HK1261758A1 - Antibody formulations - Google Patents

Description

Antibody formulations

The present application is a divisional application of PCT application PCT/US2012/062572 entitled "antibody formulation", filed on 30/10/2012 in 2012, having a date of entry into the national phase of china on 29/4/2014, No. 201280053245. X.

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 61/553,916, filed on 31/10/2011, which is hereby incorporated by reference in its entirety.

Technical Field

The invention provides formulations comprising anti-IL-13 antibodies, including pharmaceutical formulations and methods of using such formulations.

Sequence listing

This application contains a sequence listing that has been filed in ASCII format over EFS networks and is thus incorporated by reference in its entirety. The ASCII copy created on day 10, 4, 2012 was named p4786r1w.txt and was 22,776 bits in size.

Background

Interleukin (IL) -13 is a pleiotropic T helper cell subset 2(Th2) cytokine. It has been speculated that IL13 may play a more significant role in effector function associated with asthma symptoms than other Th2 cytokines (Corry, curr. opin. immunol.,11:610 (1999)). Humanized anti-IL-13 antibodies have been described. See, for example, Intn' lPub.No. 2005/062967. One particular anti-IL 13 antibody has been clinically studied for the treatment of patients with asthma dyscontrol, lebrikizumab. Some of these results have been described in Corren et al, N Engl J Med 365(12):1088-98 (2011).

formulations of such proteins present particular problems because the proteins (including antibodies) are larger and more complex than traditional organic and inorganic drugs (e.g., possess multiple functional groups in addition to complex three-dimensional structures). in order for the proteins to remain biologically active, the formulations must ensure that the conformational integrity of at least the core amino acid sequence of the protein is intact, while at the same time protecting the multiple functional groups of the protein from degradation.

For example, for therapeutic administration routes or for therapeutic applications, high concentration (e.g., >100mg/mL) liquid antibody formulations are desirable, where small volumes of drug are recommended, e.g., for subcutaneous injection. However, high concentration antibody formulations present numerous problems and problems. One problem is instability due to particle formation. For reconstituted liquid formulations, this problem has been addressed by the use of surfactants (e.g., polysorbates), but surfactants are sometimes considered unsuitable for liquid formulations because they make further processing difficult. In addition, surfactants also do not reduce the increased viscosity that results from the large molecular nature of antibodies leading to numerous intermolecular interactions.

Although surfactants have been shown to significantly reduce the extent of protein particle formation, they do not address the problem of increased viscosity leading to difficult handling and administration of concentrated antibody formulations. Antibodies tend to form viscous solutions at high concentrations due to their macromolecular nature and intermolecular interaction potential. In addition, pharmaceutically acceptable sugars are often used as stabilizers. Such sugars may enhance intermolecular interactions, thereby increasing the viscosity of the formulation. Highly viscous formulations are difficult to manufacture, draw into a syringe, and inject subcutaneously. The use of force when handling viscous formulations results in excessive foaming, which can lead to denaturation and inactivation of the active biological product.

Certain formulations of high concentrations of antibodies have been described. See, e.g., Intn' l publications 2006/065746 and 2002/30463. These publications do not specifically describe high concentrations of anti-IL 13 antibody.

It would be highly advantageous to have a formulation comprising an anti-IL-13 antibody that has extended stability and low viscosity at high antibody concentrations. Formulations with high antibody concentrations of such properties would be highly advantageous for certain routes of administration (e.g., subcutaneous administration). The formulations provided herein meet these needs and provide other useful benefits.

All references, including patent applications and publications, referred to herein are incorporated by reference in their entirety for any purpose.

SUMMARY

The compositions of the present invention are based, at least in part, on the following findings: the anti-IL 13 antibodies described herein (lekulizumab) can be formulated at high concentrations (>100mg/mL) in histidine buffer containing a polyol and a surfactant and such high antibody concentration formulations have low viscosity, have extended physical and chemical stability, and maintain potency. The compositions or formulations of the invention are useful, for example, in the treatment of asthma and other pulmonary disorders, such as idiopathic pulmonary fibrosis and certain allergic diseases, autoimmune diseases, and other inflammatory diseases. In addition, such formulations may be packaged into a subcutaneous administration device as described herein, while maintaining, for example, product stability and other desirable attributes.

Accordingly, in one aspect, a formulation comprising an anti-IL 13 antibody is provided. In certain embodiments, the concentration of the antibody in the formulation is at least 100mg/mL and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃. In another embodiment, an anti-IL 13 antibody comprises three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No. 1, a CDR-H2 having the amino acid sequence of SEQ ID No.2 and a CDR-H3 having the amino acid sequence of SEQ ID No. 3, and three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6. In one embodiment, the anti-IL 13 antibody comprises a heavy chain variable region having the amino acid sequence of seq id No. 7. In one embodiment, the anti-IL 13 antibody comprises a light chain variable region having the amino acid sequence of SEQ id No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10. In one embodiment, the anti-IL 13 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the anti-IL 13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 7 and a light chain variable region having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the concentration of antibody is 125 mg/mL. In one embodiment, the concentration of antibody is 150 mg/mL.

In another aspect, the formulation comprises a histidine acetate buffer at a pH of 5.4 to 6.0, and the histidine acetate concentration in the buffer is between 5mM and 40 mM. In certain embodiments, the formulation comprises a polyol and a surfactant, and the concentration of the polyol in the formulation is between 100mM and 200mM and the concentration of the surfactant in the formulation is between 0.01% and 0.1%. In certain embodiments, the polyol is sucrose and the surfactant is polysorbate 20. In certain embodiments, the histidine acetate buffer has a pH of 5.7 and the histidine acetate concentration in the buffer is 20mM, and the concentration of sucrose in the formulation is 175mM and the concentration of polysorbate 20 is 0.03%. In one embodiment, the concentration of antibody is 125mg/mL or 150 mg/mL. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No. 1, a CDR-H2 having the amino acid sequence of SEQ ID No.2 and a CDR-H3 having the amino acid sequence of SEQ ID No. 3, and three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6.

In yet another aspect, the formulation comprises an anti-IL 13 antibody in a histidine acetate buffer at pH5.4 to 6.0, and the histidine acetate concentration in the buffer is between 5mM and 40mM and the concentration of the antibody in the formulation is at least 100 mg/mL. In certain embodiments, the formulation further comprises a polyol and a surfactant, and the concentration of the polyol in the formulation is between 100mM and 200mM and the concentration of the surfactant in the formulation is between 0.01% and 0.1%. In one embodiment, the polyol is sucrose and the surfactant is polysorbate 20. In one embodiment, the histidine acetate buffer has a pH of 5.7 and the histidine acetate concentration in the buffer is 20mM, and wherein the concentration of sucrose in the formulation is 175mM and the concentration of polysorbate 20 is 0.03%. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No. 1, a CDR-H2 having the amino acid sequence of SEQ ID No.2 and a CDR-H3 having the amino acid sequence of SEQ ID No. 3, and three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6. In one embodiment, the anti-IL 13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 7. In one embodiment, the anti-IL 13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10. In one embodiment, the anti-IL 13 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the anti-IL 13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 7 and a light chain variable region having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the formulation has a viscosity of less than 15 centipoise (cP) at 25 ℃. In one embodiment, the concentration of antibody is 125 mg/mL. In one embodiment, the concentration of antibody is 150 mg/mL.

In yet another aspect, formulations comprising anti-IL-13 antibodies with extended stability are provided. In certain embodiments, the antibody concentration is at least 100mg/mL and the viscosity is less than 15 centipoise (cP) at 25 ℃. In one embodiment, the anti-IL-13 antibody is stable at 5 ℃ for at least one year. In one embodiment, the anti-IL-13 antibody is stable at 5 ℃ for at least two years. In one embodiment, the anti-IL 13 antibody is stable at 5 ℃ for three years. In one embodiment, the anti-IL 13 antibody is stable at 25 ℃ for at least 4 weeks, or at 25 ℃ for at least 8 weeks, or at 25 ℃ for at least 12 weeks, or at 4 ℃ for 26 weeks. In one embodiment, the formulation comprises a histidine acetate buffer at pH5.4 to 6.0, and the histidine acetate concentration in the buffer is between 5mM and 40 mM. In one embodiment, the formulation further comprises a polyol and a surfactant, and the concentration of the polyol in the formulation is between 100mM and 200mM and the concentration of the surfactant in the formulation is between 0.01% and 0.1%. In one embodiment, the polyol is sucrose and the surfactant is polysorbate 20. In one embodiment, the histidine acetate buffer has a pH of 5.7 and the histidine acetate concentration in the buffer is 20mM, and the concentration of sucrose in the formulation is 175mM and the concentration of polysorbate 20 is 0.03%. In one embodiment, the concentration of antibody is 125mg/mL or 150 mg/mL. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No. 1, a CDR-H2 having the amino acid sequence of SEQ ID No.2 and a CDR-H3 having the amino acid sequence of SEQ ID No. 3, and three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6.

In yet another aspect, a formulation comprising an anti-IL 13 antibody having extended stability in 20mM histidine acetate buffer pH5.7, 175mM sucrose, 0.03% polysorbate 20 is provided. In one embodiment, the concentration of the antibody in the formulation is 125mg/mL and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃. In one embodiment, the concentration of the antibody in the formulation is 150mg/mL and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No. 1, a CDR-H2 having the amino acid sequence of SEQ ID No.2 and a CDR-H3 having the amino acid sequence of SEQ ID No. 3, and three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6. In one embodiment, the anti-IL 13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 7 and a light chain variable region having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14.

In yet another aspect, an article of manufacture comprising a subcutaneous applicator is provided. In certain embodiments, the subcutaneous administration device delivers a near flat dose (flat dose) of anti-IL 13 antibody to the patient. In one embodiment, the near flat dose is 37.5mg of anti-IL 13 antibody. In one embodiment, the near flat dose is 75mg of anti-IL 13 antibody. In one embodiment, the near flat dose is 125mg of anti-IL 13 antibody. In one embodiment, the near-flat dose is 150mg of anti-IL 13 antibody. In certain embodiments, the anti-IL 13 antibody is secukinumab. The anti-IL 13 antibody in the subcutaneous administration device was formulated in buffers and other excipients as described above so that it was provided in a stable pharmaceutical formulation. In certain embodiments, the subcutaneous administration device is a prefilled syringe comprising a glass needleA tube, a piston rod comprising a piston stopper (plungerstopper) and a needle. In certain embodiments, the subcutaneous administration device further comprises a needle shield and optionally a needle shield device. In certain embodiments, the volume of formulation contained in the prefilled syringe is 0.3mL, 1mL, 1.5mL, or 2.0 mL. In certain embodiments, the needle is a strut (staked-in) needle comprising a 3-bevel tip or a 5-bevel tip. In certain embodiments, the needle is between 25 gauge (G) and 30G and between 1/2 inch and 5/8 inch in length. In one embodiment, the subcutaneous administration set comprises a prefilled 1.0mL low borosilicate tungsten glass (type I) syringe and a stainless steel 5 bevel 27G1/2 inch long thin-walled pillared needle. In certain embodiments, the subcutaneous administration device comprises a rigid needle shield. In certain embodiments, the rigid needle shield comprises a rubber formulation having a low zinc content. In one embodiment, the needle shield is rigid and comprises an elastomeric component FM27/0 and a rigid polypropylene shield. In certain embodiments, the piston rod comprises a rubber piston stopper. In certain embodiments, the rubber piston stopper comprises 4023/50 rubber andethylene-tetrafluoroethylene (ETFE) coating. In certain embodiments, the subcutaneous administration device comprises a needle safety device. Exemplary needle safety devices include, but are not limited to, UltrasafeNeedle sheath X100L (Safety ceramics, Inc.) and Rexam Safety Sound^TM(Rexam)。

In yet another aspect, a method of treating asthma in a patient is provided. In certain embodiments, the method comprises administering to the patient an effective amount of any one of the foregoing formulations. In certain embodiments, the effective amount is 0.3mL, 0.5mL, 1mL, or 2mL, or about 0.3mL, about 0.5mL, about 1mL, or about 2 mL. In another aspect, a method of treating idiopathic pulmonary fibrosis in a patient is provided. In certain embodiments, the method comprises administering to the patient an effective amount of any one of the foregoing formulations. In certain embodiments, the effective amount is 0.5mL, 1mL, or 2mL, or about 0.5mL, about 1mL, or about 2 mL.

In yet another aspect, methods of subcutaneously administering a formulation comprising an anti-IL 13 antibody are provided. Such methods include subcutaneous administration of any of the anti-IL 13 antibody formulations described above. In certain embodiments, the method comprises a subcutaneous administration device according to any of the devices described above.

Brief Description of Drawings

Figure 1 shows the weekly anti-IL 13 antibody monomer degradation rate as a function of pH as described in example 1.

Figure 2 shows the solution turbidity increase at 350nm of an anti-IL 13 antibody solution as a function of pH during storage at 30 ℃ as described in example 1.

