WO2009120684A1 - Formulation d’anticorps - Google Patents

Formulation d’anticorps Download PDF

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Publication number
WO2009120684A1
WO2009120684A1 PCT/US2009/038088 US2009038088W WO2009120684A1 WO 2009120684 A1 WO2009120684 A1 WO 2009120684A1 US 2009038088 W US2009038088 W US 2009038088W WO 2009120684 A1 WO2009120684 A1 WO 2009120684A1
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WIPO (PCT)
Prior art keywords
antibody
months
formulation
years
fragment
Prior art date
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PCT/US2009/038088
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English (en)
Inventor
Jenny Main
Steven Bishop
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Medimmune, Llc
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Publication of WO2009120684A1 publication Critical patent/WO2009120684A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to high concentration liquid formulations of antibodies or fragments thereof, which formulations exhibit stability, low to undetectable levels of antibody fragmentation, low to undetectable levels of aggregation, and very little to no loss of the biological activities of the antibodies, even during long periods of storage.
  • Lyophilized formulations of antibodies have a number of limitations, including a prolonged process for lyophilization and resulting high cost for manufacturing.
  • a lyophilized formulation has to be reconstituted aseptically and accurately by healthcare practitioners prior to administering to patients.
  • the reconstitution step itself requires certain specific procedures, for example: (1) a sterile diluent (i.e., water for intravenous administration and 5% dextrose in water for intramuscular administration) is added to the vial containing lyophilized antibody, slowly and aseptically, and the vial must be swirled very gently for 30 seconds to avoid foaming; (2) the reconstituted antibody may need to stand at room temperature for a minimum of 20 minutes until the solution clarifies; and (3) the reconstituted preparation must be administered within six (6) hours after the reconstitution.
  • a sterile diluent i.e., water for intravenous administration and 5% dextrose in water for intramuscular administration
  • the present invention relates to sterile, stable aqueous formulations comprising an antibody or fragment thereof, wherein the concentration of the antibody is at least about 110 mg/ml.
  • the present invention further relates to processes of making a sterile, stable aqueous formulation comprising an antibody or fragment thereof, wherein the concentration of the antibody is at least about 110 mg/ml.
  • the present invention is also directed to antibody-based therapies which involve administering to a human subject the liquid antibody formulations (or "antibody formulations” or “liquid formulations") of the present invention for preventing, treating and/or managing a disease or disorder.
  • formulations of the invention liquid formulations of the invention
  • high concentration stable liquid formulations of the invention high concentration stable liquid formulations of the invention
  • antibody liquid formulations of the invention high concentration stable liquid formulations of the invention
  • antibody formulations of the invention or “antibody formulations of the invention”.
  • the term "subject” includes any human or nonhuman animal.
  • the term “subject” includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, for example, but not limited to, mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
  • antibody as referred to herein encompasses whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C HI , Cm and C H 3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (for example, but not limited to, effector cells) and the first component (CIq) of the classical complement system.
  • Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.
  • antibody refers to monoclonal antibodies, multispecif ⁇ c antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single- chain Fvs (scFv), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, for example, but not limited to, anti-Id antibodies to antibodies of the invention), intrabodies, and epitope-binding fragments of any of the above.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 ) or subclass.
  • type e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • class e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2
  • subclass e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C HI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C HI domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and C HI domains
  • F(ab')2 fragment
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • the terms "monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the term "human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, but not limited to, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, for example, but not limited to, a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (for example, but not limited to, a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, for example, but not limited to, from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo. [0016]
  • the term "isotype" refers to the classification of an antibody's heavy or light chain constant region.
  • the constant domains of antibodies are not involved in binding to antigen, but exhibit various effector functions.
  • a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM.
  • IgA immunoglobulin
  • IgD immunoglobulin
  • IgE immunoglobulin
  • IgG immunoglobulins
  • IgM immunoglobulins
  • subclasses e.g., IgGl (gamma 1), IgG2 (gamma 2), IgG3 (gamma 3), and IgG4 (gamma 4), and IgAl and IgA2.
  • the heavy chain constant regions that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the structures and three-dimensional configurations of different classes of immunoglobulins are well-known. Human light chain constant regions may be classified into two major classes, kappa and lambda
  • An "epitope” is a term well understood in the art and means any chemical moiety that exhibits specific binding to an antibody.
  • An "antigen” is a moiety or molecule that contains an epitope, and, as such, also specifically binds to antibody.
  • Affinity of an antibody for an epitope to be used in the treatment(s) described herein is a term well understood in the art and means the extent, or strength, of binding of antibody to epitope. Affinity may be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD or Kd), apparent equilibrium dissociation constant (KD ' or Kd ' ), and IC50 (amount needed to effect 50% inhibition in a competition assay). It is understood that, for purposes of this invention, an affinity is an average affinity for a given population of antibodies which bind to an epitope.
  • antibody affinity is used as a basis for administration of the treatment methods described herein, or selection for the treatment methods described herein, antibody affinity can be measured before and/or during treatment, and the values obtained can be used by a clinician in assessing whether a human patient is an appropriate candidate for treatment.
  • the term "avidity” is a measure of the overall binding strength (i.e., both antibody arms) with which an antibody binds an antigen.
  • Antibody avidity can be determined by measuring the dissociation of the antigen-antibody bond in antigen excess using any means known in the art, such as, but not limited to, by the modification of indirect fluorescent antibody as described by Gray et al., J. Virol. Meth., 44:11-24. (1993)
  • binding refers to antibody binding to a predetermined antigen.
  • the antibody binds with a dissociation constant (K D ) of 1(T 8 M or less, and binds to the predetermined antigen with a K D that is at least two-fold less than its K D for binding to a non-specific antigen (for example, but not limited to, BSA, casein) other than the predetermined antigen or a closely- related antigen.
  • K D dissociation constant
  • an antibody recognizing an antigen and an antibody specific for an antigen are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • antibody half-life means a pharmacokinetic property of an antibody that is a measure of the mean survival time of antibody molecules following their administration.
  • Antibody half-life can be expressed as the time required to eliminate 50 percent of a known quantity of immunoglobulin from the patient's body or a specific compartment thereof, for example, as measured in serum or plasma, i.e., circulating half- life, or in other tissues.
  • Half-life may vary from one immunoglobulin or class of immunoglobulin to another. In general, an increase in antibody half-life results in an increase in mean residence time (MRT) in circulation for the antibody administered.
  • MRT mean residence time
  • excipient refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder or stabilizing agent for drugs which imparts a beneficial physical property to a formulation, such as increased protein stability, increased protein solubility, and decreased viscosity.
  • excipients include, but are not limited to, proteins (for example, but not limited to, serum albumin), amino acids (for example, but not limited to, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine), surfactants (for example, but not limited to, SDS, Tween 20, Tween 80, polysorbate and nonionic surfactants), saccharides (for example, but not limited to, glucose, sucrose, maltose and trehalose), polyols (for example, but not limited to, mannitol and sorbitol), fatty acids and phospholipids (for example, but not limited to, alkyl sulfonates and caprylate).
  • proteins for example, but not limited to, serum albumin
  • amino acids for example, but not limited to, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine
  • surfactants for example, but not limited to, SDS,
  • the phrase "pharmaceutically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the terms "stability” and “stable” as used herein in the context of a liquid formulation comprising an antibody (including antibody fragment thereof) that specifically binds to an antigen of interest (e.g., an interferon alpha polypeptide) refer to the resistance of the antibody (including antibody fragment thereof) in the formulation to aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions.
  • the “stable" formulations of the invention retain biological activity under given manufacture, preparation, transportation and storage conditions.
  • the stability of said antibody (including antibody fragment thereof) can be assessed by degrees of aggregation, degradation or fragmentation, as measured by HPSEC, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS binding techniques, compared to a reference formulation.
  • a reference formulation may be a reference standard frozen at -70 0 C consisting of 10 mg/ml of an antibody in an appropriate buffer.
  • the overall stability of a formulation comprising an antibody (including antibody fragment thereof) can be assessed by various immunological assays including, for example, ELISA and radioimmunoassay using isolated antigen molecules.
  • low to undetectable levels of aggregation refers to samples containing no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1% and no more than about 0.5% aggregation by weight of protein as measured by high performance size exclusion chromatography (HPSEC) or static light scattering (SLS) techniques.
  • HPSEC high performance size exclusion chromatography
  • SLS static light scattering
  • the term "low to undetectable levels of fragmentation” as used herein refers to samples containing equal to or more than about 80%, about 85%, about 90%, about 95%, about 98% or about 99% of the total protein, for example, in a single peak as determined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (or as many peaks as there are subunits) by reduced Capillary Gel Electrophoresis (rCGE), representing the non-degraded antibody or a non-degraded fragment thereof, and containing no other single peaks having more than about 5%, more than about 4%, more than about 3%, more than about 2%, more than about 1%, or more than about 0.5% of the total protein in each.
  • reduced Capillary Gel Electrophoresis refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody.
  • Figure 1 Flow diagram of formulation manufacturing process used to produce 100 g/L
  • Figure 2 Stability of 13H5 formulations (100 mg/ml, formulation F) at 40 0 C.
  • the formulations contain antibody generated from the same cell line using different manufacturing processes.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is plotted.
  • Figure 3 Stability of 13H5 formulations (100 mg/ml, formulation F) at 5 0 C.
  • the formulations contain antibody generated from the same cell line using different manufacturing processes.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is plotted.
  • FIG. 1 Concentration dependence of 13H5 formulation stability at 40 0 C. Chart displays the percent (%) aggregate content of formulation determined by SEC at various time points.
  • Figure 6 pH dependence of 13H5 formulation (100 mg/ml) stability at 40 0 C. Chart displays the percent (%) aggregate content of formulation determined by SEC at various time points.
  • Figure 8 Stability of 13H5 formulations at 5 0 C. The percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 10 Stability of 13H5 formulations at 40 0 C. Antibody degradation over time is ascertained by measuring the total concentration of degradation products (percent (%) fragment) via RF-
  • FIG. 12 Stability of 13H5 (100 mg/ml) formulations at pH 6.0, 4O 0 C. Antibody degradation over time is monitored by determining the Mono Q ion exchange chromatography column elution profile of various antibody formulations; percent (%) of protein eluted before the intact antibody peak is displayed in a chart. [0041] Figure 13. Stability of 13H5 (100 mg/ml) formulations at 5°C. Antibody degradation over time is monitored by determining the Mono Q ion exchange chromatography column elution profile of various antibody formulations; percent (%) of protein eluted before the intact antibody peak is plotted.
  • Figure 14 Stability of 13H5 (100 mg/ml) formulations. The visual appearance of various formulations is determined by the naked eye. Formulations were stored at 5°C for 9 months.
  • Figure 15 Stability of 125 mg/ml, 150 mg/ml, 175 mg/ml and 200 mg/ml 13H5 formulations at 40 0 C. The percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 16 Stability of 125 mg/ml, 150 mg/ml, 175 mg/ml and 200 mg/ml 13H5 formulations at 5°C. The percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 17 Stability of 125 mg/ml, 150 mg/ml, 175 mg/ml and 200 mg/ml 13H5 formulations at 40 0 C. Antibody degradation over time is monitored by determining the Dionex ion exchange chromatography column elution profile of various antibody formulations; percent (%) of protein eluted before the intact antibody peak is displayed in a chart.
  • Figure 18 Stability of 125 mg/ml, 150 mg/ml, 175 mg/ml and 200 mg/ml 13H5 formulations at 5 0 C. Antibody degradation over time is monitored by determining the Dionex ion exchange chromatography column elution profile of various antibody formulations; percent (%) of protein eluted before the intact antibody peak is displayed in a chart.
  • FIG. 19 Stability of formulations comprising 130 mg/ml antibody Z (Mab Z) at 5°C. The percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 20 Stability of formulations comprising 130 mg/ml antibody Z (Mab Z) at 40 0 C.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 21 Stability of 150 mg/ml and 175 mg/ml antibody Y (Mab Y) formulations at 5°C.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 22 Stability of 150 mg/ml and 175 mg/ml antibody Y (Mab Y) formulations at 40 0 C.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 24 Stability of 150 mg/ml and 200 mg/ml antibody X (Mab X) formulations at 4O 0 C.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 25 Stability of 150 mg/ml and 200 mg/ml antibody W (Mab W) formulations at 5°C.
  • the percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • Figure 27 Stability of a formulation comprising 150 mg/ml of antibody Q (Mab Q) at 5 0 C and 40 0 C. The percent (%) aggregate content of formulation determined by SEC at various time points is displayed in a chart.
  • the present invention relates to stable, high concentration liquid formulations of antibodies or fragments thereof, wherein the concentration of the antibody is at least about 110 mg/ml.
