JP2009531371A - Anti-IGF-1R human monoclonal antibody preparation - Google Patents

Abstract

  The present invention relates to an anti-IGF-1R human monoclonal antibody preparation, a preparation method thereof, and a use thereof.

Description

  The present invention relates to an anti-IGF-1R human monoclonal antibody preparation, a preparation method thereof, and a use thereof.

In one aspect, the present invention provides the following:
-About 1 to about 150 mg / mL of huMab IGF-1R,
About 0.001 to about 1% of at least one surfactant, and about 1 to about 100 mM buffer,
Including
A pH of about 5.0 to about 7.0,
To an IGF-1R formulation.

  IGF-IR (type 1 insulin-like growth factor receptor) is involved in promoting oncogenic transformation, proliferation, and survival of cancer cells. High levels of IGF-IR expression have been reported in a wide range of human malignancies. In addition, since high levels of IGF-I and IGF-II expression have been noted in tumors and associated stromal cells, they may stimulate cancer cell growth in an autocrine or paracrine manner. Epidemiological studies have shown a correlation between IGF-I plasma levels above quintile and the high risk of prostate, colon, lung, and breast cancer. In addition to its role in cancer cell proliferation, IGF-IR protects cells from apoptosis caused by growth factor depletion, anchorage independence, or cytotoxic drug therapy.

  One promising strategy to suppress IGF-IR function in cancer cells is to utilize human anti-IGF-IR antibodies that bind to the extracellular domain of IGF-IR and inhibit receptor activation. Represented as huMab IGF-1R, which specifically binds to the type I human insulin-like growth factor receptor (IGF-IR) and suppresses the signaling and proliferation function of that receptor in cancer cells, Fully human monoclonal antibodies having the aforementioned antagonistic properties have been developed.

The antibodies included in the formulations of the present invention are first described in PCT Patent Application No. WO 2005/005635, to which the applicant is the right holder and the contents of which are specifically incorporated herein by reference. It was. As described in WO 2005/005635, the aforementioned antibody binds to IGF-IR and inhibits the binding of IGF-I and IGF-II to IGF-IR, which is:
a) IgG1 isotype,
b) 1: 3-3: 1 inhibition of IGF-I binding to IGF-IR: shows the ratio of IC ₅₀ values for inhibition of IGF-II binding to IGF-IR,
c) In a cell phosphorylation assay using HT29 cells in medium containing 0.5% heat-inactivated fetal calf serum (FCS) when compared to such an assay without the aforementioned antibody, 5 nM IGF Inhibit at least 80%, preferably at least 90% at the concentration of -IR phosphorylation, and
d) Cell phosphorylation assay using 3T3 cells that give 400,000-600,000 molecules of IGF-IR per cell in a medium containing 0.5% heat-inactivated fetal calf serum (FCS). No IGF-IR stimulating activity measured as pkB phosphorylation at a concentration of 10 μM when compared to such an assay,
It is characterized by that.

The antibodies included in the formulations according to the invention show the benefit of patients in need of anti-tumor treatment and result in reduced tumor growth and a significant increase in time to progression. The antibodies included in the formulations according to the invention have novel and original properties that benefit patients suffering from diseases associated with IGF deregulation, in particular tumor diseases. The antibodies contained in the formulations of the present invention are characterized by the properties mentioned above. Therefore, the properties are in particular the specific binding to IGF-IR, the inhibition of binding of IGF-I and IGF-II to IGF-IR at the ratios mentioned above, the IgG1 isotype, the IC _It does not activate IGF-IR signaling even in IGF-IR overexpressing cells at a concentration 200 times the ₅₀ value. Antibodies that do not have “IGF-I-like activity” provide significant advantages when used as therapeutic agents.

  The term “anti-IGF-1R human monoclonal antibody” or “huMAb IGF-IR” is described in WO 2005/005635, the contents of which are specifically incorporated herein by reference. Means an antibody defined in the range of

  The term “antibody” is not limited to whole antibodies so long as the characteristic properties according to the present invention are retained, but various antibodies including antibody fragments, human antibodies, humanized antibodies, and genetically engineered antibodies. Is covered.