FIG. 3 shows the change of Low Molecular Weight (LMW) soluble fragments and High Molecular Weight (HMW) aggregates as measured by non-reducing CE-SDS during storage at 30 ℃ as a function of pH as described in example 1.

FIG. 4 shows the rate of formation of Acidic (AV) and basic (Peak 1) (BV) variants as a function of pH at 30 ℃ as described in example 1. The rate of formation of the charge variation is expressed as%/week shown on the vertical axis.

FIG. 5 shows the rate of formation of alkaline variant (Peak 2) (BV2) and the rate of loss of Major Peak (MP) as a function of pH at 30 ℃ as described in example 1. The rate of formation of the charge variation is expressed as%/week shown on the vertical axis.

Figure 6 shows the rheological characterization of anti-IL 13 antibody as a function of antibody concentration and solution pH as described in example 1. Solution viscosity is expressed in centipoise (cP) at 25 ℃ as shown on the vertical axis.

Figure 7 shows the rheological characterization of different monoclonal antibodies over a wide range of concentrations, as described in example 1. Solution viscosity is expressed in centipoise (cP) at 25 ℃ as shown on the vertical axis.

Figure 8 shows the quantification of the visual appearance of anti-IL 13 and anti-CD 20 antibody solutions as a function of concentration using 90 degree nephelometry (nephelometry), as described in example 1.

Figure 9 shows the turbidity measurements (a350) of anti-IL 13 and anti-CD 20 antibody solutions as a function of mAb concentration as described in example 1.

Figure 10 shows the turbidity of anti-IL 13 antibody solutions as a function of concentration and pH as described in example 1.

Figure 11 shows the sub-visible (subvisible) particle counts of anti-IL 13 solution and anti-CD 20 antibody solution as a function of mAb concentration as described in example 1.

FIG. 12 shows the measurement of scattering turbidimetry (nephelometric), transmission turbidimetric (turbidimetric) and static light scattering for a 125mg/mL anti-IL 13 antibody solution as described in example 1.

FIG. 13 summarizes the temperature dependence of opalescence of solutions of anti-IL 13 antibody at 125mg/mL and at 204mg/mL under different pH conditions, as described in example 1.

FIG. 14 summarizes the hot-melt transition peaks observed with anti-IL 13 formulation composition and solution pH as a function of two partially resolved peaks in capillary DSC as described in example 1.

FIG. 15 summarizes the measured permeability second virial coefficient (B) of anti-IL 13 antibody as a function of solution pH when the sample is in simple buffer as shown and measured from 0.1-1.0mg/mL as described in example 1₂)。

FIG. 16 shows the measured permeability second virial coefficient of anti-IL 13 antibody as a function of formulation composition and pH over the range of 1.0-10mg/mL as described in example 1.

Figure 17 shows the measured static light scattering intensity versus concentration for each of the anti-IL 13 antibody and anti-CD 20 antibody compared to the Hard Spheroid (HS) model as described in example 1.

Figure 18 shows static light scattering data for anti-IL 13 antibody as a function of formulation pH expressed as apparent molecular weight observed at concentrations up to 200mg/mL, as described in example 1.

FIG. 19 shows the apparent molecular weights of anti-IL 13 and anti-CD 20 antibodies in solution at high concentrations of up to 200mg/mL, as described in example 1.

FIG. 20 shows the shear viscosity measured at 25 ℃ for anti-IL 13 and anti-CD 20 under the corresponding formulation conditions, as described in example 1.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, Dictionary of Microbiology and molecular Biology, 2 nd edition, J.Wiley & Sons (New York, N.Y.1994) and March, advanced organic Chemistry Reactions, Mechanisms and Structure, 4 th edition, John Wiley & Sons (New York, N.Y.1992) provide the skilled person with a general guide to a number of terms used in this application.

Definition of

For the purpose of interpreting the specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any of the definitions set forth below contradict any document incorporated by reference herein, the definition set forth below controls.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" or "an antibody" includes a plurality of proteins or a plurality of antibodies, respectively; reference to "a cell" includes mixtures comprising cells and the like.

The term "pharmaceutical formulation" refers to a preparation in such a form as to allow the biological activity of the active ingredient to be effective and which does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered. Such formulations are sterile. "pharmaceutically acceptable" excipients (carriers, additives) are those excipients which can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

A "sterile" formulation is sterile or free or substantially free of all living microorganisms and their spores.

A "frozen" formulation is one that is at a temperature below 0 ℃. Typically, the frozen formulation is not lyophilized, nor has it undergone prior or subsequent lyophilization. In certain embodiments, the frozen formulation comprises a frozen drug substance (in a stainless steel canister) for storage or a frozen drug substance (in a final vial configuration).

A "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity upon storage. In certain embodiments, the formulation substantially retains its physical and chemical stability, as well as its biological activity, when stored. The storage time is typically selected based on the expected shelf life of the formulation.

As used herein, a formulation having "extended stability" means a formulation in which the proteins inside it substantially maintain their physical stability, chemical stability and biological activity when stored at 5 ℃ for one year or more. In certain embodiments, storage is at 5 ℃ for two years or more. In certain embodiments, storage is at 5 ℃ for up to three years.

A protein "retains its physical stability" in a pharmaceutical formulation if the protein does not show signs of aggregation, precipitation and/or denaturation or very little aggregation, precipitation and/or denaturation as measured by color and/or clarity or as measured by UV light scattering or by size exclusion chromatography.

A protein "retains its chemical stability" in a pharmaceutical formulation if the chemical stability is such at a given time that the protein is considered to retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Chemical alteration may involve size adjustment (e.g., trimming), which may be evaluated, for example, using size exclusion chromatography, SDS-PAGE, and/or matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOFMS). Other types of chemical changes include charge changes (e.g., arising from deamidation), which can be assessed, for example, by ion exchange chromatography or imaging capillary isoelectric focusing (icIEF).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody is within about 10% of the biological activity exhibited at the time the pharmaceutical formulation was prepared (within assay error) at a given time, e.g., as determined in an antigen binding assay or potency assay.

Herein, "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen. It may also include antibodies that bind to an antigen and produce a measurable biological response that can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

A "deamidated" monoclonal antibody is one in which one or more asparagine residues have been derivatized to, for example, aspartic acid or isoaspartic acid.

An antibody that is "susceptible to deamidation" refers to an antibody that comprises one or more residues that have been found to be susceptible to deamidation.

An antibody that is "susceptible to aggregation" refers to an antibody that has been found to aggregate with other antibody molecules, particularly when frozen and/or agitated.

An antibody that is "readily fragmented" refers to an antibody in which it has been found to be cleaved into two or more fragments, for example at its hinge region.

By "reducing deamidation, aggregation or fragmentation" is meant preventing or reducing the amount of deamidation, aggregation or fragmentation relative to a monoclonal antibody formulated at a different pH or in a different buffer.

The formulated antibody is substantially pure and desirably substantially homogeneous (e.g., free of contaminating proteins, etc.). By "substantially pure" antibody is meant a composition comprising at least about 90% by weight or at least about 95% by weight of the antibody, based on the total weight of the composition. By "substantially homogeneous" antibody is meant a composition comprising at least about 99% by weight of the antibody, based on the total weight of the composition.

By "isotonic" is meant that the formulation of interest has substantially the same osmotic pressure as human blood. An isotonic formulation will generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or freeze-type permeameter.

As used herein, the term "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugated components.

"histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, histidine succinate, and the like. In one embodiment, the histidine buffer is histidine acetate. In one embodiment, histidine acetate buffer is prepared using acetic acid (liquid) titration of L-histidine (free base, solid). In certain embodiments, the histidine buffer or histidine acetate buffer is between pH 4.5 and 6.5. In certain embodiments, the histidine buffer or histidine acetate buffer is between pH5.4 and 6.0. In one embodiment, the buffer has a pH of 5.6. In one embodiment, the buffer has a pH of 5.7. In one embodiment, the buffer has a pH of 5.8.

Herein, "surfactant" refers to a surfactant, typically a nonionic surfactant. Of surfactants hereinExamples include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or octadecyl-sulphobetaine; lauryl-, myristyl-, linoleyl-or octadecyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUAT^TMSeries (Mona Industries, inc., Paterson, n.j.); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.), and the like. In one embodiment, the surfactant is polysorbate 20.

"preservatives" are compounds that may optionally be included in the formulation to substantially reduce bacterial effects therein, thus, for example, facilitating the production of a multi-purpose formulation. Examples of possible preservatives include octadecyl dimethyl ammonium chloride, chlorhexidine di-ammonium, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides where the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. In one embodiment, the preservative herein is benzyl alcohol.

"polyol" is a substance having a plurality of hydroxyl groups and includes sugars (reduced and non-reduced sugars), sugar alcohols and sugar acids. Polyols may optionally be included in the formulation. In certain embodiments, the polyols herein have a molecular weight of less than about 600kD (e.g., in the range of about 120 to about 400 kD). A "reduced sugar" is a sugar that contains a hemiacetal group that can reduce metal ions or covalently react with lysine and other amino groups in proteins, and a "non-reduced sugar" is a sugar that does not have the properties of a reducing sugar. Examples of reduced sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol, and glycerol are examples of sugar alcohols. For sugar acids, these include L-gluconic acid and its metal salts. Where it is desired that the formulation be freeze-thaw stable, the polyol is typically one that does not crystallize at freezing temperatures (e.g., -200C) so that it destabilizes the antibodies in the formulation. In one embodiment, the polyol is a non-reducing sugar. In one such embodiment, the non-reducing sugar is sucrose.

As used herein, "asthma" refers to a complex condition characterized by variable and recurrent symptoms, reversible airflow obstruction (e.g., via bronchodilators), and bronchial hyperreactivity that may or may not be associated with underlying inflammation. Examples of asthma include aspirin-sensitive/exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, corticosteroid-naive asthma, chronic asthma, corticosteroid-resistant asthma, corticosteroid refractory asthma, newly diagnosed and untreated asthma, asthma due to smoking, corticosteroid-uncontrolled asthma, and other asthma as mentioned in J Allergy Clin Immunol (2010)126(5): 926-938.

As used herein, "treatment" refers to clinical intervention intended to alter the natural process of the individual or cell being treated, and may be performed before or during the clinical pathological process. Desirable therapeutic effects include preventing the occurrence or recurrence of a disease or condition or symptom thereof, alleviating the condition or symptom of the disease, attenuating any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, alleviating or palliating the disease state, and achieving a remission or improved prognosis.

An "effective amount" refers to an amount effective, at a desired dosage and for a desired period of time, to achieve the desired therapeutic or prophylactic result. The "therapeutically effective amount" of a therapeutic agent may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

An "individual," "subject," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human and non-human primates) and rodents (e.g., mice and rats). In certain embodiments, the mammal is a human.

A "drug" is an active drug that treats a disease, disorder, and/or condition.

"antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins having similar structural features. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that generally lack antigen specificity. The latter polypeptides are produced, for example, at low levels by the lymphatic fluid system and at increased levels by myelomas.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in more detail herein). The antibody may be chimeric, human, humanized and/or affinity matured.

The terms "full length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, not to antibody fragments as defined below. The term particularly refers to antibodies having a heavy chain comprising an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably including the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, referred to as "Fab fragments, each having a single antigen-binding site, and a residual" Fc fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced F (ab') which had two antigen binding sites and was still capable of cross-linking the antigen₂And (3) fragment.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. Overall, the 6 CDRs of the Fv confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

The Fab fragment contains the heavy and light chain variable domains and also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of an additional few residues at the carboxy-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is herein the name for Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments were originally produced as paired Fab fragments with hinge region cysteines between them. Other chemical couplings of antibody fragments are also known.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising this population are substantially identical, except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is characterized as not being a mixture of unrelated antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from multiple clones (hybridoma clones, phage clones, or pools of recombinant DNA clones). It will be appreciated that the target binding sequence selected may be further altered, for example to improve affinity for the target, to humanise the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and that antibodies comprising the altered target binding sequence are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are also advantageous in that they are generally not contaminated with other immunoglobulins.

The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, Monoclonal Antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al, Nature,256:495 (1975); Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition, 1988); Hammerling et al, cited in: Monoclonal Antibodies and T-cell hybrids, 681(Elsevier, N.Y., 1981); recombinant DNA method (see, for example, U.S. Pat. No. 4,816,567), phage display technology (see, for example, Clackson et al, Nature,352:624-628 (1991); 2004. mol. biol.222:581-597 (1992); Sidhu et al, J.mol. 338.338: 2-1242 (1991); U.52-55, 120. J.340 (Legend), U.31: 85-340. 12, 2000-99.72 (Legend), and 3. 99.72 (U.31, 120. J.31, 120. J.340-33, 2000, U.3. E.3-99, U.3, 2000, USA), And techniques for producing human antibodies or human-like antibodies in animals having part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggemann et al, Yeast in Immunol.7:33 (1993); U.S. Pat. No. 5,545,807; 5,545,806; 569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al, Bio/technology 10:779-783 (1992); Lonberg Imag et al, Nature 368: 859 (1994); Morrison, Nature: Huhl 812-812; Nature et al, Nature: 85: 14: Nature: 1994; Biotech. 14: 17: 1996; and Bioscher et al; Bioscher. J. 11: 826: 1996).