  • a stable high concentration liquid formulation of the invention is suitable for parenteral administration to a human subject.
  • a stable high concentration liquid formulation of the invention is suitable for subcutaneous administration to a human subject.
  • the present invention encompasses stable liquid formulations of antibodies, that exhibit low to undetectable levels of antibody aggregation and/or fragmentation with very little to no loss of the biological activities during manufacture, preparation, transportation, and long periods of storage, wherein the concentration of the antibody is at least about 100 mg/ml.
  • the present invention also encompasses stable liquid formulations of antibodies that have increased in vivo half-lives, said formulations exhibiting low to undetectable levels of antibody aggregation and/or fragmentation, and very little to no loss of the biological activities of the antibodies, wherein the concentration of the antibody is at least about 110 mg/ml.
  • a liquid formulation of the invention is an aqueous formulation.
  • a liquid formulation of the invention is an aqueous formulation wherein the aqueous carrier is distilled water.
  • a formulation of the invention is sterile.
  • a formulation of the invention is homogeneous.
  • a formulation of the invention is isotonic.
  • a formulation of the invention comprises at least about 100 mg/ml, at least about 110 mg/ml, at least about 120 mg/ml, at least about 130 mg/ml, at least about 140 mg/ml, at least about 150 mg/ml, at least about 160 mg/ml, at least about 170 mg/ml, at least about 180 mg/ml, at least about 190 mg/ml, at least about 200 mg/ml, at least about 210 mg/ml, at least about 220 mg/ml, at least about 230 mg/ml, at least about 240 mg/ml, at least about 250 mg/ml, at least about 260 mg/ml, at least about 270 mg/ml, at least about 280 mg/ml, at least about 290 mg/m
  • a formulation of the invention comprises at least about 100 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 110 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 120 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 130 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 140 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 150 mg/ml of an antibody of a fragment thereof.
  • a formulation of the invention comprises at least about 170 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 190 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 200 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 250 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least about 300 mg/ml of an antibody of a fragment thereof.
  • a formulation of the invention comprises between about 100 mg/ml and about 300 mg/ml, between about 110 mg/ml and about 300 mg/ml, between about 120 mg/ml and about 300 mg/ml, between about 130 mg/ml and about 300 mg/ml, between about 140 mg/ml and about 300 mg/ml, between about 150 mg/ml and about 300 mg/ml, between about 160 mg/ml and about 300 mg/ml, between about 170 mg/ml and about 300 mg/ml, between about 180 mg/ml and about 300 mg/ml, between about 190 mg/ml and about 300 mg/ml, between about 200 mg/ml and about 300 mg/ml, or between about 250 mg/ml and about 300 mg/ml of an antibody or a fragment thereof.
  • a formulation of the invention comprises about 100 mg/ml, about
  • a formulation of the invention comprises at least 100 mg/ml, at least
  • 110 mg/ml at least 120 mg/ml, at least 130 mg/ml, at least 140 mg/ml, at least 150 mg/ml, at least 160 mg/ml, at least 170 mg/ml, at least 180 mg/ml, at least 190 mg/ml, at least 200 mg/ml, at least 210 mg/ml, at least 220 mg/ml, at least 230 mg/ml, at least 240 mg/ml, at least 250 mg/ml, at least 260 mg/ml, at least 270 mg/ml, at least 280 mg/ml, at least 290 mg/ml, or at least 300 mg/ml of an antibody or a fragment thereof.
  • a formulation of the invention comprises at least 100 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 110 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 120 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 130 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 140 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 150 mg/ml of an antibody of a fragment thereof.
  • a formulation of the invention comprises at least 170 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 190 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 200 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 250 mg/ml of an antibody of a fragment thereof. In a specific embodiment, a formulation of the invention comprises at least 300 mg/ml of an antibody of a fragment thereof.
  • a formulation of the invention comprises between 100 mg/ml and
  • 300 mg/ml between 110 mg/ml and 300 mg/ml, between 120 mg/ml and 300 mg/ml, between 130 mg/ml and 300 mg/ml, between 140 mg/ml and 300 mg/ml, between 150 mg/ml and 300 mg/ml, between 160 mg/ml and 300 mg/ml, between 170 mg/ml and 300 mg/ml, between 180 mg/ml and 300 mg/ml, between 190 mg/ml and 300 mg/ml, between 200 mg/ml and 300 mg/ml, or between 250 mg/ml and
  • a formulation of the invention comprises 100 mg/ml, 110 mg/ml,
  • the formulations of the invention may further comprise common excipients and/or additives such as buffering agents, saccharides, salts and surfactants. Additionally or alternatively, the formulations of the invention may further comprise common excipients and/or additives, such as, but not limited to, solubilizers, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelators, preservatives, or the like.
  • the buffering agent is selected from the group consisting of histidine, citrate, phosphate, glycine, and acetate.
  • the saccharide excipient is selected from the group consisting of trehalose, sucrose, mannitol, maltose and raff ⁇ nose.
  • the surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 80, and
  • the salt is selected from the group consisting of NaCl, KCl,
  • the formulations of the invention may further comprise other common auxiliary components, such as, but not limited to, suitable excipients, polyols, solubilizers, diluents, binders, stabilizers, lipophilic solvents, chelators, preservatives, or the like.
  • suitable excipients polyols, solubilizers, diluents, binders, stabilizers, lipophilic solvents, chelators, preservatives, or the like.
  • the formulations of the invention include a buffering or pH adjusting agent to provide improved pH control.
  • a formulation of the invention has a pH of between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5.
  • a formulation of the invention has a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about
  • a formulation of the invention has a pH of about 6.0.
  • a formulation of the invention has a pH of between 3.0 and 9.0, between
  • a formulation of the invention has a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.5, 8.0, 8.5, or 9.0.
  • a formulation of the invention has a pH of 6.0.
  • the pH of the formulation generally should not be equal to the isoelectric point of the particular antibody (including antibody fragment thereof) to be used in the formulation and may range from about 4.0 to about 8.0, or may range from about 5.5 to about 6.5.
  • the buffering agent is a salt prepared from an organic or inorganic acid or base.
  • Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • amino acid components can also function in a buffering capacity.
  • Representative amino acid components which may be utilized in the formulations of the invention as buffering agents include, but are not limited to, glycine and histidine.
  • the buffering agent is selected from the group consisting of histidine, citrate, phosphate, glycine, and acetate. In a specific embodiment, the buffering agent is histidine.
  • the buffering agent is citrate.
  • the purity of the buffering agent should be at least 98%, or at least 99%, or at least 99.5%.
  • the term "purity" in the context of histidine refers to chemical purity of histidine as understood in the art, e.g., as described in The Merck Index, 13 th ed., O'Neil et al. ed. (Merck & Co., 2001).
  • Buffering agents are typically used at concentrations between about 1 mM and about 200 mM or any range or value therein, depending on the desired ionic strength and the buffering capacity required.
  • concentrations of conventional buffering agents employed in parenteral formulations can be found in: Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2 n Edition, Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Products.
  • the buffering agent is at a concentration of about 1 mM, or of about 5 mM, or of about 10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or of about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM, or of about 90 mM, or of about 100 mM.
  • the buffering agent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15 mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, or of 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90 mM, or of 100 mM.
  • the buffering agent is at a concentration of between about 10 mM and about 50 mM.
  • the buffering agent is at a concentration of between 10 mM and 50 mM.
  • a formulation of the invention comprises a buffering agent.
  • said buffering agent is selected from the group consisting of histidine, citrate, phosphate, glycine, and acetate.
  • a formulation of the invention comprises histidine as a buffering agent.
  • a formulation of the invention comprises a citrate buffer.
  • the formulations of the invention comprise a carbohydrate excipient.
  • Carbohydrate excipients can act, e.g., as viscosity enhancing agents, stabilizers, bulking agents, solubilizing agents, and/or the like.
  • Carbohydrate excipients are generally present at between about 1% to about 99% by weight or volume. In one embodiment, the carbohydrate excipient is present at between about 0.1% to about 20%. In another embodiment, the carbohydrate excipient is present at between about 0.1% to about 15%. In a specific embodiment, the carbohydrate excipient is present at between about 0.1% to about 5%, or between about 1% to about 20%, or between about 5% to about 15%, or between about 8% to about 10%, or between about 10% and about 15%, or between about 15% and about 20%.
  • the carbohydrate excipient is present at between 0.1% to 20%, or between 5% to 15%, or between 8% to 10%, or between 10% and 15%, or between 15% and 20%. In still another specific embodiment, the carbohydrate excipient is present at between about 0.1% to about 5%. In still another specific embodiment, the carbohydrate excipient is present at between about 5% to about 10%. In yet another specific embodiment, the carbohydrate excipient is present at between about 15% to about 20%. In still other specific embodiments, the carbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.
  • Carbohydrate excipients suitable for use in the formulations of the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose, trehalose, cellobio
  • the carbohydrate excipients for use in the present invention are selected from the group consisting of, sucrose, trehalose, lactose, mannitol, and raffinose.
  • the carbohydrate excipient is trehalose.
  • the carbohydrate excipient is mannitol.
  • the carbohydrate excipient is sucrose.
  • the carbohydrate excipient is raffinose.
  • the purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.
  • a formulation of the invention comprises an excipient.
  • a formulation of the invention comprises at least one excipient selected from the group consisting of: sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative.
  • a formulation of the invention comprises a salt.
  • a formulation of the invention comprises a salt selected from the group consisting of: NaCl, KCl, CaCl 2 , and MgCl 2 .
  • a formulation of the invention comprises NaCl.
  • a formulation of the invention comprises at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM. at least about 200 mM, or at least about 300 mM sodium chloride.
  • a formulation described herein comprises between about 10 mM and about 300 mM, between about 10 mM and about 200 mM, between about 10 mM and about 175 mM, between about 10 mM and about 150 mM, between about 25 mM and about 300 mM, between about 25 mM and about 200 mM, between about 25 mM and about 175 mM, between about 25 mM and about 150 mM, between about 50 mM and about 300 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 75 mM and about 300 mM, between about 75 mM and about 200 mM, between about 75 mM and about 175 mM, between about 75 mM and about 150 mM, between about 100 mM and about 300 mM, between about 100 mM and about 200 mM, between about 100 mM and about 200
  • a formulation of the invention comprises about 10 mM. about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM sodium chloride. In a specific embodiment, a formulation of the invention comprises 125 mM sodium chloride.
  • a formulation of the invention comprises at least 10 mM, at least 25 mM, at least 50 mM, at least 75 mM, at least 100 mM, at least 125 mM, at least 150 mM, at least 175 mM. at least 200 mM, or at least 300 mM sodium chloride.
  • a formulation described herein comprises between 10 mM and 300 mM, between 10 mM and 200 mM, between 10 mM and 175 mM, between 10 mM and 150 mM, between 25 mM and 300 mM, between 25 mM and 200 mM, between 25 mM and 175 mM, between 25 mM and 150 mM, between 50 mM and 300 mM, between 50 mM and 200 mM, between 50 mM and 175 mM, between 50 mM and 150 mM, between 75 mM and 300 mM, between 75 mM and 200 mM, between 75 mM and 175 mM, between 75 mM and 150 mM, between 100 mM and 300 mM, between 100 mM and 200 mM, between 100 mM and 175 mM, or between 100 mM and 150 mM sodium chloride.
  • a formulation of the invention comprises 10 mM. 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, or 300 mM sodium chloride. In a specific embodiment, a formulation of the invention comprises 125 mM sodium chloride.
  • the formulations of the invention may further comprise a surfactant.
  • surfactant refers to organic substances having amphipathic structures; namely, they are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical compositions and preparations of biological materials. Pharmaceutically acceptable surfactants like polysorbates (e.g. polysorbates 20 or 80); polyoxamers (e.g.
  • poloxamer 188 Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUA.TM. series (Mona Industries, Inc., Paterson, N. J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc), can optionally be added to the formulations of the invention to reduce aggregation. Surfactants are particularly useful if a pump or plastic container is used to administer the formulation.
  • the formulations of the invention comprise a polysorbate which is at a concentration ranging from between about 0.001% to about 1%, or about 0.001% to about 0.1%, or about 0.01% to about 0.1%.
  • the formulations of the invention comprise a polysorbate which is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.
  • the polysorbate is polysorbate-80.
  • a formulation of the invention comprises a surfactant.
  • a formulation of the invention comprises Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.
  • a formulation of the invention comprises Polysorbate 80.
  • the formulations of the invention may further comprise other common excipients and/or additives including, but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, or the like. Pharmaceutically acceptable excipients and/or additives may be used in the formulations of the invention.
  • excipients/additives such as pharmaceutically acceptable chelators (for example, but not limited to, EDTA, DTPA or EGTA) can optionally be added to the formulations of the invention to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation.