  "Antibody fragments" comprise a portion of the full length antibody, generally at least the antigen binding portion or variable region thereof. Examples of antibody fragments include bispecific antibodies, single chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments. In addition, antibody fragments can associate with VH chain characteristics, i.e., VL chains, or VL chain characteristics that bind to IGF-1R, i.e., with VH chains into functional antigen binding pockets; As a result, the present invention includes a single-chain polypeptide that can provide the property of inhibiting the binding of IGF-I and IGF-II to IGF-IR.

  “Antibody fragments” also include those fragments that are not themselves capable of providing effector function (ADCC / CDC), but provide this function in a manner according to the present invention after being combined with an appropriate antibody constant domain.

  The term “monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules consisting of a single amino acid composition. Thus, the term “human monoclonal antibody” refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences and exhibiting single binding specificity. In one embodiment, the human monoclonal antibody is from a non-human transgenic animal having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell, such as a transgenic mouse. It is produced by a hybridoma containing the obtained B cells.

  The term “chimeric antibody” is usually a monoclonal region comprising a variable region from one source or species, ie, a binding region and a constant region obtained from another source or species, prepared by recombinant DNA technology. Refers to an antibody. Particularly preferred are chimeric antibodies comprising a mouse variable region and a human constant region. Such a mouse / human chimeric antibody is the product of an expressed immunoglobulin gene comprising a DNA fragment encoding a mouse immunoglobulin variable region and a DNA fragment encoding a human immunoglobulin constant region. Other forms of “chimeric antibodies” covered by this invention are those in which the class or subclass has been modified or altered from that of the original antibody. Such “chimeric” antibodies are also referred to as “class switch antibodies”. Chimeric antibody production methods require conventional recombinant DNA and gene transfection techniques now well known in the art. See, for example, Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244.

  The term “humanized antibody” refers to an antibody that has been modified so that the framework or “complementarity-determining region” (CDR) contains the CDRs of an immunoglobulin of a different specificity compared to that of the parent immunoglobulin. . In a preferred embodiment, mouse CDRs are implanted within the framework region of a human antibody to prepare a “humanized antibody”. See, for example, Riechmann, L, et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens shown above for chimeric and bifunctional antibodies.

  The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The variable heavy chain is preferably derived from the germline sequence DP-50 (GenBank LO6618), and the variable light chain is preferably derived from the germline sequence L6 (GenBank X01668). The constant region of the antibody is a human IgG1 type constant region. Such regions may be allotype and are described, for example, by Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218 and the databases referenced therein, It is useful as long as it retains the property of inducing ADCC, preferably CDC, according to the present invention.

  The term “recombinant human antibody” as used herein refers to, for example, a host cell, such as SP2-0, NS0, or CHO cells into which a human immunoglobulin gene has been introduced, or an animal (eg, a mouse). Prepared, expressed, produced or isolated by recombinant means, such as isolated antibodies, or antibodies expressed using recombinant expression vectors transfected into host cells It is intended to include all human antibodies. Such recombinant human antibodies have variable and constant regions derived from rearranged forms of human germline immunoglobulin sequences. The recombinant human antibody according to the present invention undergoes somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the human germline VH and VL sequences, but may not occur naturally in the human antibody germline repertoire in vivo. There is no array.

“Binding” as used herein refers to an antibody that binds to IGF-IR with an affinity of about 10 ⁻¹³ to 10 ⁻⁸ M (K _D ), preferably about 10 ⁻¹³ to 10 ⁻⁹ M. .

  The term “nucleic acid molecule”, as used herein, is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

  “Constant domains” are not directly involved in antibody binding to antigen, but are involved in effector functions (ADCC, complement binding, and CDC). The constant domain of the antibody according to the invention is of the IgG1 type. Human constant domains with these characteristics are described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and Bruggemann, M., et al. , J. Exp. Med. 166 (1987) 1351-1361; Love, TW, et al., Methods Enzymol. 178 (1989) 515-527. Examples are shown in SEQ ID NOs 5-8 of WO 2005/005635. Another useful and preferred constant domain is that of an antibody obtained from a hybridoma cell line deposited with the DSMZ for the present invention. Constant domains useful in the present invention provide complement binding. ADCC and possibly CDC are brought about by a combination of variable and constant domains.

