HK1196570B - Anti a beta antibody formulation - Google Patents

Description

anti-Abeta antibody formulations

The present application is a divisional application filed on 27/1/2006 under application number 200680003530.5 (PCT/US 2006/002837), entitled "anti-a β antibody preparation".

RELATED APPLICATIONS

This application claims priority from U.S. provisional patent application No. 60/648,631 (filed 2005, month 1, day 28) entitled "anti-a β antibody formulations". The above-referenced application is incorporated by reference herein in its entirety.

Background

Alzheimer's disease ("AD") is a neurodegenerative disorder characterized by the presence of amyloid plaques, neurofibrillary nodules, and significant neuronal loss. Beta-amyloid protein (also known as Abeta peptide), the major component of senile plaques, has been implicated in the pathogenesis of Alzheimer's disease (Selkoe (1989) Cell58: 611-612; Hardy (1997) trends Neurosci.20: 154-159). Amyloid beta has been shown to be directly toxic to cultured neurons (Lorenzo and Yankner (1996) Ann. NYAcad. Sci.777:89-95) and indirectly toxic through various mediators (Koh et al (1990) BrainResearch533: 315-320; Mattsonetal (1992) J. neurosciens 12: 376-389). Furthermore, amyloid beta has been associated with learning deficits, alterations in cognitive function, and long-term hippocampal enhancement in both in vivo models, including the PDAPP mouse and rat models (Chen et al, (2000) Nature408: 975-. Thus, there has been much interest in therapies that effectively reduce the severity of the disease or even eliminate the disease itself by altering beta amyloid levels.

With the successful research in PDAPP mouse and rat experimental models, a therapeutic strategy for AD has recently emerged by immunizing individuals to provide immunoglobulins such as antibodies (in the case of passive immunization, therapeutic immunoglobulin administration to the patient) or to produce immunoglobulins specific for beta amyloid (active immunization, in which the patient's immune system is activated to produce immunoglobulins directed against the administered antigen). These antibodies can reduce plaque burden by preventing beta amyloid aggregation (Solomon et al, (1997) Neurobiology94:4109-4112) or stimulating microglia to phagocytose and remove plaques (Bardetal, (2000) Nature medicine6: 916-919). Also for example, a humanized anti a β peptide IgG1 monoclonal antibody (humanized 3D6 antibody) is also effective in treating AD by selectively binding to human a β peptide.

In the case of proteins, and antibodies in particular, the biological activity of the protein must be maintained in a formulation that maintains the conformational integrity of at least the core sequence of the amino acids of the protein and protects the various functional groups of the protein from degradation. Degradation pathways of proteins may involve chemical instability (i.e., any process involving the generation of new chemical entities by bond formation or cleavage) or physical instability (i.e., changes in the higher order structure of the protein). Chemical instability can be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can be caused by denaturation, aggregation, precipitation or adsorption, for example. For a general review of the stability of protein drugs, see, e.g., Manning, et al (1989) pharmaceutical research6: 903-918. In addition, it is desirable that the stability of the formulation be maintained when the formulation does not contain a carrier polypeptide.

Although the possible occurrence of protein instability is widely accepted, it is still not possible to predict the specific instability of a particular protein. Any such instability can potentially lead to the production of polypeptide by-products or the formation of derivatives with reduced activity, increased toxicity, and/or increased immunogenicity. Indeed, precipitation of the polypeptide can lead to thrombosis, non-uniformity of dosage form, and immune response. Therefore, the safety and effectiveness of any polypeptide pharmaceutical formulation is directly related to its stability.

Accordingly, there is an increasing need for formulations that maintain the stability and biological activity of biological polypeptides, e.g., a β binding polypeptides, during storage and transport, and that are suitable for a variety of therapeutic administration.

Summary of The Invention

The present invention provides formulations intended to provide stability and maintain biological activity of a contained biologically active protein, particularly an a β binding protein or polypeptide, e.g., an a β antibody or fragment or portion thereof. The invention also provides polypeptide formulations, e.g., stable liquid polypeptide formulations, that are capable of preventing the formation of undesirable polypeptide by-products.

The integrity of antigen binding polypeptides is of utmost importance for therapeutic use, since if the polypeptide forms by-products, such as aggregates or degraded fragments during storage, its biological activity may be lost, thereby compromising the therapeutic activity per unit dose molecule. Furthermore, there is also a need for stabilizing therapeutic polypeptides that are intended for specific functions, for transport and use in certain biological indications, e.g. in the treatment of neurodegenerative diseases, where the polypeptide must cross the blood-brain barrier (BBB) and bind to a target antigen.

In one aspect, the invention provides a stable formulation comprising at least one a β binding polypeptide, at least one tonicity agent (tonicityagent), wherein the tonicity agent is present in an amount sufficient to render the formulation suitable for administration, and at least one buffer in an amount sufficient to maintain a physiologically suitable pH. The formulation may be lyophilized or a liquid formulation. Certain formulations include at least one antioxidant, such as an amino acid antioxidant, e.g., methionine. In some formulations, the tonicity agent is mannitol or NaCl. In some formulations, at least one buffering agent is succinate, sodium phosphate, or an amino acid, such as histidine. Preferably the formulation further comprises at least one stabilizer, such as polysorbate 80. In some formulations, the stabilizer is polysorbate 80, the antioxidant is methionine, the tonicity agent is mannitol, sorbitol or NaCl, and the buffering agent is histidine. In some formulations, at least one a β binding polypeptide is selected from the group consisting of an anti-a β antibody, an anti-a β antibody Fv fragment, an anti-a β antibody Fab' (2) fragment, an anti-a β antibody Fd fragment, a single chain anti-a β antibody (scFv), a single domain anti-a β antibody fragment (Dab), a β -sheet polypeptide comprising at least one antibody Complementarity Determining Region (CDR) from an anti-a β antibody, and a non-globular polypeptide comprising at least one antibody CDR from an anti-a β antibody. In some formulations, at least one a β binding polypeptide is an anti-a β antibody, e.g., which can specifically bind to an epitope within residues 1-7, 1-5, 3-7, 3-6, 13-28, 15-24, 16-24, 16-21, 19-22, 33-40, 33-42 of a β, or a Fab, Fab' (2), or Fv fragment thereof. Exemplary anti-A β antibodies specifically bind to an epitope within residues 1-10 of A β, e.g., within residues 1-7, 1-5, 3-7 or 3-6 of A β. Other exemplary anti-A β antibodies specifically bind to an epitope within residues 13-28 of A β, e.g., within residues 16-21 or 19-22 of A β. Yet other exemplary A.beta.antibodies can also specifically bind to a.beta.C-terminal epitopes, e.g., residues 33-40 or 33-42 of A.beta.. Preferred a β antibodies include humanized anti a β antibodies, for example, humanized 3D6 antibody, humanized 10D5 antibody, humanized 12B4 antibody, humanized 266 antibody, humanized 12a11 antibody, or humanized 15C11 antibody.

In some formulations, the anti-a β antibody binds to a discontinuous epitope included within residues 1-7 and 13-28 of a β. In some such formulations, the antibody is a bispecific antibody or an antibody prepared according to the methods described in International patent publication No. WO 03/070760. In some such formulations, the epitope is a discontinuous epitope. In a preferred formulation, the anti a β antibody is a humanized 3D6 antibody, a humanized 10D5 antibody, a humanized 12B4 antibody, a humanized 266 antibody, a humanized 12a11 antibody, or a humanized 15C11 antibody.

The isotype of the antibody may be IgM, IgG1, IgG2, IgG3, IgG4 or any other pharmaceutically acceptable isotype. In a preferred formulation, the isotype is human IgG1 or human IgG 4. In some liquid formulations, the concentration of the anti-A β antibody is about 0.1mg/ml to about 60mg/ml, about 40mg/ml to about 60mg/ml, about 50mg/ml, about 30mg/ml, about 17mg/ml to about 23mg/ml, about 20mg/ml, about 17mg/ml, about 10mg/ml, about 5mg/ml, about 2mg/ml, or about 1 mg/ml. Preferably from about 17mg/ml to about 23 mg/ml.

In some formulations, at least one tonicity agent is D-mannitol and is present at a concentration of about 1% w/v to about 10% w/v, about 2% w/v to about 6% w/v, or preferably about 4% w/v. In some formulations, at least one buffer is histidine and the concentration is about 0.1mM to about 25mM, about 5mM to about 15mM, preferably about 5mM or about 10 mM. In other formulations, at least one buffer is succinate and is at a concentration of about 0.1mM to about 25mM, e.g., about 10 mM. In some formulations, the antioxidant is methionine and is at a concentration of about 0.1mM to about 25mM, about 5mM to about 15mM, or preferably about 10 mM. In a preferred formulation, the stabilizing agent is polysorbate 80 and is present at a concentration of about 0.001% w/v to about 0.01% w/v, about 0.005% w/v to about 0.01% w/v, or about 0.005% w/v. The pH of the formulation is about 5 to 7, about 5.5 to about 6.5, about 6.0 to about 6.5, about 6.2, about 6.0, or about 5.5, preferably about 6.0.

Preferably, the formulation has a pH of about 6.0 to about 6.5 and comprises an anti-A β antibody that specifically binds to an epitope selected from the group consisting of residues 1-7, 1-5, 3-7, 3-6, 13-28, 15-24, 16-24, 16-21, 19-22, 33-40, and 33-42 of A β, e.g., D-mannitol at a concentration of about 2% w/v to about 6%, e.g., histidine at a concentration of about 0.1mM to about 25mM, methionine at a concentration of about 0.1mM to about 25mM, and a stabilizer. Preferably, the stabilizer is polysorbate 80 at a concentration of about 0.001% to about 0.01% w/v.

The formulation may be a stable liquid polypeptide formulation designed to provide stability and maintain the biological activity of the integrated polypeptide. The formulation includes a therapeutically active a β -binding polypeptide and an antioxidant present in an amount sufficient to reduce the formation of by-products of the polypeptide during storage of the formulation.

Some liquid polypeptide formulations are stable to the formation of undesirable by-products, such as high molecular weight polypeptide aggregates, low molecular weight polypeptide degradation products, or mixtures thereof.

In formulations where the therapeutic antigen-binding polypeptide is an antibody, common high molecular weight aggregates that need to be minimized are, for example, antibody: antibody complex, antibody: antibody fragment complex, antibody fragment: antibody fragment complex, or mixtures thereof. Typically, the molecular weight of the high molecular weight complex or by-product is greater than the molecular weight of the antigen-binding polypeptide monomer, e.g., greater than about 150kD in the case of IgG antibodies. In such antibody preparations, common low molecular weight polypeptide breakdown products that need to be minimized are, for example, complexes composed of antibody light chains, antibody heavy chains, antibody light and heavy chain complexes, or mixtures thereof. Typically, the low molecular weight complexes or by-products will have a molecular weight that is lower than the molecular weight of the antigen-binding polypeptide monomer, e.g., less than about 150kD in the case of IgG antibodies.

Preferred stable formulations of anti- Α β antibodies include the antioxidant methionine in an amount sufficient to inhibit the formation of undesired by-products, such as a tonicity agent in an amount sufficient to render the formulation suitable for administration, and such as an amino acid or derivative thereof in an amount sufficient to maintain a physiologically suitable pH.

Some formulations are stable upon freezing. Such formulations may be suitable for parenteral, intravenous, intramuscular, subcutaneous, intracranial, or epidural administration, preferably intravenous or subcutaneous administration. Some formulations may be suitable for targeted delivery to the brain or spinal fluid of a subject. The formulation may be substantially free of preservatives. Some formulations may be stable for at least about 12 months, at least about 18 months, at least about 24 months, or at least about 30 months. Some formulations may be stable at about-80 ℃ to about 40 ℃, at about 0 ℃ to about 25 ℃, at about 0 ℃ to about 10 ℃, preferably at about-80 ℃ to about-50 ℃ or at about 2 ℃ to about 8 ℃.

Some formulations are stable for at least 12 months at temperatures above freezing to about 10 ℃ and at a pH of about 5.5 to about 6.5. Such formulations comprise at least one A β antibody at a concentration of about 1mg/ml to about 30mg/ml, mannitol at a concentration of about 4% w/v or NaCl at a concentration of about 150mM, histidine or succinate at a concentration of about 5mM to about 10 mM; and 10mM methionine. Certain such formulations have a pH of about 6.0, about 1mg/ml of A.beta.antibody, about 10mM histidine and about 4% w/v mannitol. Other formulations may be stable for at least about 24 months at about 2 ℃ to 8 ℃ and contain polysorbate 80 at a concentration of about 0.001% w/v to about 0.01% w/v. Some such formulations have a pH of about 6.0 to about 6.5 and contain about 10mM histidine, about 4% w/v mannitol and about 1mg/ml, about 2mg/ml or about 5mg/ml of A β antibody. Other such formulations include about 10mM histidine, about 4% w/v mannitol, about 0.005% w/v polysorbate 80 and about 10mg/ml, about 20mg/ml or 30mg/ml of an A β antibody, preferably at a pH of about 6.0 to about 6.2.