  • pharmaceutically acceptable chelators for example, but not limited to, EDTA, DTPA or EGTA
  • Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (for example, but not limited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof can optionally be added to the formulations of the invention at any suitable concentration such as between about 0.001% to about 5%, or any range or value therein.
  • concentration of preservative used in the formulations of the invention is a concentration sufficient to yield an microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
  • Other contemplated excipients/additives, which may be utilized in the formulations of the invention include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, salt- forming counterions such as sodium and the like.
  • HSA human serum albumin
  • rHA recombinant human albumin
  • the formulations of the invention may be isotonic with human blood, that is the formulations of the invention have essentially the same osmotic pressure as human blood.
  • Such isotonic formulations will generally have an osmotic pressure from about 240 mOSm to about 360 mOSm.
  • isotonic formulations will generally have an osmotic pressure from about 250 mOSm to about 350 mOSm.
  • Isotonicity can be measured by, for example, using a vapor pressure or ice-freezing type osmometer. Tonicity of a formulation is adjusted by the use of tonicity modifiers.
  • Tonicity modifiers are those pharmaceutically acceptable inert substances that can be added to the formulation to provide an isotonity of the formulation.
  • Tonicity modifiers suitable for this invention include, but are not limited to, saccharides, salts and amino acids.
  • the formulations of the present invention have an osmotic pressure from about 100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or from about 250 mOSm to about 350 mOSm.
  • Concentration of any one or any combination of various components of the formulations of the invention are adjusted to achieve the desired tonicity of the final formulation.
  • the ratio of the carbohydrate excipient to antibody may be adjusted according to methods known in the art (e.g., U.S. Patent No. 6,685,940).
  • the molar ratio of the carbohydrate excipient to antibody may be from about 100 moles to about 1000 moles of carbohydrate excipient to about 1 mole of antibody, or from about 200 moles to about 6000 moles of carbohydrate excipient to about 1 mole of antibody, or from about 100 moles to about 510 moles of carbohydrate excipient to about 1 mole of antibody, or from about 100 moles to about 600 moles of carbohydrate excipient to about 1 mole of antibody.
  • the formulations of the present invention have an osmotic pressure from 100 mOSm to 1200 mOSm, or from 200 mOSm to 1000 mOSm, or from 200 mOSm to 800 mOSm, or from 200 mOSm to 600 mOSm, or from 250 mOSm to 500 mOSm, or from 250 mOSm to 400 mOSm, or from 250 mOSm to 350 mOSm.
  • Concentration of any one or any combination of various components of the formulations of the invention are adjusted to achieve the desired tonicity of the final formulation.
  • the ratio of the carbohydrate excipient to antibody may be adjusted according to methods known in the art (e.g., U.S. Patent No. 6,685,940).
  • the molar ratio of the carbohydrate excipient to antibody may be from 100 moles to 1000 moles of carbohydrate excipient to 1 mole of antibody, or from 200 moles to 6000 moles of carbohydrate excipient to 1 mole of antibody, or from 100 moles to 510 moles of carbohydrate excipient to 1 mole of antibody, or from 100 moles to 600 moles of carbohydrate excipient to 1 mole of antibody.
  • the desired isotonicity of the final formulation may also be achieved by adjusting the salt concentration of the formulations.
  • Salts that are pharmaceutically acceptable and suitable for this invention as tonicity modifiers include, but are not limited to, sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride.
  • formulations of the inventions comprise NaCl, MgCl 2 , and/or CaCt
  • concentration of NaCl is between about 75 mM and about 150 mM.
  • concentration OfMgCl 2 is between about 1 mM and about 100 mM.
  • concentration of NaCl is between 75 mM and 150 mM.
  • concentration of MgCb is between 1 mM and 100 mM.
  • Amino acids that are pharmaceutically acceptable and suitable for this invention as tonicity modifiers include, but are not limited to, proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine, and histidine.
  • the formulations of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • the formulations of the invention should be sterile.
  • the formulations of the invention may be sterilized by various sterilization methods, including sterile filtration, radiation, etc.
  • the antibody formulation is filter-sterilized with a presterilized 0.22-micron filter.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy", 21 st ed., Lippincott Williams & Wilkins, (2005).
  • sterile compositions comprising antibodies are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • a composition of the invention is provided as a pre-filled syringe.
  • a formulation of the invention comprises an antibody or fragment thereof that is susceptible to aggregation, fragmentation and/or deamidation.
  • a formulation of the invention stabilizes an antibody of interest.
  • a formulation of the invention prevents aggregation of an antibody of interest or fragment thereof.
  • a formulation of the invention prevents fragmentation of an antibody of interest or fragment thereof.
  • a formulation of the invention prevents deamidation of an antibody of interest or fragment thereof.
  • a formulation of the invention is stable upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention is stable upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention is stable upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention is stable upon storage at about 5 0 C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention is stable upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention is stable upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention is stable upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention is stable upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • the present invention relates to sterile, stable aqueous formulations comprising an antibody or fragment thereof, wherein the concentration of the antibody is at least about 110 mg/ml.
  • the stability of said antibody can be assessed by degrees of aggregation, degradation or fragmentation, as measured by HPSEC, reverse phase chromatography, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS binding techniques, compared to a reference formulation comprising said antibody.
  • a reference formulation may be a reference standard frozen at -70 0 C consisting of 10 mg/ml of said antibody in 20 mM histidine (pH 6.0).
  • a reference formulation frozen at -70 0 C is identical to the formulation whose stability is tested.
  • the stability of said antibody may also be assessed by measuring the antibodies ability to bind to its target antigen. Any binding assay known to one of skill in the art (e.g. ELISA, flow cytometry, RIA) may be utilized to measure the target antigen binding activity. Target antigen binding activity of the antibody in a given formulation may be compared to that of the antibody in a reference formulation.
  • a reference antibody is understood to mean the antibody in a reference formulation. In certain embodiments, the reference antibody is identical to the antibody whose stability is assessed.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 25°C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 25 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 25 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody that has a target antigen binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the target antigen binding activity of a reference antibody, wherein said formulation was stored at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 25°C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 25°C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 5 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein the antibody loses less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of its target antigen binding activity during storage of the formulation at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 50% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 60% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 70% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 80% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 90% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 95% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein said antibody retains at least 99% of binding ability to its target antigen compared to a reference antibody representing the antibody prior to the storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody forms an aggregate as determined by HPSEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 1% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 2% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 3% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 4% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 7% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is fragmented as determined by RP-HPLC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 5% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 10% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 20% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 30% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 40% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 4O 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 5 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 50% of said antibody is deamidated as determined by IEC upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention comprises an antibody, wherein less than 60% of said antibody is deamidated as determined by IEC upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 40 0 C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In one embodiment, a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 4O 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 5 0 C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 4O 0 C for about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 40 0 C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • a formulation of the invention is clear and colorless as determined by visual inspection upon storage at about 5°C for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, or about 12 years.
  • the formulations of the invention maintain improved aggregation profiles upon storage, for example, for extended periods (for example, but not limited to 1 week, 1 month, 6 months, 1 year, 2 years, 3 years or 5 years) at room temperature or 4 0 C or for periods (such as, but not limited to 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months) at elevated temperatures such as 38°C-42°C.
  • the formulations maintain improved aggregation profiles upon storage while exposed to light or stored in the dark in a variety of humidity conditions including but not limited to a relative humidity of up to 10%, or up to 20%, or up to 30%, or up to 40%, or up to 50%, or up to 60%, or up to 70%, or up to 80%, or up to 90%, or up to 100%.
  • a relative humidity of up to 10%, or up to 20%, or up to 30%, or up to 40%, or up to 50%, or up to 60%, or up to 70%, or up to 80%, or up to 90%, or up to 100%.
  • ambient conditions generally refers to temperatures of about 20 0 C at a relative humidity of between 10% and 60% with exposure to light.
  • temperatures between about 2 0 C and about 8 0 C at a relative humidity of less then about 10% are collectively referred to as “4 0 C” or “5 0 C”
  • temperatures between about 23 0 C and about 27 0 C at a relative humidity of about 60% are collectively referred to as “25 0 C”
  • temperatures between about 38 0 C and about 42 0 C at a relative humidity of about 75% are collectively referred to as "40 0 C.”
  • the formulations of the invention comprise (or consists of as the aggregate fraction) a particle profile of less than about 3.4 E +5 particles/ml of diameter 2-4 ⁇ m, less than about 4.0 E +4 particles/ml of diameter 4-10 ⁇ m, less than about 4.2 E +3 particles/ml of diameter 10-20 ⁇ m, less than about 5.0 E +2 particles/ml of diameter 20-30 ⁇ m, less than about 7.5 E +1 particles/ml of diameter 30-40 ⁇ m, and less than about 9.4 particles/ml of diameter 40-60 ⁇ m as determined by a particle multisizer.
  • the formulations of the invention contain no detectable particles greater than 40 ⁇ m, or greater than 30 ⁇ m.
  • a protein formulation e.g., antibody formulation of the invention
  • SEC size exclusion chromatography
  • HPSEC high performance size exclusion chromatography
  • SLS static light scattering
  • FTIR Fourier Transform Infrared Spectroscopy
  • CD circular dichroism
  • urea-induced protein unfolding techniques intrinsic tryptophan fluorescence, differential scanning calorimetry, and l-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques.
  • SEC size exclusion chromatography
  • HP-SEC high pressure liquid chromatographic columns
  • SEC analysis HP-SEC
  • SEC SEC analysis
  • HP-SEC SEC analysis
  • Examples examples
  • AUC analytical ultracentrifugation
  • AUC is an orthogonal technique which determines the sedimentation coefficients (reported in Svedberg, S) of macromolecules in a liquid sample.
  • AUC is capable of separating and detecting antibody fragments/aggregates from monomers and is further able to provide information on molecular mass.
  • Protein aggregation in the formulations may also be characterized by particle counter analysis using a coulter counter or by turbidity measurements using a turbidimeter. Turbidity is a measure of the amount by which the particles in a solution scatter light and, thus, may be used as a general indicator of protein aggregation.
  • non-reducing polyacrylamide gel electrophoresis (PAGE) or capillary gel electrophoresis (CGE) may be used to characterize the aggregation and/or fragmentation state of antibodies or a fragment thereof in a formulation of the invention.
  • a formulation of the invention is for parenteral administration. In one embodiment, a formulation of the invention is an injectable formulation. In one embodiment, a formulation of the invention is for intravenous, subcutaneous, or intramuscular administration. [00232] In one embodiment, a formulation of the invention is for intravenous administration wherein said formulation comprises between about 100 mg/ml and about 300 mg/ml of an antibody or a fragment thereof. In another embodiment, a formulation of the invention is for intravenous administration wherein said formulation comprises between about 150 mg/ml and about 300 mg/ml of an antibody or fragment thereof.
  • a formulation of the invention is for subcutaneous administration wherein said formulation comprises between about 100 mg/ml and about 300 mg/ml of an antibody or a fragment thereof. In another embodiment, a formulation of the invention is for subcutaneous administration wherein said formulation comprises between about 150 mg/ml and about 300 mg/ml of an antibody or a fragment thereof.
  • a formulation of the invention is for aerosol administration.
  • the present invention also provides a pharmaceutical unit dosage form suitable for parenteral administration to a human which comprises an anti-interferon alpha antibody formulation in a suitable container.
  • a pharmaceutical unit dosage of the invention comprises 13H5 anti- interferon alpha antibody.
  • a pharmaceutical unit dosage of the invention comprises an intravenously, subcutaneously, or intramuscularly delivered anti-interferon alpha antibody formulation.
  • a pharmaceutical unit dosage of the invention comprises aerosol delivered anti- interferon alpha antibody formulation.
  • a pharmaceutical unit dosage of the invention comprises a subcutaneously delivered 13H5 anti-interferon alpha antibody formulation.
  • a pharmaceutical unit dosage of the invention comprises an aerosol delivered anti- interferon alpha antibody formulation.
  • a pharmaceutical unit dosage of the invention comprises an intranasally administered anti-interferon alpha antibody formulation.
  • a formulation of the invention is provided in a sealed container.
  • the present invention further provides a kit comprising an antibody formulation of the invention. 5.3.Antibodies Useful in the Formulations of the Invention
  • the present invention provides formulations comprising at least one monoclonal antibody of interest. These antibodies can be used for therapeutic, including prophylactic, purposes. Such antibodies can also be used for the diagnosis of various diseases or for the study of the evolution of such diseases.
  • the antibodies useful in the present invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulf ⁇ de-linked Fvs (sdFv), and epitope- binding fragments of any of the above.
  • antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG3, IgG 4 , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • the antibodies useful in the present invention may be from any animal origin including birds and mammals (for example, but not limited to, human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). In specific embodiments, the antibodies are human or humanized monoclonal antibodies.
