WO2017055966A1 - Compositions d'anticorps à faible viscosité - Google Patents

Compositions d'anticorps à faible viscosité Download PDF

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Publication number
WO2017055966A1
WO2017055966A1 PCT/IB2016/055612 IB2016055612W WO2017055966A1 WO 2017055966 A1 WO2017055966 A1 WO 2017055966A1 IB 2016055612 W IB2016055612 W IB 2016055612W WO 2017055966 A1 WO2017055966 A1 WO 2017055966A1
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amino acid
acid sequence
antibody
seq
sequence shown
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PCT/IB2016/055612
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Robert Henry WALTERS
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Pfizer Inc.
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • 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
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge

Definitions

  • the present invention relates to the field of pharmaceutical formulations of antibodies. Specifically, the present invention relates to a low viscosity liquid antibody formulation and its pharmaceutical preparation and use.
  • Antibody preparations intended for therapeutic or prophylactic use require stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use. These problems are exacerbated at the high concentrations of antibody often desired for therapeutic administration.
  • a major aim in the development of antibody formulations is to maintain antibody solubility, stability and potency of its antigen binding. It is particularly desirable to avoid antibody self-association, aggregates, and particulates in solution which would require sterile filtration before use for intravenous or subcutaneous injection and limit route of administration.
  • Antibody aggregates can cause pain and anaphylactoid side effects when the formulation containing them is intravenously injected.
  • self- associated antibodies and aggregates present increased difficulties for subcutaneous (sc) administration.
  • the ease of injection for sc administration is described as extrusion force, the force required to extrude the composition from the syringe.
  • the viscosity of the liquid composition is directly related to the required extrusion force: more viscous compositions require greater extrusion force.
  • a larger diameter needle or a longer injection time may be required to administer the desired dose.
  • high viscosity compositions present a greater risk for pain at the injection site, and thus may negatively impact patient compliance.
  • Self-associated antibodies exhibit high viscosity and result in difficulty in manufacturing. Tangential flow filtration is often used in manufacturing for buffer exchange and protein concentration. High viscosity compositions create additional back pressure and shear stress during this process, which can increase the processing time and destabilize the antibody.
  • One solution to self -association of antibody therapeutics is to formulate the therapeutic in a viscosity lowering composition. Lyophilization or freeze drying is an alternative to the liquid formulation of antibodies. The process has a propensity for inducing denaturation of the antibody and decreasing its antigen-binding activity particularly upon reconstitution.
  • Viscosity lowering excipients, surfactants, pH, and cryoprotectant/tonicity agents such as sugars can contribute to overcoming self-association problems.
  • Formulation of antibody preparations requires careful selection of these factors among others to avoid denaturation of the protein and loss of antigen-binding activity.
  • formulation constituents, such as sugars may further exacerbate the self-association tendency of the antibody and high concentrations of these stabilizing excipients can lead to high viscosities.
  • Some viscosity lowering excipients have been explored including arginine, histidine, lysine, and camphor-10-sulfonic acid.
  • intramuscular, intraperitoneal, intradermal or subcutaneous injection is further desirable that the formulation has minimized risk of anaphylactoid side effects.
  • compositions comprising a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, a salt of camphorsulfonic acid or a salt of sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0.
  • FIG. 1 A depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
  • FIG. 1 B depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
  • FIG. 2A depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid.
  • FIG. 2B depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid.
  • FIG. 3 depicts a graph comparing the viscosity of anti-IL7R antibody formulation with a combination of camphorsulfonic acid and arginine, and arginine HCI.
  • FIG 4 depicts a graph comparing the viscosity of anti-IL7R antibody formulation at varying concentrations of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • FIG. 5 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) formulations.
  • FIG. 6 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with camphorsulfonic acid,
  • FIG. 7A depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • FIG. 7B depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • the present invention may be understood even more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein. Unless stated otherwise, the concentrations listed herein are those concentrations at ambient conditions, [i.e., at 25°C and atmospheric pressure].
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. , through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies), and fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2, which denote differences in the constant region, particularly in the hinge and upper CH 2 domain.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Following human lgG1 EU numbering and beginning at the 216 position, the isotypes have the following sequences: lgG1 : EPKSCDKTHTCPPCP lgG2: ERKCCVE— CPPCP lgG4: ESKYGPP— CPSCP
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) that contain hypervariable regions.
  • FR framework regions
  • CDRs complementarity determining regions
  • the identity of the amino acid residues in a particular antibody that make up a CDR can be determined using methods well known in the art. For example, antibody CDRs may be identified as the
  • hypervariable regions originally defined by Kabat et al (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C., NIH Publication No. 91 -3242).
  • the positions of the CDRs may also be identified as the structural loop structures described by Chothia and others (Chothia et al., 1989, Nature 342:877-883).
  • CDRs In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding (Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1 166). Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches.
  • CDRs defined according to any of these approaches.
  • the CDRs (or other residue of the antibody) may be defined in accordance with any of Kabat, Chothia, North, extended, AbM, contact, and/or conformational definitions.
  • a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
  • the "hinge region" of an antibody consists of a flexible domain that joins the Fab arms to the Fc region.
  • the antibody of the present invention is selected from the group of monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, human antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • the antibody may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).
  • the antibody can be human but is more preferably humanized.
  • the antibody is isolated, further preferably it is substantially pure. Where the antibody is an antibody fragment this preferably retains the functional characteristics of the original antibody i.e. the ligand binding and/or antagonist or agonist activity.
  • the antibody heavy chain constant region may be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotypes, such as IgGI , lgG2, lgG3, and lgG4.
  • the antibody is an lgG2 antibody.
  • the antibody can comprise the human heavy chain lgG2a constant region.
  • the antibody comprises the human light chain kappa constant region.
  • the antibody comprises a modified constant region, such as a constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
  • the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT publication No.
  • the antibody comprises a human heavy chain lgG2a constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype lgG2a sequence), Eur. J. Immunol. (1999) 29:2613-2624.
  • the antibody does not necessarily comprise an identical amino acid sequence of the amino acid sequence described herein.
  • An antibody that has a similar amino acid sequence refers to an antibody analog that satisfies at least one of the following: (a) an amino acid sequence that is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the amino acid sequence of any of the antibodies or portions of antibodies described herein; (b) an antibody encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the antibody of at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • One, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et ah, 1990, J. Mol. Biol. 215:403.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the formulations of the present invention include at least one antibody. In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five, or more, different antibodies may be present. Generally, the two or more different antibodies have complementary activities that do not adversely affect each other. The, or each, antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the antibodies.
  • the concentration of antibody can range from about 50 mg/ml to about 450 mg/ml. In other embodiments, the concentration of antibody is about 50 mg/ml, about 70 mg/ml, about 90 mg/ml, about 100 mg/ml, about 1 10 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 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml, about 300 mg/ml, about 310 mg/ml, about 320 mg/ml, about 330 mg/ml, about 340 mg/ml, about 350 mg/m/m
  • the concentration of the antibody in the formulation is between about 50 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 350 mg/ml, between about 100 mg/ml and about 300 mg/ml, between about 100 mg/ml and about 250 mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 120 mg/ml and 200 mg/ml, between about 150 mg/ml and about 200 mg/ml, or between about 165 mg/ml and about 215 mg/ml.