In such formulations, the anti-a β antibody is preferably a humanized 3D6 antibody, a humanized 10D5 antibody, a humanized 12B4 antibody, a humanized 266 antibody, a humanized 12a11 antibody, or a humanized 15C11 antibody. Certain such formulations have a pH of about 6.0 to 6.5 and comprise about 10mM histidine, about 4% w/v mannitol and about 2mg/ml to about 20mg/ml of an A β antibody selected from the group consisting of humanized 3D6 antibody, humanized 10D5 antibody, humanized 12B4 antibody and humanized 12A11 antibody. Another such formulation has a pH of about 6.0 to 6.5 and contains about 10mM histidine, about 150mM NaCl and about 2mg/ml to about 20mg/ml of an A β antibody selected from the group consisting of humanized 12B4 antibody and humanized 12A11 antibody. While other such formulations have a pH of about 6.0 to 6.5 and contain about 10mM histidine, about 4% w/v mannitol and about 2mg/ml to about 20mg/ml of an A β antibody selected from the group consisting of a humanized 266 antibody and a humanized 15C11 antibody.

Preferably, the formulation is stable for at least about 24 months at a temperature of about 2 ℃ to about 8 ℃ and a pH of about 5.5 to about 6.5, and contains about 2mg/ml to about 23mg/ml, preferably about 17mg/ml to about 23mg/ml, of the humanized 3D6 antibody, about 10mM histidine and about 10mM methionine. Preferably, the formulation further comprises about 4% w/v mannitol. The formulation preferably includes polysorbate 80 at a concentration of about 0.001% w/v to about 0.01% w/v, more preferably about 0.005% w/v. In such formulations, the humanized 3D6 antibody is present at a concentration of about 20mg/ml to about 23 mg/ml.

Other formulations are stable for at least about 24 months at a temperature of about 2 ℃ to about 8 ℃ and a pH of about 5.5 to about 6.5, and comprise about 2mg/ml to about 23mg/ml of the humanized 3D6 antibody, about 10mM succinate, about 10mM methionine, about 4% w/v mannitol, and about 0.005% w/v polysorbate 80. In certain such formulations, the humanized 3D6 antibody is present at a concentration of about 17mg/ml to about 23 mg/ml.

Other preferred formulations are stable for at least about 24 months at a temperature of about 2 ℃ to about 8 ℃ and a pH of about 6.0 to about 6.5, and contain about 2mg/ml to about 30mg/ml of the humanized 266 antibody, about 10mM histidine and about 10mM methionine. In some such formulations, mannitol is also present at about 4% w/v. In certain such formulations, polysorbate 80 is also included at a concentration of about 0.001% w/v to about 0.01% w/v, for example about 0.005% w/v. In certain such formulations, the humanized 266 antibody is present at a concentration of about 17mg/ml to about 23mg/ml, or about 20mg/ml to about 23 mg/ml.

Other formulations may be stable at temperatures of about 2 ℃ to about 8 ℃ for at least about 24 months at a pH of about 6.0 to about 6.5, and contain about 2mg/ml to about 20mg/ml of the humanized 266 antibody, about 10mM succinate, about 10mM methionine, about 4% w/v mannitol, and about 0.005% w/v polysorbate.

Other preferred formulations are stable at temperatures of about 2 ℃ to about 8 ℃ for at least about 24 months at a pH of about 6.0 to about 6.5 and contain about 2mg/ml to about 30mg/ml of humanized 12A11 antibody, about 10mM histidine and about 10mM methionine. Some such formulations contain about 150mM NaCl. Such formulations also contain polysorbate 80 at a concentration of about 0.001% w/v to about 0.01% w/v, for example, about 0.005%. In some formulations, the humanized 12A11 antibody is present at a concentration of about 17mg/ml to about 23mg/ml, or about 20mg/ml to about 23 mg/ml.

Other formulations are stable at temperatures of about 2 ℃ to about 8 ℃ for at least about 24 months at a pH of about 6.0 to about 6.5 and comprise about 2mg/ml to about 20mg/ml of humanized 12A11 antibody, about 5mM histidine, about 10mM methionine, about 4% w/v mannitol, and about 0.005% w/v polysorbate 80.

The present invention also provides a formulation that remains stable when thawed from about-50 ℃ to about-80 ℃, has a pH of about 6.0, comprises about 40 to about 60mg/ml of an anti-a β antibody, about 1.0mg/ml to about 2.0mg/ml histidine, about 1.0mg/ml to 2.0mg/ml methionine and about 0.05mg/ml polysorbate 80. Preferably, mannitol is not present. Preferably, the a β antibody is a humanized 3D6 antibody or a humanized 266 antibody.

The invention also provides liquid formulations comprising anti-a β antibodies, mannitol and histidine. In some such formulations, the concentration of the anti-A β antibody is from about 1mg/ml to about 30 mg/ml. Preferably, mannitol is present in an amount sufficient to maintain isotonicity of the formulation. Preferably, the histidine is present in an amount sufficient to maintain a physiologically suitable pH. One such formulation contains about 20mg/ml of anti-A β antibody, about 10 mML-histidine, about 10mM methionine, about 4% mannitol and a pH of about 6. Another such formulation contains about 30mg/mL anti- Α β antibody, about 10mM succinate, about 10mM methionine, about 6% mannitol and a pH of about 6.2. Yet another such formulation contains about 20mg/mL anti-a β antibody, about 10 mL-histidine, about 10mM methionine, about 4% mannitol, about 0.005% polysorbate 80 and a pH of about 6. Still other such formulations contain about 10mg/mL of anti-a β antibody, about 10mM succinate, about 10mM methionine, about 10% mannitol, about 0.005% polysorbate 80 and a pH of about 6.5.

Still other such formulations contain about 5mg/mL to about 20mg/mL of anti-a β antibody, about 5mM to about 10mM l-histidine, about 10mM methionine, about 4% mannitol, about 0.005% polysorbate 80 and a pH of about 6.0 to about 6.5. Still other such formulations contain about 5mg/mL to about 20mg/mL of anti- Α β antibody, about 5mM to about 10mM mL-histidine, about 10mM methionine, about 150mM nacl, about 0.005% polysorbate 80 and a pH of about 6.0 to about 6.5.

The invention also provides a formulation suitable for intravenous administration comprising about 20mg/mL of an anti-a β antibody, about 10mM l-histidine, about 10mM methionine, about 4% mannitol and a pH of about 6. Preferably, such formulations contain about 0.005% polysorbate 80.

The present invention provides methods for enhancing the stability of antigen-binding polypeptides, such as antibodies, in liquid pharmaceutical formulations, wherein the polypeptides may exhibit byproduct formation during storage in the liquid formulation. Thus, the method includes incorporating an antioxidant, such as methionine or the like, into the formulation in an amount sufficient to reduce the formation of by-products.

The present invention also provides a method for maintaining the stability of a humanized anti-a β antibody by storing at a temperature of about-50 ℃ to about-80 ℃ and then storing at about 2 ℃ to about 8 ℃, the method comprising (i) combining about 40mg/ml to about 60mg/ml of the humanized anti-a β antibody, about 1mg/ml to about 2mg/ml of L-histidine, about 1mg/ml to about 2mg/ml of methionine and about 0.05mg/ml of polysorbate 80; (ii) adjusting the pH to about 6.0; (iii) filtering, putting into a freezing container, and freezing; (iv) melting; (v) adding sufficient mannitol or NaCl and a diluent to obtain a humanized anti-A β antibody having about 4% or about 150mM NaCl mannitol, about 2mg/ml to about 20 mg/ml; about 5mM to about 10mM histidine; about 10mM methionine and about 0.005% polysorbate 80 final concentration; (vi) filtering; (vii) transferring into a glass vial and sealing; and (viii) stored at a temperature of from about 2 ℃ to about 8 ℃.

The invention also provides kits comprising a container containing a formulation as described herein and instructions for use.

The invention also provides a pharmaceutical unit dosage form comprising about 10mg to about 250mg of the humanized anti-A β antibody, about 4% mannitol or about 150mM NaCl, about 5mM to about 10mM histidine or succinate, and about 10mM methionine. Some such pharmaceutical unit dosage forms comprise from about 0.001% to about 0.1% polysorbate 80. Some such pharmaceutical unit dosage forms include from about 40mg to about 60mg, from about 60mg to about 80mg, from about 80mg to about 120mg, from about 120mg to about 160mg, or from about 160mg to about 240mg of the anti-a β antibody. Some such formulations may be held in glass vials at a temperature of about 2 ℃ to about 8 ℃ prior to administration to a patient.

In addition, the invention provides a therapeutic product comprising a formulation in a glass vial containing about 10mg to about 250mg of a humanized anti-A β antibody, about 4% mannitol or about 150mM NaCl, about 5mM to about 10mM histidine, and about 10mM methionine. Some such therapeutic products also include a use label that includes an indication of the volume required to achieve a dose of 0.15mg/kg to about 5mg/kg in a patient, as appropriate. Typically, the vial is a 1mL, 2mL, 5mL, 10mL, 25mL or 50mL vial. The dosage of some such therapeutic products is from about 0.5mg/kg to about 3mg/kg, preferably from about 1mg/kg to about 2 mg/kg. In certain such therapeutic products, the anti-A β antibody concentration is from about 10mg/ml to about 60mg/ml, preferably about 20 mg/ml. The therapeutic product preferably comprises about 0.005% polysorbate 80. Certain formulations of such therapeutic products may be administered subcutaneously or intravenously.

The invention also provides a method of prophylactic or therapeutic treatment of a disease characterized by a β precipitation, which method comprises intravenous or subcutaneous administration of a pharmaceutical unit dose as described herein.

Other features and advantages of the invention will become apparent from the following detailed description, and from the claims.

Drawings

FIG. 1 is a schematic representation of the approximate positions of IgG antibodies and interchain and intrachain disulfide bonds, glycosylation sites (hexagonal symbols), Complementarity Determining Regions (CDRs), framework regions (shaded), and predicted structures of the constant regions.

Figure 2 shows the complete amino acid sequences of the light and heavy chains of humanized 3D6 version 2(hu3d6.v2) anti- Α β antibodies, seq id nos: 1 and SEQ ID NO: 2. the light chain Complementarity Determining Regions (CDRs), i.e., CDR1, CDR2, CDR3 are located at residue positions 24-39, 55-61, and 94-102, respectively (upper panel). The heavy chain Complementarity Determining Regions (CDRs), namely CDR1, CDR2 and CDR3 are located at residue positions 40-44, 50-65, and 99-108, respectively (lower panel). The predicted intermolecular disulfide bonds are shown by the linkage of the cysteine residues involved. Cysteines predicted to form intramolecular disulfide bonds are underlined and their connectivity is indicated. The N-linked glycosylation consensus sites of the antibody heavy chains are shown in italics at residue position 299-. The predicted heavy chain C-terminal lysine is indicated in parentheses.

FIG. 3 is a graphical representation of the prediction of shelf life of antibody formulations prepared according to the present invention, with and without polysorbate 80(PS80), when stored at 5 ℃.

FIG. 4 is a graphical representation of the prediction of shelf life of antibody formulations prepared according to the present invention (with and without PS80) when stored at 25 ℃.

FIG. 5 is a graphical representation of the prediction of shelf life of antibody formulations prepared according to the present invention (with and without PS80) when stored at 40 ℃.

FIG. 6 is a graphical representation of the prediction of degradation of an antibody formulation with PS80 prepared according to the present invention when stored at 5 ℃.

FIG. 7 is a schematic representation of a Size Exclusion Chromatography (SEC) assay performed on an antibody preparation with PS80 prepared according to the present invention stored at 5℃, further repeated to reduce assay variability.

FIG. 8 is a graph showing the prediction of degradation of an antibody formulation prepared according to the present invention without PS80 when stored at 5 ℃.

Figure 9 shows a chromatogram illustrating that the presence of PS80 allows the conversion of by-products formed within a stabilized polypeptide formulation from a high molecular weight species to a low molecular weight species without altering the monomeric antibody profile.

FIG. 10 is a schematic representation of the inhibition of undesirable byproduct formation in a polypeptide formulation containing IgG4, particularly high molecular weight polypeptide aggregates, after the addition of an antioxidant, such as free methionine.

FIG. 11 is a schematic showing the inhibition of the formation of undesirable by-products in a polypeptide formulation containing IgG2, particularly high molecular weight polypeptide aggregates, after the addition of an antioxidant, such as free methionine.

Detailed Description

For a clearer understanding of the specification and claims, the following definitions are provided below where appropriate.