"monoclonal antibodies" herein specifically include such "chimeric antibodies" in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6855-9855 (1984)).

"native antibody" refers to a naturally occurring immunoglobulin molecule having an indeterminate structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has one variable region (VH), also known as variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chains of antibodies can be divided into one of two types, called kappa and lambda, based on the amino acid sequences of their constant domains.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., Kindt et al, Kuby Immunology, 6 th edition, W.H.Freeman and Co., page 91 (2007)). A single VH domain or VL domain may be sufficient to confer antigen binding specificity. Alternatively, antibodies that bind a particular antigen can be isolated using VH or VL domains from antibodies that bind the antigen to screen a library of complementary VL domains or VH domains, respectively. See, e.g., Portolano et al, J.Immunol.150:880-887 (1993); clarkson et al, Nature 352:624-628 (1991).

"humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least 1, and typically 2, variable domains and all or substantially all of the FR regions corresponding to those of a human antibody, wherein all or substantially all of the HVRs (e.g., CDRs) of the variable domains correspond to those of a non-human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" (e.g., non-human antibodies) of an antibody refer to antibodies that have undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101 (H3). (Chothia and Lesk J.mol.biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al, Sequences of Proteins of immunological interest, 5 th edition Public Health Service, national institutes of Health, Bethesda, MD (1991)). The exception is CDR1 in VH, which usually comprises amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDR is contained within a CDR region called the abbreviation-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3. (see Almagro and Fransson, front. biosci.13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

A "human antibody" is an antibody that comprises an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or has been produced using any of the techniques for producing human antibodies as disclosed herein. Such techniques include screening human-derived combinatorial libraries, such as phage display libraries (see, e.g., Marks et al, J.mol.biol.,222:581-597(1991) and Hoogenboom et al, Nucl.acids Res.,19:4133-4137 (1991)); human myeloma and mouse-human hybrid myeloma cell lines used to produce human Monoclonal antibodies are used (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and applications, pp.55-93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)); and monoclonal antibodies in transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci USA,90:2551 (1993); Jakobovits et al, Nature,362:255 (1993); Bruggemann et al, Yeast in Immunol, 7:33 (1993)). Such human antibody definitions specifically exclude humanized antibodies comprising antigen binding residues from non-human animals.

An "affinity matured" antibody is one in which one or more alterations in one or more CDRs are present, wherein the alterations result in an improvement in the affinity of the antibody for the antigen as compared to a parent antibody without the alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies were generated by methods known in the art. Marks et al, Bio/Technology 10:779-783(1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVRs and/or framework residues was performed by Barbas et al ProcNat.Acad.Sci.USA 91:3809-3813 (1994); schier et al Gene 169:147-155 (1995); yelton et al J.Immunol.155:1994-2004 (1995); jackson et al, J.Immunol.154(7):3310-9 (1995); and Hawkins et al, J.mol.biol.226:889-896 (1992).

A "blocking antibody" or "antagonist antibody" is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Certain blocking or antagonistic antibodies partially or completely inhibit the biological activity of an antigen.

there are five major classes of antibodies, IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2.

As used herein, an "anti-IL 13 antibody," also known as lylizumab, means a humanized IgG4 antibody that binds human IL 13. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: CDR-H1(SEQ ID NO.:1), CDR-H2(SEQ ID NO.:2) and CDR-H3(SEQ ID NO.: 3). In one embodiment, the anti-IL 13 antibody comprises three light chain CDRs: CDR-L1(SEQ ID NO.:4), CDR-L2(SEQ ID NO.:5) and CDR-L3(SEQ ID NO.: 6). In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs and three light chain CDRs: CDR-H1(SEQ ID No.:1), CDR-H2(SEQ ID No.:2), CDR-H3(SEQ ID No.:3), CDR-L1(SEQ ID No.:4), CDR-L2(SEQ ID No.:5) and CDR-L3(SEQ ID No.: 6). In one embodiment, the anti-IL 13 antibody comprises a variable heavy chain region VH having an amino acid sequence selected from SEQ ID nos.7 and 8. In one embodiment, the anti-IL 13 antibody comprises a variable light chain region VL having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a variable heavy chain region VH selected from the amino acid sequences of SEQ ID nos.7 and 8, and a variable light chain region VL having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13. In one embodiment, the anti-IL 13 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13 and a light chain having an amino acid sequence of SEQ ID No. 14. anti-IL 13 antibodies are also described in International publication No. 2005/062967.

An "isolated" biomolecule, such as a nucleic acid, polypeptide, or antibody, is a molecule that has been identified and separated and/or recovered from at least one component of its natural environment.

References herein to "about" a certain value or parameter include (and describe) embodiments that refer to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

"subcutaneous administration device" refers to a device adapted or designed to administer a drug (e.g., a therapeutic antibody or pharmaceutical formulation) by the subcutaneous route. Exemplary subcutaneous administration devices include, but are not limited to, syringes, including pre-filled syringes, injection devices, infusion pumps, injection pens, needle-less devices, and patch delivery systems. The subcutaneous administration device administers a certain volume of the pharmaceutical formulation, for example, about 1.0mL, about 1.25mL, about 1.5mL, about 1.75mL, or about 2.0 mL.

"package insert" or "label" is used to refer to instructions customarily included in commercial packages of therapeutic products or drugs, containing information about the indication, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products or drugs, etc.

A "kit" is any article of manufacture (e.g., a package or container) that contains at least one agent, e.g., a medicament for treating asthma or other lung disorders. In certain embodiments, the article is marketed, distributed or sold as a unit for performing the methods of the present invention.

A "target audience" is a group of people or institutions to whom a particular drug is promoted or intended, such as through sales or advertising, particularly for a particular use, treatment or indication, such as an individual patient, a patient population, a newspaper, a medical literature and magazine reader, a television or internet viewer, a radio or internet listener, a physician, a pharmaceutical company, and so forth.

The term "serum sample" refers to any serum sample obtained from an individual. Methods for obtaining serum from a mammal are well known in the art.

The term "whole blood" refers to any whole blood sample obtained from an individual. Generally, whole blood contains all blood components, e.g., cellular components and plasma. Methods for obtaining whole blood from a mammal are well known in the art.

The "amount" or "level" of a biomarker that is associated with an increased clinical benefit for a patient with a disease or disorder or that is predictive of response to a particular therapeutic agent or treatment regimen is a detectable level in a biological sample. These can be measured by methods known to those skilled in the art and also disclosed herein. The level or amount of expression of the biomarker assessed can be used to determine a response or predict response to a treatment or therapeutic agent.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of an amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoding a gene is converted into a structure that is present and operational in a cell. Thus, as used herein, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein.

Asthma and other pulmonary diseases and certain allergic, autoimmune and other inflammatory diseases

Asthma is described as a chronic lung disease involving airway inflammation, hyperresponsiveness and obstruction. Physiologically, airway hyperreactivity was recorded as decreased bronchial airflow following bronchial challenge with methacholine or histamine. Other triggers that trigger airway obstruction include cold air, exercise, viral upper respiratory infections, smoking, and respiratory allergens. Bronchial challenge due to allergen induces a rapid immunoglobulin E (IgE) -mediated decline in early bronchial airflow followed by an IgE-mediated late response in many patients with 4-8 hours of bronchial airflow decline. The early response is caused by inflammatory substances (such as histamine,acute release of leukotrienes, tryptase and Platelet Activating Factor (PAF)) and late phase responses are caused by de novo synthesized pro-inflammatory cytokines (e.g., TNF α, IL4, IL13) and chemokines (e.g., MCP-1 and MIP-1 α) (Busse et al, cited in Allergy: Principles and Practice, Middleston, 1173(1998)) in chronic asthma patients persistent lung symptoms are mediated by an elevated Th2 cellular response it is believed that Th2 cytokines play an important role in disease (Larche et al, J. Allergy Clin. Immunol.,111:450(2003)), in particular, MedIL 13 and IL4 produced by 2 cells (NKT) with NK phenotype in the airways, as shown in the rodent model of asthma (Akbii et al, Nature, 9: Th 582 (2003)). Th of asthma in generalLesions show lung hyperinflation, smooth muscle hypertrophy, thickening of the reticular basal lamina, mucosal edema, epithelial cell exfoliation, ciliary cell destruction, and hypersecretion of mucous glands. Microscopically, asthma is characterized by an increase in the number of eosinophils, neutrophils, lymphocytes and plasma cells, bronchial secretions and mucus present in bronchial tissue. Initially, there is CD4 activated by⁺T-lymphocytes recruit leukocytes from the bloodstream to the airways. Activated T-lymphocytes also direct the release of inflammatory mediators from eosinophils, mast cells, and lymphocytes. In addition, Th2 cells produced IL4, IL5, IL9 and IL 13. IL4, together with IL13, signals the conversion from IgM antibodies to IgE antibodies.

Membrane-bound IgE molecules are cross-linked by allergens resulting in degranulation of mast cells, releasing histamine, leukotrienes and other mediators of persistent airway inflammation. IL5 activates recruitment and activation of eosinophils. Activated mast cells and eosinophils also produce their cytokines that contribute to perpetuating inflammation. These repeated inflammatory cycles in the lung are accompanied by damage to lung tissue, which is subsequently repaired, possibly resulting in long-term structural changes ("remodeling") of the airways.

moderate asthma is currently treated with daily inhaled anti-inflammatory corticosteroids or mast cell inhibitors such as cromolyn sodium or nedocromil, plus inhaled β 2-agonists (3-4 times per day) as needed to alleviate the emergent symptoms (breakthrough symptoms) or allergen-induced or exercise-induced asthma cromolyn sodium and nedocromil block bronchospasm and inflammation, but are generally only effective against allergen-or exercise-related asthma and generally only against juvenile asthma.

Even mildly ill patients exhibit airway inflammation, including infiltration of the mucosa and epithelium by activated T cells, mast cells, and eosinophils. T cells and mast cells release cytokines that promote eosinophil growth and maturation and IgE antibody production, and these cytokines in turn increase microvascular permeability, disrupt the epithelium and stimulate the neural reflex and mucous secretory glands. The result is airway hyperresponsiveness, bronchoconstriction and hypersecretion, manifested as wheezing, coughing, and dyspnea.

Traditionally, asthma has been treated with oral and inhaled bronchodilators. These agents help alleviate asthma symptoms, but do nothing to the underlying inflammation. The recognition of the importance of inflammation in the etiology of asthma has led to increased use of corticosteroids during the last decade, but many patients continue to suffer from uncontrolled asthma.

In addition to asthma, other diseases that may be treated by the formulations of the present invention include allergies, autoimmune diseases, or other inflammatory diseases. Other allergic diseases including allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria; immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis; autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile chronic arthritis; inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease); other diseases associated with IL13 include idiopathic interstitial pneumonia, goblet cell metaplasia, inflammatory and fibrotic lung diseases such as cystic fibrosis, gluten sensitive bowel disease, and Whipple disease; immune diseases of the lung, such as eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis; chronic obstructive pulmonary disease, RSV infection, uveitis (uveitis), scleroderma, osteoporosis, and hodgkin's lymphoma.

Idiopathic Pulmonary Fibrosis (IPF) is a condition that can be addressed with the formulations of the present invention. IPF is a restrictive lung disease characterized by progressive interstitial fibrosis of the lung parenchyma, affecting approximately 100,000 patients in the United states (Raghu et al, Am J RespirCrit Care Med 174:810-816 (2006)). This IPF-associated interstitial fibrosis leads to a progressive loss of lung function, resulting in death from respiratory failure in most patients. Median survival from diagnostic time was 2-3 years (Raghu et al, Am J Respir Crit Care Med 183:788-824 (2011)). The etiology and key molecular and pathophysiological driving forces of IPF are unknown. The only treatment that proved to prolong survival in IPF patients was lung transplantation (thabout et al, Annals of internal medicine 151:767-774 (2009)). However, lung transplantation is associated with a significant morbidity, not all IPF patients are suitable candidates for this therapy, and there is a relative lack of suitable donor lungs. Despite numerous attempts, however, no drug therapy has been shown to greatly prolong survival in IPF patients in randomized, placebo-controlled interventional therapy trials, but some interventions appear to have slowed The rate of decline of lung function in some patients (Raghu et al, Am J Respir Crit Care Med 183:788-824 (2011); Richeldi et al, The new england J. of med.365:1079-1087 (2011)).