  • Isolated when used to describe the various antibodies disclosed herein, means a polypeptide or antibody that has been identified, separated and/or recovered from a component of its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody in the antibody formulation is purified prior to being added to the antibody formulation.
  • At least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about or 99.9% (w/w) of an impurity is purified from the antibody.
  • CSA camphor-10-sulphonic acid or the compound of formula CSA.
  • CSA is represented by the chemical formula:
  • CSA includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form.
  • CSA includes all enantiomers (e.g. ,
  • (+)camphorsulphonic acid and (-)camphorsulphonic acid any combination of enantiomers (e.g., 50% (+)camphorsulphonic acid and 50% (-)camphorsulphonic acid; 90%-100% (+)camphorsulphonic acid and 10%-0% (-)camphorsulphonic acid, etc.).
  • the term "CSA” includes greater than 99% (+)camphorsulphonic acid and less than 1 % (-)camphorsulphonic acid.
  • the term "CSA” includes an enantomerically pure (+)camphorsulphonic acid.
  • Exemplary pharmaceutically acceptable acid salts may be formed from counterions and include sodium, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof.
  • the present invention includes various amounts of CSA.
  • the amount of CSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM.
  • the amount of CSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM.
  • the concentration of CSA and the counterion can be the same or different.
  • a salt of CSA formed with arginine (CSA-Arg) with a concentration of 50mM has a CSA concentration of 50mM and an arginine concentration of 50mM.
  • more or less arginine (counterion) could be present, whereby not all of the CSA or counterion is in the salt form.
  • Arginine (Arg) is an amino acid that can be represented by the chemical formula:
  • Arginine includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation Arginine
  • Arg-HCI hydrochloride
  • Arginine includes all enantiomers (e.g., L-arginine and D- arginine), and any combination of enantiomers (e.g., 50% L-arginine and 50% D- arginine; 90%-100% L-arginine and 10%-0% D-arginine, etc.).
  • the term "arginine” includes greater than 99% L-arginine and less than 1 % D-arginine.
  • the term "arginine” includes an enantomerically pure L-arginine.
  • the arginine is a pharmaceutical grade arginine.
  • Histidine is an amino acid that can be represented by the chemical formula:
  • Histidine includes all hydrate/so I vate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation histidine hydrochloride.
  • Histidine includes all enantiomers (e.g. , L-histidine and D-histidine), and any combination of enantiomers (e.g., 50% L- histidine and 50% D- histidine; 90%- 100%) L- histidine and 10%-0% D- histidine, etc.).
  • the term “histidine” includes greater than 99%) L- histidine and less than 1 % D- histidine.
  • the term "histidine” includes an enantomerically pure L- histidine. In some embodiments, the histidine is a pharmaceutical grade histidine. "Lysine” is an amino acid that can be represented by the chemical formula:
  • “Lysine” includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation lysine hydrochloride
  • “Lysine” includes all enantiomers (e.g., L-lysine and D-lysine), and any combination of enantiomers (e.g., 50% L- lysine and 50% D- lysine; 90%-100% L- lysine and 10%-0% D- lysine, etc.).
  • the term “lysine” includes greater than 99% L- lysine and less than 1 % D- lysine.
  • the term “lysine” includes an enantomerically pure L- lysine.
  • the lysine is a pharmaceutical grade lysine.
  • chelating agent is meant an optional composition component that is a
  • Such chelating agents can reduce or prevent degradation of an antibody that is formulated in comparision to the antibody without the protection of a chelating agent.
  • Exemplary chelating agents include aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2- (2- amino-2-oxocthyl) aminoethane sulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and desoxycholates and mixtures thereof.
  • the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2- acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycolether, ⁇ , ⁇ , ⁇ ', ⁇ '-tetraacetic acid (EGTA), trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, and aspartic acid, N- hydroxyethyliminodiacetic acid (HIMDA), N, N-bis-hydroxyethylglycine (bicine) and N- (trishydroxymethylmethyl) 10 glycine (tricine), glycylglycine, sodium desoxycholate, ethylenediamine; propylenediamine; diethylenetriamine;
  • EDTA ethylenediaminetetraacetic
  • the chelating agent is selected from the group consisting of salts of EDTA including dipotassium EDTA, disodium EDTA, EDTA calcium disodium, sodium EDTA, trisodium EDTA, and potassium EDTA; and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or mixtures thereof.
  • DEF deferoxamine
  • DMF deferoxamine mesylate
  • Chelating agents used in the invention can be present, where possible, as the free acid or free base form or salt form of the compound, also as an anhydrous, solvated or hydrated form of the compound or corresponding salt.
  • the concentration of chelating agent when present, generally ranges from about 0.01 mg/ml to about 50 mg/ml, from about 0.01 mg/ml to about 10.0 mg/ml, from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, or from about 0.01 mg/ml to about 0.3 mg/ml.
  • the concentration of chelating agent generally ranges from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.09 mM, from about 0.01 mM to about 0.08 mM, from about 0.01 mM to about 0.07 mM, from about 0.01 mM to about 0.06 mM, from about 0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from about 0.01 mM to about 0.02 mM or from about 0.005 mM to about 0.01 mM.
  • the concentration of chelating agent can be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07 mg/ml, about 0.10 mg/ml, about 0.20 mg/ml.
  • cryoprotectant is an optional composition component that is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage.
  • cryoprotectants include sugars and their corresponding sugar alcohols; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g.
  • cryoprotectants include glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose.
  • reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose.
  • non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols including sucrose, dextrose, mannose and trehalose (including all forms of trehalose such as trehalose monohydrate and trehalsoe dihydrate).
  • Example sugar alcohols are monoglycosides, especially those compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose.
  • the glycosidic side group can be either glucosidic or galactosidic. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and iso-maltulose.
  • the concentration of the cryoprotectant, when present in the liquid composition ranges from about 0.1 mg/ml to about 150 mg/ml, from about 0.1 mg/ml to about 100 mg/ml, or from about 1 mg/ml to about 100 mg/ml.
  • the concentration of the cryoprotectant in the liquid composition is about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 1 10 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, or about 150 mg/ml.
  • the concentration of the salt in the liquid composition ranges from about 1 mg/ml to about 20 mg/ml. Salts that are
  • pharmaceutically acceptable and suitable for this invention include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride.
  • Exemplary salts include sodium chloride and magnesium chloride, magnesium chloride may also improve the antibody stability by protecting the protein from deamidation.
  • the salt in the liquid composition is selected from a range of concentrations of any of about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8, mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml and 20 mg/ml.
  • injection force is the amount of pressure (in Newtons) required to pass the antibody formulation through a needle.
  • the injection force is correlated with the amount of resistance provided by the antibody formulation when administering the antibody formulation to a subject.
  • the injection force will be dependent on the gauge of the administering needle, as well as temperature.
  • the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 27 Ga thin wall PFS needle such as defined in the International Organization for Standardization (ISO) document "Stainless steel needle tubing for the manufacture of medical devices" (ISO 9626:1991 ) and manufactured by BD Medical, Pharmaceutical Systems (Franklin Lakes, N.J.).