In the present specification, the term "amyloid disease" includes any disease associated with (or caused by) formation or precipitation of insoluble amyloid fibrils. Exemplary amyloid diseases include, but are not limited to, systemic amyloidosis, alzheimer's disease, mature diabetic attacks, parkinson's disease, huntington's disease, frontotemporal dementia, and prion-related transmissible spongiform encephalopathies (kuru and creutzfeldt-jakob disease in humans, scrapie and BSE in sheep and cattle, respectively). Different amyloidogenic diseases are defined or characterized by the nature of the deposited fibrous polypeptide component. For example, for subjects or patients with alzheimer's disease, beta-amyloid (e.g., wild-type, variant, or truncated beta-amyloid) is a characteristic polypeptide component of the amyloid deposit. Thus, alzheimer's disease is an example of a "disease characterized by a β deposits" or a "disease associated with a β deposits", for example, in the brain of a subject or patient.

The terms "β -amyloid protein", "β -amyloid peptide", "β -amyloid protein", "a β" and "a β peptide" may be used interchangeably in this specification.

The term "a β binding polypeptide" includes polypeptides capable of specifically binding to a β peptide or to an internal epitope of said a β peptide. Generally, an a β binding polypeptide comprises at least a functional portion of an immunoglobulin or immunoglobulin-like domain, e.g., a receptor comprising one or more variable regions or Complementarity Determining Regions (CDRs) that confer specific binding characteristics to the polypeptide. Preferred antigen binding polypeptides include antibodies, e.g., IgM, IgG1, IgG2, IgG3, or IgG 4.

The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (molecules that contain an antigen binding site that specifically binds to an antigen), including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies, and single chain antibodies (scFvs). In the present specification, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a population of antibody molecules that contain only one antigen binding site capable of recognizing and binding to a particular epitope of a target antigen, e.g., an epitope of a β. Monoclonal antibody compositions thus typically exhibit individual binding specificity and affinity for a particular target antigen with which they are immunoreactive. The term "single chain antibody" refers to a protein having a two polypeptide chain structure consisting of one heavy chain and one light chain, which chains are stable, e.g., via an intrachain polypeptide linker, with the ability to specifically bind an antigen. Techniques for making single chain antibodies specific for a target antigen have been described, for example, in U.S. Pat. No. 4,946,778. The term "antibody fragment" includes F (ab ') 2 fragments, Fab' fragments, Fd fragments, Fv fragments, and single domain antibody fragments (DAbs). Immunologically active portions of immunoglobulins include, for example, F (ab) and F (ab') 2 fragments. Methods for the construction of Fab fragments have been described, for example, in Huse, et al (1989) Science246: 12751281). Other antibody fragments can be prepared by techniques well known in the art, including, but not limited to, (i) F (ab') 2 fragments produced by pepsin digestion of antibody molecules; (ii) fab fragments produced by reducing the disulfide bond of the F (ab') 2 fragment; (iii) treating the Fab' fragments produced by the antibody molecule with papain and a reducing agent; and (iv) Fv fragments. Various fragments can also be prepared using recombinant engineering techniques recognized in the art. Non-human antibodies can be "humanized" by methods such as those described in U.S. Pat. No. 5,225,539. In one approach, non-human CDRs can be inserted into human antibodies or consensus antibody framework sequences. Other changes can then be introduced into the antibody framework to modulate its affinity or immunogenicity.

The term "domain" refers to a globular region of a heavy or light chain polypeptide containing an immunoglobulin fold. The immunoglobulin fold comprises the beta-sheet secondary structure and contains a single disulfide bond. Domains may be further referred to herein as "constant" or "variable" in the context of sequence variations that are relatively absent from the various types of domains in the case of "constant" domains, or significant variations within the various types of domains in the case of "variable" domains. Antibody or polypeptide "domains" are often used interchangeably in the art with antibody or polypeptide "regions". The "constant" domain of an antibody light chain may be used interchangeably with a "light chain constant region", "light chain constant domain", "CL" region or "CL" domain. Antibody heavy chain "constant" domains can be used interchangeably with "heavy chain constant region", "heavy chain constant domain", "CH" region or "CH" domain. The "variable" domain of an antibody light chain may be used interchangeably with the "light chain variable region", "light chain variable domain", "VL" region or "VL" domain. The "variable" domain of an antibody heavy chain may be used interchangeably with "heavy chain variable region", "heavy chain variable domain", "VH" region or "VH" domain.

The term "region" may also refer to portions of an antibody chain or antibody chain domain (e.g., portions of a heavy or light chain or portions of a constant or variable domain, as defined herein), as well as more discrete portions of the chain or domain. For example, light and heavy chains or light and heavy chain variable domains include "complementarity determining regions" or "CDRs" as defined herein, spaced apart in "framework regions" or "FRs".

The term "anti-a β antibody" includes antibodies (and fragments thereof) that are capable of binding to an epitope of a β peptide. Anti-a β antibodies include, for example, antibodies described in U.S. patent publication No. 20030165496a1, U.S. patent publication No. 20040087777a1, international patent publication No. WO02/46237A3, and international patent publication No. WO04/080419a 2. Other anti- Α β antibodies can be found, for example, in the descriptions of international patent publication nos. WO03/077858a2 and WO04/108895a2, entitled "humanized antibodies recognizing amyloid β peptides", international patent publication No. WO03/016466a2, entitled "anti- Α β antibodies", international patent publication No. WO0162801a2, entitled "humanized antibodies capable of chelating amyloid β peptides", and international patent application No. WO02/088306a2, entitled "humanized antibodies", and international patent application No. WO 03/070a 2, entitled "anti- Α β antibodies and uses thereof".

The term "fragment" refers to an antibody or portion of an antibody chain that contains fewer amino acid residues than the entire or complete antibody or antibody chain. The fragments may be obtained by chemical or enzymatic treatment of the intact or complete antibody or antibody chain. Fragments may also be obtained by recombinant means. Exemplary fragments include Fab, Fab ', F (ab') 2, Fabc, and/or Fv fragments. The term "antigen-binding fragment" refers to an immunoglobulin or polypeptide fragment of an antibody that binds to an antigen or competes for specific antigen binding with the intact antibody from which the fragment was obtained.

The term "conformation" refers to the tertiary structure of a protein or polypeptide, e.g., an antibody, antibody chain, domain or region thereof. For example, the phrase "light (or) heavy chain conformation" refers to the tertiary structure of the light (or heavy) chain variable region, while the phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiary structure of an antibody or fragment thereof.

In exemplary embodiments, the antibody does not exhibit cross-reactivity (e.g., does not cross-react with the non-A β peptide or with a distal or distal epitope on A β.) an "apparent" or preferred binding includes at least 10^-6，10^-7，10^-8，10^-9M, or 10^-10The affinity of M binds. Affinity greater than 10^-7M, preferably greater than 10^-8M is more preferred. All of these numerical ranges set forth in this specification are included within the scope of the present invention, and preferably, binding affinity can be expressed as a range of affinities, e.g., 10^-6To 10^-10M, preferably 10^-7To 10^-10M, more preferably 10^-8To 10^-10And M. An antibody that "does not exhibit significant cross-reactivity" is one that does not significantly bind to an undesired entity (e.g., an antibody that does not exhibit significant cross-reactivity)For example, an antibody that specifically binds to A β can significantly bind to A β but does not significantly react with a non-A β protein or peptide (e.g., a non-A β protein or peptide contained in a plaque).

Binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab ', F (ab') 2, Fabc, Fv, single chain, and single chain antibodies. With the exception of "bispecific" or "bifunctional" immunoglobulins or antibodies, it is generally accepted that the binding sites of the immunoglobulins or antibodies are identical. A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including techniques of hybridoma fusion or Fab' fragment ligation. See, for example, Songsivilai & Lachmann, Clin. exp. Immunol.79: 315-; kostelnyetal, J.Immunol.148, 1547-1553 (1992).

An "antigen" is a molecule (e.g., a protein, polypeptide, peptide, carbohydrate, or small molecule) that contains an antigenic determinant to which an antibody is capable of specifically binding.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody (or antigen-binding fragment thereof) is capable of specifically binding. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids that are contiguous by tertiary folding of the protein. Epitopes formed from contiguous amino acids are generally retained when exposed to denaturing solvents, whereas epitopes formed from tertiary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids present in a unique spatial conformation. Methods for determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, epitomeppapingprotocolsinmolecular biology, vol.66, g.e.morris, Ed. (1996).

The term "stable formulation" or "stable liquid polypeptide formulation" includes formulations in which the polypeptide substantially maintains its physical and chemical identity and integrity when stored. Various analytical techniques for determining protein stability are available in the art and are described herein (reviewed in PeptindProteinDrugDelivery, 247-. Stability can be measured at a selected temperature for a selected period of time. For rapid assays, the formulations may be stored at higher or "accelerated" temperatures, for example at 40 ℃ for 2 weeks to 1 month or more, during which time stability is measured. In exemplary embodiments, the formulation has difficulty forming by-products of the component polypeptides, such as high molecular weight aggregate products, low molecular weight degradation or fragmentation products, or mixtures thereof. The term "stability" refers to the length of time that a molecular species, such as an antibody, retains its original chemical identity, e.g., primary structure, secondary structure, and/or tertiary structure.

The term "by-product" includes undesirable products that impair or reduce the proportion of a therapeutic polypeptide in a given formulation. Common by-products include aggregates of the therapeutic polypeptide, fragments of the therapeutic polypeptide (e.g., produced by degradation due to deamidation or hydrolysis of the polypeptide), or mixtures thereof.

The term "high molecular weight polypeptide aggregate" includes aggregates of a therapeutic polypeptide, fragments of a therapeutic polypeptide (e.g., resulting from degradation of the polypeptide, e.g., by hydrolysis), or mixtures thereof, which subsequently aggregate. Generally, the high molecular weight aggregates are complexes having a molecular weight higher than the molecular weight of the therapeutic monomeric polypeptide. In the case of antibodies, e.g., IgG antibodies, such aggregates are typically greater than about 150 kD. However, in the case of other therapeutic polypeptides, e.g., single chain antibodies having a molecular weight of typically 25kD, such aggregates typically have a molecular weight greater than about 25 kD.

The term "low molecular weight polypeptide degradation product" includes, for example, fragments of therapeutic polypeptides, e.g., fragments resulting from deamidation or hydrolysis. Generally, the low molecular weight degradation products are complexes having a molecular weight less than the molecular weight of the therapeutic monomeric polypeptide. In the case of antibodies, e.g., IgG antibodies, such degradation products are below about 150 kD. However, in the case of other therapeutic polypeptides, e.g., single chain antibodies having a molecular weight of typically 25kD, such aggregates typically have a molecular weight of less than about 25 kD.

The term "route of administration" includes art-recognized routes of administration for delivering a therapeutic polypeptide, e.g., parenteral, intravenous, intramuscular, subcutaneous, intracranial, or epidural. For the treatment of neurodegenerative diseases of therapeutic polypeptide administration, intravenous, epidural or intracranial administration is desired.

In this specification, the term "treatment" is defined as the administration or administration of a therapeutic agent to a patient, or to an isolated tissue or cell line from a patient, who has a disease, exhibits symptoms of a disease, or is susceptible to a disease, with the aim of curing, treating, ameliorating, delaying, alleviating, altering, correcting, ameliorating, improving, or affecting the disease, the symptoms of the disease, or the susceptibility to the disease.

The term "effective dose" or "effective amount" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest a disease and its complications in a patient already suffering from the disease. Effective amounts for this use depend on the severity of the infection and the general state of the patient's own immune system.

The term "patient" includes human and other mammalian subjects who have received prophylactic or therapeutic treatment.

In this specification, the term "dosage unit form" (or "unit dosage form") refers to physically discrete units suitable as unitary dosages for the patients to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, diluent or excipient. The description of the unit dosage forms of the invention is provided by and directly dependent upon the active compound and the particular therapeutic effect to be achieved, as well as the inherent deficiencies in the art of compounding such active compounds for the treatment of patients.

Actual dosage levels of active ingredient (e.g., a β polypeptide) in the formulations of the invention can be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, an amount of the composition, and a mode of administration without toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition employed in the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition being administered, the age, sex, weight, health status of the patient, the general health and prior medical history of the subject patient, and like factors well known in the medical arts.

In the present specification, the term "diluent" refers to a solution suitable for altering or achieving the exemplary or appropriate concentrations as described herein.

SUMMARY

The present invention provides formulations suitable for A β binding polypeptides, in particular, anti-A β antibodies, and portions and/or fragments thereof. In certain aspects, the invention provides stable liquid polypeptide formulations suitable for therapeutic use. In particular, the invention provides for the stabilization of Α β binding polypeptides, e.g., antibodies, and antigen-binding fragments thereof, for use in the treatment of amyloid diseases and/or disorders. In particular, the present invention provides stable formulations, such that the active therapeutic polypeptide remains stable for extended periods of time, and can be administered by a variety of routes of administration. This is crucial for those a β binding polypeptides (e.g., antibodies) that are intended for the treatment of amyloid diseases and/or disorders. In other aspects, the invention provides unique stable antibody formulations, e.g., which are capable of remaining stable under various stress conditions, such as freezing, lyophilization, heating, and/or reduction. In addition, exemplary formulations of the invention are capable of maintaining antibody stability, biological activity, purity and quality over extended periods of time (e.g., a year or even longer during formulation storage), even under unfavorable temperature conditions. In addition, exemplary formulations of the invention can be suitable for administration to (e.g., intravenously to) a subject or patient, e.g., a human suffering from or predicted to suffer from an amyloidogenic disease and/or disorder.