Although the prognosis for all IPF patients is unfavorable, there is a great inconsistency in the course of the disease (disease diagnosis) (Raghu et al, Am J Respir Crit Care Med 183:788-824 (2011)). Some patients show a relatively inert process, losing lung function at a relatively constant rate over a range of up to 10 years or more, while others suffer a more rapid decline in lung function, approaching death within one or two years of diagnosis. In addition, some patients suffer from acute exacerbations, generally characterized by a sudden and dramatic decline in lung function. Overall, these patients do not recover adequately following an acute event and often die during exacerbations or shortly thereafter. This inconsistency in the course of the disease suggests that different IPF patients may have different pathophysiological factors as their basis of the disease, which factors may be differentially sensitive to molecular targeted therapies, such as the formulations of the present invention.

Eosinophilic inflammation is associated with a variety of allergic and non-allergic diseases (Gonlugur (2006) immunological. invest.35(1): 29-45). Inflammation is the restorative response of living tissue to injury. One characteristic of the inflammatory response is that leukocytes accumulate in damaged tissue due to certain chemicals produced in the tissue itself. Eosinophils accumulate in a wide variety of conditions such as allergic disease, helminth infection and neoplastic disease (Kudlacz et al, (2002) Inflammation26: 111-119). Eosinophils, a component of the immune system, are defensive elements of mucosal surfaces. They react not only to antigens but also to parasites, chemicals and wounds.

Tissue eosinophils occur in skin diseases such as eczema, pemphigus, acute urticaria and toxic epidermal necrolysis, as well as in atopic dermatitis ([ Rzany et al, 1996] ]). Eosinophils accumulate in tissues and empty the particulate proteins in an IgE-mediated allergic skin reaction ([ Nielsen et al, 2001 ]). Mast cell-bound eosinophils are a possible cause of joint inflammation (Miosec et al, 1997). Eosinophilic inflammation is sometimes associated with joint trauma. Synovial eosinophilia may be associated with a variety of diseases, such as rheumatoid arthritis, parasitic diseases, hypereosinophilic syndrome, lyme disease and allergic processes as well as hemarthrosis and arthroscopy (Atanes et al, 1996). Eosinophilic inflammation can also affect bone ([ Yetiser et al, 2002 ]). Examples of eosinophilic muscle diseases include eosinophilic pericyatitis, eosinophilic polymyositis, and focal eosinophilic myositis ([ Lakhapal et al, 1988 ]). Eosinophilic inflammatory conditions affecting skeletal muscle may be associated with parasitic infections or with features of drugs or some systemic disorders of eosinophilia (e.g., idiopathic hypereosinophilic syndrome and eosinophil-myalgia syndrome). Eosinophils are involved in inflammatory responses to epitopes recognized by autoimmune antibodies ([ Engineer et al, 2001 ]). Connective tissue diseases can lead to neutrophilic, eosinophilic or lymphocytic vasculitis ([ Chen et al, 1996 ]). Tissue and peripheral blood eosinophilia can occur in active rheumatic diseases. Elevated serum ECP levels in ankylosing spondylitis, a class of connective tissue diseases, indicate that eosinophils are also involved in the underlying process (Feltelius et al, 1987). Wegener's granulomatosis may rarely occur with lung nodules, pleural effusion, and peripheral blood eosinophilia (Krupsky et al, 1993).

At least 400/mm³Peripheral eosinophilia can occur in 7% of cases with systemic sclerosis, 31% of cases with localized scleroderma and 61% of cases with eosinophilic fasciitis ([ Falanga and Medsger, 1987)]). Scleroderma produces an inflammatory process very similar to the mysterious and austenitic plexuses and consists of mast cells and eosinophils in the gastrointestinal system. Eosinophil-derived neurotoxins can promote gastrointestinal motility dysfunction as occurs in scleroderma ([ de Schryver Kecskemeti and Clouse, 1989)])。

Eosinophils can be associated with either localized ([ Varga and Kahari,1997]) or systemic ([ Bouros et al, 2002]) proliferation of connective tissue. They can provoke fibrosis by inhibiting proteoglycan degradation in fibroblasts ([ Hernnas et al, 1992]), and fibroblasts mediate eosinophil survival by secreting GM-CSF ([ Vancheri et al, 1989 ]). Eosinophils can be present in nasal tissue ([ Bacherct et al, 2001]), bronchial tissue ([ Arguelles and Blanco,1983]), and gastrointestinal polyp tissue ([ Assarian and Sundareson,1985 ]). Similarly, eosinophils can be localized to inflammatory pseudotumors (myofibroblast tumors). Eosinophils are often associated with inflammatory pseudotumors in the orbital region, in which case the condition may mimic angioedema or allergic rhinoconjunctivitis ([ Li et al, 1992 ]).

Eosinophilic inflammation may be present in tissue wounds (e.g., due to surgery or injury). Eosinophilic inflammation may also be associated with cardiovascular disease (e.g., eosinophilic myocarditis, eosinophilic coronary arteritis, ischemic heart disease, acute myocardial infarction, cardiac rupture). Necrotic inflammatory processes may also involve eosinophilic inflammation (polymyositis, coronary artery dissection, necrotic foci of neuro-Behcet disease, dementia, cerebral infarction).

Certain therapeutic agents

Provided herein are therapeutic agents for the treatment of asthma and other pulmonary diseases. In one embodiment, the therapeutic agent is an anti-IL 13 antibody, also known as lylizumab. Lenjunuzumab is an IgG4 antibody. In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs: CDR-H1(SEQ ID NO.:1), CDR-H2(SEQ ID NO.:2) and CDR-H3(SEQ ID NO.: 3). In one embodiment, the anti-IL 13 antibody comprises three light chain CDRs: CDR-L1(SEQ ID NO.:4), CDR-L2(SEQ ID NO.:5) and CDR-L3(SEQ ID NO.: 6). In one embodiment, the anti-IL 13 antibody comprises three heavy chain CDRs and three light chain CDRs: CDR-H1(SEQ ID No.:1), CDR-H2(SEQ ID No.:2), CDR-H3(SEQ ID No.:3), CDR-L1(SEQ ID No.:4), CDR-L2(SEQ ID No.:5) and CDR-L3(SEQ ID No.: 6). In one embodiment, the anti-IL 13 antibody comprises a variable heavy chain region VH having an amino acid sequence selected from SEQ ID nos.7 and 8. In one embodiment, the anti-IL 13 antibody comprises a variable light chain region VL having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a variable heavy chain region VH selected from the amino acid sequences of SEQ ID nos.7 and 8, and a variable light chain region VL having the amino acid sequence of SEQ ID No. 9. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13. In one embodiment, the anti-IL 13 antibody comprises a light chain having the amino acid sequence of SEQ ID No. 14. In one embodiment, the anti-IL 13 antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13 and a light chain having an amino acid sequence of SEQ ID No. 14. anti-IL 13 antibodies are also described in International publication No. 2005/062967.

In another aspect, an anti-IL-13 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 8. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-IL-13 antibody comprising that sequence retains the ability to bind to human IL-13. In certain embodiments, a total of 1 to 10 amino acids have been substituted, altered, inserted, and/or deleted in SEQ ID No. 8. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDR (i.e., in the FR). Optionally, the anti-IL 13 antibody comprises the VH sequence in SEQ ID No. 8, including post-translational modifications of that sequence.

In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 9. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-IL-13 antibody comprising that sequence retains the ability to bind to IL-13. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID No. 9. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDR (i.e., in the FR). Optionally, the anti-IL-13 antibody comprises the VL sequence in SEQ ID No. 9, including post-translational modifications of this sequence.

In yet another embodiment, an anti-IL-13 antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 9 and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 8.

Certain molecular biomarkers

In some cases, biomarkers, e.g., serum biomarkers, are quantified in a biological sample obtained from a patient as a means of selecting a patient for treatment with a given therapeutic agent. U.S. application Nos. 61/459760, 61/465425, 61/484650, and 61/574485 ("Diagnosis and treatment associated with TH2 Inhibition") describe an periostin assay and method of selecting patients for treatment with an anti-IL 13 antibody formulation as described herein.

General techniques for formulation

Formulations comprising anti-IL 13 antibodies can be prepared and analyzed using certain excipients and techniques known in the art and as further described herein. In certain embodiments, the antibody to be formulated has not been subjected to prior lyophilization and the formulation of interest herein is an aqueous formulation. In certain embodiments, the antibody is a full length antibody. In one embodiment, the antibody in the formulation is an antibody fragment, such as F (ab')₂In such cases, it may be desirable to address issues that may not exist for full-length antibodies (e.g., trimming antibodies to Fab). A therapeutically effective amount of the antibody present in the formulation is determined, for example, by considering the desired dose volume and mode of administration. About 0.1mg/mL to about 250mg/mL, or about 10mg/mL to about 200mg/mL or about 50mg/mL to about 175mg/mL are exemplary antibody concentrations in the formulation. In one embodiment, the anti-IL 13 antibody is formulated at a concentration of 125 mg/mL. In one embodiment, the anti-IL 13 antibody is formulated at a concentration of 150 mg/mL.

An aqueous formulation is prepared comprising the antibody in a pH buffered solution. In certain embodiments, the buffer has a pH in the range of about 4.5 to about 6.5. In certain embodiments, the pH is in the range of pH 5.0 to 6.0, or in the range of pH 5.25 to 5.75, or in the range of pH 5.3 to 5.6. In certain embodiments of the invention, the formulation has a pH of 5.6 or about 5.6. In certain embodiments of the invention, the formulation has a pH of 5.7 or about 5.7. In certain embodiments of the invention, the formulation has a pH of 5.8 or about 5.8. Examples of buffers that control the pH within this range include acetates (e.g., histidine acetate, arginine acetate, sodium acetate), succinates (e.g., histidine succinate, arginine succinate, sodium succinate), gluconates, citrates and other organic acid buffers and combinations thereof. The buffer concentration may be from about 1mM to about 600mM, for example, depending on the buffer and the desired isotonicity of the formulation. In certain embodiments, the buffer contains histidine at a concentration of about 5mM to 40 mM. In one embodiment, the buffer is 20mM histidine acetate, pH 5.7. In certain embodiments, the buffer is 20mM histidine succinate, pH 5.7.

A surfactant may optionally be added to the antibody preparation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of particulate matter in the formulation and/or reduces adsorption. For example, the surfactant may be present in the formulation in an amount of about 0.001% to about 0.5% or about 0.005% to about 0.2% or about 0.01% to about 0.1%. In one embodiment, the surfactant is polysorbate 20 present in the formulation in an amount of 0.03%.

In one embodiment, the formulation contains the agents identified above (e.g., antibodies, buffers, and surfactants) and is substantially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium chloride. In one embodiment, the formulation does not comprise a preservative. In another embodiment, a preservative may be included in the formulation, particularly where the formulation is a multi-dose formulation. The concentration of the preservative may range from about 0.1% to about 2% or from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers may be included in the formulation, such as those described in Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980), provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include additional buffers; a co-solvent; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions.

metal ion chelators are well known to those skilled in the art and include, but are not necessarily limited to, aminopolycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis (β -aminoethyl ether) -N, N' -tetraacetic acid), NTA (nitrilotriacetic acid), EDDS (ethylenediaminedisuccinate), PDTA (1, 3-propylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), ADA (β -alanine diacetic acid), MGCA (methylglycinediacetic acid), and the like.

Tonicity agents (sometimes referred to as "stabilizers") are present to adjust or maintain the tonicity of the liquid composition. When used with large, charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thus mitigating the potential for intermolecular or intramolecular interactions. Tonicity agents may be present in any amount between 0.1% to 25% or 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Tonicity agents include polyhydric, trihydric or higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

additional stabilizers include a wide variety of excipients ranging in function from fillers to solubility enhancers to substances that prevent denaturation or adherence to the container wall, stabilizers may be present in the range of 0.1 to 10,000 parts per weight of active protein or antibody common stabilizers include polyhydric sugar alcohols (listed above), amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like, organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoisitose, inositol (myoisitol), galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycols, sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α -monothioglycerol and sodium thiosulfate, low molecular weight proteins such as albumin, bovine serum albumin, sugars or other immunoglobulins, carbohydrates such as polyethylene glycols, polysaccharides such as lactose, glucose, oligosaccharides, and hydrophilic polysaccharides such as raffinose, oligosaccharides, and disaccharides, oligosaccharides such as maltodextrins, dextrans, and dextrans (e.g., dextrans, and oligosaccharides such as glucose.