  • the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 25 or 26 Gauge needle.
  • an “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to about 350 mOsm.
  • the term “hypotonic” describes a formulation with an osmotic pressure below that of human blood.
  • the term “hypertonic” is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • the compositions of the present invention are isotonic.
  • IV bag protectant refers to the surfactant added to the intravenous bag prior to dilution of the antibody formulation described herein into the intravenous bag.
  • the IV bag protectant can also be added to the intravenous bag prior to addition of other antibody formulations known to those of skill in the art, e.g. , a lyophilized antibody formulation.
  • Surfactants suitable for use as an IV bag protectant will generally be those suitable for use in IV formulations.
  • the surfactant used in the IV bag protectant is the same buffer used in the antibody formulation. For example, if the antibody formulation comprises polysorbate 80 as a surfactant, then polysorbate 80 would be added to the intravenous bag prior to adding the antibody formulation to the intravenous bag.
  • the surfactant concentration in the IV bag resulting from addition of the IV protectant will be about the same or only a portion of the surfactant concentration in the antibody formulation. Knowing the desired final concentration of surfactant in the IV bag, one can formulate the desired concentration of the surfactant in the IV bag protectant.
  • K off is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • Kd is intended to refer to the dissociation constant of an antibody-antigen interaction.
  • One way of determ ining the K d or binding affinity of antibodies to the antigen is by measuring binding affinity of monofunctional Fab fragments of the antibody.
  • an antibody for example, IgG
  • IgG antibody
  • the affinity of a Fab fragment of an antibody can be determ ined by surface plasmon resonance (BIAcorC1 GM000TM surface plasmon resonance (SPR) system , BIAcore, INC, Piscaway NJ).
  • CM5 chips can be activated with N-ethyl-N'-(3-dimethylam inopropyl)-carbodiinide hydrochloride (EDC) and N-hydroxysuccinim ide (NHS) according to the supplier's instructions.
  • EDC N-ethyl-N'-(3-dimethylam inopropyl)-carbodiinide hydrochloride
  • NHS N-hydroxysuccinim ide
  • Human antigen can be diluted into 1 0 mM sodium acetate pH 4.0 and injected over the activated chip at a concentration of 0.005 mg/mL. Using variable flow time across the individual chip channels, two ranges of antigen density can be achieved: 1 00-200 response units (RU) for detailed kinetic studies and 500-600 RU for screening assays. Serial dilutions (0.1 -1 Ox estimated K d ) of purified Fab samples are injected for 1 m in at 100 m icroliters/min and dissociation times of up to 2 hours are allowed.
  • concentrations of the Fab proteins are determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of known concentration (as determ ined by amino acid analysis) as a standard.
  • Kinetic association rates (k on ) and dissociation rates (k 0 ff) are obtained simultaneously by fitting the data to a 1 : 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-1 10) using the BIAevaluation program.
  • Equilibrium dissociation constant (Kd) values are calculated as k 0 ff/k on . This protocol is suitable for use in determ ining binding affinity of an antibody to any antibody.
  • the antibody formulations can have different "osmolarity" concentrations.
  • Methods of measuring osmolarity of antibody formulations are known to those in the art, and can include, e.g. , an osmometer (e.g., an Advanced Instrument Inc 2020 freezing point depression osmometer).
  • the formulation has an osmolarity of between 200 and 600 mosm/kg, between 260 and 500 mosm/kg, or between 300 and 450 mosm/kg.
  • the formulation does not comprise an osmolyte.
  • phrases "pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • a “pharmaceutically acceptable acid” includes inorganic and organic acids which are non toxic at the concentration and manner in which they are formulated.
  • suitable inorganic acids include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric, sulfonic, sulfinic, sulfanilic, phosphoric, carbonic, etc.
  • Suitable organic acids include straight and branched-chain alkyl, aromatic, cyclic, cyloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono, di- and tri-carboxylic, including for example, form ic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, t-butyl acetic, anthranilic, propanoic, 2-hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic, cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic, benzoic, 3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic, cinnam ic, lauryl sulfuric, stearic, muconic, mandelic, succinic, embonic, fumaric, malic, maleic, hydroxymale
  • “Pharmaceutically-acceptable bases” include inorganic and organic bases which are non-toxic at the concentration and manner in which they are formulated.
  • suitable bases include those formed from inorganic base forming metals such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine and organic nontoxic bases including, primary, secondary and tertiary amine, substituted amines, cyclic amines and basic ion exchange resins, [e.g., N(R').sub.4.sup.+ (where R 1 is independently H or C. sub.1 -4 alkyl, e.g., ammonium, Tris)], for example,
  • ethanolamine 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like.
  • Additional pharmaceutically acceptable acids and bases useable with the present invention include those which are derived from the amino acids, for example, histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, arginine and asparagine.
  • “Pharmaceutically acceptable” buffers and salts which are an optional composition component, include those derived from both acid and base addition salts of the above indicated acids and bases. According to the present invention, the buffer is used to adjust the pH and it may also enhance the antibody stability.
  • the buffer is selected from the group consisting of acetate, succinate, gluconate, citrate, histidine, arginine, acetic acid, phosphate, phosphoric acid, ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate (EDTA), acetate, malate, imidazole, tris, phosphate, and mixtures thereof.
  • the concentration of the buffer can range from about 0.1 millimolar (mM) to about 200 mM. In one embodiment, the concentration of the buffer is from about 0.5 mM to about 200 mM, from about 1 mM to about 100 mM, from about 1 mM to about 65 mM, or from about 1 mM to about 30 mM.
  • the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.
  • the pH can be in the range of about 4.0 to about 9.0. In another embodiment, the pH is between about 4.5 and about 8.0, between about 5.0 and about 7.5, between about 5.5 and about 7.0, or between about 6.0 and 7.5.
  • the pH is about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, 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 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
  • viscosity lowering excipient comprising CSA, SSA, CSA salts or SSA salts
  • CSA viscosity lowering excipient
  • SSA CSA salts
  • SSA salts CSA salts
  • a "preservative” is a compound which can be added to the formulations herein to reduce bacterial activity.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential
  • preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.
  • Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
  • the composition does not include a preservative.
  • a “stable” formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991 ) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).
  • Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured.
  • a “stable" formulation may be one wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 2% of the antibody is degraded, denatured, aggregated or unfolded as determined by SEC HPLC when the antibody is stored at 2- 8°C for 6 months, or 12 months, or 18 months, or 24 months.
  • the antibody stability is determined by size exclusion
  • SEC chromatography
  • analytes e.g., macromolecules such as proteins and antibodies
  • SEC can separate antibodies in their natural three-dimensional conformation from antibodies in various states of denaturation, and/or antibodies that have been degraded.
  • the stationary phase is generally composed of inert particles packed into a dense three- dimensional matrix within a glass or steel column.
  • the mobile phase can be pure water, an aqueous buffer, an organic solvent, mixtures of these, or other solvents.
  • the stationary-phase particles have small pores and/or channels which will only allow species below a certain size to enter.
  • SEC is combined with an identification technique to identify or characterize proteins, or fragments thereof.