  • the antibodies useful in the present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may specifically bind to different epitopes of a polypeptide or may specifically bind to both a polypeptide as well a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793 Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148: 1547-1553.
  • the antibodies useful in the present invention can be single-chain antibodies.
  • the design and construction of a single-chain antibody is described in Marasco et al, 1993, Proc Natl Acad Sci 90:7889- 7893, which is incorporated herein by reference in its entirety.
  • a formulation of the invention may comprise an antibody of any specificity. Accordingly, in a specific embodiment, a formulation of the invention may comprise an antibody that specifically binds to any antigen including, but not limited to, the following list of proteins, as well as subunits, domains, motifs and epitopes belonging to the following list of proteins: renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha- 1 - antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasm
  • cancer related proteins including, but not limited to, ALK receptor (pleiotrophin receptor), pleiotrophin; KS 1/4 pan-carcinoma antigen; ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostate specific antigen (PSA); prostate specific membrane antigen (PSMA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonic antigen (CEA); carcinoembryonic antigen-related cell adhesion molecule (CEACAMl); cytokeratin tumor-associated antigen; human milk fat globule (HMFG) antigen; CanAg antigen; tumor-associated antigen expressing Lewis Y related carbohydrate; colorectal tumor-associated antigens such as: CEA, tumor-associated glycoprotein-72 (TAG-72), CO17-1A, GICA 19-9, CTA
  • protein targets contemplated include but are not limited to the following list of proteins, as well as subunits, domains, motifs, and epitopes belonging to the following list of microbial proteins: B. anthracis proteins or toxins; human cytomegalovirus (HCMV) proteins such as, envelope glycoprotein, gB, internal matrix proteins of the virus, pp65 and ppl50, immediate early (IE) proteins; human immunodeficiency virus (HIV) proteins such as, Gag, Pol, Vif and Nef (Vogt et al., 1995, Vaccine 13: 202-208); HIV antigens gpl20 and gpl60 (Achour et al., 1995, Cell. MoI. Biol.
  • HCMV human cytomegalovirus
  • IE immediate early
  • HIV human immunodeficiency virus
  • HCV hepatitis C virus
  • HCV hepatitis C virus
  • core protein pC
  • El pEl
  • E2 pE2
  • SARS severe acute respiratory syndrome corona virus proteins
  • S (spike) glycoprotein small envelope protein E (the E protein), the membrane glycoprotein M (the M protein), the hemagglutinin esterase protein (the HE protein), and the nucleocapsid protein (the N-protein) See, e.g., Marra et al., "The Genome Sequence of the SARS-Associated Coronavirus," Science Express, May 2003); Mycobacterium tuberculosis proteins such as S (spike) glycoprotein, small envelope protein E (the E protein), the membrane glycoprotein M (the M protein), the hemagglutinin esterase protein (the HE protein), and the nucleocapsid protein (the N-protein) See, e.g., Marra et al., "The Genome Sequence of the SARS-Associated Corona
  • antigen 85 alpha-antigen
  • a lipoglycoprotein on the cell surface a 65-kDa heat shock protein, and a 36-kDa proline -rich antigen
  • Ag85A Ag85b
  • 65-kDa heat shock protein hsp65
  • MPB/MPT51 Miki et al., 2004, Infect. Immun.
  • HSV Herpes simplex virus
  • gD glycoprotein gB glycoprotein
  • proteins from intracellular parasites such as Leishmania include LPG, gp63 (Xu and Liew, 1994, Vaccine 12: 1534- 1536; Xu and Liew, 1995, Immunology 84: 173-176), P-2 (Nylen et al., 2004, Scand. J. Immunol. 59:294-304), P-4 (Kar et al. 2000, J Biol. Chem.
  • microbial toxin proteins such as Clostridium perfringens toxin; C. difficile toxin A and B; in addition, exemplary antigen peptides of human respiratory syncytial virus (hRSV), human metapneumovirus (HMPV) and Parainfluenza virus (PIV) are detailed in: Young et al., in Patent publication WO04010935A2.
  • antibodies and/or antigen binding fragments thereof which bind to a protein, the ligands or receptors that bind them, or other members of their corresponding biochemical pathway, may be derived by methods well known in the art, such as those described below, and that such antibodies and/or antigen binding fragments may be engineered to comprise a variant Fc region or fragment thereof.
  • the present invention provides for formulations of antibodies and antibody fragments that specifically bind to an antigen of interest (e.g. , an interferon alpha polypeptide) which have an extended half- life in vivo.
  • an antigen of interest e.g. , an interferon alpha polypeptide
  • the present invention provides formulations of antibodies and antibody fragments that specifically bind to an antigen of interest which have a half-life in a mammal (for example, but not limited to, a human), of greater than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.
  • inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to the antibodies (including antibody fragments thereof) with or without a multifunctional linker either through site -specific conjugation of the PEG to the N- or C- terminus of the antibodies or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • PEG high molecular weight polyethyleneglycol
  • the degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion-exchange chromatography. PEG-derivatized antibodies (including antibody fragments thereof) can be tested for binding activity as well as for in vivo efficacy using methods known to those of skill in the art, for example, by immunoassays described herein.
  • Antibodies having an increased half-life in vivo can also be generated introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (e.g., Fc or hinge-Fc domain fragment). See, e.g., International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375, each of which is incorporated herein by reference in its entirety.
  • antibodies (including antibody fragments thereof) can be conjugated to albumin in order to make the antibody (including antibody fragment thereof) more stable in vivo or have a longer half life in vivo.
  • the present invention provides formulations of antibodies (including antibody fragments thereof) that specifically binds to an antigen of interest recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment of a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the invention provides formulations of fusion proteins comprising an antigen-binding fragment of an antibody described herein (for example, but not limited to, a Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide.
  • an antibody described herein for example, but not limited to, a Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR
  • a heterologous protein polypeptide, or peptide.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (for example, but not limited to, antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.
  • Antibodies including antibody fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • a polynucleotide encoding an antibody (including antibody fragment thereof) thereof may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the antibodies can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence may be a hexa- histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.
  • HA hemagglutinin
  • antibodies of the present invention or fragments thereof conjugated to a diagnostic or detectable agent can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder (for example, but not limited to, an autoimmune disorder) as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • a disease or disorder for example, but not limited to, an autoimmune disorder
  • Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidinlbiotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine ( 131 I, 125 I, 123 I, and
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • the therapeutic moiety or drug conjugated to an antigen of interest e.g.
  • an interferon alpha polypeptide) or fragment thereof should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disease or disorder, for example, a disease or disorder associated with or characterized by aberrant expression and/or activity of an interferon alpha polypeptide, a disease or disorder associated with or characterized by aberrant expression and/or activity of the interferon alpha receptor or one or more subunits thereof, an autoimmune disease, an autoimmune disease, transplant rejection, graft versus host disease, or one or more symptoms thereof, in a subject.
  • a disease or disorder associated with or characterized by aberrant expression and/or activity of an interferon alpha polypeptide a disease or disorder associated with or characterized by aberrant expression and/or activity of the interferon alpha receptor or one or more subunits thereof
  • an autoimmune disease an autoimmune disease, transplant rejection, graft versus host disease, or one or more symptoms thereof, in a subject.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the present invention provides methods for preparing liquid formulations of antibodies or derivatives, analogues, or fragments thereof that specifically bind to an antigen of interest.
  • the methods for preparing liquid formulations of the present invention may comprise: purifying the antibody (including antibody fragment thereof) from conditioned medium (either single lots or pooled lots of medium) and concentrating a fraction of the purified antibody (including antibody fragment thereof) to a final concentration of about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 250 mg/ml, about 300 mg/ml, about 350 mg/ml, about 400 mg/ml, or about 500 mg/ml.
  • Conditioned medium containing the antibody (including antibody fragment thereof), for example, an antibody that specifically binds to an interferon alpha polypeptide may be subjected to CUNO filtration and the filtered antibody is subjected to HS50 cation exchange chromatography. The fraction from the HS50 cation exchange chromatography is then subjected to low pH treatment followed by MEP Hypercel chromatography. The fraction from the MEP Hypercel chromatography is subject to nanofiltration. The purified antibody or a fragment thereof obtained after nanof ⁇ ltration is then subjected to diaf ⁇ ltration and ultrafiltration to buffer exchange and concentrate into the formulation buffer using the same membrane.
  • the antibody including antibody fragment thereof
  • the liquid formulations of the present invention can be prepared as unit dosage forms by preparing a vial containing an aliquot of the liquid formulation for a one-time use.
  • a unit dosage per vial may contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml of different concentrations of an antibody (including antibody fragment thereof). If necessary, these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial.
  • the liquid formulations of the present invention may be sterilized by various sterilization methods, including sterile filtration, radiation, etc.
  • the diafiltrated antibody formulation is filter-sterilized with a presterilized 0.2 micron filter.
  • Sterilized liquid antibody formulations of the present invention may be administered to a subject to prevent, treat and/or manage a disease or disorder associated with or characterized by aberrant expression and/or activity of the antigen recognized by the antibody.
  • the invention is directed to liquid non-lyophilized formulations, it should be noted for the purpose of equivalents that the formulations of the invention may be lyophilized if desired. Thus, the invention encompasses lyophilized forms of the formulations of the invention.
  • the antibodies (including antibody fragments thereof) that specifically bind to an antigen can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques (see, US Patent Publication 2007/0014724A1).
  • Polyclonal antibodies specific for an antigen can be produced by various procedures well- known in the art. For example, a human antigen can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the human antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a non-murine antigen and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
  • a RIMMS (repetitive immunization multiple sites) technique can be used to immunize an animal (Kilpatrack et al., 1997, Hybridoma 16:381-9, incorporated herein by reference in its entirety).
  • the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with a non-murine antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind to the antigen.
  • Antibody fragments which recognize specific particular epitopes may be generated by any technique known to those of skill in the art.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g. , human or murine cDNA libraries of affected tissues).
  • the DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M 13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g. , using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • VH constant region e.g. , the human gamma 4 constant region
  • the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g. , human kappa or lamba constant regions.
  • the vectors for expressing the VH or VL domains may comprise an EF- l ⁇ promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, for example, but not limited to, IgG, using techniques known to those of skill in the art.
  • humanized antibodies or chimeric antibodies For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be appropriate to use humanized antibodies or chimeric antibodies. Completely human antibodies and humanized antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then be bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al, 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,8 16397, and 6,331,415, which are incorporated herein by reference in their entirety.
  • a humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2 , Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHl, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG2, IgG 3 and IgG 4 .
  • the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically IgGi.
  • the constant domain may be of the IgG2 class.
  • the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework and CDR sequences, more often 90%, and greater than 95%.
  • Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al, 1994, PNAS 91 :969-973), chain shuffling (U.S. Patent No.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, for example, but not limited to, by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, which are incorporated herein by reference in their entireties).
  • Single domain antibodies for example, antibodies lacking the light chains, can be produced by methods well-known in the art. See Riechmann et al., 1999, J. Immuno. 231 :25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. l(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302; U.S. Patent No. 6,005,079; and International Publication Nos. WO 94/04678, WO 94/25591, and WO 01/44301, each of which is incorporated herein by reference in its entirety.
  • the antibodies that specifically bind to an antigen can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" an antigen using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).
  • Recombinant expression of an antibody contained in a formulation of the invention may require construction of an expression vector containing a polynucleotide that encodes the antibody.
  • an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule, heavy or light chain of an antibody, or fragment thereof has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well-known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody (including antibody fragment thereof), or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication No.
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • a variety of host-expression vector systems may be utilized to express the antibody molecules of the invention (see, e.g., U.S. Patent No. 5,807,715).
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (for example, but not limited to, E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (for example, but not limited to, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (for example, but not limited to, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (for example, but not limited to, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (for example, but not limited to, Ti plasmid) containing antibody coding sequences; or mammalian cell systems (for example, but not limited to, COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (
  • Bacterial cells such as Escherichia coli, and eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al, 1990, Bio/Technology 8:2).
  • the expression of nucleotide sequences encoding antibodies of the invention, derivative, analog, or fragment thereof is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • GST glutathione 5-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • AcNPV promoter for example the polyhedrin promoter
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome ⁇ e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts ⁇ e.g. , see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355-359).
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544). [00283] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Such modifications for example, but not limited to, glycosylation
  • processing for example, but not limited to, cleavage
  • protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • stable expression is may be used.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements ⁇ e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplif ⁇ able
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, MoI. Cell. Biol. 3:257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • rCGE and HPSEC are the most common and simplest methods to assess the formation of protein aggregates, protein degradation, and protein fragmentation. Accordingly, the stability of the liquid formulations of the present invention may be assessed by these methods.