Preparation

In one aspect, the invention provides a stable formulation comprising an a β binding polypeptide. A tonicity agent, wherein the tonicity agent is present in an amount sufficient to render the stable formulation suitable for intravenous infusion, and an amino acid or derivative thereof, wherein the amino acid or derivative thereof is present in an amount sufficient to maintain a physiologically suitable pH. In an exemplary embodiment, the present invention provides a stable formulation comprising an anti-a β antibody, mannitol and histidine.

In one embodiment, the invention provides a stable formulation comprising an a β binding polypeptide. A tonicity agent, wherein the tonicity agent is present in an amount sufficient to render the formulation suitable for intravenous infusion, and an amino acid or derivative thereof, wherein the amino acid or derivative thereof is present in an amount sufficient to maintain a physiologically suitable pH. In an exemplary embodiment, the tonicity agent is mannitol. In other exemplary embodiments, the amino acid is histidine.

In another aspect, the invention provides stable formulations comprising an a β binding polypeptide. Suitable a β binding polypeptides for stabilization in the formulations of the invention include antibodies and fragments thereof, and in particular, antibodies capable of binding to a therapeutic target involved in an amyloid disease or disorder. Thus, by adding an antioxidant in an amount sufficient to inhibit the formation of such by-products, the therapeutic polypeptide is stabilized in accordance with the present invention, avoiding the formation of by-products, typically high molecular weight aggregates, low molecular weight degradation fragments, or mixtures thereof. Antioxidants include methionine and its analogs at concentrations sufficient to achieve the desired inhibition of undesirable by-products, as described below. Optionally, the stabilized polypeptide formulation of the invention may further comprise a tonicity agent, wherein the tonicity agent is present in an amount sufficient to render the stabilized formulation suitable for several routes of administration, e.g., intravenous infusion, and an amino acid or derivative thereof, wherein the amino acid or derivative thereof is present in an amount sufficient to maintain a physiologically suitable pH. In an exemplary embodiment, the present invention provides a stable formulation comprising an anti-a β antibody, methionine, mannitol and histidine.

In one embodiment, the present invention provides a stable liquid formulation comprising a therapeutically active a β binding polypeptide, wherein the polypeptide is capable of forming by-products during storage, and an antioxidant, wherein the antioxidant is present in an amount sufficient to reduce the formation of by-products during storage of the formulation. In an exemplary embodiment, the antioxidant is methionine or an analog thereof.

In some embodiments of the invention, the a β binding polypeptide is selected from the group consisting of an antibody, an antibody Fv fragment, an antibody Fab' (2) fragment, an antibody Fd fragment, a single chain antibody (scFv), a single domain antibody fragment (Dab), a β -sheet polypeptide comprising at least one antibody Complementarity Determining Region (CDR), and a non-globular polypeptide comprising at least one antibody complementarity determining region. In exemplary embodiments of the invention, A β binding polypeptide may be about 0.1mg/ml to about 60 mg/ml. In other exemplary embodiments, the formulations of the invention include about 30mg/ml of A β binding polypeptide. In other exemplary embodiments, the formulations of the invention include about 20mg/ml of A β binding polypeptide. In yet another exemplary embodiment, a formulation of the invention includes about 17mg/ml of A β binding polypeptide.

In an exemplary embodiment of the invention, the a β binding polypeptide is an anti-a β antibody. In some embodiments of the invention, the anti a β antibody is selected from a humanized 3D6 antibody, a humanized 10D5 antibody, a humanized 12B4 antibody, a humanized 266 antibody, a humanized 12a11 antibody, or a humanized 15C11 antibody. In an exemplary embodiment of the invention, the epitope to which the anti-A β antibody binds comprises residues selected from the group consisting of A β amino acids 1-7, 1-5, 3-7, 3-6, 13-28, 16-21, 19-22, 33-40 and 33-42. In some embodiments of the invention, the anti-a β antibody is of a subtype selected from the group consisting of human IgG1, IgG2, IgG3 and IgG 4. In a particular embodiment of the invention, the anti-a β antibody is of the human IgG1 subtype.

The a β polypeptide is capable of forming a byproduct selected from the group consisting of high molecular weight polypeptide aggregates, low molecular weight polypeptide degradation products, and combinations thereof. The high molecular weight aggregate may comprise an antibody to antibody complex, an antibody to antibody fragment complex, an antibody fragment to antibody fragment complex, or a combination thereof. The low molecular weight polypeptide degradation products can include antibody light chains, antibody heavy chains, antibody light and heavy chain complexes, antibody fragments, and combinations thereof.

In one embodiment of the invention, a liquid formulation according to the invention comprises an a β -binding polypeptide, mannitol and histidine. In an exemplary embodiment of the invention, the a β binding polypeptide is an anti-a β antibody. In some exemplary embodiments of the invention, the anti a β antibody is selected from the group consisting of a humanized 3D6 antibody, a humanized 10D5 antibody, a humanized 12B4 antibody, a humanized 266 antibody, a humanized 12a11 antibody, and a humanized 15C11 antibody. In other embodiments of the invention, the epitope to which the anti-A β antibody binds comprises residues selected from the group consisting of A β amino acids 1-7, 1-5, 3-7, 3-6, 13-28, 16-21, 19-22, 33-40, and 33-42. In some embodiments of the invention, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG 4. In a particular embodiment of the invention, the antibody is of the IgG1 subtype.

In exemplary embodiments of the invention, the anti-A β antibody may be about 0.1mg/ml to about 200 mg/ml. In other exemplary embodiments, the anti A β antibody may be about 20 mg/ml.

In some embodiments of the invention, the formulations of the invention include mannitol in an amount sufficient to maintain isotonicity of the formulation. In an exemplary embodiment of the invention, the mannitol is about 2% w/v to about 10% w/v. In other exemplary embodiments of the invention, mannitol is about 4%. In other exemplary embodiments, the mannitol is about 6%. In other exemplary embodiments, the mannitol is about 10%.

In some embodiments of the invention, the formulations of the invention include a sufficient amount of histidine to maintain a physiologically suitable pH. In exemplary embodiments of the invention, histidine is in the range of about 0.1mM to about 25 mM. In other exemplary embodiments, histidine is about 10 mM.

In some embodiments of the invention, the formulations of the invention comprise from about 0.1mM to about 25mM succinate. In an exemplary embodiment, succinate is about 10 mM.

In some embodiments of the invention, the formulation of the invention further comprises an antioxidant. In an exemplary embodiment, the antioxidant is methionine or an analog thereof. In one embodiment of the invention, methionine or an analog is about 0.1mM to about 25 mM. In other embodiments, methionine or the like is about 10 mM.

In some embodiments of the invention, the formulations of the invention further comprise a stabilizer. In an exemplary embodiment of the invention, the stabilizer is polysorbate 80. In some embodiments, the polysorbate 80 is about 0.001% w/v to about 0.01% w/v. In other embodiments, the polysorbate 80 is about 0.005% w/v. In other embodiments of the invention, the polysorbate 80 is about 0.01% w/v.

In some embodiments of the invention, the pH of the formulation is from about 5 to about 7. In an exemplary embodiment of the invention, the pH of the formulation is about 5.5. In other exemplary embodiments, the pH of the formulation is about 6.0. In other exemplary embodiments, the pH of the formulation is about 6.2. In other exemplary embodiments, the pH of the formulation is about 6.5.

In some embodiments, the formulation is stable to freezing conditions. In other embodiments of the invention, the formulation is suitable for intravenous administration. In exemplary embodiments of the invention, the formulations may be suitable for intramuscular or subcutaneous administration. In exemplary embodiments, the formulation may be suitable for delivery to the brain of a subject.

In some embodiments, the formulation may be suitable for delivery to the spinal fluid of a subject. In other embodiments, the formulation is substantially free of preservatives.

In some embodiments of the invention, the formulation is stable for at least about 12 months. In some embodiments, the formulation is stable for at least about 18 months. In some embodiments of the invention, the formulation is stable for at least about 24 months. In some embodiments of the invention, the formulation is stable for at least about 30 months.

In exemplary embodiments of the invention, the formulations may remain stable at about-80 ℃ to about-40 ℃. In exemplary embodiments, the formulation may remain stable at about 0 ℃ to about 25 ℃. Preferably, the formulation can remain stable at 2 ℃ to about 8 ℃.

In a particular embodiment of the invention, a formulation suitable for intravenous administration comprises about 20mg/ml anti-A β antibody, about 10 mML-histidine, about 10mM methionine, about 4% mannitol and a pH of about 6. In other particular embodiments, formulations suitable for intravenous administration include about 30mg/ml anti-a β antibody, about 10mM l-histidine, about 10mM methionine, about 6% mannitol and a pH of about 6.2. A preferred formulation suitable for intravenous administration comprises about 20mg/ml anti-a β antibody, about 10mM l-histidine, about 10mM methionine, about 4% mannitol, about 0.005% polysorbate 80, and a pH of about 6. In yet another exemplary embodiment of the invention, a formulation suitable for intravenous administration comprises about 10mg/ml of an anti-a β antibody, about 10mM l-histidine, about 10mM methionine, about 10% mannitol, about 0.005% polysorbate 80, and a pH of about 6.5.

In some embodiments of the aforementioned formulations according to the present invention, the anti-a β antibody is selected from a humanized 3D6 antibody, a humanized 10D5 antibody, a humanized 12B4 antibody, a humanized 266 antibody, a humanized 12a11 antibody, or a humanized 15C11 antibody. In exemplary embodiments, the anti-A β antibody binds to an epitope selected from within amino acid residues 1-7, 1-5, 3-7, 3-6, 13-28, 16-21, 19-22, 33-40, and 33-42 of A β. In some formulations, anti-A β antibodies bind to a discontinuous epitope within residues 1-7 of A β 13-28. In some such formulations, the antibody is a bispecific antibody or an antibody prepared by a method described in International patent publication No. WO 03/070760. In some such formulations, the epitope is a discontinuous epitope.

In yet another aspect of the invention, a pharmaceutical unit dosage form comprises an effective amount of the formulation of any of the preceding embodiments for treating a disease by administering the dosage form to a patient. In an exemplary embodiment, the pharmaceutical unit dosage form is a container containing a formulation according to the present invention. In exemplary embodiments, the container is a vial containing from about 1mg to about 2000mgA β binding polypeptide. In other exemplary embodiments, the vial contains from about 50mg to about 1500mg of a β binding polypeptide. In yet another exemplary embodiment, the vial contains from about 5mg to about 50mg of a β binding polypeptide.

In exemplary embodiments, the vial has a volume of about 2 to about 100 ml. In other embodiments, the vial has a volume of about 2 to about 10 ml.

In some embodiments, the pharmaceutical unit dosage form according to the invention is suitable for intravenous infusion into a patient.

Also described in this specification are kits comprising pharmaceutical unit dosage forms according to the invention, and instructions for their use. In one embodiment of the invention, the container containing the pharmaceutical unit dosage form is a container having a use label. In exemplary embodiments, the container is labeled for prophylactic use. In other exemplary embodiments, the container is labeled for therapeutic use.

The present invention provides a method of enhancing the stability of an a β -binding polypeptide in a liquid pharmaceutical formulation, wherein said polypeptide exhibits byproduct formation during storage in the liquid formulation, comprising incorporating into said formulation an antioxidant in an amount sufficient to reduce byproduct formation of said polypeptide. In exemplary embodiments, the a β binding polypeptide component is selected from the group consisting of an antibody, an antibody Fv fragment, an antibody Fab' (2) fragment, an antibody Fd fragment, a single chain antibody (scFv), a single domain antibody fragment (Dab), a β -sheet polypeptide comprising at least one antibody Complementarity Determining Region (CDR), and a non-globular polypeptide comprising at least one antibody complementarity determining region. In one embodiment, the by-product is selected from the group consisting of high molecular weight polypeptide aggregates, low molecular weight polypeptide degradation products, and combinations thereof. In another embodiment, the antioxidant is selected from methionine and its analogs.

In some embodiments, the method of making a formulation of any of the preceding embodiments of the invention comprises combining excipients of the formulation. In an exemplary embodiment, a method of making a formulation as described in any of the preceding embodiments comprises combining an a β binding polypeptide with one or more diluents, wherein the one or more diluents include excipients of the formulation.

In an exemplary embodiment, a method of making a pharmaceutical unit dosage form comprises combining the formulations of any of the preceding embodiments in a suitable container. In another exemplary embodiment, a method of making a formulation of any of the preceding embodiments comprises combining a solution comprising an a β binding polypeptide and at least a portion of an excipient of the formulation with a diluent comprising a residual excipient.

