 ° C for about 14 days. The turbidity was measured at an optical density (OD) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The turbidity difference can be ignored for all samples. The percentage of total recovered mAb1 was determined by reverse phase HPLC (RP-HPLC). The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). An acidic or basic substance is defined as the total amount of mAb1 peaks that are dialyzed from the cation exchange (CEX-HPLC) column earlier than the main peak or at a later residence time. The SE-HPLC results shown in the "Starting Materials" table are the average of the formulations that are not subjected to heat stress.

Formulation test shows alkaline conditions (pH 6.5) mAb1 in the lower solution can be deamidated. Conversely, it has been found to increase the rate of mAb1 that forms a variable molecular weight below pH 5.0. Based on these data, the pH of the mAb1 formulation was maintained between about pH 5.6 and pH 6.2. It has been found that mAbl can be stably present in this pH range.

The effect of the pH and buffer species of 20 mM histidine pH 6, 12.5 mM acetate pH 5.3 or mixed 20 mM histidine and 12.5 acetate pH 5.9 on the stability of mAb 1 was further evaluated in the formulation (Table 6). Comparing the individual buffer systems, the formulation contained histidine and mAb1 at about pH 5.9 acetate was the most stable. The slowest aggregation rate was obtained when mAbl was 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 in a 2 ml glass vial mixed with various buffers were stored at about 45 ° C for about 14 days. The turbidity was measured at an optical density (OD) of 405 nm and the relative change in OD of the starting material at 405 nm was compared. The turbidity difference can be ignored for all samples. The percentage of total recovered mAb1 was determined by reverse phase HPLC (RP-HPLC). The percentage of native and aggregated mAb 1 was determined by size exclusion HPLC (SE-HPLC). An acidic or basic substance is defined as the total amount of mAb1 peaks that are dialyzed from the cation exchange (CEX-HPLC) column earlier than the main peak or at a later residence time. The SE-HPLC results shown in the "Starting Materials" table are the average of the formulations that are not subjected to heat stress.

Example 4. Control of viscosity and tension

The viscosity and tension (expressed as osmolality) of various excipients mixed with high concentrations of mAbl (i.e., 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 easily and comfortably and rapidly infused with high dose of mAb1 (Table 7). Liquid formulation 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 pH 5.9 (Formulation A ) is the best formulation with low viscosity (about 8.5 cPoise) and physiological isotonic (about 293 mOsm/kg) while still maintaining the stability of mAbl.

Example 5. Characterization of Formulation A

The main degradation pathways in the formation of the mab1 liquid formulation are the generation of aggregates, cleavage products and charge variants. The 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 formulated 150 mg/ml mAb1 were found to be in a clear to slightly milky white liquid solution with substantially no visible particles.

The formulated mAb 1 has physical and chemical stability under various pressing conditions (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. Furthermore, it has been found that the pH of the solution, the turbidity or the recovery of mAbl is not affected. After storage of the prepared mAb 1 at 25 ° C for 3 months, the antibody determined by SE-HPLC was not significantly degraded and the degradation by CEX-HPLC was only 3.3% higher. It has been found that increasing the amount of degradation as determined by SE-HPLC and CEX-HPLC after storage for 8 weeks at 45 °C shows that formation of aggregation and charge variation is the major degradation pathway for this mAb1 antibody molecule. No degradation was found when the prepared mAb1 antibody was 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. An acidic or basic substance is defined as the total amount of mAb1 peaks that are dialyzed from the cation exchange (CEX-HPLC) column earlier than the main peak or at a later residence time.

Example 6. Container

The filter-sterilized mAb1 containing formulation has been tested to demonstrate stability. The clinically supplied manufacturing system uses a microporous MILLIPAK filtration device, while the same composition of the filter (microporous Millex DURAPORE) is used in the laboratory.

For a minimum of 2.5 ml of pH 5.9, 150 mg/ml mAb1, 5% (w/v) sucrose, 25 mM L-arginine hydrochloride, 0.2% (w/v) polysorbate 20, 12.5 mM acetate, 20 mM histamine The acid is filled into a 5 ml glass bottle. A 0.5 ml excess formulation was used in a 5 ml vial to ensure that 2.0 ml of the formulation could be withdrawn. This excess is not used to compensate for loss during manufacture of the mAb 1 or mAb containing formulation, degradation during manufacturing, degradation during storage (shelf life), or extended expiration.

The stability of the formulated mAb1 (formulation A) was not affected when stored in polypropylene glycol tubes, polystyrene tubes, polycarbonate tubes, or in glass bottles containing stainless steel blocks compared to storage in glass bottles. (Table 9).

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

<110> Regeneron Pharmaceuticals, Inc.

<120> Stabilization Formulation Containing Anti-Interleukin-4 Receptor (IL-4R) Antibody

<130> 6032

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<170> PatentIn version 3.5

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Claims (13)

 A stable liquid pharmaceutical formulation comprising: (i) a human antibody at a concentration ranging from 15 mg/ml to less than 100 mg/ml, wherein the antibody specifically binds to human interleukin-4 receptor alpha (hIL-4Rα) And comprising 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; ) 10 mM to 15 mM acetate; (iii) 15 mM to 25 mM histidine; (iv) 2.5% weight / volume to 10% weight / volume sucrose; (v) 0.1% weight / volume to 0.3% weight / volume Polysorbate; and (vi) 25 mM to 100 mM arginine; wherein the liquid pharmaceutical formulation has a pH of 5.6 to 6.2.    A liquid pharmaceutical formulation according to claim 1 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.    A liquid pharmaceutical formulation according to claim 1 wherein the polysorbate is polysorbate 20 or polysorbate 80.    A liquid pharmaceutical formulation according to claim 1 wherein the polysorbate is polysorbate 20 and has a concentration of 0.2% ± 0.03% w/v.    A liquid pharmaceutical formulation according to claim 1 wherein the polysorbate is polysorbate 80 and has a concentration of 0.2% ± 0.03% w/v.    A liquid pharmaceutical formulation according to claim 1 wherein sucrose is present at a concentration of 5% ± 0.75% w/v.    A liquid pharmaceutical formulation according to claim 1 wherein arginine is present in a concentration of 25 mM ± 3.75 mM.    A liquid pharmaceutical formulation according to claim 1 wherein arginine is present at a concentration of 50 mM ± 7.5 mM.    The liquid pharmaceutical preparation of claim 1, wherein the pharmaceutical preparation is contained in a glass bottle.    A liquid pharmaceutical formulation according to claim 1 wherein the pharmaceutical formulation is contained in a syringe.    A liquid pharmaceutical formulation according to claim 1 wherein the formulation is contained in a microinfusion set.    A liquid pharmaceutical formulation according to claim 10, wherein the syringe comprises a fluorocarbon coated plunger.    A liquid pharmaceutical formulation according to claim 10 or 12, wherein the syringe is a low tungsten syringe.