  “Variable region” (light chain variable region (VL), heavy chain variable region (VH)) is used herein to refer to the combination of light and heavy chains that are directly involved in binding of an antibody to an antigen. Each means. The variable human light and heavy chain domains have the same general structure, and each domain is connected by three “hypervariable regions” (or complementarity determining regions, CDRs) sequences Includes four framework (FR) regions that are extensively conserved. Framework regions take a β-sheet conformation, and CDRs may form loops connecting the β-sheet structures. CDRs in each chain are folded into their three-dimensional structure by framework regions and form antigen binding sites with CDRs from other chains. The heavy and light chain CDR3 regions of the antibody play a particularly important role in the binding specificity / affinity of the antibody according to the invention and therefore provide a further object of the invention.

  As used herein, the term “hypervariable region” or “antigen-binding portion of an antibody” refers to the amino acid residues of an antibody that are responsible for antigen-binding. Hypervariable regions include amino acid residues from “complementarity determining regions” or “CDRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Thus, the light and heavy chains of an antibody include the N-terminal to C-terminal domains, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, the heavy chain CDR3 is the region most contributing to antigen binding. CDR and FR regions are standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and / or “hypervariable loops”. Are determined according to their residues from

As used herein, the term “binding to IGF-IR” refers to an assay in a test tube, preferably an antibody that binds to the surface and that measures the binding of IGF-IR by surface plasmon resonance (SPR). Refers to antibody binding to IGF-IR in the assay. Binding means a binding affinity (K _D ) of 10 ⁻⁸ M or less, preferably 10 ⁻¹³ to 10 ⁻⁹ M.

Binding to IGF-IR can be investigated by BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). The affinity of binding is defined by the terms ka (rate constant for antibody binding from antibody / antigen complex), kd (dissociation constant), and K _D (kd / ka). The antibody according to the invention exhibits a K _D of 10 ⁻¹⁰ M or less.

The binding of IGF-I and IGF-II to IGF-IR is also inhibited by the antibody according to the invention. Said inhibition is measured as IC ₅₀ by an assay for binding of IGF-I / IGF-II to IGF-IR on tumor cells. Such an assay is described in Example 7. In such assays, the amount of radiolabeled IGF-I or IGF-II, or IGF-IR binding fragment thereof, bound to IGF-IR prepared on the surface of the aforementioned tumor cells (eg, HT29) is determined by the antibody concentration Measured without increasing and with increasing antibody concentration. The IC ₅₀ value of the antibody according to the invention for binding of IGF-I and IGF-II to IGF-IR is 2 nM or less, and the ratio of IC ₅₀ values for binding of IGF-I / IGF-II to IGF-IR Is about 1: 3 to 3: 1. IC ₅₀ values are measured as the mean or median of at least three independent measurements. A single IC ₅₀ value may be out of range.

The term “inhibition of IGF-I and IGF-II binding to IGF-IR” refers herein to IGF-IR prepared on the surface of HT29 (ATCC HTB-38) tumor cells in an in vitro assay. Inhibiting the binding of I ¹²⁵ -labeled IGF-I or IGF-II. Inhibition means an IC ₅₀ value of 2 nM or less.

  The term “surfactant” means herein a pharmaceutically acceptable surfactant. In the preparation of the present invention, the amount of the surfactant is described as a percentage expressed in weight / volume. The most commonly used weight / volume unit is mg / mL. Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyethylene-sorbitan-fatty acid esters, polyethylene-polypropylene glycol, polyoxyethylene-stearate, and sodium dodecyl sulfate. Preferred polyethylene-sorbitans are polyethylene (20) -sorbitan esters (also known as polysorbate 20, sold under the trademark Tween 20®), and polyoxyethylene (20) sorbitan monooleate (also known as polysorbate 80). Sold under the trademark Tween 80 (registered trademark). Preferred polyethylene-polypropylene glycols are those sold under the names Pluronic® F68 or Poloxamer 188®. Preferred polyoxyethylene-stearates are those sold under the trademark Myrj®. Preferred polyoxyethylene monolauryl ethers are those sold under the trademark Brij®. When polyethylene-sorbitan-polyethylene (20) -sorbitan-ester (Tween 20) and polyoxyethylene (20) sorbitan monooleate (Tween 80) are used, they are generally about 0.001 to about 1%, preferably Is used in an amount of about 0.005 to about 0.1%, and more preferably about 0.01% to about 0.02% w / v.