  • the stability of the liquid formulations of the present invention may be evaluated by HPSEC, wherein the percent area of the peaks represents the non-degraded antibody or non- degraded antibody fragments.
  • approximately 250 ⁇ g of the antibody (including antibody fragment thereof) (approximately 25 ⁇ l of a liquid formulation comprising 10 mg/ml said antibody or antibody fragment) is injected onto a TosoH Biosep TSK G3000SW XL column (7.8 mm x 30 cm) fitted with a TSK SW xl guard column (6.0 mm CX 4.0 cm).
  • the antibody (including antibody fragment thereof) is eluted isocratically with 0.1 M disodium phosphate containing 0.1 M sodium sulfate and 0.05% sodium azide, at a flow rate of 0.8 to 1.0 ml/min. Eluted protein is detected using UV absorbance at 280 nm. Reference standards are run in the assay as controls, and the results are reported as the area percent of the product monomer peak compared to all other peaks excluding the included volume peak observed at approximately 12 to 14 minutes. Peaks eluting earlier than the monomer peak are recorded as percent aggregate.
  • the liquid formulations of the present invention exhibit low to undetectable levels of aggregation as measured by any of the methods described above, that is, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and no more than 0.5% aggregate by weight protein, and low to undetectable levels of fragmentation, that is, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or higher, or 99.5% or higher of the total peak area in the peak(s) representing intact antibodies (including antibody fragments thereof).
  • the density or the radioactivity of each band stained or labeled with radioisotope can be measured and the % density or % radioactivity of the band representing non-degraded antibodies (including antibody fragments thereof) can be obtained.
  • the stability of the liquid formulations of the present invention can be also assessed by any assays which measure the biological activity of the antibody in the formulation.
  • the biological activities of antibodies include, but are not limited to, antigen-binding activity, blocking of ligand-receptor interaction, and so forth (see infra).
  • Antigen-binding activity of the antibodies can be measured by any method known to those skilled in the art, including but not limited to ELISA, radioimmunoassay, Western blot, and the like. Also see Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • the purity of the liquid antibody formulations of the invention may be measured by any method well-known to one of skill in the art such as, for example, but not limited to, HPSEC.
  • the sterility of the liquid antibody formulations may be assessed by any method well-known to one of skill in the art such as, e.g.: sterile soybean-casein digest medium and fluid thioglycollate medium are inoculated with a test liquid antibody formulation by filtering the liquid antibody formulation through a sterile filter having a nominal porosity of 0.45 ⁇ m.
  • each filter device is aseptically filled with approximately 100 ml of sterile soybean-casein digest medium or fluid thioglycollate medium.
  • the challenged filter is aseptically transferred to 100 ml of sterile soybean-casein digest medium or fluid thioglycollate medium.
  • the media are incubated at appropriate temperatures and observed three times over a 14 day period for evidence of bacterial or fungal growth.
  • the present invention is also directed to antibody-based therapies which involve administering to a human subject the liquid antibody formulations (or "antibody formulations” or “liquid formulations") of the present invention for preventing, treating and/or managing a disease or disorder.
  • the liquid formulations of the present invention may be used locally or systemically in the body as a therapeutic. Particularly, the liquid formulations of the invention may be used in the prevention, treatment and/or management of a disease or disorder.
  • the formulations of the present invention may also be utilized in combination with one or more other therapies (e.g., one or more other prophylactic or therapeutic agents), for example, therapies useful in the prevention, treatment and/or management of a disease or disorder.
  • therapeutic or prophylactic agents include, but are not limited to, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (for example, but not limited to, DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides) antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules.
  • nucleic acids for example, but not limited to, DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides
  • Any therapy e.g., prophylactic or therapeutic agents
  • therapy which is known to be useful, or which has been used or is currently being used for the prevention, treatment and/or management of one or more symptoms associated with a disease or disorder
  • any therapy e.g., prophylactic or therapeutic agents
  • Any therapy which is known to be useful, or which has been used or is currently being used for the prevention, treatment and/or management of one or more symptoms associated with a disease or disorder
  • therapy e.g., prophylactic or therapeutic agents
  • prophylactic and therapeutic agents include, but are not limited to, immunomodulatory agents, anti-inflammatory agents (for example, but not limited to, adrenocorticoids, corticosteroids (for example, but not limited to, beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methlyprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steriodal anti-inflammatory drugs (for example, but not limited to, aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), and leukotreine antagonists (for example, but not limited to, montelukast, methyl xanthines, zafirlukast, and zileuton), beta2-agonists (for example, but not limited to, albuterol, biterol, fenoterol, isoetharie, metaproterenol, pir
  • a liquid formulation of the invention may be administered to a human concurrently with one or more other therapies (e.g. , one or more other prophylactic or therapeutic agents) useful for the prevention, treatment and/or management of a disease or disorder, or one or more symptoms thereof.
  • therapies e.g. , one or more other prophylactic or therapeutic agents
  • the term “concurrently” is not limited to the administration of prophylactic or therapeutic agents/therapies at exactly the same time, but rather it is meant that a liquid formulation of the invention and the other agent/therapy are administered to a mammal in a sequence and within a time interval such that the antibody (including antibody fragment thereof) contained in the liquid formulation can act together with the other agent/therapy to provide an increased benefit than if they were administered otherwise.
  • a liquid formulation of the invention and one or more other therapies are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • therapies e.g., one or more other prophylactic or therapeutic agents
  • a liquid formulation of the invention and one or more other therapies are administered within the same patient visit.
  • a liquid formulation of the invention and one or more other therapies are administered at about 2 to 4 days apart, at about 4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeks apart.
  • a liquid formulation of the invention and one or more other therapies are administered in a time frame where both agents are still active.
  • One skilled in the art would be able to determine such a time frame by determining the half- life of the administered agents.
  • a liquid formulation of the invention and one or more other therapies are cyclically administered to a subject.
  • Cycling therapy involves the administration of a first agent for a period of time, followed by the administration of a second agent and/or third agent for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improves the efficacy of the treatment.
  • a liquid formulation of the invention and one or more other therapies are administered in a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days or about once every week.
  • One cycle can comprise the administration of a therapeutic or prophylactic agent by infusion over about 90 minutes every cycle, about 1 hour every cycle, about 45 minutes every cycle.
  • Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest.
  • the number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.
  • liquid formulation of the invention and one or more other therapies are administered in metronomic dosing regimens, either by continuous infusion or frequent administration without extended rest periods.
  • metronomic administration can involve dosing at constant intervals without rest periods.
  • the prophylactic or therapeutic agents in particular cytotoxic agents, are used at lower doses.
  • dosing regimens encompass the chronic daily administration of relatively low doses for extended periods of time.
  • the use of lower doses can minimize toxic side effects and eliminate rest periods.
  • the prophylactic and therapeutic agents are delivered by chronic low-dose or continuous infusion ranging from about 24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3 weeks to about 1 month to about 2 months, to about 3 months, to about 4 months, to about 5 months, to about 6 months.
  • a liquid formulation of the invention is administered in a dosing regimen that maintains the plasma concentration of the antibody (including antibody fragment thereof) at a desirable level (e.g., about 0.1 to about 100 ⁇ g/ml).
  • the plasma concentration of the antibody (including antibody fragment thereof) is maintained at 0.2 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml, 3 ⁇ g/ml, 4 ⁇ g/ml, 5 ⁇ g/ml, 6 ⁇ g/ml, 7 ⁇ g/ml, 8 ⁇ g/ml, 9 ⁇ g/ml, 10 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 25 ⁇ g/ml, 30 ⁇ g/ml, 35 ⁇ g/ml, 40 ⁇ g/ml, 45 ⁇ g/ml or 50 ⁇ g/ml.
  • a liquid formulation of the invention is administered intermittently to a subject, wherein the liquid formulation comprises an antibody (including antibody fragment thereof) conjugated to a moiety.
  • the liquid formulations of the invention and the other therapy can act additively or synergistically.
  • the invention contemplates administration of a liquid formulation of the invention in combination with other therapies (e.g., prophylactic or therapeutic agents) by the same or different routes of administration, for example, but not limited to, oral and parenteral.
  • a liquid formulation of the invention is administered concurrently with one or more therapies (e.g.
  • prophylactic or therapeutic agents that potentially produce adverse side effects (including, but not limited to, toxicity)
  • the therapies e.g., prophylactic or therapeutic agents
  • the therapies can advantageously be administered at a dose that falls below the threshold that the adverse side effect is elicited.
  • Various delivery systems are known and can be used to administer a liquid formulation of the present invention or a prophylactic or therapeutic agent.
  • Methods of administering antibody liquid formulations of the present invention or a therapy include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and, and subcutaneous), epidural administration, topical administration, and mucosal administration (for example, but not limited to, intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and, and subcutaneous
  • epidural administration e.g., epidural administration
  • topical administration e.g., topical administration
  • mucosal administration for example, but not limited to, intranasal and oral routes.
  • liquid formulations of the present invention are administered intramuscularly, intravenously, or subcutaneously.
  • the liquid formulations of the invention are administered subcutaneously.
  • the formulations may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • a liquid formulation of the present invention is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of antibody (including antibody fragment thereof).
  • a liquid formulation of the present invention is in a hermetically sealed container indicating the quantity and concentration of the antibody (including antibody fragment thereof).
  • a liquid formulation of the present invention is supplied in a hermetically sealed container and comprises about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 250 mg/ml, or about 300 mg/ml of an antibody (including antibody fragment thereof), in a quantity of about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, 6
  • a liquid formulation of the invention is supplied in a hermetically sealed container and comprises at least about 15 mg/ml, at least about 20 mg/ml, at least about 25 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 125 mg/ml, at least about 150 mg/ml, at least about 175 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml or at least about 300 mg/ml of an antibody (including antibody fragment thereof) for intravenous injections, and at least about 15 mg/ml, at least about 20 mg/ml, at least about 50 mg/ml, at least about 80 mg/ml, at least about 100 mg/ml, at least about 125 mg/ml, at least about 150 mg/ml, at least about 175 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml or at least about 300 mg/ml of an antibody (including antibody fragment thereof) for intravenous injections, and at
  • the amount of a liquid formulation of the present invention which will be effective in the prevention, treatment and/or management of a disease or disorder can be determined by standard clinical techniques well-known in the art or described herein.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the inflammatory disorder, or autoimmune disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • the required volume (in mL) to be given is then determined by taking the mg dose required divided by the concentration of the antibody formulation.
  • the final calculated required volume will be obtained by pooling the contents of as many vials as are necessary into syringe(s) to administer the antibody formulation of the invention.
  • the final calculated required volume will be obtained by pooling the contents of as many vials as are necessary into syringe(s) to administer the drug.
  • a maximum volume of 2.0 mL of the antibody formulation can be injected per site.
  • Dose (mL) [volunteer weight] (kg) x [dose] mg/kg ⁇ 100 mg/mL of the antibody formulation.
  • the dosage, volume and frequency of administration of liquid formulations of the present invention may be reduced by increasing the concentration of an antibody (including antibody fragment thereof) in the formulations, increasing affinity and/or avidity of the antibody (including antibody fragment thereof), and/or increasing the half- life of the antibody (including antibody fragment thereof).
  • the dosage administered to a patient will be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • the required volume (in mL) to be given is then determined by taking the mg dose required divided by the concentration of the antibody (including antibody fragment thereof) in the formulations (100 mg/mL).
  • the final calculated required volume will be obtained by pooling the contents of as many vials as are necessary into syringe(s) to administer the drug.
  • a maximum volume of 2.0 mL of antibody (including antibody fragment thereof) in the formulations can be injected per site.
  • 0.1 to 20 mg/kg/week, 1 to 15 mg/kg/week, 2 to 8 mg/week, 3 to 7 mg/kg/week, or 4 to 6 mg/kg/week of an antibody (including antibody fragment thereof) in a liquid formulation of the invention is administered to a subject with an inflammatory disorder or an autoimmune disorder.
  • a subject is administered one or more doses of a prophylactically or therapeutically effective amount of a liquid formulation of the invention, wherein the prophylactically or therapeutically effective amount is not the same for each dose.
  • a liquid formulation of the invention is administered in a dosing regimen that maintains the plasma concentration of the antibody at a desirable level (e.g., from about 0.1 to about 100 ⁇ g/ml).
  • the plasma concentration of the antibody is maintained at about 0.2 ⁇ g/ml, about 0.5 ⁇ g/ml, about 1 ⁇ g/ml, about 2 ⁇ g/ml, about 3 ⁇ g/ml, about 4 ⁇ g/ml, about 5 ⁇ g/ml, about 6 ⁇ g/ml, about 7 ⁇ g/ml, about 8 ⁇ g/ml, about 9 ⁇ g/ml, about 10 ⁇ g/ml, about 15 ⁇ g/ml, about 20 ⁇ g/ml, about 25 ⁇ g/ml, about 30 ⁇ g/ml, about 35 ⁇ g/ml, about 40 ⁇ g/ml, about 45 ⁇ g/ml or about 50 ⁇ g/ml
  • a liquid formulation of the invention comprising a conjugated antibody (including antibody fragment thereof) is administered intermittently.