Nonionic surfactants or detergents (also known as "wetting agents") are present to help solubilize the therapeutic agent, as well as to protect the therapeutic protein from agitation-induced aggregation, and they also allow the formulation to be exposed to shear surface stress without denaturing the active therapeutic protein or antibody. The nonionic surfactant is present in the range of about 0.05mg/ml to about 1.0mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.),A polyhydric alcohol,Polyoxyethylene sorbitan monoether (-20、-80, etc.),Lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and protein Drug Delivery,247-301, Vincent Lee, Marcel Dekker, inc., New York, n.y., Pubs. (1991) and Jones, a.adv.drug Delivery rev.10:29-90 (1993). Stability may be measured at a selected temperature for a selected duration. In certain embodiments, the formulation is stable at about 40 ℃ for at least about 2-4 weeks, and/or at about 5 ℃ for at least 3 months, and/or at about 5 ℃ for at least 6 months, and/or at about 5 ℃ for at least 12 months and/or at about-20 ℃ for at least 3 months or at least 1 year. In certain embodiments, the formulation is stable at about 25 ℃ for at least 6 months and/or at about 25 ℃ for 12 months, and/or at about 5 ℃ for 6 months, and/or at about 5 ℃ for 12 months, and/or at about-20 ℃ for at least 6 months, and/or at about-20 ℃ for at least 12 months, and/or at about-20 ℃ or-20 ℃ for at least 2 years. In certain embodiments, the formulation is stable after the formulation is frozen (frozen to, e.g., -70 ℃) and thawed, e.g., after 1,2, or 3 cycles of freezing and thawing. Stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); charge heterogeneity is assessed by using cation exchange chromatography, imaged capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; comparing SDS-PAGE analysis of reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; evaluating the biological activity or antigen binding function of the antibody. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal elongation, C-terminal processing, glycosylation differences, and the like.

The formulation to be used for in vivo administration should be sterile. This is easily accomplished by filtration through sterile filtration membranes before or after preparation of the formulation.

The therapeutic agent may be administered according to known methods, such as intravenous administration as an infusion or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Optionally, administration can be by micro-pump infusion using various commercially available devices.

The formulations herein may also contain more than one active compound, preferably those active compounds that have complementary activities that do not adversely affect each other, as required by the particular indication being treated. Alternatively or additionally, the composition may comprise a cytotoxic drug, cytokine or growth inhibitory agent. Such molecules are suitably present in an amount effective for the intended purpose.

The active ingredients may also be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly- (methylmethacylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or emulsions, for example, prepared by coacervation techniques or interfacial polymerization methods, respectively, such techniques are disclosed above in Remington's pharmaceutical sciences, 18 th edition.

Sustained release articles can be prepared. Suitable examples of sustained-release articles include solid hydrophobic polymer semipermeable matrices containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), L-glutamic acid, and γ -ethyl-glutamateCopolymers of (A) and (B) non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. Human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rpg120 have been successfully used for the microencapsulation of recombinant proteins for sustained release. Johnson et al, nat. Med.2:795-799 (1996); yasuda et al, biomed.Ther.27:1221-1223 (1993); hora et al, Bio/Technology 8:755-758(1990), "Design and Production of Single ImmunationVaccines Using Polylactide polyol microspheres Systems", from Vaccine Design: The Subunit and Adjuvant apparatus, Powell and Newman eds (Plenum Press: New York,1995), pages 439-462; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. patent No. 5,654,010.

Sustained release formulations of these proteins can be developed using polylactic-co-glycolic acid (PLGA) polymers due to their biocompatibility and a wide variety of biodegradable properties. Can rapidly remove PLGA degradation products, lactic acid and glycolic acid in human body. In addition, the degradability of such polymers can be adjusted from months to years, depending on their molecular weight and composition. Lewis, "Controlled release of biologically active agents from cellulose/glycolic polymer", from Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker; New York,1990), M.Chasin and R.Langer (eds.) pages 1-41.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in loss of biological activity and possible alteration of immunogenicity. Rational strategies for stabilization can be conceived according to the mechanisms involved. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation due to sulfhydryl-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Liposome compositions or proteoid compositions can also be used to formulate the proteins or antibodies disclosed herein. See U.S. Pat. nos. 4,925,673 and 5,013,556.

The stability of the proteins and antibodies described herein can be enhanced by the use of non-toxic "water-soluble multivalent metal salts". Examples include Ca²⁺、Mg²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Cu²⁺、Sn²⁺、Sn⁴⁺、Al²⁺And Al³⁺. Examples of anions that can form water-soluble salts with the above polyvalent metal cations include those formed from inorganic acids and/or organic acids. Such water-soluble salts have a solubility in water (at 20 ℃) of at least about 20mg/mL, alternatively at least about 100mg/mL, and additionally at least about 200 mg/mL.

Suitable inorganic acids that may be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that may be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids within this definition may be defined as saturated or unsaturated C_2-9Carboxylic acids (e.g., aliphatic mono-, di-, and tri-carboxylic acids). For example, exemplary monocarboxylic acids within this definition include saturated C2-9 monocarboxylic acids: acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid and capryonic acid, and unsaturated C2-9 monocarboxylic acids: acrylic acid, propiolic acid (proprolic acid), methacrylic acid, crotonic acid, and isocrotonic acid. Exemplary dicarboxylic acids include saturated C2-9 dicarboxylic acids: malonic, succinic, glutaric, adipic and pimelic acids, and unsaturated C_2-9Dicarboxylic acids include maleic acid, fumaric acid, citraconic acid and mesaconic acid. Exemplary tricarboxylic acids include saturated C_2-9Tricarboxylic acid: propanetricarboxylic acid and 1,2, 3-butanetricarboxylic acid. In addition, the carboxylic acids of this definition may also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, and citric acid. Aromatic acids within this definition include benzoic and salicylic acids.

Commonly used water-soluble multivalent metal salts that may be used to help stabilize the encapsulated polypeptides of the invention include, for example: (1) inorganic acid metal salt: halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) metal salts of aliphatic carboxylic acids (e.g., calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) metal salts of aromatic carboxylic acids: benzoates (e.g., zinc benzoate) and salicylates.

In certain embodiments, the anti-IL 13 antibody is administered, for example, using a self-injection device, an autoinjector device, or other device designed for self-administration. In certain embodiments, the anti-IL 13 antibody is administered using a subcutaneous administration device. Various self-injection devices and subcutaneous administration devices, including autoinjector devices, are known in the art and are commercially available. Exemplary devices include, but are not limited to, prefilled syringes (e.g., BD HYPAK from Becton DickinsonREADYFILL^TMAnd STERIFILL SCF^TM(ii) a CLEARSHOT from Baxter^TMA copolymer prefilled syringe; and Daikyo Seiko CRYSTAL available from Westpharmaceutical ServicesA prefilled syringe); disposable Pen injection devices such as BD Pen from Becton Dickinson; ultra-sharp and microneedle devices (e.g., injection-ase from Becton Dickinson^TMAnd microinfusion devices (microinfusers); and H-PATCH available from Valeritas^TM) And needleless injection devices (as available from Bioject)Andand available from MedtronicAnd a patch device). Certain embodiments of subcutaneous administration devices are further described herein. It is contemplated that the anti-IL 13 antibody is co-formulated or co-administered with such self-injection devices or subcutaneous administration devices along with at least a second therapeutic compound.

Recombination method

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-IL 13 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising an antibody VL and/or an amino acid sequence comprising an antibody VH (e.g., a light chain and/or a heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VL of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-IL 13 antibody is provided, wherein the method comprises culturing a host cell as provided above comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-IL 13 antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, NJ,2003), pp.245-254, which describes the expression of antibody fragments in E.coli). After expression, the antibody peptide can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22:1409-1414(2004), and Li et al, nat. Biotech.24:210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculoviral strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing antibody-producing PLANTIBODIIES in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension culture may be useful. Useful inOther examples of mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse support cells (such as TM4 cells as described in, for example, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; Bufaro rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo., Humana Press, Totowa, NJ), pp.255-268 (2003).

Assay method

The anti-IL 13 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

Binding assays and other assays

In one aspect, anti-IL 13 antibodies are tested for antigen binding activity, e.g., by known methods such as ELISA, western blotting, and the like.

In another aspect, a competition assay can be used to identify antibodies that compete with anti-IL 13 antibodies for binding to IL 13. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear epitope or a conformational epitope) that lekuzumab or another anti-IL 13 antibody described herein binds to. A detailed exemplary method for locating epitopes bound by antibodies is provided in Morris (1996) "Epitope Mapping Protocols", referenced in Methods in molecular Biology Vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized IL13 is incubated in a solution comprising a first labeled antibody (e.g., secukinumab) that binds to IL13 and a second unlabeled antibody that is being tested for the ability to compete with the first antibody for binding to IL 13. The second antibody may be present in the hybridoma supernatant. As a control, immobilized IL13 was incubated in a solution comprising the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to IL13, excess unbound antibody is removed and the amount of label bound to immobilized IL13 is measured. If the amount of label bound to immobilized IL13 is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody is competing with the first antibody for binding to IL 13. See Harlow and Lane (1988) Antibodies, Chapter 14 of A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Activity assay

In one aspect, provided is an assay for identifying an anti-IL-13 antibody having biological activity. Biological activity may for example include activity in asthma. Antibodies having such biological activity in vivo and/or in vitro are also provided. In certain embodiments, antibodies of the invention are tested for such biological activity.

Article of manufacture and kit

An article of manufacture is provided which contains the formulation and instructions for its use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (e.g., single chamber or dual chamber syringes), and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container contains the formulation and a label on or associated with the container may indicate reconstitution and/or instructions for use. The label may also indicate that the formulation is useful or intended for subcutaneous administration. The container holding the formulation may be a multi-use vial which allows repeated administration (e.g. 2-6 administrations) of the reconstituted formulation. The article of manufacture may also comprise a second container comprising a suitable diluent (e.g., BWFI). When the diluent and lyophilized formulation are mixed, the final protein concentration in the reconstituted formulation will typically be at least 50 mg/mL. The article of manufacture may also comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

In certain embodiments, an article of manufacture is provided comprising a subcutaneous administration device that delivers a near-flat dose of an anti-IL 13 antibody to a patient, wherein the near-flat dose is, for example, but not limited to, 37.5mg, 75mg, or 125mg, or 150 mg. In certain embodiments, the anti-IL 13 antibody is secukinumab. The anti-IL 13 antibody in the subcutaneous administration device was formulated in a buffer (e.g., histidine pH 5.7) and other excipients (e.g., sucrose and polysorbate) to provide it in a stable pharmaceutical formulation. In certain embodiments, the subcutaneous administration device is a prefilled syringe comprising a glass needle cannula with a needle and optionally a needle shield and also optionally a needle shield device. In certain embodiments, the volume contained in the syringe is 0.5mL, 1mL, 1.5mL, or 2.0mL, or about 0.5mL, about 1mL, about 1.5mL, or about 2.0 mL. In certain embodiments, the needle is a strut (staked-in) needle comprising a 3-bevel tip or a 5-bevel tip. In certain embodiments, the needle is between 25 gauge (G) and 30G. In some embodiments, the needle is between 1/2 inch and 5/8 inch in length. In one embodiment, the subcutaneous administration device comprises a prefilled 1.0mL low borosilicate tungsten glass (type I) syringe and a stainless steel 5 bevel 27G1/2 inch long thin-walled pillared needle. In certain embodiments, the subcutaneous administration device comprises a rigid needle shield. In certain embodiments, the rigid needle shield comprises a rubber formulation having a low zinc content, for example, FM27/0(Daetwyler) and comprises a rigid polypropylene shield. In certain embodiments, the piston rod comprises a rubber piston stopper. In certain embodiments, the rubber piston stopper comprises 4023/50 rubber andethylene-tetrafluoroethylene (ETFE) coatings (West Pharmaceutical Services, Inc.). In certain embodiments, the subcutaneous administration device comprises a needle safety device. Exemplary needle safety devices include, but are not limited to, UltrasafeNeedle sheath X100L (Safety ceramics, Inc.) and Rexam Safety n Sound^TM(Rexam)。

Additional devices suitable for subcutaneous delivery include, for example, but are not limited to, injection devices such as injection-ase^TMAnd GENJECT^TMA device; infusion pumps, e.g. ACCU-CHECK^TM(ii) a Injection pens such as GENPEN^TM(ii) a Needleless devices such as MEDDTORs^TMAnd BIOJECTOR^TM(ii) a Autoinjectors and subcutaneous patch delivery systems.

The kit will generally comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. A label may be present on the container to indicate that the composition is for a particular therapy.

Examples

The following are examples of the formulations and methods of the present invention. It should be understood that various other embodiments may be implemented in view of the general description provided above.

Example 1

Materials and methods

Materials and sample preparation methods

All other antibodies used in the experiments described below were humanized IgG1monoclonal antibody, except anti-IL 13, which is a humanized IgG4 monoclonal antibody. Monoclonal antibodies were expressed in Chinese Hamster Ovary (CHO) cell lines and purified by a series of standard chromatographic steps, including protein a and ion exchange chromatography methods. Purified antibody was obtained as a concentrated solution from tangential flow filtration with added solution buffer and stabilizer. These are the antibody stocks used as starting materials for the studies described below.