  The term “buffer” as used herein means a pharmaceutically acceptable buffer. Suitable pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and phosphate buffer. Preferred buffers include L-histidine, or a mixture of L-histidine and L-histidine hydrochloride, with isotonic agents known in the art, and pH adjusters using acids or bases. It is. The histidine buffer mentioned above is generally used in an amount of about 1 mM to about 100 mM, preferably about 5 mM to about 50 mM, and even more preferably about 20 mM. Regardless of the buffer used, the pH is adjusted to a value including about 5.0 to about 7.0, preferably about 5.5 to about 6.5, and more preferably about 6.0.

  The term “isotonicity agents” as used herein means pharmaceutically acceptable tonicity agents. Isotonic agents are used to provide isotonic formulations. An isotonic formulation is a liquid or a solid, eg, a liquid reconstituted from a lyophilized state, and compared to several such as, for example, physiological saline or serum It means a solution having the same tonicity as other solutions. Suitable tonicity agents include, but are not limited to, any component from the group consisting of sodium chloride, potassium chloride, glucose, glycerin, and amino acids, sugars, and combinations thereof. Tonicity agents are generally used in a total amount of about 5 mM to about 350 mM.

  The term “liquid” as used herein in connection with a formulation according to the invention means a formulation that is a liquid at a temperature of at least about 2 to about 8 ° C.

  The term “lyophilized” as used herein with respect to a formulation according to the invention is formulated by any lyophilization method known in the art, eg by a commercial lyophilization device. Means a formulation that has been frozen by freezing followed by transpiration of ice from the frozen contents.

  The term `` amino acid '' as used herein includes, but is not limited to arginine, glycine, ornithine, lysine, histidine, glutamic acid, asparagine aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline. Means an amino acid in an amount of about 1 to about 100 mg / mL.

  The term “sugar” as used herein means a pharmaceutically acceptable sugar used in an amount of about 25 mM to about 500 mM. Suitable sugars include, but are not limited to, trehalose, saccharose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (so-called “meglumine”), galactosamine, and neuraminic acid It is.

The term “stabilizer” is pharmaceutically acceptable, such as, but not limited to, the amino acids and sugars described in the previous section, and commercially available cyclodextrins and dextrans of all types and molecular weights known in the art. Refers to a stabilizer.
The term “antioxidant” means a pharmaceutically acceptable antioxidant.

As mentioned earlier, in one aspect, the present invention includes the following:
-About 1 to about 150 mg / mL of huMab IGF-1R,
About 0.001 to about 1% of at least one surfactant, and about 1 to about 100 mM buffer,
Including
A pH of about 5.0 to about 7.0,
To an IGF-1R formulation.

  The formulations according to the invention preferably comprise from about 0.001 to about 1% of at least one surfactant.

In a particular embodiment, the formulation according to the invention has the following:
-About 1 to about 150 mg / mL of huMab IGF-1R,
About 0.005 to about 0.05% of at least one surfactant, and about 1 to about 100 mM buffer,
Including
A pH of about 5.0 to about 7.0,
It is.

  Formulations according to the present invention may be liquid, lyophilized, or liquid reconstituted from a lyophilized state.

  In a particular embodiment, the formulation according to the invention is a lyophilized formulation. The lyophilized formulation according to the present invention comprises particulate matter formation and higher molecular weight, which is generally difficult to achieve with liquid formulations at that concentration of the described anti-IGF-1R human monoclonal antibody. There is an advantage of improved stability with respect to aggregation.

  The formulations according to the invention may be administered intravenously (i.v.), subcutaneously (s.c.) or by other parental administration means such as, for example, those known in the pharmaceutical art.

  The formulations of the present invention may further comprise one or more tonicity agents in an amount of about 5 mM to about 350 mM. Suitable tonicity agents may be selected from the group consisting of sodium chloride (NaCl), potassium chloride, sugars including glucose, glycerin, amino acids, and combinations thereof.

  The formulations of the present invention may further comprise sugar in an amount of about 25 mM to about 500 mM. Suitable sugars may be selected from the group consisting of trehalose, saccharose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, neuraminic acid, and combinations thereof.

  The preparation of the present invention comprises the following components: antioxidants, ascorbic acid, glutathione, preservatives such as m-cresol, phenol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, thiomersal, benzalkonium chloride, cyclohexane Dextrins such as hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin, polyethylene glycols such as PEG 3000, 3350, 4000, 6000, albumin, human serum albumin (HSA), bovine Further includes one or more of serum albumin (BSA), polyhydric alcohol, glycerol, ethanol, mannitol, salt, acetate (eg, sodium acetate), magnesium chloride, calcium chloride, tromethamine, EDTA (eg, Na-EDTA) But There.