  • a conjugated antibody or antibody fragment refers to an antibody (including antibody fragment thereof) that is conjugated or fused to another moiety, including but not limited to, a heterologous peptide, polypeptide, another antibody (including antibody fragment thereof), a marker sequence, a diagnostic agent, a polymer, albumin, and a solid support.
  • a human subject is administered one or more doses of a prophylactically or therapeutically effective amount of an antibody (including antibody fragment thereof) in a liquid formulation of the invention, wherein the dose of a prophylactically or therapeutically effective amount of the antibody (including antibody fragment thereof) in the liquid formulation of the invention administered to said subject is increased by, e.g., about 0.01 ⁇ g/kg, about 0.02 ⁇ g/kg, about 0.04 ⁇ g/kg, about 0.05 ⁇ g/kg, about 0.06 ⁇ g/kg, about 0.08 ⁇ g/kg, about 0.1 ⁇ g/kg, about 0.2 ⁇ g/kg, about 0.25 ⁇ g/kg, about 0.5 ⁇ g/kg, about 0.75 ⁇ g/kg, about 1 ⁇ g/kg, about 1.5 ⁇ g/kg, about 2 ⁇ g/kg, about 4 ⁇ g/kg, about 5 ⁇ g/kg, about 10 ⁇ g/kg, about 15 ⁇ g/kg, about 20 ⁇
  • a subject e.g., a human
  • a liquid formulation of the invention wherein the dose of a prophylactically or therapeutically effective amount of the antibody (including antibody fragment thereof) in the liquid formulation of the invention administered to said subject is decreased by, e.g., about 0.01 ⁇ g/kg, about 0.02 ⁇ g/kg, about 0.04 ⁇ g/kg, about 0.05 ⁇ g/kg, about 0.06 ⁇ g/kg, about 0.08 ⁇ g/kg, about 0.1 ⁇ g/kg, about 0.2 ⁇ g/kg, about 0.25 ⁇ g/kg, about 0.5 ⁇ g/kg, about 0.75 ⁇ g/kg, about 1 ⁇ g/kg, about 1.5 ⁇ g/kg, about 2 ⁇ g/kg, about 4 ⁇ g/kg, about 5 ⁇ g/kg, about 10 ⁇ g/kg, about 15
  • the toxicity and/or efficacy of the prophylactic and/or therapeutic protocols of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Therapies that exhibit large therapeutic indices are preferred. While therapies that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by ELISA.
  • Antibodies including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof
  • Antibodies of the liquid formulations of the invention can be used for diagnostic purposes to detect, diagnose, prognose, or monitor a disease or disorder or one or more symptoms thereof.
  • Antibodies of the liquid formulations of the invention can be used to assay the level of their specific target in a biological sample using classical immunohistological methods known to those of skill in the art.
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RlA).
  • ELISA enzyme linked immunosorbent assay
  • RlA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. 5.10.Kits
  • the invention provides a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation of the invention.
  • the liquid formulations of the invention comprise antibodies (including antibody fragments thereof) recombinantly fused or chemically conjugated to another moiety, including but not limited to, a heterologous protein, a heterologous polypeptide, a heterologous peptide, a large molecule, a small molecule, a marker sequence, a diagnostic or detectable agent, a therapeutic moiety, a drug moiety, a radioactive metal ion, a second antibody, and a solid support.
  • the invention also provides a pharmaceutical pack or kit comprising in one or more first containers a liquid formulation of the invention and in one or more second containers one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of a disease or disorder.
  • the formulations of the invention may be supplied in 3 cc USP Type I borosilicate amber vials (West Pharmaceutical Serices - Part No. 6800-0675) with a target volume of 1.2 mL.
  • the formulations of the invention may also be supplied in clear borosilicate vials with a target volume of 1.2 mL or 3 mL.
  • a container filled with a liquid formulation of the invention is a pre-filled syringe. Any pre-filled syringe known to one of skill in the art may be used in combination with a liquid formulation of the invention.
  • Pre-filled syringes that may be used are described in, for example, but not limited to, PCT Publications WO05032627, WO08094984, WO9945985, WO03077976, US Patents US6792743, US5607400, US5893842, US7081107, US7041087, US5989227, US6807797, US6142976, US5899889, US Patent Publications US20070161961A1, US2005007561 IAl, US20070092487A1, US20040267194A1, US20060129108A1.
  • Pre-filled syringes may be made of various materials.
  • a pre-filled syringe is a glass syringe.
  • a pre-filled syringe is a plastic syringe.
  • One of skill in the art understands that the nature and/or quality of the materials used for manufacturing the syringe may influence the stability of a protein formulation stored in the syringe. For example, it is understood that silicon based lubricants deposited on the inside surface of the syringe chamber may affect particle formation in the protein formulation.
  • a pre-filled syringe comprises a silicone based lubricant.
  • a pre-filled syringe comprises baked on silicone.
  • a pre-filled syringe is free from silicone based lubricants.
  • a pre-f ⁇ lled syringe may comprise tungsten at a level above 500 ppb.
  • a pre-filled syringe is a low tungsten syringe.
  • a pre- f ⁇ lled syringe may comprise tungsten at a level between about 500 ppb and about 10 ppb, between about 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb, between about 100 ppb and about 10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb and about 10 ppb.
  • a pre-filled syringe may comprise tungsten at a level lower than 500 ppb, lower than 400 ppb, lower than 300 ppb, lower than 200 ppb, lower than 100 ppb, lower than 75 ppb, lower than 50 ppb, lower than 25 ppb, lower than 10 ppb, lower than 55 ppb, lower than 2 ppb, or lower than 1 ppb.
  • the liquid formulations of the present invention can be prepared as unit dosage forms by preparing a pre-f ⁇ lled syringe containing an aliquot of the liquid formulation for a one-time use.
  • a unit dosage per pre-filled syringe may contain 0.1ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml of different concentrations of an antibody (including antibody fragment thereof) ranging from about 10 mg/ml to about 300 mg/ml.
  • the liquid formulations of the present invention are formulated into single dose pre-f ⁇ lled syringes as a sterile liquid that contains 25 mM histidine buffer at pH 6.0, 8% trehalose and 0.02% polysorbate 80.
  • Each 1.0 mL of solution contains 100 mg of the antibody (including antibody fragment thereof).
  • Each 1.0 mL of solution contains 125 mg of the antibody (including antibody fragment thereof).
  • Each 1.0 mL of solution contains 150 mg of the antibody (including antibody fragment thereof).
  • Each 1.0 mL of solution contains 175 mg of the antibody (including antibody fragment thereof).
  • Each 1.0 mL of solution contains 200 mg of the antibody (including antibody fragment thereof).
  • the present invention provides kits that can be used in the above methods.
  • a kit comprises a liquid formulation of the invention, in one or more containers.
  • a kit comprises a liquid formulation of the invention, in one or more containers, and one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of a disease or disorder.
  • the kit may further comprise instructions for preventing, treating and/or managing a disorder (e.g., using the liquid formulations of the invention alone or in combination with another prophylactic or therapeutic agent), as well as side effects and dosage information for method of administration. 5.11.Articles of Manufacture
  • the present invention also encompasses a finished packaged and labeled pharmaceutical product.
  • This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed.
  • the unit dosage form is provided as a sterile particulate free solution comprising an anti-interferon alpha antibody that is suitable for parenteral administration.
  • the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery.
  • the invention encompasses sterile solutions suitable for each delivery route.
  • the packaging material and container are designed to protect the stability of the product during storage and shipment.
  • the products of the invention include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question.
  • the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, and other monitoring information.
  • the invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, pre-f ⁇ lled syringe, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within said packaging material, wherein said pharmaceutical agent comprises a liquid formulation containing an antibody.
  • packaging material includes instruction means which indicate that said antibody can be used to prevent, treat and/or manage one or more symptoms associated with a disease or disorder by administering specific doses and using specific dosing regimens as described herein.
  • the invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, pre-filled syringe, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of each pharmaceutical agent contained within said packaging material, wherein one pharmaceutical agent comprises a liquid formulation containing an antibody and the other pharmaceutical agent comprises a prophylactic or therapeutic agent other than an antibody, and wherein said packaging material includes instruction means which indicate that said agents can be used to prevent, treat and/or manage one or more symptoms associated with a disease or disorder by administering specific doses and using specific dosing regimens as described herein.
  • packaging material such as a box, bottle, tube, vial, container, sprayer, pre-filled syringe, insufflator, intravenous (i.v.) bag, envelope and the like
  • at least one unit dosage form of each pharmaceutical agent contained within said packaging material wherein one pharmaceutical agent comprises a liquid formulation containing an antibody and the other pharmaceutical
  • the present invention provides that the adverse effects that may be reduced or avoided by the methods of the invention are indicated in informational material enclosed in an article of manufacture for use in preventing, treating and/or managing one or more symptoms associated with a disease or disorder.
  • Adverse effects that may be reduced or avoided by the methods of the invention include, but are not limited to, vital sign abnormalities (fever, tachycardia, bardycardia, hypertension, hypotension), hematological events (anemia, lymphopenia, leukopenia, thrombocytopenia), headache, chills, dizziness, nausea, asthenia, back pain, chest pain (chest pressure), diarrhea, myalgia, pain, pruritus, psoriasis, rhinitis, sweating, injection site reaction, and vasodilatation.
  • the information material enclosed in an article of manufacture described herein can indicate that foreign proteins may also result in allergic reactions, including anaphylaxis, or cytosine release syndrome.
  • the information material should indicate that allergic reactions may exhibit only as mild pruritic rashes or they may be severe such as erythroderma, Stevens- Johnson syndrome, vasculitis, or anaphylaxis.
  • the information material should also indicate that anaphylactic reactions (anaphylaxis) are serious and occasionally fatal hypersensitivity reactions.
  • Allergic reactions including anaphylaxis may occur when any foreign protein is injected into the body. They may range from mild manifestations such as urticaria or rash to lethal systemic reactions. Anaphylactic reactions occur soon after exposure, usually within 10 minutes.
  • a sterile, stable aqueous formulation comprising an antibody or fragment thereof, wherein the concentration of the antibody is at least 110 mg/ml.
  • a pharmaceutical unit dosage form suitable for parenteral administration to a human which comprises an antibody formulation of any one of embodiments 1 to 57 in a suitable container.
  • a kit comprising the formulation of any one of embodiments 1 to 60.
  • a method of preventing, managing, treating or ameliorating a disease or disorder comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of an antibody formulation of any one of embodiments 1 to 60.
  • 13H5 is a human IgGl monoclonal antibody produced by recombinant DNA technology that that binds to IFN alpha and inhibits the biological activity of multiple IFN alpha subtypes, but does not substantially inhibit the biological activity of IFN alpha subtype 21, or of IFN beta or IFN omega.
  • the 13H5 antibody used for the examples described herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO:2. 7.1.1.Experimental methods
  • Purified 13H5 antibody is generated following standard industrial scale protocols. Details of cell culture condition and antibody purification are described in the co-pending US Provisional Patent Application 60/909,232, filed on March 30, 2007. The purified antibody is eluted from the final column in 10 mM sodium acetate (pH 5.2) at an approximate concentration of 2.5 mg/ml. Protein concentration is estimated from optical density measurement at 280 nm.
  • Purified 13H5 antibody is nanofiltered using a Planova 2ON filter to remove particulate matter. 13H5 formulations are prepared using Tangential Flow Filtration (TFF). The nanofiltered 13H5 antibody is concentrated to approximately 25 mg/ml on a Millipore Labscale TFF device.
  • the antibody is then 5x diafiltered into the appropriate buffer (e.g., 25 mM histidine-HCL (pH 6.0)). Once the buffer exchange is complete, the antibody is concentrated to approximately 150 mg/ml. Excipients are introduced by spiking the concentrated antibody preparation with the appropriate concentrated stock solutions. For example, a final concentration of 8% Trehalose is achieved by adding 25 ml of 25 mM histidine-HCl, 40% Trehalose (pH 6.0) to every 100 ml of concentrated antibody preparation. Multiple excipients may be introduced in consecutive steps.
  • the appropriate buffer e.g., 25 mM histidine-HCL (pH 6.0)
  • a final concentration of 0.02% Polysorbate 80 is introduced after the addition of Trehalose by diluting 100 fold a 25 mM histidine-HCl, 8% Trehalose, 2% Polysorbate 80 (pH 6.0) stock solution with the Trehalose containing antibody preparation.