These stock mAb starting materials were stored at 2-8 ℃ until further use. Additional preparation of mAb solutions included dialysis against low ionic strength buffers and filtration through 0.22 μm modified PVDF (polyvinylidene fluoride) filters (Millipore Steriflip, Millipore corp. Typically, mAb concentrations of 140-150mg/mL are obtained after dialysis. To obtain higher mAb concentrations, 10mL of mAb was concentrated in an Amicon YM30Centriprep (Millipore Corp, MA) concentrator centrifuged at 2700 rpm. The final concentration of mAb in the dialyzed and centrifugally concentrated preparations was determined by spectrophotometrically measuring the UV absorbance at 280nm (a280) using a gravimetric dilution and absorbance at 280nm (a280) and using an Agilent diode array model 8453 with a quartz cuvette having a 1cm path length. The extinction coefficient was determined by quantitative amino acid analysis.

Monoclonal antibody solutions for light scattering experiments were prepared in 20mL scintillation counting vials in the range of 0.5-275mg/mL by re-diluting known stock solution concentrates in a laminar flow clean bench. All scintillation counting vials were carefully cleaned with deionized water and dried in a filtered stream of compressed nitrogen. All buffer and reagent solutions were additionally filtered through a 0.10 μm Whatman Anotop 25 filter before addition to the protein solution. After preparation or dilution of the samples, the mAb solution was mixed to homogeneity and allowed to reach thermal and chemical equilibrium at controlled room temperature for 2 hours. The protein solution was centrifuged at 3000 rpm for 20-30 minutes at room temperature to remove extraneous dust and air bubbles from the solution prior to use for light scattering. Higher concentration solutions (mAb >170mg/mL) were centrifuged for longer periods until the light scattering signal showed minimal noise. The outer surface of the scintillation counting vial was lightly coated with silicone oil to reduce unwanted scattering from vial surface defects. The sample prepared as above was directly placed in a light scattering instrument for measurement.

Determination of B by Multi-Angle light Scattering₂

Sample preparation for light scattering a 20mL teflon-lined septum-capped vial was cleaned with MilliQ water and dried under a filtered stream of nitrogen. Samples were prepared at various concentrations by: an appropriate volume of about 80mg/mL of mAb stock was first diluted with an appropriate buffer to about 8mg/mL and then a final dilution was made with 20mL of 0.2 μm filtered buffer. A total of 8 protein concentrates (0.05-1.1mg/mL mAb) per buffer condition were equilibrated at room temperature for 14-18 hours before starting the measurements. All measurements were performed as a series of solutions of increasing white matter concentration, each experiment being performed in triplicate. An Agilent solvent degasser with a 25mm Millipore (Millipore, Billerica, MA) solvent filter (PVDF, 0.1 μm) and an equal intensity pump (Agilent, Palo Alto, Calif.) were used at a continuous flow rate of 0.5 ml/min. Loading was automated with a Gilson GX281(Gilson, inc., Middleton WI) liquid handling apparatus equipped with a 2mL injection ring and Wyatt technology deutschland in-line microfilter with a 0.1 μm, 10mM PVDF membrane. A series of concentration and light scattering measurements were performed, with Agilent MWD UV detector measurements at 280nm followed by 18-angle EOS MALS detector (Wyatt Technology Corporation, santa barbara, CA) measurements and the gain was reduced to 21 x. Obtaining data and using Astra^TM4.90.07(WTC) software processes and performs other analyses by outputting the segmentation (slice) results. Using linear regression fitting of data, K c/R (theta 0)/K c/or 1/M_WappThe slope 2B is generated on the concentration curve₂And intercept 1/M_w0(weight average molecular weight at infinite dilution).

High concentration multi-angle Static Light Scattering (SLS)

An 18-angle Dawn EOS light scattering detector from Wyatt Technology (Santa Barbara, CA) with a 30mW solid state laser (λ 690nm) was used for all static light scattering measurements, while a water cooled Peltier temperature controller was set at 23 ℃. Will be provided withThe instrument was calibrated with 99.9% toluene (chromatographic grade). For a common scintillation counting vial experiment, the detector gain was set 1x for all photodiodes at a fixed angle of 38 ° to 148 °. Since the radius of gyration (Rg) of the anti-CD 11a was less than 10nm, the angle dependence (angular dependence) of the photodiode was normalized to the 90 ° detector using a dilute solution of anti-CD 11a (1-2mg/mL) at each salt concentration to use the photodiode detector gain setting 21x at the end of each experiment. The measurement of static light scattering intensity was performed as the concentration of mAb varied from 0.5mg/mL to 275mg/mL and as the concentration of NaCl (0-600 mM). Scatter data was collected for each sample/vial at intervals ranging from 5-10 minutes, with a data collection frequency of 12 points/minute. Static light scattering data was obtained and processed using Astra 4.90.07 software (Wyatt Technology Corporation, Santa Barbara, CA), with a dn/dc value of 0.185 appropriate for calculations that may be output as a result of the segmentation. Additional analyses and calculations using this output were performed in Microsoft Excel, Origin v7.5 and MATLAB R14. For high concentration light scattering data, M is often easier to interpret_WappResults in the relative mAb concentration format, where an increase in molecular weight corresponds to the presence of concentration-dependent reversible self-association. (see, e.g., Scherer, T.M. et al, The Journal of Physical Chemistry B114 (40):12948-12957 (2010); Minton, A.P., Jpharm Sci 96(12):3466-9 (2007); Minton, A.P. Biophysical Journal 93(4):1321-1328 (2007)).

Haze obtained by UV Spectroscopy

The turbidity of the tested protein solutions from the high concentration light scattering experiments and the protein solutions from the pH screening experiments (each as described below) was measured at ambient temperature by using an Agilent 8453 spectrophotometer. Turbidity was calculated as the average of the absorbance at a wavelength of 350nm, where the sum of the absorbance values in 5nm increments over the wavelength range 340nm to 360nm was divided by 5. The measurement of the protein solution was performed in a small volume quartz cuvette with a 1cm optical path length. The absorbance at 690nm was also recorded.

Capillary Differential Scanning Calorimetry (DSC) characterization of melting temperature (Tm)

Protein thermal conformational stability was assessed by using a MicroCal capillary differential scanning calorimeter. MAb was diluted to 1mg/mL in buffer. 500 microliters of protein and its matching buffer were loaded into a 96-well plate. The heat capacity was monitored as the temperature increased 95 ℃ from 15 ℃ at a scan rate of 60 ℃/hr. VPViewer 2000Cap DSC was used to obtain Data and MicroCal, LLC DSC Data Analysis was used to analyze Data. See Yadav, S. et al, J Pharm Sci.99(3):1152-68 (2010).

Nephelometry of nephelometry

Nephelometric turbidimetry measurements were performed using a HACH (model 2100AN IS) laboratory turbidimeter with 90 degree scattering intensity detection. The detector was calibrated with formalin standard 4000 scatter turbidimetric turbidity units (NTU) stock at relative turbidity standard concentrations of 0-0.5. Samples were placed in cuvettes and measured in duplicate, and the average NTU of the samples was reported.

Rheology of

The viscosity of the samples was measured with an MCR300 rheometer (Anton Paar, Ashland, VA) using a cone and plate measurement system. The sample was loaded onto the lower measurement plate and allowed to reach thermal equilibrium at 25 ℃. A solvent trap was used to prevent solvent evaporation. The sample underwent two cycles of shear rate scanning (each cycle included from 10 seconds)^-1Increment to 1000 seconds^-1At 1000 seconds^-1Hold for 1 minute from 1000 seconds^-1Decreasing to 10 seconds^-1). There was a1 minute residence time between cycles. The reported value is 1000 seconds^-1An average of two shear rate scans was performed on one sample. Error bars represent the standard deviation of two runs in milliPascal-seconds (mPas). The total time of the sample is 1000 seconds^-1Under shear stress for 2 minutes. We choose 1000 seconds^-1Since the viscosity is relatively independent of shear rate in this range (200 seconds)^-1<Shear rate<2000 seconds^-1). The difference in viscosity between two aliquots of one sample was 1000 seconds^-1Within a range of. + -. 0.5 mPa. Excellent using US200 software (Anton Paar, Ashland, Va.)The duration of the measurement at each shear rate is quantified.

Cloud point temperature (cloud temperature) determination

For systems that undergo liquid-liquid phase separation (LLPS), lowering the temperature results in the formation of droplets of one liquid phase in the other. The temperature at which these droplets are formed is called the cloud point temperature and can be determined experimentally by microscopy or by monitoring solution transmittance. For the experiments described herein, the cloud point temperature was determined by monitoring the loss of transmission at 600nm with temperature changes in an Aviv 14DS spectrophotometer (Aviv biological, Lakewood, NJ). A5 mm square cuvette was filled with approximately 0.6mL of antibody solution. The temperature was reduced from 25 ℃ to 0 ℃ in 0.5 ℃ steps using a thermoelectric cooler. The samples were equilibrated at each temperature for 10 minutes before transmission was recorded. The cloud point temperature was named the temperature at which% transmission dropped to 50% of the original value (Asherie, 2004). The Tc of anti-IL 13 phase separation in different protein concentrations and in different study solutions was measured by using an Aviv Biomedical 14S type UV-Vis spectrophotometer. Scanning at a temperature of 25-0.5 ℃ in step length, balancing for 600 seconds and a wavelength of 600nm, and acquiring transmittance percentage versus temperature data. The measurement of the protein solution was carried out in a quartz cuvette with a 1cm path length.

Size exclusion chromatography

Size exclusion chromatography was used to quantify aggregates and fragments. This assay utilizes a TSK G3000SWXLTM, 7.8X300mm column and is run on an HP 1100TMHPLC system at 25 ℃. The sample was diluted to 2mg/mL with mobile phase and the amount sampled was 25. mu.L. The mobile phase was 0.2M K₂HPO₄0.25M KCl, pH6.2, and protein eluted at a steady flow rate of 0.5 ml/min for 30 minutes. The eluent absorbance was monitored at 280 nm. Using HP CHEMSATIONM^TMAnd integrating software.

Imaging capillary isoelectric focusing (icIEF)

Samples were analyzed using icIEF to quantify charge (acidic and basic) variation of anti-IL 13 antibody stability samples. This method uses a fluorocarbon coated capillary (Convergent Bioscience) in an iCE280 analyzer (Convergent Bioscience) with a PringCE microinjector. Solutions of anolyte and catholyte were purchased from GE healthcare biosciences; pI-labeled solutions were purchased from Convergent Bioscience.

Capillary electrophoresis method-dodecyl sodium sulfate (CE-SDS)

CE-SDS was performed using a Beckman P/ACE MDQ or PA800 capillary electrophoresis system with excitation at 488nm, capable of controlling capillary temperature from 20 to 40+2 ℃.

anti-IL 13 antibody potency assay

Evaluation of biological Activity or potency of anti-IL 13 antibody solutions Using cell culture assays that measure anti-IL 13 antibody solutions in L-Beas-2B cells, a human bronchial epithelial cell line (available from ATCC under ATCC accession number CRL-9609)^TM) The ability to inhibit IL-13 induced luciferase expression. Different concentrations of anti-IL 13 antibody standards, controls, and samples were mixed with a fixed concentration of IL-13 (e.g., rhu-IL13, Peprotech, catalog No. 200-13) and added to 96-well plates at 2x10⁵L-Beas-2B cells were seeded at a concentration of one cell/ml. After incubation, a luminescent luciferase substrate was used, according to the manufacturer's instructions (Bright-Glo)^TMLuciferase assay systems, Promega catalog No. E2620, E2650 or Brite-Lite Plus, Perkin Elmer catalog No. 6016761) quantitate luciferase expression. Dilution curves for each antibody solution were generated and compared to a reference material. The results are expressed in Relative Luminescence Units (RLU). Relative efficacy estimates were calculated using the least squares method and a parallel line analysis program. The% specific activity is calculated by multiplying the relative potency estimate by the specific activity of the reference substance.

anti-IL 13 antibody (Leijunumab) amino acid sequence

The following table shows the amino acid sequences of the monoclonal anti-mAb CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 regions, along with the VH, VL, heavy chain and light chain sequences. As shown in table 1 below, the VH and heavy chain may include an N-terminal glutamine and the heavy chain may also include a C-terminal lysine. As is well known in the art, the N-terminal glutamine residue can form pyroglutamic acid and the C-terminal lysine residue can be trimmed during the manufacturing process.

TABLE 1 amino acid sequence of anti-IL 13 antibody (Leijunumab)

Results

Physical and chemical stability of anti-IL 13 antibody formulations at various pHs

Buffers with different pH were generated using 20mM histidine acetate or 20mM sodium phosphate to cover the range of pH 5.4-7.8. Histidine acetate buffer covered the pH range 5.4-6.0 and sodium phosphate buffer covered the pH range 6.6-7.8. For each buffer pH, the following were maintained constant: anti-IL 13 antibody concentration of 150mg/mL, 175mM sucrose and 0.3mg/mL (0.03%) polysorbate 20.

The antibody solutions were stored in vials at the temperatures and for the time periods shown in table 2 below. At various times indicated by "X" in table 2, samples were analyzed by various methods of assessing physical stability, including SEC, a350 turbidity and non-reducing CE-SDS, and various methods of chemical stability, including icIEF.