Polypeptides for use in the stabilized formulations of the invention

The polypeptides for use in the formulations of the invention as described herein may be prepared using techniques which are applicable to those established in the art and include, for example, synthetic techniques (e.g., recombinant techniques as well as polypeptide synthesis or combinations of these techniques), or may be isolated from endogenous sources of the polypeptides. In certain embodiments of the invention, the selected polypeptide is an antigen-binding polypeptide, more preferably, an antibody, and particularly, an anti-a β antibody. Techniques for the preparation of antigen-binding polypeptides, particularly antibodies, are described below.

Polyclonal antibodies

Polyclonal antibodies can be prepared by immunizing an appropriate subject with an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized target antigen. If desired, antibody molecules directed against the target antigen can be isolated from the mammal (e.g., from blood) and further purified by well-known techniques, such as protein ASepharose chromatography to obtain the antibody, e.g., an IgG fraction. At an appropriate time after immunization, e.g., when the anti-antigen antibody titer is highest, antibody-producing cells are obtained from the subject and purified by standard techniques, e.g., initially described by Kohlerand Milstein (1975) Nature256:495-497) (see also, Brown et al (1981) J.Immunol.127: 539-46; brown et al (1980) J.biol.chem.255: 4980-83; yehead (1976) Proc.Natl.Acad.Sci.USA76: 2927-31; and Yehetal, (1982) int.J. cancer29:269-75) to prepare monoclonal antibodies. For the preparation of chimeric polyclonal antibodies, see Buechler, U.S. patent No. 6,420,113.

Monoclonal antibodies

The generation of monoclonal antibodies can be accomplished by fusing lymphocytes with an immortalized cell line using any of a number of well-known methods (see, e.g., G.Galfreetal (1977) Nature266: 55052; Geftertel.somaticCellGenet, supra; Lerner, YaleJ.biol.Med., supra; Kenneth, monoclonal antibodies, supra). In addition, one of ordinary skill in the art will appreciate that many variations of this method can be successfully practiced. Typically, the immortalized cell line (e.g., myeloma cell line) is also from the same mammalian species as the lymphocytes. For example, murine hybridomas may be prepared by fusing lymphocytes from mice immunized with an immunogenic formulation of the invention with an immortal mouse cell line. The preferred immortalized cell line is a murine myeloma cell line that is sensitive to a medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a variety of myeloma cell lines may be used as fusion partners according to standard techniques, for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma cell lines. These myeloma cell lines are available from ATCC. Generally, HAT-sensitive mouse myeloma cells can be fused to mouse spleen cells using polyethylene glycol ("PEG"). Hybridoma cells resulting from such fusions can then be screened by killing HAT medium unfused and without proliferating fused osteoblast myeloma cells (unfused splenocytes die after several days because of inability to transform). Hybridoma cells producing a monoclonal antibody of the invention can be assayed by screening for antibodies derived from hybridoma culture supernatants that bind to a target antigen, e.g., a β, using standard ELISA assays.

Recombinant antibodies

In addition to preparing hybridomas that secrete monoclonal antibodies, monoclonal antibodies can also be identified and isolated by screening recombinant combinatorial immunoglobulin libraries (e.g., antibody phage display libraries) having a target antigen to isolate immunoglobulin library members that bind to the target antigen. Kits for generating and screening phage display libraries are commercially available (e.g., pharmacia Recombinant Phageanthroenclosure System, CatalogNo. 27-9400-01; and Stratagene SurfZAP^TMPhageDisplaykit, CatalogNo.240612). Furthermore, examples of methods and reagents particularly suited for generating and screening antibody display libraries can be found in, for example, U.S. Pat. Nos. 5,223,409 to Ladner et al; PCT International publication No. WO92/18619 by Kang et al; PCT International publication No. WO91/17271 to Dower et al(ii) a PCT International publication No. WO92/20791 by Winter et al; PCT International publication No. WO92/15679 to Markland et al; PCT International publication No. WO93/01288 to Breitling et al; PCT International publication No. WO92/01047 to McCafferty et al; PCT International publication No. WO92/09690 to Garrrad et al; PCT International publication No. WO90/02809 to Ladner et al; fuchsetal (1991) Bio/Technology9: 1370-; hayetal (1992) hum.antibody.hybridoma 3: 81-85; huseeet (1989) Science246: 1275-; griffithtotal (1993) EMBOJ12: 725-; hawkinsetal (1992) J.mol.biol.226: 889-896; clarkson et al (1991) Nature352: 624-; gramet (1992) Proc.Natl.Acad.Sci.USA89: 3576-3580; garradet (1991) Bio/Technology9: 1373-1377; hoogenbometal (1991) Nuc. acid Res.19: 4133-; barbasetal (1991) Proc. Natl. Acad. Sci. USA88: 7978-; and McCaffertyetal. Nature (1990)348: 552-.

Chimeric and humanized antibodies

In addition, recombinant antibodies, e.g., containing human and non-human portions, chimeric and humanized monoclonal antibodies, which can be prepared using standard recombinant DNA techniques, are also within the scope of the invention.

The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that contains at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., light chain or heavy chain, respectively) having variable regions that include variable framework regions derived substantially from a human immunoglobulin or antibody and Complementarity Determining Regions (CDRs) derived substantially from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further having constant regions (e.g., at least one constant region or portion thereof in the case of a light chain and three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes variable framework regions derived substantially from a human immunoglobulin or antibody and Complementarity Determining Regions (CDRs) derived substantially from a non-human immunoglobulin or antibody.

The phrase "substantially from a human immunoglobulin or antibody" or "substantially human" means that the region is at least 80-90%, 90-95%, or 95-99% identical (i.e., local sequence identity) to a human framework or constant region sequence when compared to the human immunoglobulin or antibody amino acid sequence for comparison purposes, thereby allowing, for example, conservative substitutions, consensus substitutions, germline substitutions, back mutations, and the like. The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, back mutations, etc., is often referred to as "optimization" of the humanized antibody or chain. The phrase "substantially from non-human immunoglobulins and antibodies" or "substantially non-human" means having at least 80-95%, preferably at least 90-95%, more preferably 96%, 97%, 98% or 99% identity to immunoglobulin or antibody sequences of a non-human organism, e.g., a non-human mammal.

Thus, all regions or residues of a humanized immunoglobulin or antibody, or humanized immunoglobulin or antibody chain, except the CDRs, are substantially identical to corresponding regions or residues of one or more native human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue on a second amino acid or nucleotide sequence that occupies the same (i.e., equal) position region or residue on the first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparison purposes.

The term "significant identity" refers to two polypeptide sequences that, when optimally aligned, e.g., using default GAP (GAP) weights by the GAP or BESTFIT programs, have at least 50-60% sequence identity, preferably at least 60-70% sequence identity, more preferably at least 70-80% sequence identity, more preferably at least 80-90% sequence identity, even more preferably at least 90-95% sequence identity, and even more preferably at least 95% or even higher (e.g., 99% or higher) sequence identity. The term "substantial identity" refers to two polypeptide sequences that, when optimally aligned, e.g., using default GAP weights by the programs GAP or BESTFIT, have a sequence identity of at least 80-90%, preferably at least 90-95%, more preferably at least 95% or even greater (e.g., 99% or greater). For sequence alignment, one sequence is typically used as a reference sequence to which test sequences are aligned. When using a sequence alignment algorithm, the test and reference sequences are input into a computer, subsequent coordinates are specified, and sequence algorithm program parameters may be specified if desired. The sequence alignment algorithm can then calculate the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

The optimal alignment for sequence comparison can be performed by, for example, local homology algorithms of Smith & Waterman, adv.Appl.Math.2:482(1981), homology alignment algorithms of Needleman & Wunsch, J.Mol.biol.48:443(1970), similar methods of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA85:2444(1988), computerized application of these algorithms (GAP, BESTFIT, FASTA and TFASTA, Wisconsin genetics software Package, genetics computer group, 575Scien Dr., Madison, Wis)), or by visual inspection (see generally Ausubentl., CurrentProtocol.Scien. Biotechnology). Among these algorithms, one example that may be useful for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al, J.mol.biol.215:403 (1990). Software for performing BLAST analysis is publicly available through the national center for biotechnology information (national institutes for biotechnology information), which is available through the national institutes of health NCBI internet server (national institutes of health NCBI internet server). Typically, default program parameters can be used for sequence comparisons, although custom parameters can also be used as well. For amino acid sequences, the BLASTP program uses a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix as default parameters (see Henikoff & Henikoff, proc. natl. acad. sci. usa89:10915 (1989)).

Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. For classification purposes, amino acid substitutions may be classified as conservative, non-conservative, and amino acids are classified as: group I (hydrophobic side chains): leu, met, ala, val, leu, ile; group II (neutral hydrophilic side chains): cys, ser, thr; group III (acidic side chain): asp, glu; group IV (basic side chain): asn, gln, his, lys, arg; group V (residues affecting chain orientation): gly, pro; and group VI (aromatic side chains): trp, ryr, phe. Conservative substitutions involve substitutions between amino acids of the same group. Non-conservative substitutions involve exchanging members of one of these groups for members of the other group.

Preferably, the humanized immunoglobulin or antibody binds antigen with an affinity that is a factor of three, four or five relative to the affinity of the corresponding non-humanized antibody. For example, if the non-humanized antibody binding affinity of 10^-9M, the binding affinity of the humanized antibody is at least 3x10^-8M，4x10^-8M，5x10^-8M, or 10^-9When it is possible to confer specific binding properties or binding affinities to an intact immunoglobulin or antibody (or antigen-binding fragment thereof), then that chain is termed "direct antigen-binding". if the mutation affects (e.g., reduces) the binding affinity of the intact immunoglobulin or antibody (or antigen-binding fragment thereof) comprising that chain by at least one order of magnitude, then the mutation (e.g., a back-mutation) is believed to affect to a large extent the ability of the heavy chain or light chain to bind directly to antigen, compared to an antibody (or antigen-binding fragment thereof) comprising the same chain lacking the mutation, then the mutation is able to affect (e.g., reduce) the binding affinity of the intact immunoglobulin or antibody (or antigen-binding fragment thereof) comprising that chain by a factor of only 2, 3,4, then the mutation is able to affect to a large extent, e.g., by a factor of only 2, 3, and/or by a factor of less than the binding affinity of an antibody (or antigen-binding fragment thereof) comprising the same chain lacking the mutationLow) chain direct antigen binding capacity. "

The term "chimeric immunoglobulin" or antibody refers to an immunoglobulin or antibody whose variable regions are derived from one species and whose constant regions are derived from another species. Chimeric immunoglobulins or antibodies can be constructed by, for example, genetic engineering of immunoglobulin gene fragments belonging to different species. The term "humanized immunoglobulin" or "humanized antibody" is not intended to include chimeric immunoglobulins or antibodies, as described below. Although humanized immunoglobulins or antibodies are chimeric in their structure (i.e., contain regions from more than one protein), they also encompass additional features not found in chimeric immunoglobulins or antibodies (i.e., variable regions containing donor CDR residues and acceptor framework residues), as described herein.

Such chimeric and humanized monoclonal antibodies can be prepared by recombinant DNA techniques well known in the art, e.g., using Robinson et al, International patent application No. PCT/US 86/02269; akira et al, European patent application No. 184,187; taniguchi, M european patent application No. 171,496; morrison et al, European patent application No. 173,494; neuberger et al, International patent application publication No. WO 86/01533; cabilly et al, U.S. Pat. No. 4,816,567; cabilly et al, European patent application No. 125,023; betteret (1988) Science240: 1041-; liuetal (1987) Proc.Natl.Acad.Sci.USA84: 3439-; liuetal (1987) J.Immunol.139: 3521-3526; sunetal (1987) Proc.Natl.Acad.Sci.USA84: 214-; nishimuraet (1987) Canc. Res.47: 999-; woodetal (1985) Nature314: 446-449; and Shawet (1988) J.Natl.cancer Inst.80: 1553-1559); morrison, S.L. (1985) Science229: 1202-1207; oietal (1986) BioTechniques4: 214; winteru.s.patent5,225,539; joneset al (1986) Nature321: 552-525; verhoeyanental (1988) Science239: 1534; and Beidleretal (1988) J.Immunol.141: 4053-4060.

Human antibodies from transgenic animals and phage display

Alternatively, it is possible to prepare transgenic animals (e.g., mice) that are capable of producing human antibodies with intact components when immunized in the absence of endogenous immunoglobulin production. For example, it has been reported that the antibody heavy chain junction region (J) is thought to be present in chimeric and germline mutant mice_H) The homozygous deletion of the gene results in complete suppression of endogenous antibody production. The transfer of human germline immunoglobulin gene arrangements in such germline mutant mice results in the production of human antibodies following antigen challenge. See, for example, U.S. patent nos. 6,150,584; 6,114,598, and 5,770,429.

Fully human antibodies can also be obtained from phage display libraries (Hoogenbometer, J.mol.biol., 227:381 (1991); Marksetal, J.mol.biol., 222:581-597 (1991)). Chimeric polyclonal antibodies can also be obtained from phage display libraries (Buechler et al, U.S. Pat. No. 6,420,113).