  • the final concentration of 13H5 is adjusted to 100 ⁇ 5 mg/ml with the final formulation buffer (e.g., 25 mM histidine-HCl, 8% Trehalose, 0.01% Polysorbate 80 (pH 6.0)).
  • a flow chart for the preparation of 13H5 formulations is presented in Figure 1.
  • Additional storage conditions that may affect stability of a formulation include, but are not limited to, light intensity, light wavelength, humidity, vial composition, and stopper composition. The effect of these parameters on formulation stability may also be determined using the methods described herein.
  • Size exclusion chromatography was utilized to measure the amount of antibody aggregates in the formulation.
  • SEC was performed using the Agilent 1100 Series High Performance Liquid Chromatography (HPLC) system. Samples were diluted to 10 mg/ml. 25 ⁇ l diluted sample containing 250 ug protein was injected onto a TSK-GeI 3000 column (size 7.8 mm x 30.0 cm.; Tosoh Biosciences Corporation). Protein elution profile was determined by following the eluate's optical density at 280 nm. Data analysis was performed using ChemStation (Agilent) auto integration parameters. The percent of protein in aggregate form in various formulations is plotted in Figures 1 -9
  • RP-HPLC Reversed Phase High Performance Liquid Chromatography
  • HPLC High Performance Liquid Chromatography
  • Samples were analyzed on a PLRP-S (8 um, 4000 A, 2.0 x 150 mm) column from Michrom Bioresources. Protein elution profile was determined by following the eluate's optical density at 280 nm. Data analysis was performed using ChemStation (Agilent) auto integration parameters. The percent of fragmented antibody in various formulations is plotted in Figure 10 and 11.
  • IEC Ion exchange chromatography
  • Stability of 100 mg/ml 13H5 in formulation E is higher that the stability of 50 mg/ml 13H5 in formulation B or D.
  • Figure 6 shows that the formulation pH (within the examined range of pH 5-7) has no effect on stability.
  • Figure 7 and 9 shows that the stability of 13H5 in formulation E is very similar to that of two unrelated clinical candidate high concentration liquid antibody formulations.
  • Figure 8 presents a stability comparison of various high concentration 13H5 formulations stored at 5 0 C; formulations B, E, and D display identical stability characteristics that are better than that of formulation F.
  • 10 and 11 show the fragmentation rates of 13H5 in formulation B and E stored at 4O 0 C and 5 0 C, respectively.
  • the observed fragmentation rates are identical in the two formulations and very similar to the fragmentation rate of an unrelated antibody in high concentration liquid formulation.
  • Formulation E displayed the highest stability under all conditions examined. Formulation E is colorless and clear after two month storage at 5°C. Based on its superior stability, formulation E was selected to be used as a clinical candidate high concentration 13H5 formulation. 13H5 in formulation E at a concentration of 100 ⁇ 5 mg/ml is hereinafter referred to as "13H5 fE" formulation.
  • Size exclusion chromatography may be performed to analyze the antibody formulation for the presence of antibody aggregates and fragments.
  • the test samples are injected onto a high resolution size exclusion column (e.g., G3000 SW XL 5 ⁇ m, 300 A, 7.8 x 300 mm, TosoHaas).
  • the mobile phase is 0.1 M di-sodium phosphate, 0.1 M sodium sulphate and 0.05 % sodium azide (pH 6.7), running isocratically at a flow rate of 0.25 - 1.0 mL/min.
  • Eluted protein may be detected by UV absorbance at 280 nm and collected for further characterization.
  • the relative amount of any protein species detected is reported as the area percent of the product peak as compared to the total area of all other detected peaks excluding the initial excluded volume peak. Peaks eluting earlier than the antibody monomer peak are recorded in the aggregate percentile, while peaks eluting later than the antibody monomer peak, but earlier than the buffer peak, are recorded in the fragment percentile.
  • the hydrodynamic radius and molecular weight of the individual peaks may be obtained with a coupled multiangle light scattering detector.
  • SEC may be used to monitor antibody aggregate formation and antibody fragmentation in a formulations stored for extended time periods (e.g., multiple measurements performed over 9 months).
  • the formulation may be stored at different temperature ranges (e.g., 2-8 0 C, 20-24 0 C and 38-42 0 C). Temperature ranges above the proposed clinical storage temperature (2-8 0 C) are used to stress the formulation with the goal of simulating the effects of storage beyond 9 months. The ratio of fragments and aggregates is expected to increase over time; this increase is likely to be accelerated at elevated temperatures. A finding that fragmentation and aggregation rates are constant within each temperature range would show that higher storage temperatures accurately simulate an accelerated time scale. [00420] The logarithm of the estimated rates of fragmentation/aggregation (log(rate)) may also be determined.
  • AUC Analytical ultracentrifugation
  • SEC sedimentation coefficients
  • AUC is capable of separating and detecting antibody fragments/aggregates from monomers and is further able to provide information on molecular mass.
  • AUC eliminates the possibility of aggregate loss due to solid-phase interaction and is better able to resolve differing species of a given macromolecule.
  • Sedimentation velocity experiments may be performed using an analytical ultracentrifuge, for example, Beckman Optima XL-A.
  • Test samples are diluted to an antibody concentration of 0.5 mg/ml with reference buffer (e.g., 20 mM citric acid, 100 mM NaCl, 1.5% mannitol, 50 ⁇ M diethylenetriamine- pentaacetic acid, 0.02% Polysorbate 80, pH 6.0).
  • 415 ⁇ l of the diluted antibody sample and 412 ⁇ l or the reference buffer is loaded into a 12 mm centrifuge cell in the sample and reference channels, respectively. Loaded cells are placed into an AN-50Ti analytical rotor and equilibrated to 25 0 C.
  • Samples are scanned at 280 nm with a rotor speed of 42000 rpm at full vacuum. A total of 80 scans for each cell are collected for analysis. The first scan for each sample is excluded from downstream data processing to avoid artifacts caused by meniscus.
  • the data is analyzed using the c(s) method developed by Peter Shuck at N. I. H. and the SEDFIT (version 8.8) program with implemented c(s).
  • c(s) method raw data scans are directly fit to a Lamm function of S in order to derive a distribution of sedimentation coefficients.
  • the parameters used for the fitting procedure are resolution, 400; confidence interval, 0.75; grid size, 1000; partial specific volume, 0.7245; buffer density, 1.000; and buffer viscosity, 0.1002. Frictional ratio, meniscus and bottom positions are set as fitted parameters. Time independent noise is also fitted.
  • the detected peaks are integrated and classified as follows: from 0 to 6 S, fragments; from 6 to 9 S, monomer; and from 9 to 20 S, aggregates.
  • AUC may be used to characterize antibody formulations with low relative levels of aggregation and fragmentation. AUC may be able to better resolve antibody fragments and aggregates from the monomer species in situations that are beyond the resolution capabilities of SEC. peaks. AUC estimates of the molecular mass of an aggregate peak may also be used as an indicator of its composition
  • AUC may also able to better resolve differing species of a given macromolecule. It is, however, necessary to establish first the proper sample dilution rate, as the noise/signal ratio of AUC is dependent on the antibody concentration in the sample.
  • Protein aggregation in the antibody formulation may also be characterized by turbidity measurement.
  • Turbidity is a measure of the amount by which the particles in a solution scatter light and, thus, may be used as a general indicator of protein aggregation or denaturation. Elevated turbidity may indicate a higher level of aggregation or an increased number/ increased size of particles.
  • Turbidity measurement may be performed with a turbidimeter (e.g. , 2100AN or 2100N,
  • Turbidity standard Hatch at 40, 200, 1000 and 4000 NTU (nephelometric turbidity unit) and verified by analyzing control suspensions of formazin at 3, 6, 18, 30 and 60 NTU.
  • the number and size of particles in a particular formulation may be determined using a particle counter (e.g., Beckman Coulter Multisizer 3) according to the manufacturers instruction.
  • a particle counter e.g., Beckman Coulter Multisizer 3
  • Viscosities of antibody formulations may be measured using a viscometer (e.g. , ViscoLab
  • the viscometer is calibrated before use with the appropriate standards (e.g., S6S Reference
  • Piston is also checked using RODI H 2 O (1.00 cP @ 20.0 0 C).
  • the piston is cleaned and rinsed thoroughly with soap and water between measurements of each different solution type. Subsequently the system is cooled to ⁇ 2 0 C. Once the system temperature is at or below 2 0 C, sample is loaded into the chamber and the piston is lowered into the sample. After sample is equilibrated to the temperature of the chamber, measurement is initiated. The temperature is increased at I 0 C increments every 7-10 minutes to a final temperature of > 25 0 C. The viscosity result is recorded immediately prior to increasing the temperature. The piston remains in motion during measurements to minimize the need for re- equilibration.
  • Differential Scanning Calorimetry may be used to ascertain changes over time in the thermal stability of an antibody in a particular formulation.
  • Thermal melting temperatures T m
  • Thermal melting temperatures T m
  • VP-DSC differential scanning calorimeter
  • VP-DSC is used at a scan rate of 1.0°C/min and with a temperature range of 25 -120 0 C.
  • a filter period of 8 seconds is used along with a 5 minute pre-scan thermostating.
  • Samples are prepared by dialysis into 10 mM Histidine-HCl, pH 6 using Pierce dialysis cups (3.5 kD). Average Mab concentrations are 50 ⁇ g/mL as determined by A 2 So- Melting temperatures are determined following the manufacturer's instructions using software supplied with the system.
  • LC-MS Liquid Chromatography Mass Spectrometry
  • Peak SEC column fractions containing the degradation fragment are collected and digested with N-Glycosidase F, also known as PNGase F, at 37°C overnight.
  • PNGase F is an amidase used to deglycosylate protein samples.
  • the enzyme cleaves between the innermost GIcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides on N-linked glycoproteins.
  • the deglycosylated samples mixed with a reducing buffer (e.g., 2.5 mg/mL DTT, 6.0 M guanindine HCl, pH 8.2) and kept at 56 0 C in a water bath for 60 minutes.
  • a reducing buffer e.g., 2.5 mg/mL DTT, 6.0 M guanindine HCl, pH 8.2
  • Neat 4-vinylpyridine (e.g., Aldrich Chem. Co., WI) is then added to the sample, and the reaction mixture is held at ambient temperature for 30 minutes.
  • the deglycosylated, reduced and alkylated sample is immediately loaded onto a reversed phase column in order to separate the modified samples from the reactants.
  • Deglycosylated, reduced, and alkylated samples are fractionated using a reversed phase column (e.g., Jupiter 5 ⁇ m C4, 300 A, 250 x 2.00 mm, Phenomenex) with a binary gradient HPLC system (Agilent 1100).
  • Mobile phase A consists of 30% acetonitrile in water with 0.1% trifluoroacetic acid and mobile phase B consists of 50% acetonitrile in water with 0.1% trifluoroacetic acid.
  • the samples are separated using a linear gradient of 30-50% acetonitrile in water, over 16 min. with a flow rate of approximately 200 ⁇ L/min.
  • the column effluent is directed to a UV detector and then split 1 :1, one half going through a switching valve on an Ion Trap mass spectrometer (e.g., LTQ, ThermoElectro, San Jose, CA), and the remaining half to waste.
  • Ion Trap mass spectrometer e.g., LTQ, ThermoElectro, San Jose, CA
  • the ion-trap mass spectrometer is calibrated before the experimental run using a mixture of caffeine, L-methionyl-arginyl-phenylalanyl-alanine acetate H 2 O, and Ultramark 162.
  • the Electrospray Iomsation Mass Spectrometry (ESI-MS) data is acquired in positive ESI full scan mode.
  • the Bio Work deconvolution program (ThermoFmnigan) may be used to reconstruct the mass spectra and obtain the molecular masses of the peptides/proteins from their original mass spectra The mass data subsequently is used to determine the identity of the degradation fragment 7 3.2.
  • Differential Scanning Calorimetry may be used to reconstruct the mass spectra and obtain the molecular masses of the peptides/proteins from their original mass spectra. The mass data subsequently is used to determine the identity of the degradation fragment 7 3.2.
  • Differential Scanning Calorimetry may be used to ascertain changes over time m the thermal stability of an antibody in a particular formulation
  • Thermal melting temperatures T m
  • T m Thermal melting temperatures
  • VP-DSC is used at a scan rate of 1.0°C/min and with a temperature range of 25 -12O 0 C
  • a filter period of 8 seconds is used along with a 5 minute pre-scan thermostating.