  • Protein identification and characterization can be accomplished by various techniques, including but not limited chromatographic techniques, e.g., high-performance liquid chromatography (HPLC), immunoassays, electrophoresis, ultra-violet/visible/infrared spectroscopy, raman spectroscopy, surface enhanced raman spectroscopy, mass spectroscopy, gas chromatography, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS protein binding.
  • HPLC high-performance liquid chromatography
  • immunoassays electrophoresis
  • ultra-violet/visible/infrared spectroscopy raman spectroscopy
  • surface enhanced raman spectroscopy mass spectroscopy
  • gas chromatography gas chromatography
  • SLS static light scattering
  • FTIR Fourier Transform In
  • HPLC high-pressure liquid chromatography
  • Various instruments and apparatuses are known to those of skill in the art to perform HPLC.
  • HPLC involves loading a liquid solvent containing the protein of interest onto a separation column, in which the separation occurs.
  • the HPLC separation column is filled with solid particles (e.g. silica, polymers, or sorbents), and the sample mixture is separated into compounds as it interacts with the column particles.
  • HPLC separation is influenced by the liquid solvent's condition (e.g. pressure, temperature), chemical interactions between the sample mixture and the liquid solvent (e.g. hydrophobicity, protonation, etc.), and chemical interactions between the sample mixture and the solid particles packed inside of the separation column (e.g. ligand affinity, ion exchange, etc.).
  • the SEC and protein identification occurs within the same apparatus, or simultaneously.
  • SEC and HPLC can be combined, often referred to as SE-HPLC.
  • the stability of the antibody in the antibody formulation can be determined.
  • the term “stability” generally is related to maintaining the integrity or to minimizing the degradation, denaturation, aggregation or unfolding of a biologically active agent such as a protein, peptide or another bioactive macromolecule.
  • improved stability generally means that, under conditions known to result in degradation, denaturation, aggregation or unfolding, the protein (e.g., antibody), peptide or another bioactive macromolecule of interest maintains greater stability compared to a control protein, peptide or another bioactive macromolecule.
  • stability refers to an antibody formulation having low to undetectable levels of aggregation.
  • low to undetectable levels of aggregation refers to samples containing 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% aggregation by weight of protein as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and 1- anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques.
  • 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
  • the antibody formulation has low to undetectable levels of fragmentation.
  • low to undetectable levels of fragmentation refers to samples containing equal to or more than 80%, 85%, 90%, 95%, 98% or 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 5%, more than 4%, more than 3%, more than 2%, more than 1 %, or more than 0.5% of the total protein in each.
  • rCGE reduced Capillary Gel Electrophoresis
  • reduced Capillary Gel Electrophoresis refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody.
  • stability of a protein is dependent on other features in addition to the composition of the formulation. For example, stability can be affected by temperature, pressure, humidity, and external forms of radiation. Thus, unless otherwise specified, stability referred to herein is considered to be measured at 2-8. degree. C, one atmosphere pressure, 60% relative humidity, and normal background levels of radiation.
  • various components can be omitted from the antibody formulation, or can be “substantially free” of that component.
  • the term “substantially free” as used herein refers to an antibody formulation, said formulation containing less than 0.01 %, less than 0.001 %, less than 0.0005%, less than 0.0003%, or less than 0.0001 % of the designated component.
  • SSA Sulfosalicyclic acid
  • SSA includes hydrate/solvate forms including the anhydrous form as well as the free base form and any salt form.
  • exemplary pharmaceutically acceptable acid salts may be formed from counter-ions and include sodium, hydrochloride, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof.
  • the present invention includes various amounts of SSA.
  • the amount of SSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM.
  • the amount of SSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM.
  • “Surfactant” is an optional component to the composition which is a pharmaceutically acceptable agent that reduces the tendency for the formation of bubbles in the formulation during preparation and handling of the formulation and preparation for parenteral administration and especially from stress related to shaking and agitation during preparation and also during shipping.
  • exemplary surfactants include
  • the surfactant is selected from the group consisting of polysorbate 20, polysorbate 21 , polysorbate 40, polysorbate 60, polysorbate 61 , polysorbate 65, polysorbate 80, polysorbate 81 , polysorbate 85, and mixtures thereof.
  • the concentration of the surfactant generally ranges from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 2.0 mg/ml, from about 0.01 mg/ml to about 1.5 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, from about 0.01 mg/ml to about 0.5 mg/ml, from about 0.01 mg/ml to about 0.4 mg/ml, from about 0.01 mg/ml to about 0.3 mg/ml, from about 0.01 mg/ml to about 0.2 mg/ml, from about 0.01 mg/ml to about 0.15 mg/ml, from about 0.01 mg/ml to about 0.1 mg/ml, or from about 0.01 mg/ml, to about 0.05 mg/ml.
  • the concentration of the surfactant is about 0.5 mg/ml, about 0.05 mg/ml about 0.06 mg/ml about 0.07 mg/ml about 0.08 mg/ml about 0.09 mg/ml about 0.1 mg/ml about 0.11 mg/ml about 0.12 mg/ml about 0.13 mg/ml about 0.14 mg/ml about 0.15 mg/ml about 0.16 mg/ml about 0.17 mg/ml about 0.18 mg/ml about 0.19 mg/ml, or about 0.2 mg/ml.
  • terapéuticaally effective amount means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • treating embrace both preventative, i.e., prophylactic, and palliative treatment.
  • animal refers to humans (male or female), companion animals (e.g., dogs, cats and horses), food-source animals, zoo animals, marine animals, birds and other similar animal species.
  • Edible animals refers to food-source animals such as cows, pigs, sheep and poultry.
  • Viscosity as used herein may be “kinematic viscosity” or “absolute viscosity.”
  • Kininematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less viscous fluid to flow through the capillary.
  • Absolute viscosity is the product of kinematic viscosity and fluid density: Absolute
  • Viscosity Kinematic Viscosity x Density
  • the dimension of kinematic viscosity is L 217 where L is a length and T is a time.
  • kinematic viscosity is expressed in centistokes (cSt).
  • the SI unit of kinematic viscosity is mm 2/s , which is 1 cSt.
  • Absolute viscosity is expressed in units of centipoise (cP).
  • the formulation may be in either aqueous or lyophilized form.
  • the formulation may have a viscosity of no greater than about 60 cP.
  • the formulation has a viscosity of no greater than about 50 cP, or no greater than about 40 cP, or no greater than about 30 cP, or no greater than about 20 cP, or no greater than about 40 cP, or no greater than about 15 cP.
  • the composition comprising antibody has a viscosity of between about 1 cP and about 50 cP, between about 1 cP and 40 cP, between about 1 cP and about 30 cP, between about 1 cP and about 20 cP, between about 1 cP and about 15 cP, or between about 1 cP and about 10 cP at 25°C.
  • the formulation has a viscosity of about 50 cP, about 45 cP, about 40 cP, about 35 cP, about 30 cP, about 25 cP, about 20 cP, about 15 cP, or about 10 cP, or about 5 cP.
  • the formulation has a viscosity of between about 10 cP and 50 cP, between about 10 cP and 30 cP, between about 10 cP and 20 cP, or between about 5 cP and 15 cP.
  • the present invention is directed at pharmaceutical compositions that include: a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, or a salt of camphorsulfonic acid or sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0.
  • the composition also includes a pharmaceutically acceptable buffer.