Bispecific antibodies, antibody fusion polypeptides, and single chain antibodies

Bispecific antibodies (BsAbs) are antibodies with at least 2 different epitope binding specificities. Such antibodies can be obtained from full-length antibodies or antibody fragments (e.g., f (ab)' 2 bispecific antibodies). Methods for making bispecific antibodies are well known in the art. Conventional preparation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, the two chains having different specificities (Millsteinetet, Nature, 305:537-539 (1983)). These hybridomas (cell hybridomas) produce a potential mixture of different antibody molecules due to the random arrangement of the immunoglobulin heavy and light chains (see WO93/08829 and Traveckereral, EMBOJ., 10:3655-3659 (1991)).

Bispecific antibodies also include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heteroconjugate may be conjugated to avidin, while the other moiety is conjugated to biotin or other payload. Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with various crosslinking techniques.

In yet another embodiment, the antibody can be chemically or genetically fused to a payload, e.g., a reactive, detectable, or functional moiety, e.g., an immunotoxin, to produce an antibody fusion polypeptide. The payload includes, for example, an immunotoxin, a chemotherapeutic agent, and a radioisotope, all of which are well known in the art.

Single chain antibodies may also be used for stabilization as described herein. The fragments comprise a heavy chain variable domain (VH) linked by a linker to a light chain variable domain (VL) which is capable of allowing interaction between each of the variable regions and reconstituting the antigen binding pocket of the parent antibody from which the VL and VH are obtained. See Gruberet al, J.Immunol., 152:5368 (1994).

It will be appreciated that any of the above polypeptide molecules, alone or in combination, may be suitable for use in the preparation of stable formulations according to the invention.

anti-A β antibodies

In general, the formulations of the invention include a variety of antibodies for the treatment of amyloid diseases, particularly Alzheimer's disease by targeting A β peptides.

The terms "A β antibody," "anti-A β antibody," and "anti-A β," which are used interchangeably in this specification, refer to antibodies that are capable of binding to one or more epitopes or epitopes of human Amyloid Precursor Protein (APP), A β protein, or both⁶⁹⁵，APP⁷⁵¹And APP⁷⁷⁰. The amino acids within APP are according to APP⁷⁷⁰The sequence of the isoforms (see, e.g., genbank accession No. p05067) obtains the indicated number. An example of the APP specific isoforms currently known to exist in humans is 6 as described in Kanget.al. (1987) Nature325:733-95 amino acid polypeptides designated as "normal" APP, the 751 amino acid polypeptides described in "Pontetal. (1988) Nature331:525-527(1988) and Tanzietal. (1988) Nature331:528-530, and the 770 amino acid polypeptides described in Kitaguchi et al (1988) Nature331: 530-532A β can be found both in" short form ", 40 amino acids long, and" long form ", 42-43 amino acids long as a result of in vivo or in situ proteolytic processing of APP by different secretases₄₀Consisting of the 672-711 residues of APP the long forms, e.g., A β₄₂Or A β₄₃A part of the APP hydrophobic region, which is responsible for the ability of A β to aggregate, can be found at the carboxy-terminus of A β, particularly in the case of long format A β peptide can be found in, or purified from, body fluids, such as cerebrospinal fluid, of humans and other mammals, including normal individuals and individuals suffering from amyloidogenic diseases.

As used herein, the terms "β -amyloid", "β -amyloid", "β 0-amyloid", "A β 1" and "A β 2 peptide" are used interchangeably A β 3 peptide (e.g., A β 439, A β 40, A β 41, A β 42 and A β 43) is a 4kD internal fragment of APP39-43 amino acid residues A β 40, e.g., consisting of residues 672 and 711 of APP and A β 42 consisting of residues 672 and 713 of APP A β peptide includes peptides cleaved from APP secretase and peptides synthesized with the same or substantially the same sequence as the cleaved products A β peptide can be obtained from a variety of sources, e.g., from tissue, cell lines, or body fluids (e.g., serum or cerebrospinal fluid.) for example, APP-expressing cells can be obtained, e.g., stably transfected with APP_717V→FObtained from a tissue source using prior art methods (see, e.g., Johnson-woodactual., (1997), proc.natl.acad.sci.usa94: 1550.) alternatively, the a β peptide can be synthesized using methods well known in the art&Co., NewYork, NY, 1992, p 77. Thus, it is possible to provideFor example, polynucleotides encoding the peptides or fusion peptides can be synthesized or molecularly cloned and inserted into an appropriate expression vector for transfection and heterologous expression in an appropriate host cell.A β peptide also refers to the relevant A β sequence generated by mutation from the A β region of a normal gene.

Exemplary epitopes or antigenic determinants to which the a β antibody can bind that can be found in human Amyloid Precursor Protein (APP), but preferably can be found in the a β peptide of APP. Exemplary epitopes or antigenic determinants within A β are located at the N-terminus, middle region, or C-terminus of A β. An "N-terminal epitope" is an epitope or antigenic determinant located at or including the N-terminus of the A.beta.peptide. Exemplary N-terminal epitopes include residues in the range of amino acids 1-10 or 1-12 of A.beta.and preferably 1-3, 1-4, 1-5, 1-6, 1-7, 2-6, 2-7, 3-6 or 3-7 from A.beta.42. Other exemplary N-terminal epitopes begin at residues 1-3 and terminate at residues 7-11 of A β. Other exemplary N-terminal epitopes include residues 2-4, 5, 6,7 or 8 of a β, residues 3-5, 6,7, 8 or 9 of a β, or residues 4-7, 8, 9 or 10 of a β 42. A "meditope" is an epitope or antigenic determinant that contains residues located within the middle or middle of an A β peptide. Exemplary intermediate epitopes include residues within amino acids 13-28 of A.beta.and preferably 14-27, 15-26, 16-25, 17-24, 18-23 or 19-22 from A.beta.. Other exemplary intermediate epitopes include residues within A.beta.amino acids 16-24, 16-23, 16-22, 16-21, 18-21, 19-21, 19-22, 19-23 or 19-24. A "C-terminal" epitope or antigenic determinant is located at or includes the C-terminus of the A.beta.peptide and includes residues within amino acids 33-40, 33-41 or 33-42 of A.beta.. A "C-terminal epitope" is an epitope or antigenic determinant that contains residues located at the C-terminus of the A.beta.peptide (e.g., within about 30-40 or 30-42 amino acids of A.beta.). Other exemplary C-terminal epitopes or antigenic determinants include residues 33-40 or 33-42 of A β.

When an antibody is described as binding to a particular residue, such as an epitope within a β 3-7, it is meant that the antibody specifically binds to a polypeptide containing the particular residue (i.e., in this case, a β 3-7). Such antibodies do not necessarily contact every residue within a β 3-7. Likewise, substitutions or deletions of each amino acid within a β 3-7 do not necessarily significantly affect its binding affinity. In various embodiments, the a β antibody is end-specific. In the present specification, the term "terminal specificity" means that an antibody is capable of specifically binding to the N-terminal or C-terminal residues of the a β peptide, but is not capable of recognizing the same residues when present in the longer a β class containing said residues or in APP. In various embodiments, the a β antibody has "C-terminal specificity". In the present specification, the term "C-terminal specificity" means that the antibody specifically recognizes the free C-terminus of the a β peptide. Examples of C-terminal specific a β antibodies include: an a β peptide that is capable of recognizing an a β peptide that terminates at residue 40 but does not recognize an a β peptide that terminates at residues 41, 42, and/or 43; (ii) is capable of recognizing a β peptides terminating at residue 42 but is incapable of recognizing a β peptides terminating at residues 40, 41, and/or 43; etc. of

In one embodiment, the Α β antibody may be A3D 6 antibody or variant thereof, or a 10D5 antibody or variant thereof, both disclosed in U.S. patent publication No. 20030165496a1, U.S. patent publication No. 20040087777a1, international patent publication No. WO02/46237A3, and international patent publication No. WO04/080419a 2. Descriptions of 3D6 and 10D5 may also be found, for example, in International patent publication Nos. WO02/088306A2 and WO02/088307A 2. Additional descriptions of 3D6 antibodies can be found in U.S. patent application No. 11/303,478 and International patent application No. PCT/US 05/45614. 3D6 is a monoclonal antibody (mAb) that is capable of specifically binding to an epitope located at the N-terminus of human β -amyloid peptide, particularly, at residues 1-5. In contrast, 10D5 is a monoclonal antibody that specifically binds to the N-terminal epitope of human β -amyloid peptide, particularly residues 3-6. Cell lines producing the 3D6 monoclonal antibody (RB963D6.32.2.4) were deposited at American Type Culture Collection (ATCC), Manassas, VA20108, usa under accession number PTA-5130 on 8/4/2003 under the provisions of the budapest treaty. The cell line producing the 10D5 monoclonal antibody (RB4410D5.19.21) was deposited at ATCC as PTA-5129 on day 8, 4/2003 under the provisions of the Budapest treaty.

Exemplary variant 3D6 antibodies are those, for example, humanized light chains comprising the variable region amino acid sequence shown as seq id No. 3 or seq id No. 5, and humanized heavy chain antibodies comprising the variable region amino acid sequence shown as seq id No. 4 or seq id No. 6. Other exemplary variant 3D6 antibodies are those, for example, antibodies having the humanized light chain amino acid sequence shown as SEQ ID NO. 7 and the humanized heavy chain amino acid sequence shown as SEQ ID NO. 8.

Exemplary variant 10D5 antibodies are those, for example, humanized light chains comprising the variable region amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 11 and humanized heavy chains comprising the variable region amino acid sequence shown as SEQ ID NO. 10 or SEQ ID NO. 12. Other exemplary variant 10D5 antibodies are those, for example, antibodies having the humanized light chain amino acid sequence shown as SEQ ID NO. 13 and the humanized heavy chain amino acid sequence shown as SEQ ID NO. 14. Such variant antibodies can also be found in WO02/088306A 2.

In yet another embodiment, the antibody may be a 12B4 antibody or a variant thereof, as described in U.S. patent publication No. 20040082762a1 and international patent publication No. WO03/077858a 2. 12B4 is a monoclonal antibody capable of specifically binding to an epitope located at the N-terminus of human β -amyloid peptide, in particular, the epitopes at residues 3-7.

Exemplary variant 12B4 antibodies are those, for example, humanized light chains (or light chains) comprising a variable region amino acid sequence as set forth in SEQ ID NO. 15 or SEQ ID NO. 17, and humanized heavy chains comprising a variable region amino acid sequence as set forth in SEQ ID NO. 16, SEQ ID NO. 18, or SEQ ID NO. 19.

In yet another embodiment, the antibody may be a 12A11 antibody or a variant thereof, as described in U.S. patent publication No. 20050118651A1 and U.S. patent application No. 11/303,478, International patent publication No. WO04/108895A2, and International patent application No. PCT/US 05/45614. 12A11 is a monoclonal antibody capable of specifically binding to an epitope located at the N-terminus, in particular, residues 3-7, of human β -amyloid peptide. The cell line producing the 12A11 monoclonal antibody was deposited at ATCC at 13.12.2005 under the Budapest treaty, accession number PTA-7271.

Exemplary variant 12A11 antibodies are those, for example, humanized light chains comprising the variable region amino acid sequence shown as SEQ ID NO. 20 and humanized heavy chains comprising the variable region amino acid sequence shown as SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO.24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO.27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 or SEQ ID NO. 41.

In yet another embodiment, the antibody may be a 6C6 antibody or a variant thereof, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide". 6C6 is a monoclonal antibody capable of specifically binding to an epitope located at the N-terminus of human β -amyloid peptide, in particular, residues 3-7. The cell line producing 6C6 antibody was deposited at ATCC as accession number PTA-7200 on day 1, 11/2005 under the Budapest treaty.

In yet another embodiment, the antibody can be a 2H3 antibody, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide. 2H3 is a monoclonal antibody capable of specifically binding to an epitope located at the N-terminus of human β -amyloid peptide, in particular, residues 2-7.

In yet another embodiment, the antibody can be a 3a3 antibody, as described in U.S. patent application No. __________. 3a3 is a monoclonal antibody capable of specifically binding to an epitope located at the N-terminus of human β -amyloid peptide, in particular, residues 3-7.

Cell lines producing the 2H3 and 3A3 monoclonal antibodies were deposited at ATCC at 13.12.2005 under the Budapest treaty, accession Nos. PTA-7267 and PTA-7269, respectively.

In yet another embodiment, the antibody may be a 15C11 antibody or a variant thereof, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide". 15C11 is a monoclonal antibody that is capable of specifically binding to an epitope located in the middle of human β -amyloid peptide, in particular, residues 19-22. The 15C11 monoclonal antibody-producing cell line was deposited at ATCC at 13.12.2005 under the Budapest treaty accession number PTA-727.