Table 2 stability time points and conditions used to determine the physical and chemical stability of anti-IL 13 antibody solutions.

Figure 1 shows the percent monomer loss per week at the indicated pH in buffer as determined by SEC. As shown in figure 1, the% monomer loss is lower in the lower pH range than in the higher pH range, with the lowest% monomer loss at pH5.7, which shows a% monomer loss per week of 0.056.

Another physical stability assay measures the change in turbidity (as determined by a350) with change in pH at 30 ℃ over time. As shown in fig. 2, the initial turbidity and change of the buffer between pH5.4 and 6.0 was higher compared to the higher pH range. In fig. 2, the net haze is a350 ═ 1/T, where T is the transmitted light intensity at 350nm with a given optical path length of 1 cm.

A third physical stability assay measures the increase of Low Molecular Weight (LMW) soluble fragments and High Molecular Weight (HMW) aggregates with pH change in anti-IL 13 antibody solutions during six weeks of storage at 30 ℃. As shown in fig. 3, the fragmentation rate and aggregation rate were lowest in the lower pH range pH 5.4-6.6.

Using icIEF, we also evaluated chemical stability to determine the change in the rate of formation of acidic and basic varieties over time with pH at 30 ℃ (fig. 4) and the change in the rate of loss of basic varieties and major peaks over time with pH at 30 ℃ (fig. 5). As shown in fig. 4, the ratio of acidic variants was lowest in the low pH range and highest in the high pH range, while the ratio of basic variants (BV1 peak) was lowest in the high pH range and highest in the low pH range. The results shown in figure 5 indicate that the major peak loss is minimized between pH5.4 and 6.0.

To determine if pH affects solution viscosity, we performed rheological characterization of different anti-IL 13 antibody concentrates (ranging from 0 to 200mg/mL antibody) at different pH (ranging from pH 5.5-7.2). Each solution had 175mM sucrose and 0.3mg/mL polysorbate 20. The results are shown in fig. 6. Those results indicate that a consistent viscosity profile is maintained regardless of the solution pH for a given antibody concentration. In particular, the results show that viscosity at higher antibody concentrations is not affected by pH.

In summary, the data presented in FIGS. 1-6 show that at pH 5.4-6.0, there is a shallow gradient (shape gradient) for most physical and chemical changes. Therefore 20mM histidine acetate buffer pH5.7 was selected for subsequent studies and formulation evaluation.

Rheological characterization of high concentration monoclonal antibody solutions

To explore whether the viscosity observed with anti-IL 13 antibody formulated in 150mg/mL in 20mM histidine acetate pH5.7, 175mM sucrose, 0.3mg/mL polysorbate 20 (at 25 ℃ <15cP) would be observed for a variety of different antibodies in general, we examined the viscosity of three additional antibodies at 150mg/mL in similar formulations. Such a viscosity profile as observed for anti-IL 13 antibodies is desirable for manufacture at high antibody concentrations and for certain routes of drug administration (e.g., subcutaneous injection). As shown in figure 7, the anti-IL 13 antibody maintained a viscosity profile similar to that of the anti-CD 11a antibody, i.e., a viscosity of <15cP at 25 ℃. In contrast, the anti-CD 20 antibody and mAb-1 antibody showed considerably different viscosity profiles. The viscosity of anti-CD 20 antibody at 150mg/mL was >15cP at 25 ℃, whereas mAb-1 could not be formulated in this buffer at 150mg/mL due to obvious problems with viscosity as can be seen in fig. 7. FIG. 7 shows that the viscosity of mAb-1 at 125mg/mL is >45cP at 25 ℃. Thus, it is clear from this data that different antibodies have different rheological characteristics when formulated at 150mg/mL in 20mM histidine acetate pH5.7, 175mM sucrose, 0.3mg/mL polysorbate 20.

Characterization of visual appearance and opalescence

Using 90 degree nephelometry and a350 turbidity measurements, we characterized the visual appearance and opalescence of the anti-IL 13 antibody formulation compared to the anti-CD 20 antibody formulation. Fig. 8 shows quantification of visual appearance in Nephelometric Turbidity Units (NTU) of two different antibody formulations. In fig. 8, R1, R2, R3 and R4 refer to reference standards, with R4 having the highest degree of visual opalescence and R1 the lowest degree of visual opalescence. The a350 turbidity values for anti-IL 13 and anti-CD 20 antibodies are shown in figure 9. As shown in figure 9, turbidity increased with increasing protein concentration for each antibody formulation. The results shown in these figures indicate that the two different visual appearance values for the two antibodies have different trends, especially at higher protein concentrations, due to the intrinsic measured differences. The data also shows that the magnitude trend is consistent between the two antibodies that appear to have elevated solution opalescence.

We also examined the concentration of anti-IL 13 antibody as a function of concentration and pH. The results are shown in fig. 10. The solution showing the maximum turbidity is around the isoelectric point (pI) of the mAb.

While not being bound by theory, we interpret these results to indicate that the absorbance at 350nm wavelength (turbidity) increases with increasing protein concentration, since the protein absorption band absorbs light with a maximum near 280nm due to the broad tail of this peak. A second contributing factor to increasing a350 vs mAb solution concentration is the non-linear increase in light scattering, thereby reducing total transmitted light.

Furthermore, we evaluated sub-visible particle counts as a function of mAb concentration and these results are shown in fig. 11. No significant increase in sub-visible particles >2 μm in size was observed by HIAC light blocking analysis, indicating that particles >2 μm do not contribute to opalescence or turbidity of the anti-IL 13 antibody solution. FIG. 12 shows the magnitude of the scattering turbidimetry, transmission turbidimetry, and static light scattering for a 125mg/mL anti-IL 13 antibody solution when the antibody solution was filtered with progressively smaller pore sizes (down to 0.1 μm or 100 nm). These results, shown in fig. 11 and 12, generally indicate that anti-IL 13 antibody solutions and pharmaceutical formulations appearance are not determined by concentration-dependent sub-visible or sub-micron particulate matter that causes light scattering.

Next, we investigated the dependence of the appearance of the solution as a function of the pH of the solution at 125mg/mL and 204 mg/mL. The solution appearance was evaluated using a temperature scan of transmitted light intensity at 600 nm. The results are shown in fig. 13 and demonstrate that the opalescence of anti-IL 13 antibody solutions that remained constant with decreasing temperature changes was not due to critical phenomena such as liquid-liquid phase separation, where the solution components had divergent solubility and formed two separate phases of different composition. This indicates that solution homogeneity (phase separation) is not affected by temperature over the usual storage and/or exposure temperature range, regardless of the opalescence/visual appearance (room temperature) of the initial solution.

Thermal stability (Tmelt) study

We measured two partially resolved peaks e of thermal melting transition as a function of formulation composition and solution pH in a capillary differential scanning calorimeter. The results are shown in fig. 14. As shown in FIG. 14, the maximum value of anti-IL 13 melt transition behavior as a function of pH was observed between pH 6.0 and 7.5. The prevailing scientific opinion is that the lower the melting transition occurs, the lower the expected physical stability of the system at any time of storage. See, e.g., Chi et al, Protein Science 12(5):903-913 (2003); chi et al, Pharmaceutical Research 20(9):1325-1336 (2003); goldberg et al, J.Pharm.sciences 100(4):1306-1315 (2011). Thus, the physical stability data shown herein are surprising and unexpected for an anti-IL 13 antibody formulation (pH 5.7).

Colloidal stability

Colloidal stability was measured by static light scattering using dilute antibody solutions (between 0.10-1 mg/mL) and by light scattering at antibody concentrations above 200 mg/mL. Colloidal stability refers to the solution behavior of macromolecules suspended in solution and allows one to study equilibrium, time-averaged interactions between macromolecules such as monoclonal antibodies. Without being bound by theory, when the interaction is repulsive, then it can be expected that the solution composition remains stable. However, net attractive interactions occur between solute molecules and are often associated with phase transition and protein solubility problems.

Using samples in simple buffer, we measured the osmotic second virial coefficient of anti-IL 13 antibody as a function of solution pH (B)₂) (at concentrations ranging from 0.1 to 1.0 mg/mL). Note that in fig. 15 and 16, a value higher than 0 is a positive permeability second virial coefficient indicating a net repulsive intermolecular interaction, and a value lower than 0 is a negative permeability second virial coefficient indicating a net attractive intermolecular interaction. The data in figure 15 show that anti-IL 13 antibody has properties across this pH rangeAttractive interactions, but the most intense ones occur between pH 5.5-6.5. For the results shown in fig. 16, formulation additions were added to solutions at different pH. As can be seen in fig. 16, the permeability second dimension coefficient measured at pH 5.5-6.5 remains negative and therefore attractive. The light scatter measurements for the multi-angle light scatter detector over the concentration range 1-200mg/ml, which extrapolates the intensity to a scatter angle of 0, are shown in FIG. 17. These data reveal that the scattering intensity distribution is highly similar to that observed for the HACH turbidimeter (compare fig. 8-17). Both instruments measure scattered light intensity and thus both measure rayleigh scattering. This scattering is dominant in solutions that do not contain particulate matter and is caused by low density and concentration fluctuations of the solution, which also depend on interactions between the scattering molecules. As the molecules come into increasingly intimate contact with each other and their temporal/spatial positions become correlated, resulting in destructive interference with scattered light, there is a drop in scattered light intensity (see, for example, Bettelheim et al, Biophysical Journal 41(1):29-33(1983), Xia et al, Biophysical Journal 66(3_ Pt-1): 861-872(1994), and Xia et al, biophysical journal 41(1):29-33 (1996). fig. 18 shows static light scattering data for anti-IL 13 antibody as a function of formulation pH the data in fig. 18 is expressed as the apparent molecular weight observed at antibody concentrations up to 200mg/mL the data shown in fig. 18 shows weak (pH 7.2) to moderately attractive colloid (pH 6.5) interactions and anti-IL 13 antibody self-association across the concentration range relative to theoretical scattering of a simple hard sphere species model excluding mAb volume (dashed lines in fig. 18).

Both anti-IL 13 and anti-CD 20 showed comparable levels of turbidity caused by attractive colloidal interactions and mAb self-association, as shown in figure 19. Surprisingly, such attractive colloidal interactions do not manifest as high viscosity problems (e.g., >15cP at 150mg/mL) or rheology problems with anti-IL 13 antibody formulations, as shown in fig. 20. However, the colloidal interaction of the anti-CD 20 antibody did have an effect on solution rheology, producing not only solution opalescence (fig. 8), but also a high viscosity of >15cP at 25 ℃ and 150mg/mL (fig. 20).

Long term physical, chemical and efficacy stability

To test long-term stability and efficacy, anti-IL 13 antibody was formulated at 125mg/mL in 20mM histidine acetate pH5.7, 175mM sucrose and 0.3mg/mL polysorbate 20 and subsequently subjected to various storage conditions. The vials were stored at 5 ℃ or 25 ℃ for the weeks indicated in table 3 (up to 156 weeks at 5 ℃ and up to 26 weeks at 25 ℃). At each time point as shown in table 3, the samples were analyzed for Color Appearance and Clarity (CAC), pH, and the indicated chemical or physical stability values. In addition, biological activity (potency) was also assessed at each time point. As shown by the data in table 3, anti-IL 13 antibody formulated in 125mg/mL in 20mM histidine acetate ph5.7, 175mM sucrose and 0.3mg/mL polysorbate 20 maintained potency and demonstrated good chemical and physical stability at 5 ℃ for all 156 weeks (three years) and at 25 ℃ for all 26 weeks. These data demonstrate that this formulation maintains the desired chemical, physical and potency attributes of the anti-IL 13 antibody for an extended period of time.

Table 3 stability and conditions used to determine long-term physical stability, chemical stability and potency stability of anti-IL 13 antibodies.

CAC: color appearance and clarity

SY slightly yellow

LIQ ═ liquid

Slightly opalescent SOPL

Conclusion

We have shown that anti-IL 13 antibodies have been successfully formulated with excipients at pH and solution conditions that promote long-term chemical and physical stability and maintain potency. Specifically, the formulation contains the antibody at a concentration of 100mg/mL and above (including 125mg/mL and 150mg/mL) in 20mM histidine acetate pH5.7, 175mM sucrose and 0.3mg/mL polysorbate 20. Surprisingly, we found that this formulation has a desirable viscosity profile at 25 ℃ <15 cP. Such a viscosity profile is desirable for ease of manufacture and also ease of administration, e.g., subcutaneous injection, where a small volume of a high concentration drug is optimal for several reasons, including patient comfort and compliance. We observed that other antibodies in the same or similar formulations had unfavorable viscosity profiles (> 15cP at 25 ℃), which highlights the unpredictability of the viscosity profile of the anti-IL 13 antibody formulations.