  • the pharmaceutically acceptable buffer comprises arginine, histidine, tris, phosphate or lysine, or a salt thereof.
  • the pharmaceutically acceptable buffer comprises histidine, tris, or phosphate, or a salt thereof.
  • the concentration of pharmaceutically acceptable buffer is from about 1.0 to about 200 mM.
  • the composition also includes a surfactant.
  • a surfactant in one embodiment
  • the surfactant is polysorbate 20 or polysorbate 80. In another embodiment, the surfactant is polysorbate 20 or polysorbate 80. In another
  • the concentration of surfactant is from about 0.01 to about 0.3 mg/ml.
  • the composition also includes a chelating agent.
  • the chelating agent is EDTA or disodium EDTA.
  • the concentration of chelating agent is from about 0.01 to about 0.3 mg/ml.
  • the composition also includes a cryoprotectant.
  • the cryoprotectant is sucrose, dextrose, mannose or trehalose.
  • the concentration of the cryoprotectant is from about 1 mg/ml to about 100 mg/ml.
  • the viscosity lowering agent is (+)camphorsulfonic acid or (-)camphorsulfonic acid or a salt thereof. In another embodiment, the viscosity lowering agent concentration is between about 50 mM to about 150 mM. In another
  • the viscosity lowering agent concentration is between about 70 mM to about 1 10 mM.
  • the viscosity lowering agent is a salt of camphorsulfonic acid comprising camphorsulfonic acid and arginine. In another embodiment, the
  • camphorsulfonic acid concentration is between about 50 mM and about 150 mM and the arginine concentration is between about 50 mM and about 150 mM. In another embodiment, the camphor sulfonic acid concentration is between about 70 mM and about 1 10 mM and the arginine concentration is between about 70 mM and about 110 mM.
  • the antibody is a human or humanized monoclonal lgG1 , lgG2 or lgG4 antibody. In another embodiment, the antibody is an anti-IL7R, anti-PCSK9 or anti-glucagon receptor antibody.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 5, or 6; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or 8; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 1 1 , and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 3.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 13, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 14.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, 18 or 19; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21 ; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 15, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 16.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 26 or 27, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 28.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32, 33, or 34; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35 or 36; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 40.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 41 , and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 42.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 30, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 31.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the composition is lyophilized. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is between about 250 mg/ml and about 400 mg/ml. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is higher than the antibody concentration before lyophilization. In yet another embodiment, the composition has a viscosity of less than about 50 cP at 25°C. In yet another embodiment, the composition is isotonic.
  • IL-7R refers to any form of IL-7R and variants thereof that retain at least part of the activity of IL-7R. Unless indicated differently, such as by specific reference to human IL-7R, IL-7R includes all mammalian species of native sequence IL-7R, e.g., human, canine, feline, equine, and bovine. One exemplary human IL-7R is found as Uniprot Accession Number P16871 (SEQ ID NO: 1 ).
  • Antagonist IL-7R antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) IL-7R biological activity, including downstream pathways mediated by IL-7R signaling, such interaction with IL-7 and/or elicitation of a cellular response to IL-7.
  • IL-7R antibody (interchangeably termed “IL-7R antagonist antibody,” “antagonist anti-IL-7R antibody” or “anti-IL-7R antagonist antibody”) encompasses all the previously identified terms, titles, and functional states and characteristics whereby the IL-7R itself, an IL-7R biological activity (including but not limited to interaction with IL-7, its ability to mediate any aspect of phosphorylation of STAT5, phosphatidylinositol-3-kinase (PI3K)-Akt pathway activation, p27Kip1 downregulation, Bcl-2 upregulation, Rb hyperphosphorylation, and CXCR4
  • an antagonist IL-7R antibody binds IL-7R and prevents interaction with IL-7.
  • antagonist IL-7R antibodies are provided herein.
  • Anti-IL-7R antagonist antibodies for use in the invention can be identified or characterized using methods known in the art, whereby reduction, amelioration, or neutralization of an IL-7R biological activity is detected and/or measured.
  • C1 GM is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • C1 GM The generation and characterization of C1 GM is described in the Examples of
  • C1 GM refers to
  • the antibody is an anti-IL-7R antibody that binds IL-7Ra (such as human IL-7Ra) with a high affinity.
  • high affinity is (a) binding IL- 7R with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies (a) bind IL-7R (such as human IL-7R) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k 0 ff of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same IL-7R epitope as antibody C1 GM.
  • the antibody can be anti-IL-7R antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4
  • GFTFDDSVMH extended or in SEQ ID NO: 5 (DSVMH) (Kabat) or in SEQ ID NO: 6 (GFTFDDS) (Chothia);
  • the antibody can be an anti-IL-7R antibody comprising a light chain variable region comprising:
  • the antibody can be anti-IL-7R antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.
  • the antibody can be anti-IL-7R antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 2 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 3, wherein the antibody binds specifically to human IL-7Ra.
  • the anti-IL-7R antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 2 and 3.
  • the anti-IL-7R antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%), 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14, wherein the antibody binds specifically to human IL-7Ra.
  • the anti-IL-7R antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 13 and 14.
  • the anti-IL-7R antibody may compete for IL-7R binding with an anti-IL-7R antibody as defined herein.
  • the anti-IL-7R antibody may compete for IL-7R binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may be a human and affinity matured antibody, C1 GM, which specifically binds human IL-7Ra.
  • Antibody C1 GM is described in WO2011/104687, the content of which is hereby incorporated by reference in its entirety.
  • the amino acid sequences of the heavy chain and light chain variable regions of C1 GM are shown in SEQ ID NOs: 2 and 3, respectively.
  • the CDR portions of antibody C1 GM (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 of
  • Antibody C1 GM is highly potent in blocking IL-7R biological activity.
  • the anti-IL-7R antibody may also comprise a fragment or a region of the antibody C1 GM.
  • the fragment is a light chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 14 herein.
  • the fragment is a heavy chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 13 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody C1 GM.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody C1 GM comprising the amino acid sequences as shown in SEQ ID NOS: 14 and 13, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody C1 GM shown in SEQ ID NOs: 4-12.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-IL-7R antibodies described herein is deleted.
  • the heavy and/or light chain of the anti-IL-7R antibodies described herein may optionally include a signal sequence.
  • the antibody may be selected from an anti-IL-7R antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO201 1/104687 (including, for example without limitation, any of the antibodies listed in Table 1 ), WO/201 1/094259 (including, for example without limitation, antibodies H3L4, BPC4401 , BPC4398, BPC1 142, BPC4399, BPC4402, BPC4403, and BPC1 142), WO/2013/056984 (including, for example without limitation, antibodies MD707-1 , MD707-2, MD707-3, MD707-4, MD707-5, MD707-6, MD707-9, MD707-12, and MD707-13), and WO2010/017468 (including, for example without limitation, antibodies 9B7, R34.34, 6A3 and 1A11 ).
  • the antibody may bind to the same epitope as an anti-IL-7R antibody known in the art and/or may compete for binding to
  • Glucagon Receptor refers to any form of glucagon receptor and variants thereof that retain at least part of the activity of the glucagon receptor. Unless indicated differently, such as by specific reference to human glucagon receptor, glucagon receptor includes all mammalian species of native sequence glucagon receptor, e.g., human, canine, feline, equine, and bovine. One exemplary human glucagon receptor is found as Uniprot Accession Number P47871 (SEQ ID NO: 29).