  The formulations of the present invention may further comprise one or more of the stabilizers previously defined herein, and likewise sugars, sugar alcohols, amino acids, known in the art, And may further comprise components known in the art as “lyoprotectants” such as dextran.

In a particular embodiment, the formulation of the present invention is in the following formulation, which is either liquid, lyophilized, or liquid reconstituted from lyophilized:
-About 1 to about 150 mg / mL of huMab IGF-1R,
−0.01% Tween 20 w / v,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
including.

Formulations according to the invention also include the following specific formulations:
−25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or -25 mg / mL huMab IGF-1R,
-0.03% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or -25 mg / mL huMab IGF-1R,
-0.05% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or

−10 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or −10 mg / mL huMab IGF-1R,
-0.03% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or

−10 mg / mL huMab IGF-1R,
-0.05% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
-PH 6.0,
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−140 mM NaCl,
-PH 5.5,
Or

−25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
-PH 5.5,
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
-PH 6.0,
Or

−25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
-PH 6.0,
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
-0.05% polysorbate 20,
-PH 6.0,
Or

−25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−60 mM trehalose dihydrate,
-0.01% polysorbate 20,
-PH 6.0,
Or -25 mg / mL huMab IGF-1R,
-20 mM succinate,
-250 mM trehalose dihydrate,
-0.01% polysorbate 20,
-PH 5.5,
Or

−25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−60 mM trehalose dihydrate,
-0.01% polysorbate 20,
-PH 6.0.

In a preferred embodiment of the formulation according to the invention, the formulation is lyophilized and after reconstitution with an appropriate amount of water for injection the following:
−25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
Including
-PH 5.5,
It is.

  This formulation has less than 1.5% high molecular weight species even after storage at 2-8 ° C. and 25 ° C. for about 6 months, and good stability without formation of visible and invisible particles Showing gender. Shaking and multiple freeze-thaw steps were applied to the liquid formulation to simulate physical stress conditions that may occur during production, for example, by pressure filtration and filling, lyophilization, and finishing operations . This formulation was found to be stable after 1 week of shaking at 5 ° C and 25 ° C. The formation of visible and invisible particles could not be detected, and no significant increase in the fraction of high molecular weight species indicating the formation of soluble aggregates was observed.

Liquids for intravenous administration according to the present invention and lyophilized pharmaceutical formulations were developed as follows:
Preparation of liquid formulation About 10 and 25 mg / mL huMab IGF-1R solution in manufacturing buffer (about 10 mM histidine buffer containing about 150 mM NaCl), a large amount of water, and for the final formulation Dialyzed against each buffer salt system (see table for exact formulation composition). If necessary, the protein concentration was increased by filtration using a commercially available centrifugal filtration device prior to dialysis and then adjusted to the desired protein concentration by dilution with dialysis buffer. Sugars and salts to stabilize the protein and to adjust tonicity were added to the dialysis buffer as needed. Surfactant was added to the formulation as a 2 to 40-fold stock solution after dialysis. Alternatively, buffer exchange and concentration was performed using a commercially available tangential flow filtration device such as AKTA CF (GE Healthcare) with Sartorius Hydrosart membrane (30'000 Da molecular weight cut-off). For example, components such as sugars, salts, or surfactants were added by using an appropriate amount of concentrated stock solution after buffer exchange. All formulations were aseptically filtered through a 0.22 μm low protein adsorption filter and aseptically dispensed into sterile 6 mL glass vials closed by Teflon-coated rubber stoppers and alucrimp caps. These formulations are stored at different temperatures for different time intervals and for analysis 1) UV spectroscopy, 2) size exclusion chromatography (SEC), and 3) turbidity of the solution Were taken at the time indicated in the individual terms by light obscuration to measure. In addition, analysis of visible particles was performed on each sample using a Seidenader V90-T instrument. The presence of particles invisible to the naked eye was evaluated with a HIAC Royco device.