In addition, two frequently used criteria for Protein formulation selection include thermal and colloidal stability (see Chi et al, Protein Science 12(5):903-913 (2003); Chi et al, Pharmaceutical Research 20(9):1325-1336 (2003)). Thermal analysis of the unfolding temperature of the anti-IL 13 antibody solution showed that physical stability at pH 5.4-6.0 would not be optimal for the physical stability of the anti-formulation. Colloidal stability analysis of anti-IL 13 antibody solutions also showed that formulation conditions in the pH range 5.5-6.5 would be the minimum requirement to maintain a low aggregation rate. However, surprisingly, as the data presented herein show, anti-IL 13 antibodies formulated at pH5.7 exhibit good physical stability at 5 ℃ over an extended period of time and also under accelerated conditions. It is also surprising that the product stability under these conditions is both physically and chemically superior to that observed at higher pH, even in the presence of lower thermal melting transitions and colloidal stability. Although the appearance (and turbidity) of the formulated anti-IL 13 antibody solution was more milky under the selected formulation conditions than under some non-selected conditions, the molecular characteristics and formulation composition maintained optimal stability, maintained efficacy and provided the required solution rheology for delivering high concentrations of the drug in small volumes under both real-time and accelerated storage conditions.

Subcutaneous administration device

One subcutaneous administration device including a prefilled syringe with a needle, a piston with a piston stopper, a needle shield, and a needle safety device for administering the anti-IL 13 formulation described above was selected by evaluating various commercially available components. For example, the parts evaluated included glass rods, formed syringes with staked needles, pistons and piston stoppers, rigid needle shields, and needle safety devices.

The various components were evaluated in various combinations according to methods known to those skilled in the art for their effect on formulation properties including, but not limited to, stability and other considerations: this includes factors such as the effect of needle gauge and needle inside diameter on injection time and glide force when the formulation has certain viscosities as described herein, such as patient comfort and convenience. These studies led us to select a prefilled 1.0mL low tungsten borosilicate glass (type I) syringe and a stainless steel 5 bevel 27G1/2 inch thin walled pillared needle with a rigid needle shield (including FM27/0(Daetwyler) and rigid polypropylene shield) as the optimal subcutaneous administration device for administering the lekumab formulated at high concentrations as described herein. In addition, the piston rod contains a rubber piston stopper comprising 4023/50 rubber andethylene-tetrafluoroethylene (ETFE) coatings (West Pharmaceutical Services, Inc.). The subcutaneous administration device further comprises a needle safety device, UltrasafeNeedle sheath X100L (Safety ceramics, Inc.). The subcutaneous administration device detailed above is hereinafter referred to as a pillared needle prefilled syringe or "SIN PFS".

To show the stability of the letrozumab drug product in vials comparable to the selected SIN PFS, we evaluated GMP drug substance manually filled into 2cc vials or 1mL SIN PFS at 40 ℃/ambient relative humidity. We assessed the degradation rate as characterized by monomer changes by Size Exclusion Chromatography (SEC) and the change in the percentage of major peaks by imaging capillary isoelectric focusing (ICIEF) and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).

These studies revealed that after 4 weeks of storage at 40 ℃, there was no significant difference in monomer reduction as measured by SEC (each showing 0.6-0.9% reduction) or in the percent reduction of the major peak (each showing 18-21% reduction as measured by ICIEF, and 0.9-1.5% reduction as measured by CE-SDS) between the vials and the SIN PFS. Furthermore, the chromatograms were comparable to each other and no new peaks were observed in the SIN PFS samples compared to the vial samples.

There was a slight difference in degradation rate (after 4 weeks at 40 ℃, the high molecular weight species of the vials increased by 0.5% -0.6%, compared to 0.8% for the SIN PFS). It is believed that this slight difference is unlikely to affect product quality during real-time storage.

Therefore, we conclude that: the data described above show that the stability of high concentration of the secukinumab drug product formulated as described above in a vial is comparable to the stability in the selected SIN PFS described above.

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Sequence listing

<110> Fuffman-Raroshi Co., Ltd

<120> antibody preparation

<130>P4786R1-WO

<140>

<141>

<150>61/553,916

<151>2011-10-31

<160>14

<170>PatentIn version 3.5

<210>1

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>1

Ala Tyr Ser Val Asn

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<210>2

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>2

Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser

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<210>3

<211>10

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<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

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Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn

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<210>4

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<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

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Arg Ala Ser Lys Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His

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<210>5

<211>7

<212>PRT

<213> Artificial sequence

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<223> description of Artificial sequences synthetic peptides

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Leu Ala Ser Asn Leu Glu Ser

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<210>6

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<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

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Gln Gln Asn Asn Glu Asp Pro Arg Thr

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<213> Artificial sequence

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<223> description of Artificial sequences synthetic polypeptides

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Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser

20 25 30

Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala

3540 45

Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gly

85 90 95

Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser Leu

100 105 110

Val Thr Val Ser Ser

115

<210>8

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400>8

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr

20 25 30

Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser

100 105 110

Leu Val Thr Val Ser Ser

115

<210>9

<211>112

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Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly

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Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

<210>10

<211>443

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<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

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Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser

20 25 30

Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala

35 40 45

Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gly

85 90 95

Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp GlyVal Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210>11

<211>444

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400>11

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr

20 25 30

Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly ProPro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210>12

<211>444

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400>12

Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr

15 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser

20 25 30

Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala

35 40 45

Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gly

85 90 95

Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln PheAsn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>13

<211>445

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400>13

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr

20 25 30

Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro SerVal Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>14

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400>14

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims (36)

1. A formulation comprising an anti-IL 13 antibody in histidine acetate buffer at ph5.4 to 6.0, wherein the formulation does not comprise arginine, wherein the concentration of the antibody in the formulation is at least 100mg/mL, and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃, and wherein:

(i) the anti-IL 13 antibody comprises:

a. a heavy chain and a light chain having the amino acid sequences of SEQ ID No. 10, the light chain having three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6;

b. a light chain and a heavy chain having the amino acid sequence of SEQ ID No. 14, said heavy chain having three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No.:1, a CDR-H2 having the amino acid sequence of SEQ ID No.:2 and a CDR-H3 having the amino acid sequence of SEQ ID No.: 3; or

c. A heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14; and

(ii) the histidine acetate concentration in the buffer is between 5mM and 40 mM.

2. The formulation of claim 1 wherein the anti-IL 13 antibody comprises a heavy chain and a light chain having the amino acid sequence of SEQ ID No. 10, the light chain having three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5 and CDR-L3 having the amino acid sequence of SEQ ID No. 6.

3. The formulation of claim 1 wherein the anti-IL 13 antibody comprises a light chain and a heavy chain having the amino acid sequence of SEQ ID No. 14, the heavy chain having three heavy chain CDRs: CDR-H1 having the amino acid sequence of SEQ ID No. 1, CDR-H2 having the amino acid sequence of SEQ ID No.2 and CDR-H3 having the amino acid sequence of SEQ ID No. 3.

4. The formulation of claim 1 wherein the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14.

5. The formulation of claim 1, wherein the concentration of antibody is 125 mg/mL.

6. The formulation of claim 1, wherein the concentration of antibody is 150 mg/mL.

7. The formulation of claim 1, further comprising a polyol and a surfactant, wherein the concentration of the polyol in the formulation is between 100mM and 200mM, and the concentration of the surfactant in the formulation is between 0.01% and 0.1%.

8. The formulation of claim 7, wherein the polyol is sucrose and the surfactant is polysorbate 20.

9. The formulation of claim 8, wherein the pH of the histidine acetate buffer is pH5.7 and the concentration of histidine acetate in the buffer is 20mM, and wherein the concentration of sucrose in the formulation is 175mM and the concentration of polysorbate 20 is 0.03%.

10. The formulation of claim 9, wherein the concentration of antibody is 125 mg/mL.

11. The formulation of claim 9, wherein the concentration of antibody is 150 mg/mL.

12. The formulation of claim 1, wherein the anti-IL-13 antibody is stable at 5 ℃ for at least one year.

13. The formulation of claim 12, wherein the anti-IL-13 antibody is stable at 5 ℃ for at least two years.

14. The formulation of claim 13, wherein the anti-IL-13 antibody is stable at 5 ℃ for three years.

15. The formulation of claim 1, wherein the anti-IL 13 antibody is stable at 25 ℃ for at least 4 weeks, or at 25 ℃ for at least 8 weeks, or at 25 ℃ for at least 12 weeks, or at 4 ℃ for 26 weeks.

16. A formulation comprising an anti-IL 13 antibody in a histidine acetate buffer at pH5.4 to 6.0, wherein the formulation does not comprise arginine, wherein the concentration of histidine acetate in the buffer is between 5mM to 40mM, wherein the concentration of the antibody in the formulation is at least 100mg/mL, and wherein the anti-IL 13 antibody comprises:

a. a heavy chain and a light chain having the amino acid sequences of SEQ ID No. 10, the light chain having three light chain CDRs: a CDR-L1 having the amino acid sequence of SEQ ID No. 4, a CDR-L2 having the amino acid sequence of SEQ ID No. 5, and a CDR-L3 having the amino acid sequence of SEQ ID No. 6;

b. a light chain and a heavy chain having the amino acid sequence of SEQ ID No. 14, said heavy chain having three heavy chain CDRs: a CDR-H1 having the amino acid sequence of SEQ ID No.:1, a CDR-H2 having the amino acid sequence of SEQ ID No.:2 and a CDR-H3 having the amino acid sequence of SEQ ID No.: 3; or

c. A heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14.

17. The formulation of claim 16, further comprising a polyol and a surfactant, wherein the concentration of the polyol in the formulation is between 100mM and 200mM, and the concentration of the surfactant in the formulation is between 0.01% and 0.1%.

18. The formulation of claim 17, wherein the polyol is sucrose and the surfactant is polysorbate 20.

19. The formulation of claim 18, wherein the pH of the histidine acetate buffer is pH5.7 and the concentration of histidine acetate in the buffer is 20mM, and wherein the concentration of sucrose in the formulation is 175mM and the concentration of polysorbate 20 is 0.03%.

20. The formulation of claim 16 wherein the anti-IL 13 antibody comprises a heavy chain and a light chain having the amino acid sequence of SEQ ID No. 10, the light chain having three light chain CDRs: CDR-L1 having the amino acid sequence of SEQ ID No. 4, CDR-L2 having the amino acid sequence of SEQ ID No. 5, and CDR-L3 having the amino acid sequence of SEQ ID No. 6.

21. The formulation of claim 16 wherein the anti-IL 13 antibody comprises a light chain and a heavy chain having the amino acid sequence of SEQ ID No. 14, the heavy chain having three heavy chain CDRs: CDR-H1 having the amino acid sequence of SEQ ID No. 1, CDR-H2 having the amino acid sequence of SEQ ID No.2 and CDR-H3 having the amino acid sequence of SEQ ID No. 3.

22. The formulation of claim 16 wherein the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10 and a light chain having the amino acid sequence of SEQ ID No. 14.

23. The formulation of claim 16, having a viscosity of less than 15 centipoise (cP) at 25 ℃.

24. The formulation of claim 16, wherein the concentration of antibody is 125 mg/mL.

25. The formulation of claim 16, wherein the concentration of antibody is 150 mg/mL.

26. A formulation comprising an anti-IL 13 antibody having extended stability in 20mM histidine acetate buffer pH5.7, 175mM sucrose, 0.03% polysorbate 20, wherein the formulation does not comprise arginine, wherein the concentration of the antibody in the formulation is 125mg/mL and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃, and wherein the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10, and a light chain having the amino acid sequence of SEQ ID No. 14.

27. A formulation comprising an anti-IL 13 antibody having extended stability in 20mM histidine acetate buffer pH5.7, 175mM sucrose, 0.03% polysorbate 20, wherein the formulation does not comprise arginine, wherein the concentration of the antibody in the formulation is 150mg/mL and the viscosity of the formulation is less than 15 centipoise (cP) at 25 ℃, and wherein the anti-IL 13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 10, and a light chain having the amino acid sequence of SEQ ID No. 14.

28. An article of manufacture comprising the formulation of any one of claims 1, 16, 26, or 27 and a subcutaneous administration device.

29. The article of manufacture according to claim 28, wherein the subcutaneous administration device comprises a prefilled syringe.

30. The article of manufacture of claim 29, wherein the prefilled syringe comprises a glass needle cannula, a needle, and a needle shield.

31. The article of manufacture of claim 30, further comprising a piston rod and a piston stopper.

32. The article of manufacture of claim 31, further comprising a needle safety device.

33. The article of manufacture of claim 31 wherein the piston stopper comprises 4023/50 rubber and an ethylene-tetrafluoroethylene coating.

34. The article of manufacture of claim 30, wherein the glass needle tubing comprises borosilicate glass and contains about 0.3mL, about 1.0mL, or about 2.0mL of formulation.

35. The article of manufacture of claim 30, wherein the needle is a staked-in, stainless steel, 27G thin wall, 1/2 inch long, and 5 bevel needle tip.

36. The article of manufacture of claim 30, wherein the needle shield is rigid and comprises an elastomeric component FM27/0 and a rigid polypropylene shield.