  • an "anti-glucagon receptor antagonist antibody” refers to an antibody that is able to inhibit glucagon receptor biological activity and/or downstream events(s) mediated by glucagon receptor.
  • Anti-glucagon receptor antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) glucagon receptor biological activity, including downstream events mediated by glucagon receptor, such glucagon binding and downstream signaling, adenylate cyclase activation, increased levels of intracellular cAMP, glycogenolysis stimulation, gluconeogenesis activation, glycogenesis inhibition, glycolysis inhibition, and hepatic glucose production.
  • anti-glucagon receptor antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby the glucagon receptor itself, a glucagon receptor biological activity (including but not limited to its ability to bind glucagon, increase intracellular cAMP, stimulate glycogenolysis, activate gluconeogenesis, and promote relase of hepatic glucose), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti-glucagon receptor antagonist antibody binds glucagon receptor and lowers plasma glucose levels. Examples of anti-glucagon receptor antagonist antibodies are provided herein.
  • mAb5 is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • mAb5 heavy chain variable region is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • immunoglobulin encoded by (a) a polynucleotide encoding mAb5 light chain variable region that has a deposit number of ATCC No. PTA-120164 and (b) a polynucleotide encoding mAb5 heavy chain variable region that has a deposit number of ATCC No. PTA-120165.
  • the antibody is an anti- glucagon receptor antibody that binds glucagon receptor (such as human glucagon receptor) with a high affinity.
  • high affinity is (a) binding IL-7R with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies bind glucagon receptor (such as human glucagon receptor) with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k 0 ff Of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same glucagon receptor epitope as antibody mAb5.
  • the antibody can be anti- glucagon receptor antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17
  • GYTFTDFSVH extended or in SEQ ID NO: 18 (GYTFTDF) (Chothia) or in SEQ ID NO: 19 (DFSVH) (Kabat);
  • the antibody can be an anti-glucagon receptor antibody comprising a light chain variable region comprising:
  • the antibody can be anti-glucagon receptor antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the antibody can be anti-glucagon receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO.
  • a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 16, wherein the antibody binds specifically to human glucagon receptor.
  • the anti-glucagon receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 15 and 16.
  • the anti-glucagon receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28, wherein the antibody binds specifically to human glucagon receptor.
  • the amino acid sequence of mAb5 full-length heavy chain (SEQ ID NO: 26) is shown below:
  • amino acid sequence of mAb5 full-length heavy chain without the C-terminal lysine is shown below:
  • amino acid sequence of mAb5 full-length light chain (SEQ ID NO: 28) is shown below:
  • the anti-glucagon receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28.
  • the anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 26 and 28.
  • the anti-glucagon receptor antibody may compete for glucagon receptor binding with an anti-glucagon receptor antibody as defined herein.
  • the anti-glucagon receptor antibody may compete for glucagon receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may be a human and affinity matured antibody, mAb5, which specifically binds human glucagon receptor.
  • Antibody mAb5 is described in WO2014/181229, the content of which is hereby incorporated by reference in its entirety for all purposes.
  • the amino acid sequences of the heavy chain and light chain variable regions of mAb5 are shown in SEQ ID NOs: 15 and 16, respectively.
  • the CDR portions of antibody mAb5 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 A of WO2014/181229.
  • Antibody mAb5 is highly potent in blocking glucagon receptor biological activity.
  • the anti-glucagon receptor antibody may also comprise a fragment or a region of the antibody mAb5.
  • the fragment is a light chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 28 herein.
  • the fragment is a heavy chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 26 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody mAb5.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody mAb5 comprising the amino acid sequences as shown in SEQ ID NOS: 28 and 26, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody mAb5 shown in SEQ ID NOs: 17-25.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-glucagon receptor antibodies described herein is deleted, as in SEQ ID NO. 27.
  • the heavy and/or light chain of the anti-glucagon receptor antibodies described herein may optionally include a signal sequence.
  • the antibody may be selected from an anti-glucagon receptor antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO2014/181229 (including, for example without limitation, any of the antibodies listed in Tables 1 A and 1 B including, for example without limitation antibodies mAb1 , mAb2, mAb3, mAb4, mAb5, mAb6, H2-A8, H2-A1 1 , H2-C8, H2-E7, H2-F10, H2-F11 , H3-C5, H3-C10, H3-F5, H3- H9, H2-A1 1 -H3-1 , H2-A1 1 -H3-2, H2-A1 1 -H3-3, H2-A1 1 -H3-4, H2-C8-H3-1 , H2-C8-H3- 2, H2-C8-H3-3, H2-C8-H3-4, H2-E7-H3-1 , H2-E7-H3-2,
  • the antibody may bind to the same epitope as an anti-glucagon receptor antibody known in the art and/or may compete for binding to glucagon receptor with such an antibody.
  • PCSK9 refers to any form of PCSK9 and variants thereof that retain at least part of the activity of PCSK9. Unless indicated differently, such as by specific reference to human PCSK9, PCSK9 includes all mammalian species of native sequence PCSK9, e.g., human, canine, feline, equine, and bovine. One exemplary human PCSK9 is found as Uniprot Accession Number Q8NBP7. (SEQ ID NO: 43)
  • PCSK9 antagonist refers to an antibody, peptide, or aptamer that is able to inhibit PCSK9 biological activity and/or downstream pathway(s) mediated by PCSK9 signaling, including PCSK9-mediated down-regulation of the LDLR, and
  • PCSK9-mediated decrease in LDL blood clearance A PCSK9 antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and/or elicitation of a cellular response to PCSK9.
  • PCSK9 signaling such as LDLR interaction and/or elicitation of a cellular response to PCSK9.
  • PCSK9 antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby the PCSK9 itself, a PCSK9 biological activity (including but not limited to its ability to mediate any aspect of interaction with the LDLR, down regulation of LDLR, and decreased blood
  • a PCSK9 antagonist antibody binds PCSK9 and prevents interaction with the LDLR.
  • Examples of PCSK9 antagonist antibodies are provided herein.
  • the term "L1 L3" is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively.
  • L1 L3 DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 42)
  • SEQ ID NO: 42 The generation and characterization of L1 L3 is described in the Examples of
  • L1 L3 refers to
  • immunoglobulin encoded by (a) a polynucleotide encoding L1 L3 light chain variable region that has a deposit number of ATCC No. PTA-10303 and (b) a polynucleotide encoding L1 L3 heavy chain variable region that has a deposit number of ATCC No. PTA-10302.
  • the antibody is an anti-PCSK9 antibody that binds PCSK9 receptor (such as human PCSK9 receptor) with a high affinity.
  • high affinity is (a) bindingPCSK9 receptor with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies bind PCSK9 receptor (such as human PCSK9 receptor) with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k off of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same PCSK9 receptor epitope as antibody L1 L3.
  • the antibody can be anti-PCSK9 receptor antibody comprising a heavy chain variable region comprising:
  • the antibody can be an anti-PCSK9 receptor antibody
  • a light chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38 (RASQGISSALA);
  • the antibody can be anti-PCSK9 antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41.