Preparation of lyophilized formulation
A solution of 25 mg / mL huMab IGF-1R was prepared as described above for the liquid formulation. All formulations were sterile filtered through a 0.22 μm low protein adsorption filter and dispensed aseptically into sterile glass vials. The vial was partially closed using a Teflon® coated rubber stopper suitable for use in the lyophilization process and transferred to the drying chamber of the lyophilizer. Any lyophilization method known in the art is intended to be within the scope of the present invention. For example, the lyophilization process used for this study included heat removal (pre-cooling) of the formulation from room temperature to about 5 ° C. with an ascending rate of about 1 ° C./min to 5 ° C./min, followed by − Includes freezing at 40 ° C. (Freezing I). The first drying step is applied from −40 ° C. to −30 ° C. at a rising rate of 0.3-0.5 ° C./min, and then at a chamber pressure of about 75-80 mTorr (about 10.0-100.7 Pa), −30 ° C. May be maintained for at least 50 hours. The second drying step is performed from −30 ° C. to 25 ° C. at a rising rate of 0.1-0.3 ° C./min, and at least at a chamber pressure of about 50-80 mTorr (about 6.7-10.7 Pa), 15 ° C. May be maintained for 5 hours. The huMab IGF-1R formulation dried using the described lyophilization process was found to have a convenient and rapid reconstitution time of about <5 minutes. Lyophilization was performed with a LyoStar II Freeze-dryer (FTS Systems, Stone Ridge, NY, USA and Usifroid Orion, Maurepas, France). All of the lyophilized cakes in this study had a residual moisture content of about 0.1-5.0% as measured by the Karl Fischer method. The lyophilized vials were stored at different temperatures for different time intervals. Before the lyophilized preparation, 1) UV spectroscopic measurement, 2) Reconstitution time measurement, 3) Size exclusion chromatography (SEC), and 4) Light opacity analysis to measure solution turbidity Reconstituted with each water for injection (WFI) volume. In addition, analysis of the visible particles was performed on each sample using a Seidenader V90-T instrument (Seidenader, Marktschwaben, Germany). The presence of particles invisible to the naked eye was evaluated with a HIAC Royco device.

Size exclusion chromatography (SEC) was performed to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysis products in the formulation. In the method, a Merck Hitachi 7000 HPLC instrument or a Waters Alliance 2795 equipped with a UV detector (detection wavelength λ (280 nm)) was used. Both instruments were equipped with a TSK G3000 SWXL column; the method used 0.2 M K ₂ HPO ₄ /0.25 M KCL, pH 7.0 as the mobile phase. The flow rate was 0.5 mL / min (isotonic) and the runtime was 30 minutes at a column temperature of 25 ° C. UV spectroscopy for protein concentration quantification, after dilution of the sample to an antibody concentration of 0.5 mg / mL, with Uvikon 932 (Kontron Instruments) at 278 nm wavelength and Varian Cary Bio UV spectroscopy at 280 nm, respectively Performed with a photometer. For turbidity measurements, light opacity was measured in units of FTU (turbidity units) using a HACH 2100AN turbidimeter at room temperature.

Composition of liquid huMAb IGF-1R pharmaceutical formulation according to the present invention and stability data after 3 months storage at 2-8 ° C. Formulation A consists of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM A liquid formulation with a composition of NaCl, 0.01% polysorbate 20, pH 6.0.

  Formulation B is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, 0.03% polysorbate 20, pH 6.0.

  Formulation C is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, 0.05% polysorbate 20, pH 6.0.

  Formulation D is a liquid formulation having a composition of 10 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, 0.01% polysorbate 20, pH 6.0.

  Formulation E is a liquid formulation having a composition of 10 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, 0.03% polysorbate 20, pH 6.0.

  Formulation F is a liquid formulation having a composition of 10 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, 0.05% polysorbate 20, pH 6.0.

  Formulation G is a liquid formulation having a composition of 10 mg / mL huMab IGF-1R, 20 mM L-histidine, 140 mM NaCl, pH 5.5.

  Formulation H is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 250 mM trehalose dihydrate, 0.01% polysorbate 20, pH 5.5.

  Formulation I is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 250 mM trehalose dihydrate, pH 6.0.

  Formulation J is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 250 mM trehalose dihydrate, 0.01% polysorbate 20, pH 6.0.

  Formulation K is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 250 mM trehalose dihydrate, 0.05% polysorbate 20, pH 6.0.

  Formulation L is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 60 mM trehalose dihydrate, 0.01% polysorbate 20, pH 6.0.

  Formulation M is a liquid formulation having a composition of 25 mg / mL huMab IGF-1R, 20 mM succinate, 250 mM trehalose dihydrate, 0.01% polysorbate 20, pH 5.5.