  • Samples are prepared by dialysis into 10 mM Histidine-HCl, pH 6 using Pierce dialysis cups (3 5 kD). Average Mab concentrations are 50 ⁇ g/mL as determined by A280. Melting temperatures are determined following the manufacturer's instructions using software supplied with the system. 7 3.3. Isoelectric Focusing Gel Electrophoresis
  • Isoelectric point measurements of 13H5 may be used to ascertain the antibody's chemical stability in a given formulation. Isoelectric points are determined using a Pharmacia Biotech Multiphor 2 electrophoresis system with a multi temp 3 refrigerated bath recirculation unit and an EPS 3501 XL power supply Pre-cast ampholine gels (Amersham Biosciences, pi range 2 5-10) are loaded with 5 ⁇ g of protein Broad range pi marker standards (Amersham, pi range 3-10, 8 ⁇ L) are used to determine relative pi for the Mabs Electrophoresis is performed at 1500 V, 50 mA for 105 minutes The gel is fixed using a Sigma fixing solution (5x) diluted with purified water to Ix.
  • Pre-cast ampholine gels (Amersham Biosciences, pi range 2 5-10) are loaded with 5 ⁇ g of protein Broad range pi marker standards (Amersham, pi range 3-10, 8 ⁇ L) are used to determine relative pi for the Mabs
  • Electrophoresis is performed at 1500 V
  • Disulfude bond determination protocols may be used to monitor the stability of disulfide bridge crosslinks in a particular antibody formulation
  • Antibody samples are denatured, for example, in 10 mM phosphate buffer, 250 mM NaCl, 5 mM NEM, 6 M Guamdme, pH 7.0 at 37 0 C for 1 to 3 hr.
  • the denatured samples are diluted 6 fold with 100 mM phosphate buffer, 0 1 mM EDTA, pH 7 0, to which Endoproteinase Lys-C (e.g., Roche) is added at a 1 :10 enzyme to protein ratio.
  • the reaction mixtures are incubated at 37 0 C for 16 to 24 hours.
  • disulfide bridges are reduced by adding 5-10 ⁇ L of 100 mM DTT followed by incubation at 37 0 C for 1 hr.
  • Lys-C digested samples are fractionated by reverse-phase HPLC (e.g., Phenomenex Jupiter 5m C 18 column; 250 x 2.1 mm). Eluant is analyzed by an UV-detector and an in-line LCQ or LTQ Ion Trap mass spectrometer (e.g., ThermoElectron).
  • the RP-HPLC mobile phase A is 0.1 % TFA in H2 ⁇ and mobile phase B is 0.1 % TFA in acetonitrile.
  • the peptides are eluted at a flow rate of 0.2 mL/min with the following step gradient: 1) 0- 2 min, 5% Mobile Phase B; 2) 2-32 min, 5-20% Mobile Phase B; 3) 32-132 min, 20-40% Mobile Phase B; 4) 132-152 min, 40-60% Mobile Phase B; 5)152-155 min, 60-95% Mobile Phase B.
  • the ion-trap mass spectrometer is calibrated before the experimental run using a mixture of caffeine, L-methionyl-arginyl-phenylalanyl-alanine acetate ' H 2 O, and Ultramark 162.
  • Electrospray Ionisation Mass Spectrometry (ESI-MS) data is acquired in positive ESI full scan mode.
  • the Bio Work deconvolution program (ThermoFinnigan) may be used to reconstruct the mass spectra and obtain the molecular masses of the peptides from their original mass spectra. Comparison of the mass data acquired using the DTT reduced and non-reduced samples allows the identification of the disulfide crosslinked peptides.
  • Binding affinity of 13H5 monoclonal antibody recovered form 13H5 fE formulation may be determined by surface plasmon resonance (see, e.g., Jonsson et al, Biotechniques 11(5):620-627 (1991); Johne, B., Molecular Biotechnology 9(1):65-71(1989)) using a BIAcore 3000 instrument (BIAcore, Inc., Piscataway, NJ). 13H5 antibody is captured on a Prot-G coated CM5 chip. A Prot-G coated CM5 chip with captured isotype control human-IgG (Sigma) antibody is used for reference purposes.
  • Interferon alpha dissolved in HBS-EP running buffer is passed over the chip at a rate of 25 ul/min. 5 minutes of association time is followed by a 10 minute dissociation period.
  • Independent measurements are performed by exposing the chips to different concentrations of interferon alpha (e.g. concentrations between 10 nM and 80 nM). Chips are regenerated by a 0.4 minute wash with 20 mM NaOH + 40OmM NaCl at a flow rate of 1 OOul/min.
  • Polysorbate 80 was introduced by spiking (1 : 100 dilution) the concentrated antibody preparation with a concentrated stock solution of 25 mM histidine-HCl (ph 6.0), 5% sucrose, 2% Polysorbate 80. The final concentration of 13H5 was adjusted to the target concentration with the final formulation buffer (e.g., 25 mM histidine-HCl (pH 6.0), 5% sucrose, 0.02% Polysorbate 80).
  • the final formulation buffer e.g., 25 mM histidine-HCl (pH 6.0), 5% sucrose, 0.02% Polysorbate 80.
  • the stability of the ultra concentrated antibody Z was tested in three different liquid formulations: (1) 10 mM histidine pH 6.0, (2) 10 mM Histidine, 50 mM Sodium Chloride pH 6.0, (3) 10 mM Histidine, 150 mM Sodium Chloride pH 6.0. All three formulations were prepared by concentrating the purified antibody Z to approximately 20 mg/ml, diaf ⁇ ltering (5-6X) the antibody into the buffer of choice, and concentrating the formulation to a final concentration of 130 mg/ml antibody Z. [00447] The stability of ultra concentrated Mab Y, Mab X, and Mab W antibodies in 10 mM histidine was compared.
  • the formulations were prepared by concentrating the purified antibody to approximately 20 mg/ml, diafiltering (5-6X) the antibody into 10 mM histidine pH 6.0, and concentrating the formulation to a final concentration of 150 mg/ml or 200 mg/ml antibody.
  • the stability of the ultra concentrated Mab Q antibody was tested in 5OmM Sodium Acetate, 85 mM Sodium Chloride, 0.01% Polysorbate 80 pH 5.5.
  • the formulation was prepared as follows: the viral filtration product was concentrated by ultrafiltration to approximately 180 mg/ml; the concentrated solution was spiked with Polysorbate 80; and the formulation was diluted to final concentration of 150 mg/ml antibody.

Abstract

La présente invention concerne des formulations liquides à concentration ultra-élevée d’anticorps ou de fragments de ceux-ci, qui se lient spécifiquement à un polypeptide d’interféron alpha humain.
PCT/US2009/038088 2008-03-25 2009-03-24 Formulation d’anticorps WO2009120684A1 (fr)

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Cited By (13)

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WO2011104381A2 (fr) 2010-02-26 2011-09-01 Novo Nordisk A/S Compositions stables contenant des anticorps
US8613919B1 (en) 2012-08-31 2013-12-24 Bayer Healthcare, Llc High concentration antibody and protein formulations
US9592297B2 (en) 2012-08-31 2017-03-14 Bayer Healthcare Llc Antibody and protein formulations
EP2691112B1 (fr) 2011-03-31 2018-05-23 Merck Sharp & Dohme Corp. Formulations stables d'anticorps dirigés contre le récepteur humain pd-1 de la mort programmée et traitements associés
WO2018116198A1 (fr) 2016-12-23 2018-06-28 Serum Institute Of India Private Limited Procédés améliorés pour augmenter la productivité d'anticorps dans une culture de cellules de mammifère et minimiser l'agrégation pendant des processus de formulation en aval, procédés de formulation et formulations d'anticorps stables obtenus à partir de ceux-ci
USRE47150E1 (en) 2010-03-01 2018-12-04 Bayer Healthcare Llc Optimized monoclonal antibodies against tissue factor pathway inhibitor (TFPI)
US10179172B2 (en) 2013-09-11 2019-01-15 Eagle Biologics, Inc. Liquid pharmaceutical formulations for injection comprising yellow 5 or orange G and uses thereof
US10835602B2 (en) 2010-05-28 2020-11-17 Novo Nordisk A/S Stable multi-dose compositions comprising an antibody and a preservative
US11471479B2 (en) 2014-10-01 2022-10-18 Eagle Biologics, Inc. Polysaccharide and nucleic acid formulations containing viscosity-lowering agents
US11634485B2 (en) 2019-02-18 2023-04-25 Eli Lilly And Company Therapeutic antibody formulation
US11633476B2 (en) 2017-05-02 2023-04-25 Merck Sharp & Dohme Llc Stable formulations of programmed death receptor 1 (PD-1) antibodies and methods of use thereof
US11845798B2 (en) 2017-05-02 2023-12-19 Merck Sharp & Dohme Llc Formulations of anti-LAG3 antibodies and co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
US11857625B2 (en) 2017-11-23 2024-01-02 UCB Biopharma SRL Pharmaceutical compositions

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070110757A1 (en) * 2005-06-23 2007-05-17 Ziping Wei Antibody formulations having optimized aggregation and fragmentation profiles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070110757A1 (en) * 2005-06-23 2007-05-17 Ziping Wei Antibody formulations having optimized aggregation and fragmentation profiles

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WO2011104381A2 (fr) 2010-02-26 2011-09-01 Novo Nordisk A/S Compositions stables contenant des anticorps
EP3216462A2 (fr) 2010-02-26 2017-09-13 Novo Nordisk A/S Compositions contenant un anticorps stable
US9795674B2 (en) 2010-02-26 2017-10-24 Novo Nordisk A/S Stable antibody containing compositions
EP3409289A2 (fr) 2010-02-26 2018-12-05 Novo Nordisk A/S Compositions contenant un anticorps stable
EP3708190A1 (fr) 2010-02-26 2020-09-16 Novo Nordisk A/S Compositions contenant un anticorps stable
US10709782B2 (en) 2010-02-26 2020-07-14 Novo Nordisk A/S Stable antibody containing compositions
USRE47150E1 (en) 2010-03-01 2018-12-04 Bayer Healthcare Llc Optimized monoclonal antibodies against tissue factor pathway inhibitor (TFPI)
US10835602B2 (en) 2010-05-28 2020-11-17 Novo Nordisk A/S Stable multi-dose compositions comprising an antibody and a preservative
EP2691112B1 (fr) 2011-03-31 2018-05-23 Merck Sharp & Dohme Corp. Formulations stables d'anticorps dirigés contre le récepteur humain pd-1 de la mort programmée et traitements associés
US8613919B1 (en) 2012-08-31 2013-12-24 Bayer Healthcare, Llc High concentration antibody and protein formulations
US9592297B2 (en) 2012-08-31 2017-03-14 Bayer Healthcare Llc Antibody and protein formulations
US9849181B2 (en) 2012-08-31 2017-12-26 Bayer Healthcare Llc High concentration antibody and protein formulations
US10646571B2 (en) 2013-09-11 2020-05-12 Eagle Biologics, Inc. Liquid protein formulations containing cimetidine
RU2710542C2 (ru) * 2013-09-11 2019-12-27 Игл Байолоджикс, Инк. Жидкие составы белков, содержащие средства для снижения вязкости
US10179172B2 (en) 2013-09-11 2019-01-15 Eagle Biologics, Inc. Liquid pharmaceutical formulations for injection comprising yellow 5 or orange G and uses thereof
US10821184B2 (en) 2013-09-11 2020-11-03 Eagle Biologics, Inc. Liquid protein formulations containing thiamine pyrophosphate (TPP)
US10821183B2 (en) 2013-09-11 2020-11-03 Eagle Biologics, Inc. Liquid protein formulations containing 4-(3-butyl-1-imidazolio)-1-butane sulfonate (BIM)
US10849977B2 (en) 2013-09-11 2020-12-01 Eagle Biologics, Inc. Liquid Protein Formulations Containing Thiamine
US11819550B2 (en) 2013-09-11 2023-11-21 Eagle Biologics, Inc. Liquid protein formulations containing cyclic adenosine monophosphate (cAMP) or adenosine triphosphate (ATP)
US11471479B2 (en) 2014-10-01 2022-10-18 Eagle Biologics, Inc. Polysaccharide and nucleic acid formulations containing viscosity-lowering agents
WO2018116198A1 (fr) 2016-12-23 2018-06-28 Serum Institute Of India Private Limited Procédés améliorés pour augmenter la productivité d'anticorps dans une culture de cellules de mammifère et minimiser l'agrégation pendant des processus de formulation en aval, procédés de formulation et formulations d'anticorps stables obtenus à partir de ceux-ci
US11633476B2 (en) 2017-05-02 2023-04-25 Merck Sharp & Dohme Llc Stable formulations of programmed death receptor 1 (PD-1) antibodies and methods of use thereof
US11845798B2 (en) 2017-05-02 2023-12-19 Merck Sharp & Dohme Llc Formulations of anti-LAG3 antibodies and co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
US11857625B2 (en) 2017-11-23 2024-01-02 UCB Biopharma SRL Pharmaceutical compositions
US11634485B2 (en) 2019-02-18 2023-04-25 Eli Lilly And Company Therapeutic antibody formulation

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