  • the antibody can be anti-PCSK9 receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 41 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 42, wherein the antibody binds specifically to human PCSK9 receptor.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 41 and 42.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 , wherein the antibody binds specifically to human PCSK9 receptor.
  • amino acid sequence of L1 L3 full-length heavy chain (SEQ ID NO: 30) is shown below:
  • the anti- PCSK9 receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 .
  • the anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 30 and 31.
  • the anti-PCSK9 receptor antibody may compete for PCSK9 receptor binding with an anti- PCSK9 receptor antibody as defined herein.
  • the anti- PCSK9 receptor antibody may compete for PCSK9 receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may be a human and affinity matured antibody, L1 L3, which specifically binds human PCSK9 receptor.
  • Antibody L1 L3 is described in WO2010/029513, the content of which is hereby incorporated by reference in its entirety for all purposes.
  • the amino acid sequences of the heavy chain and light chain variable regions of L1 L3 are shown in SEQ ID NOs: 41 and 42, respectively.
  • the CDR portions of antibody L1 L3 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 7 of WO2010/029513.
  • Antibody L1 L3 is highly potent in blocking PCSK9 receptor biological activity.
  • the anti-PCSK9 receptor antibody may also comprise a fragment or a region of the antibody L1 L3.
  • the fragment is a light chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 31 herein.
  • the fragment is a heavy chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 30 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody L1 L3.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody L1 L3 comprising the amino acid sequences as shown in SEQ ID NOS: 31 and 30, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody L1 L3 shown in SEQ ID NOs: 32-40.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-PCSK9 receptor antibodies described herein is deleted.
  • the heavy and/or light chain of the anti-PCSK9 receptor antibodies described herein may optionally include a signal sequence.
  • the anti-PCSK9 receptor antibody is alirocumab
  • PRALUENTTM evolocumab
  • REPATHATM evolocumab
  • REGN728 LGT209
  • RG7652 evolocumab
  • the anti-PCSK9 antibody is bococizumab, alirocumab
  • the antibody may bind to the same epitope as an anti-PCSK9 receptor antibody known in the art and/or may compete for binding to PCSK9 receptor with such an antibody.
  • camphorsulfonic acid or sulfosalicyclic acid are mixed together and the pH of the mixture is measured and if necessary, adjusted with the use of a buffer or basic component.
  • Other optional components may also be added to the mixture including one or more surfactants, chelating agents, and cryopretectants.
  • the formulation may be utilized in the liquid state, or lyophilized.
  • Many different freeze-dryers are available for this purpose such as Hull50.TM. (Hull, USA) or GT20.TM. (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying.
  • the product temperature is below the eutectic point or the collapse temperature of the formulation.
  • the shelf temperature for the primary drying will range from about -30 to 25°C (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr.
  • the formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs).
  • a secondary drying stage may also be performed depending upon the desired residual moisture level in the product.
  • the temperature at which the secondary drying is carried out ranges from about 0-4OC, depending primarily on the type and size of container and the type of protein employed.
  • the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30C (e.g., about 20°C).
  • the time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters.
  • the secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours).
  • the pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.
  • the formulations described herein may also be prepared as reconstituted lyophilized formulations.
  • the compositions described herein are lyophilized and then reconstituted to produce the reduced-viscosity stable liquid formulations of the invention.
  • a "pre-lyophilized formulation” is produced after preparation of the antibody of interest as described above.
  • the amount of antibody present in the pre- lyophilized formulation is determined taking into account the desired dose volumes and mode(s) of administration.
  • the concentration of an antibody in the pre- lyophilized formulation and in the reconstituted formulation can be as described previously, and may differ such that the reconstituted formulation may have an increased antibody concentration as compared to the pre-lyophilized formulation.
  • a "reconstituted" formulation is one which has been prepared by dissolving a lyophilized formulation in a diluent such that the antibody is distributed throughout the reconstituted formulation.
  • the reconstituted formulation is suitable for administration (e.g. parenteral administration) to a patient to be treated with the antibody of interest and, in certain embodiments of the invention, may be one which is suitable for subcutaneous administration.
  • diluent of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization.
  • exemplary diluents include sterile water, bacteriostatic water for injection (BWFI or WFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • diluents can include aqueous solutions of salts and/or buffers and/or surfactants.
  • Reconstitution generally takes place at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired.
  • the time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein.
  • the reconstituted formulation has less than 3000 particles per vial which are less than 10 pm per vial and 300 particles per vial which are less than 25 pm per vial for vials containing less than 100 ml. of solution.
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g.
  • injectable and infusible solutions dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general.
  • One mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes).
  • kits comprising antibodies of the invention and pharmaceutical compositions comprising these antibodies.
  • a kit may include, in addition to the antibody or pharmaceutical composition, diagnostic or therapeutic agents.
  • a kit may also include instructions for use in a diagnostic or therapeutic method.
  • the kit includes the antibody or a
  • kits includes the antibody or a pharmaceutical composition thereof and one or more therapeutic agents, such as an additional antineoplastic agent, anti-tumor agent or chemotherapeutic agent.
  • Liposomes containing compounds of the invention are prepared by methods known in the art, such as described in US4485045 and US4544545. Liposomes with enhanced circulation time are disclosed in US5013556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • oil e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion can comprise fat droplets between 0.1 and ⁇ . ⁇ , particularly 0.1 and 0.5pm, and have a pH in the range of 5.5 to 8.0.
  • the emulsion compositions can be those prepared by mixing a compound of the invention with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example,
  • hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or 'poly(vinylalcohol)), polylactides (US3773919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate), or 'poly(vinylalcohol)
  • polylactides US3773919
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate non
  • compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic compounds of the invention are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. Compositions of the invention may be used in conjunction with established treatments for the relevant indication.
  • the present invention also provides various therapeutic applications for the
  • compositions of the invention can be used for treating various diseases (e.g. cancer, autoimmune diseases, or viral infections) by binding the first protein (e.g. first human antibody variable domain) to an effector antigen and by binding the second protein (e.g. second human antibody variable domain) to a target antigen.
  • the compositions of the invention can be used for redirecting cytotoxicity, delivering thrombolytic agents to clots, for delivering immunotoxins to tumor cells, or for converting enzyme activated prodrugs at a target site (e.g. a tumor).
  • compositions of the invention can be used for increasing specificity of a therapeutic agent and/or modulating synergistic or additive pathways (e.g. metabolic or biochemical pathways).
  • the compositions of the invention can engage receptor/receptor, receptor/ligand, ligand/ligand, cell/cell, ligand/payload, receptor/payload, or single receptor.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The Use of
  • formulations of the present invention are administered to a mammal in need of treatment with the protein, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular,
  • the formulations are administered to the mammal by:
  • an article of manufacture which contains the formulation and preferably provides instructions for its use.
  • the article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g.
  • the container may be formed from a variety of materials such as glass or plastic.
  • the container holds the formulation and the label on, or associated with, the container may indicate directions for reconstitution and/or use.
  • the label may further indicate that the formulation is useful or intended for subcutaneous administration.