Composition of lyophilized huMAb IGF-1R pharmaceutical formulation according to the present invention and stability data after storage for 3 months at 2-8 ° C. Formulation N consists of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 60 mM A lyophilized formulation with the composition of a reconstituted solution of 10% trehalose dihydrate, 0.01% polysorbate 20, pH 6.0.

  Formulation O is a lyophilized formulation with a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 60 mM sucrose, 0.01% polysorbate 20, pH 6.0 reconstituted solution.

  Formulation P is a lyophilized formulation with a composition of 25 mg / mL huMab IGF-1R, 20 mM succinate, 250 mM trehalose dihydrate, 0.01% polysorbate 20, pH 5.5 reconstituted solution.

  Formulation Q is a lyophilized formulation with a composition of 25 mg / mL huMab IGF-1R, 20 mM L-histidine, 250 mM trehalose dihydrate, 0.01% polysorbate 20, pH 5.5 reconstituted solution .

Claims (14)

-About 1 to about 150 mg / mL of huMab IGF-1R,
About 0.001 to about 1% of at least one surfactant, and about 1 to about 100 mM buffer,
Including
A pH of about 5.0 to about 7.0,
A formulation that is   The formulation of claim 1, comprising from about 0.001 to about 1% of at least one surfactant.   The preparation according to claim 1, which is a liquid, a lyophilized state, or a liquid reconstituted from a lyophilized state.   The formulation of claim 1, which can be administered intravenously (i.v.) or subcutaneously (s.c.) or by other parenteral administration means.   5. The formulation of any one of claims 1-4, further comprising one or more tonicity agents in an amount of about 5 mM to about 350 mM.   6. The formulation of claim 5, wherein the tonicity agent is selected from the group consisting of sodium chloride (NaCl), potassium chloride, sugars including glucose, glycerin, amino acids, and combinations thereof.   7. The formulation of any one of claims 1-6, further comprising a sugar in an amount of about 25 mM to about 500 mM.   The sugar is selected from the group consisting of trehalose, saccharose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine ("meglumine"), galactosamine, neuraminic acid. 7. The preparation according to 7. Pharmaceutically acceptable antioxidants, ascorbic acid, glutathione, preservatives, especially m-cresol, phenol, benzyl alcohol, methyl paraben, propyl paraben, chlorbutanol, thiomersal, benzalkonium chloride, cyclodextrin, especially hydroxypropyl -β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin, polyethylene glycol, especially PEG 3000, 3350, 4000, 6000, albumin, human serum albumin (HSA), bovine serum albumin (BSA), many Monohydric alcohol, glycerol, ethanol, mannitol, salt, acetate, especially sodium acetate, magnesium chloride, calcium chloride, tromethamine, EDTA, especially Na-EDTA
The formulation of claim 7, further comprising one or more components selected from the group consisting of: -About 1 to about 150 mg / mL of huMab IGF-1R,
−0.01% Tween 20 w / v,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0,
The preparation according to any one of claims 1 to 9. −25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
-0.03% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
-0.05% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0
Or −10 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0
Or −10 mg / mL huMab IGF-1R,
-0.03% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
huMab IGF-1R at pH 6.0 or −10 mg / mL,
-0.05% polysorbate 20,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−140 mM NaCl,
Comprising
pH 5.5
Or -25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
Comprising
pH 5.5
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
-0.01% polysorbate 20,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
Comprising
huMab IGF-1R at pH 6.0 or -25 mg / mL,
−20 mM L-histidine,
-250 mM trehalose dihydrate,
-0.05% polysorbate 20,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−60 mM trehalose dihydrate,
-0.01% polysorbate 20,
Comprising
pH 6.0
Or -25 mg / mL huMab IGF-1R,
-20 mM succinate,
-250 mM trehalose dihydrate,
-0.01% polysorbate 20,
Comprising
pH 5.5
Or -25 mg / mL huMab IGF-1R,
−20 mM L-histidine,
−60 mM trehalose dihydrate,
-0.01% polysorbate 20,
Comprising
pH 6.0,
The preparation according to any one of claims 1 to 9.   Use of a formulation according to any one of claims 1 to 11 for the preparation of a medicament useful for treating a disease modulated by an IGF-IR receptor.   13. Use according to claim 12, wherein the disease is selected from the group consisting of breast cancer, colon cancer, non-small cell lung cancer (NSCLC) and prostate cancer.   The invention described herein.