In yet another embodiment, the antibody can be a 266 antibody, as described in U.S. patent publication No. 20050249725a1 and international patent publication No. WO01/62801a 2. 266 is a monoclonal antibody capable of specifically binding to an epitope located in the middle of human β -amyloid peptide, in particular, residues 16-24. The 266 monoclonal antibody-producing cell line was deposited at ATCC at 20/7 of 2004 under the provisions of the Budapest treaty and was deposited under accession number PTA-6123.

Exemplary variant 266 antibodies are those, for example, antibodies having a humanized light chain comprising the variable region amino acid sequence shown as SEQ ID NO:42 or SEQ ID NO:44 and a humanized heavy chain comprising the variable region amino acid sequence shown as SEQ ID NO:43 or SEQ ID NO: 45. Other exemplary variant 266 antibodies are those, for example, having the humanized light chain amino acid sequence shown as SEQ ID NO. 46 and the humanized heavy chain amino acid sequence shown as SEQ ID NO. 47. Such variant antibodies can also be found in U.S. patent publication No. 20050249725A1 and International patent publication No. WO01/62801A 2.

In yet another embodiment, the antibody may be a 2B1 antibody or a variant thereof, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide". 2B1 is a monoclonal antibody capable of specifically binding to an epitope located in the middle of human β -amyloid peptide, in particular, residues 19-23.

In yet another embodiment, the antibody may be a 1C2 antibody or variant thereof, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide". 1C2 is a monoclonal antibody capable of specifically binding to an epitope located in the middle of human β -amyloid peptide, in particular, residues 16-23.

In yet another embodiment, the antibody may be a 9G8 antibody or a variant thereof, as described in U.S. patent application No. 11/304,986 and international patent application No. PCT/US05/45515 entitled "humanized antibody that recognizes beta amyloid peptide". 9G8 is a monoclonal antibody capable of specifically binding to an epitope located in the middle of human β -amyloid peptide, in particular, residues 16-21.

Cell lines producing antibodies 2B1, 1C2 and 9G8 were deposited at ATCC on 1/11 of 2005 under the Budapest treaty, accession numbers PTA-7202, PTA-7199 and PTA-7201, respectively.

Antibodies that specifically bind to epitopes located at the C-terminus of human β -amyloid peptide that may be suitable for use in the present invention include, but are not limited to 369.2B, as described in U.S. patent No. 5,786,180 entitled "monoclonal antibody 369.2B specific for β a4 peptide". Additional descriptions of antibodies suitable for use in the invention may be found, for example, in Busieneetal., (am.J.Pathol.165(3):987-95(2004)) Bardetal. (PNAS100(4):2023-8(2003)), Kajkowskietal. (J.biol.chem.276(22):18748-56(2001)), Gamesetal. (Ann.NYAcad.Sci.920:274-84(2000)), Bardetal. (Nat.Med.6(8):916-9(2000)), and International patent application No. WO03015691A2 entitled "effective and rapid improvement in cognition in patients with Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, or mild cognitive impairment, including administration of anti-A β antibodies". Additional descriptions of antibody fragments suitable for use in the present invention are found, for example, in Balesetal (Abstract P4-396, pageS587, presentedPtoster Session P4: Therapeutic Stregthenes-Therapeutic Stregthenes, Amyloid-Based) and Zamertal (Abstract P4-420, pageS593, presentedPtoster Session P4: Therapeutic and Therapeutic Stregthenes-phylogens, Amyloid-Based).

Antibodies suitable for use in the present invention may be recombinantly or synthetically prepared. For example, the cells can be cultured by recombinant cell culture methods using, for example, CHO cells, NIH3T3 cells,in particular embodiments, the antibody is a humanized anti-A β peptide 3D6 antibody that selectively binds to the A β peptide, more particularly, a humanized anti-A β peptide 3D6 antibody is intended to specifically bind to an NH 2-terminal epitope, e.g., amino acid residues 1-5 of the human β -amyloid 1-40 or 1-42 peptide in plaque deposits of the brain (e.g., in patients suffering from Alzheimer's disease).

FIG. 1 provides a schematic representation of the predicted structure of an exemplary humanized anti-A β peptide antibody the total amino acid sequences of the h3D6v2 light and heavy chains predicted from the DNA sequences of the corresponding expression vectors are in FIG. 2 (where the residues are in the NH of the light and heavy chains₂Residue number 1) at the-terminal start position is represented by SEQ ID NO:1 and SEQ ID NO:2, respectively. The last amino acid residue encoded by the heavy chain DNA sequence, Lys⁴⁴⁹Without wishing to be bound by any theory, it is speculated that the last amino acid is removed by CHO cell proteases during intracellular processing, which is not observed in the mature, secreted form of h3D6v 2. Thus, the COOH-terminus of the h3D6v2 heavy chain may optionally be Gky⁴⁴⁸. COOH-terminal lysine processing was observed in recombinant antibodies and plasma membrane derived antibodies and was not shown to affect their function (Harris (1995) J.Chromatogr.A.705: 129-134). Purified h3D6v2 can be post-translationally modified by adding N-linked glycans to the Fc portion of the heavy chain, which is known to contain a single N-glycosylation consensus site. The N-sugarThe glycosylation sites exhibit three major complex biantennary neutral oligosaccharide structures that are commonly observed at similar N-glycosyl acid sites on mammalian IgG proteins.

Other exemplary humanized anti-a β peptide antibodies are humanized 3D6 version 1(hu3D6v1) having the sequence shown in figure 2, but with a D in position 1 in the light chain substituted for Y.

In various embodiments of the invention, the anti-a β antibody (e.g., a humanized anti-a β peptide 3D6 antibody) can be about 0.1mg/ml to about 100mg/ml, about 0.1mg/ml to about 75mg/ml, about 0.1mg/ml to about 50mg/ml, about 0.1mg/ml to about 40mg/ml, about 0.1mg/ml to about 30mg/ml, about 10mg/ml to about 20mg/ml, about 20mg/ml to 30mg/ml, or higher, e.g., up to about 100mg/ml, about 200mg/ml, about 500mg/ml, or about 1000mg/ml or more. Preferably, the concentration of the anti A β antibody is from about 17mg/ml to about 23 mg/ml. In various embodiments, the anti-a β antibody can be about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 mg/ml. In a particular embodiment, the antibody (e.g., a humanized anti a β peptide 3D6 antibody) is about 17 mg/ml. In other embodiments, the antibody (e.g., a humanized anti a β peptide 3D6 antibody) is about 20 mg/ml. In yet another embodiment, the antibody (e.g., a humanized anti A β peptide 3D6 antibody) is about 30 mg/ml. Intermediate concentrations, such as from about 12mg/ml to about 17mg/ml, are also part of the invention. For example, concentration value ranges using combinations of the above values as upper and/or lower limits are also included within the scope of the present invention.

Excipient

In various embodiments, the present invention provides formulations containing a variety of excipients, including, but not limited to, buffers, antioxidants, tonicity agents and stabilizers. In addition, the formulation also contains other agents for pH adjustment (e.g., HCl) and diluents (e.g., water). In other embodiments, different forms of histidine may be used for pH adjustment. In part, the excipient may maintain the stability and biological activity of the antibody (e.g., by maintaining the appropriate conformation of the protein), and/or maintain pH.

Buffering agent

In various aspects of the invention, the formulation includes a buffer (buffer). The buffer can maintain a physiologically suitable pH. In addition, the buffer may enhance the isotonicity and chemical stability of the formulation. Generally, the formulation should have a physiologically suitable pH. In various embodiments of the invention, the formulation has a pH of from about 5 to about 7, from about 5.5 to about 6.5, preferably from about 6.0 to about 6.5. In a particular embodiment, the formulation has a pH of about 6. Intermediate ranges of the above pH levels, for example, about pH5.2 to about pH6.3, preferably 6.0 or pH6.2, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. The pH can be adjusted as needed using techniques well known in the art. For example, the pH can be adjusted to the desired level by adding HCl as needed or by using various forms of histidine.

The buffer may include, but is not limited to, succinate (sodium or phosphate), histidine, phosphate (sodium or potassium), Tris (hydroxymethyl) aminomethane), diethanolamine, citrate, other organic acids, and mixtures thereof. In a preferred embodiment, the buffer is histidine (e.g., L-histidine). In other particular embodiments, the buffer is succinate. In other embodiments, the formulation includes an amino acid, such as histidine, present in an amount sufficient to maintain the formulation at a physiologically suitable pH. Histidine is an exemplary amino acid with buffering capacity at physiological pH ranges. Histidine has a buffering capacity ranging from its imidazole group. In an exemplary embodiment, the buffer is L-histidine (base) (e.g., C)₆H₉N₃O₂FW: 155.15). In other embodiments, the buffer is L-histidine monohydrochloride monohydrate (e.g., C)₆H₉N₃O₂.HCl.H₂O, FW: 209.63). In yet other embodiments, the buffer is L-histidine (base) and an L-histidine monosaltA mixture of acid monohydrate.

In one embodiment, the buffer (e.g., L-histidine or succinate) may be at a concentration of about 0.1mM to about 50mM, about 0.1mM to about 40mM, about 0.1mM to about 30mM, about 0.1mM to about 25mM, about 0.1mM to about 20mM, or about 5mM to about 15mM, preferably 5mM or 10 mM. In various embodiments, the buffer may be about 6mM, 7mM, 8mM, 9mM, 11mM, 12mM, 13mM, 14mM, or 15 mM. In a particular embodiment, the buffer is about 10 mM. Intermediate ranges of the above concentrations, for example, from about 12mM to about 17mM, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. In certain embodiments, the buffer is present in an amount sufficient to maintain a physiologically suitable pH.

Tension agent

In various aspects of the invention, the formulation includes a tonicity agent. In part, the tonicity agent helps to maintain the isotonicity of the formulation, as well as to maintain protein levels. In part, the tonicity agent helps preserve the level, ratio or proportion of the therapeutically active polypeptide in the formulation. In this specification, the term "stress" refers to the behaviour of a biologically active ingredient in a liquid environment or solution. An isotonic solution has the same osmotic pressure as plasma and is thus capable of intravenous infusion into a subject without altering the osmotic pressure of the subject's plasma. Indeed, in one embodiment according to the present invention, the tonicity agent is present in an amount sufficient to render the formulation suitable for intravenous infusion. Generally, tonicity agents may also act as bulking agents. Also, the formulation may allow the protein to overcome various stresses, such as freezing and shearing.

The tonicity agent may include, but is not limited to, CaCl₂，NaCl，MgCl₂Lactose, sorbitol, sucrose, mannitol, trehalose, raffinose, polyethylene glycol, hydroxyethyl starch, glycine and mixtures thereof. In a preferred embodiment, the tonicity agent is mannitol (e.g., D-mannitol, e.g., C)₆H₁₄O₆，FW:182.17)。

In one embodiment, the tonicity agent may be about 2% to about 6% w/v, or about 3% to about 5% w/v. In other embodiments, the tonicity agent may be about 3.5% to about 4.5% w/v. In other embodiments, the tonicity agent may be from about 20mg/ml to about 60mg/ml, from about 30mg/ml to about 50mg/ml, or from about 35mg/ml to about 45 mg/ml. Preferably, the tonicity agent may be about 4% w/v or about 40 mg/ml. In other particular embodiments, the tonicity agent may be about 6% w/v. In yet another particular embodiment, the tonicity agent may be about 10% w/v.

Intermediate ranges of the above concentrations, for example, from about 3.2% to about 4.3% w/v or from about 32 to about 43mg/ml, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. The tonicity agent should be present in an amount sufficient to maintain isotonicity of the formulation.

Antioxidant agent

In various aspects of the invention, the formulation includes an antioxidant to, in part, preserve the formulation (e.g., by preventing oxidation).

The antioxidants include, but are not limited to, GLA (gamma-linolenic acid) -lipoic acid, DHA (docosahexaenoic acid) -lipoic acid, GLA-tocopherol, di-GLA-3, 3' -thiodipropionic acid and any of the generally chemically linkable, e.g., GLA, DGLA (dihomo-gamma-linolenic acid), AA (arachidonic acid), SA (salicylic acid), EPA (eicosabienoic acid) or DHA (docosahexaenoic acid) as well as any natural or synthetic antioxidant. These include phenolic antioxidants (e.g., eugenol, carnosic acid, caffeic acid, BHT (butylated hydroxyanisole), gallic acid, tocopherols, tocotrienols, and flavonoid (flavanoid) antioxidants (e.g., myricetin and fisetin)), polyenes (e.g., retinoic acid), unsaturated sterols (e.g., Δ @), and mixtures thereof⁵-avenosterol), organic sulfur compounds (e.g. allicin), terpenes (e.g. geraniol, abietic acid) and amino acid antioxidants (e.g. methionineAcid, cysteine, carnosine). In one embodiment, the antioxidant is ascorbic acid. Preferably, the antioxidant is methionine, or an analogue thereof, for example, selenomethionine, hydroxymethylbutyric acid, ethionine or trifiuoromethionine.