  • the container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • the article of manufacture may further comprise a second container comprising a suitable diluent (e.g. BWFI).
  • the final protein concentration in the reconstituted formulation will generally be at least 50 mg/ml.
  • the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • patches that release the medications directly into a subject's skin.
  • patches can contain the compound of the present invention in an optionally buffered, liquid solution, dissolved and/or dispersed in an adhesive, or dispersed in a polymer.
  • the compound may be administered once daily, but may also be administered multiple times. For example, the compound may be administered from once daily to once every six months or longer.
  • the administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
  • the compound may also be administered continuously via a minipump.
  • the compound may be administered at the site of the diseased body part or at a site distant from the site of the diseased body part.
  • the compound may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured.
  • the compound generally may be administered for as long as the disease is present.
  • the compound typically would be administered as part of a pharmaceutical composition as described supra.
  • Exemplary, non-limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of the antibody administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg.
  • an average human subject weighs about 70 kg.
  • Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg - 199 mg/kg are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.
  • Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound or portion and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.
  • the liquid compositions of the present invention can be prepared as unit dosage forms.
  • a unit dosage per vial may contain from 1 to 1000 milliliters (mis) of different concentrations of the compound of Formula (A).
  • a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the compound of Formula (A).
  • these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial.
  • the liquid compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.
  • Formulation 1 was amenable to achieve concentrations of approximately 50-70 mg/mL C1 GM antibody (in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8), with suitable stability characteristics.
  • the drug product was formulated as a lyophilized powder for reconstitution with sWFI (Table 1 ). Viscosity was evaluated using an Anton-Paar rheometer in cone- plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s "1 ).
  • FIGS. 1 A and 1 B viscosity of formulation at pH 5.8 and pH 5.0 (A) up to approximately 200 mg/mL C1 GM; (B) y-axis scale limited to 100 cP).
  • camphor-10-sulfonic acid also described as camphorsulfonic acid or CSA
  • CSA camphorsulfonic acid
  • Formulation 2 shown in Table 2 below, includes 200 mM CSA. Due to the acidic nature of CSA, nearly an equimolar amount of NaOH was used to neutralize the acid and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM.
  • Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s "1 ). Viscosity data are summarized in Table 3 below and FIG. 2A and 2B.
  • Viscosity of formulation 2 containing 200 mM CSA showed significantly reduced viscosity, i.e., approximately 20-40 fold reduction in viscosity, compared to formulation 1 across concentrations tested (Table 3 and FIG. 2A and 2B).
  • viscosity of formulation 2 was 3.2 cP, compared to viscosity of formulation 1 , which was 55.1 cp, a 17.2 fold reduction.
  • viscosity of formulation 2 was 4.4 cP, compared to viscosity of formulation 1 , which was 89.5 cP, a 20.3 fold reduction.
  • viscosity of formulation 2 was 7.5 cP, compared to viscosity of formulation 1 , which was 221.8 cP, a 29.6 fold reduction.
  • viscosity of formulation 2 was 13.1 cP, compared to viscosity of formulation 1 , which was 506.3 cP, a 38.6 fold reduction.
  • viscosity reduction on a fold basis increases as antibody concentration increases.
  • Formulation 2 which contains 200 mM CSA and has pH 7, allows C1 GM protein concentrations of greater than 170 mg/mL with viscosity behavior suitable for use in therapeutic treatment. This was not possible for C1 GM in formulation 1 because of high viscosity.
  • Example 3 Impact of counterion on viscosity reduction This example illustrates how varying the counterion ion for CSA can also impact viscosity.
  • Example 2 NaOH was used to neutralize the acidic nature of CSA in creating formulation 2, and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM.
  • other molecules basic in nature may be utilized to neutralize the acidic nature of CSA, effectively forming a salt of CSA.
  • One such species is arginine (Arg).
  • Formulation 3 shown in the table below, includes 200 mM CSA and 200 mM arginine. The basic nature of the arginine largely neutralizes the acidic nature of the CSA, and minimal acid or base needs to be added to adjust the pH to 7.0, avoiding the
  • Formulation 4 utilizes 200 mM arginine-HCI (Arg-HCI).
  • Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ . The samples were measured with a constant shear rate (898 s "1 ). Viscosity data are summarized in Table 5 below and FIG. 3. Table 5
  • This example illustrates the impact of varying excipient concentration on viscosity in an anti-IL-7R antibody formulation.
  • cryoprotectants, surfactants, and chelating agents may be necessary to include in the formulation, and will contribute to the tonicity of the formulation. Therefore, reduction of CSA and Arg levels may be required to allow these other excipients to be included in the formulation. Even at lower levels of CSA and Arg, formulations of C1 GM that include these excipients have significantly lower viscosity than formulations without these excipients. These results demonstrate that CSA-Arg appears to provide some robust protection against viscosity increases over the range of the ionic strength of the formulation. Example 5. Viscosity Reduction by Camphorsulfonic acid and CSA-Arg formulations of other antibodies
  • mAb5 and L1 L3 were reformulated with the above formulations by adding 200 mM Arginine HCI, 200 mM CSA, or 200 mM CSA-Arg. Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s " ). Viscosity data are summarized in Table 9 below and FIG. 6 (XX 1 is mAb5 antibody and XX2 is L1 L3 antibody).
  • the CSA-Arg combination offers the most effective reduction of viscosity for the mAb5 and L1 L3 antibody formulations.
  • This example illustrates the impact of varying excipient concentration on viscosity on mAb5 and L1 L3 antibody formulations.
  • Excipient concentrations of CSA and Arginine were reduced to either 100 mM or 50 mM from the previous level of 200 mM. Viscosities of these formulations were evaluated at pH 7 using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s " ). Results are summarized in FIG. 7A and 7B (XX1 is mAb5 antibody and XX2 is L1 L3 antibody) and Table 10. Lower amounts of excipients led to higher viscosities than observed for the 200 mM excipients.
  • This example illustrates the impact of the choice of hinge region structure on the viscosity of a formulation of monoclonal antibodies.
  • Monoclonal antibodies with identical CDR regions but differing hinge regions were produced and characterized using methods known in the art. Identical CDR regions were incorporated into frameworks of the lgG1 , lgG2, and lgG4 subtypes, and referred to as lgG1 , lgG2, and lgG4, respectively.
  • the lgG4 contained a hinge stabilizing S228P mutation (serine at position 228 of the heavy chain is replaced with proline).
  • Each antibody was formulated at 130 mg/ml_ in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8.
  • Viscosity of these formulations was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s " ). Viscosity data are summarized in Table 1 1 .

Abstract

La présente invention concerne de manière générale le domaine des formulations pharmaceutiques d'anticorps. Spécifiquement, la présente invention concerne une formulation liquide stable d'anticorps à faible viscosité, ainsi que sa préparation et son utilisation pharmaceutique.
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CN112105343A (zh) * 2018-03-07 2020-12-18 辉瑞公司 抗-pd-1抗体组合物
WO2021053001A1 (fr) * 2019-09-17 2021-03-25 Merck Patent Gmbh Acide camphosulfonique et leurs combinaisons avec des excipients cationiques utilisés en tant qu'agents réducteurs de viscosité dans des formulations de protéines concentrées élevées
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