In one embodiment, the antioxidant (e.g., methionine, e.g., L-methionine, e.g., CH)₃SCH₂CH₂CH(NH₂)CO₂H, FW =149.21) may be about 0.1mM to about 50mM, about 0.1mM to about 40mM, about 0.1mM to about 30mM, about 0.1mM to about 20mM, or about 5mM to about 15 mM. In various embodiments, the antioxidant may be about 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, or 15 mM. Preferably, the antioxidant may be about 10 mM. In other particular embodiments, the antioxidant may be about 15 mM. Intermediate ranges of the above concentrations, for example, from about 12mM to about 17mM, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. In certain embodiments, the antioxidant is present in an amount sufficient to preserve the formulation, in part, by preventing oxidation.

Stabilizer

In various aspects of the invention, the formulation includes a stabilizer, which may also be referred to as a surfactant. Stabilizers are specific chemical compounds that are capable of interacting with and stabilizing biomolecules in a formulation and/or common pharmaceutical excipients. In certain embodiments, the stabilizer may be used in conjunction with lower temperature storage conditions. Stabilizers generally protect proteins from air/solution interface-induced tensions and solution/surface-induced tensions, which typically lead to protein aggregation.

The stabilizer may include, but is not limited to, glycerin, polysorbates, e.g., polysorbate 80, dicarboxylic acids, oxalic acid, succinic acid, adipic acid, fumaric acid, phthalic acid, and combinations thereof. In a preferred embodiment, the stabilizer is polysorbate 80.

In one embodiment, the stabilizer (e.g., polysorbate 80) concentration is about 0.001% w/v to about 0.01% w/v, about 0.001% w/v to about 0.009% w/v, or about 0.003% w/v to about 0.007% w/v. Preferably, the stabilizer concentration is about 0.005% w/v. In other particular embodiments, the stabilizer concentration is about 0.01% w/v. Intermediate ranges of the above concentrations, for example, from about 0.002% w/v to about 0.006% w/v, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. The stabilizing agent is present in an amount sufficient to stabilize the a β binding polypeptide (e.g., an anti-a β antibody).

Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington pharmaceutical sciences, 16 th edition, Osol, A.Ed. (1980), may also be included in the formulation, provided that they do not adversely affect the desired properties of the formulation. In certain embodiments, the formulation is substantially free of preservatives, although, in other embodiments, preservatives may be added as needed. For example, antifreeze or freeze-drying agents (lyoprotectants) may also be included, for example, if the formulation is freeze-dried.

In various aspects of the invention, the formulation optionally includes some or all of the above types of excipients. In one aspect, the formulations of the invention include an a β binding polypeptide (e.g., an anti-a β antibody), mannitol, and histidine. In particular embodiments, the formulation may include an antioxidant, such as methionine, and/or a stabilizer, such as polysorbate 80. In certain embodiments, the formulation has a pH of about 6. In other aspects, the formulation includes an a β binding polypeptide (e.g., an anti-a β antibody), mannitol, histidine, and methionine. In yet another aspect, the formulation includes an a β binding polypeptide (e.g., an anti-a β antibody), mannitol, histidine, methionine, and polysorbate 80. In a particular aspect of the invention, the formulation comprises about 20mg/ml Α β binding polypeptide (e.g., anti Α β antibody), about 10mM histidine, about 10mM methionine, about 4% mannitol and a pH of about 6. In other aspects of the invention, the formulation comprises about 20mg/ml of an A β binding polypeptide (e.g., an anti-A β antibody), about 10mM histidine, about 10mM methionine, about 4% w/v mannitol, 0.005% w/v polysorbate 80 and a pH of about 6. A preferred formulation comprises about 17mg/ml to about 23mg/ml of humanized 3D6 antibody, about 10mM histidine, about 10mM methionine, about 4% w/v mannitol, about 0.005% polysorbate 80 and a pH of about 5.5 to about 6.5. Other preferred formulations include about 10mg/ml to about 30mg/ml of the humanized 266 antibody, about 10mM histidine or succinate, about 10mM methionine, about 4% w/v mannitol or sorbitol, and a pH of about 5.5 to about 6.5. Yet another preferred formulation comprises about 10mg/ml to about 30mg/ml of the humanized 12A11 antibody, about 5mM histidine, about 10mM methionine, about 4% mannitol or 150mM NaCl, and a pH of about 5.5 to about 6.5. Other formulations may be stable for at least 12 months at temperatures above freezing to about 10 ℃, at a pH of about 5.5 to about 6.5, and including at least one anti-a β antibody at a concentration of about 1mg/ml to about 30mg/ml, mannitol at a concentration of about 4% w/v or NaCl at a concentration of about 150mM, histidine or succinate at a concentration of about 5mM to about 10mM, and methionine at 10 mM. Preferably, the formulation may further comprise polysorbate at a concentration of about 0.001% w/v to about 0.01% w/v.

Exemplary embodiments of the present invention also provide concentrated formulations of a β binding polypeptides (e.g., anti-a β antibodies) that are generally useful as bulk formulation products. Furthermore, exemplary embodiments of the present invention may be stable to freezing, lyophilization, and/or reconstitution. Furthermore, exemplary embodiments of the present invention may remain stable for extended periods of time. For example, the formulations of the present invention are stable for at least about 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 months. In particular embodiments, the formulations of the present invention are stable for at least about 12 months, at least about 18 months, at least about 24 months, or at least about 30 months.

As described herein, the formulation may be stored at about-80 ℃ to about 40 ℃, about 0 ℃ to about 25 ℃, about 0 ℃ to about 15 ℃, or about 0 ℃ to about 10 ℃, preferably about 2 ℃ to about 8 ℃. In various embodiments, the formulation may be stored at about 0 ℃,1 ℃,2 ℃,3 ℃,4 ℃,5 ℃,6 ℃,7 ℃,8 ℃,9 ℃, or 10 ℃. In particular embodiments, the formulation may be stored at about 5 ℃. In general, the formulations are stable and retain biological activity in these ranges. Intermediate ranges of the above temperatures, for example, from about 2 ℃ to about 17 ℃, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention.

The formulations of the present invention may be adapted for release by a variety of techniques. In certain embodiments, the formulation may be administered parenterally, for example, intravenously or intramuscularly. In addition, one skilled in the art can target the release of the formulation to the brain (e.g., so that the antibody can cross the blood brain barrier) or spinal fluid. In particular embodiments, the formulation may be administered intravenously.

The effective dosage of the formulations of the invention will depend upon a variety of factors including the mode of administration, the target, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Titration of the therapeutic dose is required to optimize its safety and efficacy.

For passive immunization with antibodies, exemplary doses can be from about 0.0001mg/kg to about 100mg/kg, from about 0.01mg/kg to about 5mg/kg, from about 0.15mg/kg to about 3mg/kg, from 0.5mg/kg to about 2mg/kg, preferably from about 1mg/kg to about 2mg/kg of host body weight. In some exemplary embodiments, the dose may be about 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, or 2.0 mg/kg. Other exemplary passive immunization doses are about 1mg/kg to about 20 mg/kg. In some exemplary embodiments, the dose may be about 5, 10, 15 or 20 mg/kg. Such doses may be administered to a subject daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments include multiple doses administered over an extended period of time, e.g., at least 6 months. Other exemplary treatment regimens include once every two weeks or once a month or once every 3-6 months. Exemplary dosage schedules include administration of 1-10mg/kg or 15mg/kg on consecutive days, 30mg/kg on alternate days, or 60mg/kg weekly. In some methods, two or more monoclonal antibodies having different binding specificities can be administered simultaneously, in which case the dosage of each antibody administered is within the ranges described above.

Antibodies are typically administered multiple times. The interval between single doses may be weeks, months or years. The intervals may be irregular according to the determination of blood levels of antibodies corresponding to a β in the patient. In some methods, the dose can be adjusted to achieve a plasma antibody concentration of 1-1000. mu.g/ml and in some methods 25-300. mu.g/ml. Alternatively, the antibody may be administered in a sustained release formulation, in which case a lower frequency of administration is required. Both dose and frequency vary with the half-life of the antibody in the patient. Generally, human antibodies have the longest half-life, followed by humanized, chimeric, and non-human antibodies.

The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. Under conditions of prophylactic use, a formulation comprising an antibody of the invention or a mixture thereof is administered to a patient who has not entered a disease state in order to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose". In this use case, the precise amount again depends on the health and normal immunity of the patient, and is usually from 0.1 to 25mg per dose, in particular from 0.5 to 2.5mg per dose. Relatively low doses may be administered over a long period of time at relatively infrequent intervals. Some patients may continue to receive treatment for life.

In some therapeutic applications, it is sometimes desirable to have a relatively high dose (e.g., about 0.5 or 1 to about 200mg/kg antibody per dose (e.g., 0.5, 1, 1.5, 2, 5, 10, 20, 25, 50 or 100mg/kg), with 5-25kg/mg being more common) over a relatively short interval until the progression of the disease is reduced or halted, preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the patient may be administered a prophylactic treatment.

It is particularly advantageous to provide the formulations of the invention in unit dosage form for ease of administration and uniformity of dosage. The formulations of the present invention may be presented in capsules, ampoules, lyophilized forms, or in multi-dose containers. The term "container" refers to an article for storage, such as a holder, container or vessel, in which an object or liquid may be placed or contained. The unit dosage form may comprise any of the formulations described in this specification, including suspensions, solutions or emulsions of the active ingredient together with formulating agents such as suspending, stabilizing and/or dispersing agents. In exemplary embodiments, the pharmaceutical dosage form can be added to an intravenous drip bag (e.g., a 50ml, 100ml, or 250ml, or 500ml drip bag) with an appropriate diluent, such as sterile pyrogen-free water or saline solution, prior to administration to a patient, e.g., intravenous infusion. Some pharmaceutical unit dosage forms require reconstitution with a suitable diluent prior to addition to an intravenous drip bag, particularly in lyophilized form. In an exemplary embodiment, the pharmaceutical unit dosage form is a container containing the above-described formulation. For example, the container may be a 10mL glass, type I, tubular vial. Typically, the container is required to maintain the sterility and stability of the formulation. For example, the vial may be closed using a serum stopper. Furthermore, in various embodiments, the container may be designed to allow removal of 100mg of formulation or active ingredient (e.g., for a single use). Alternatively, the container may be adapted for larger quantities of the formulation to obtain the active ingredient, e.g., from about 10mg to about 5000mg, from about 100mg to about 1000mg, and from about 100mg to about 500mg, from about 40mg to about 250mg, from about 60mg to about 80mg, from about 80mg to about 120mg, from about 120mg to about 160mg, or ranges or intervals thereof, e.g., from about 100mg to about 200 mg. Ranges intermediate to the above amounts, for example, from about 25mg to about 195mg, are also part of the invention. For example, ranges of values using combinations of the above values as upper and/or lower limits are also included within the scope of the invention. In particular embodiments, the formulations are typically supplied in liquid unit dosage forms.

In other aspects, the invention provides kits comprising a pharmaceutical unit dosage form (e.g., a container with a formulation disclosed herein), and instructions for use. Thus, the containers and kits may be designed to provide sufficient formulation for multiple uses. In various embodiments, the kit further comprises a diluent. The diluent may comprise excipients alone or in combination. For example, the diluent may include a tonicity modifier, such as mannitol, a buffering agent, such as histidine, a stabilizer, such as polysorbate 80, an antioxidant, such as methionine, and/or combinations thereof. The diluent may contain other excipients, for example Lyoprotectant, if deemed necessary by the person skilled in the art.

Other useful embodiments of the invention have been described in the section of this application entitled "summary of the invention".

The invention is further illustrated by the following examples, which are not intended to be limiting. All references, patents and published patent applications cited in this application, as well as the figures, are incorporated by reference in their entirety.

Examples

In general, the practice of the present invention involves, unless otherwise indicated, standard techniques of chemistry, molecular biology, recombinant DNA technology, immunology (particularly, e.g., antibody technology), and polypeptide preparation. See, e.g., Sambrook, FritschandManiatis, molecular cloning, Cold spring harbor laboratory Press (1989); antibodies engineering protocols (methods molecular biology), 510, Paul, s., HumanaPr (1996); (iii) antibody engineering, APractcalAproach (practical Aproachseries, 169), McCafferty, Ed., IrlPr (1996); antibodies, ALaboratoryManual, Harlowetal, C.S.H.L.Press, Pub. (1999); and CurrentProtocol molecular biology, eds. Ausubeletal., John Wiley & Sons (1992).

Example I cloning and expression of humanized anti-A β antibody

An exemplary antibody for use in the formulation of the methods described herein is the 3 D6.3D6 monoclonal antibody that is specific for the N-terminus of A β and has been shown to mediate phagocytosis of amyloid plaques (e.g., to induce phagocytosis). 3D6 does not recognize secreted APP or full-length APP, but detects the A β type with aspartic acid at the amino terminus.3D 6 is thus an end-specific antibody.ATCC accession number PTA-5130 for the cell line designated RB963D6.32.2.4 producing antibody 3D6, deposited at 8/4/2003 the cloning, characterization and humanization of the 3D6 antibody can be found in U.S. patent application publication No. 20030165496A 1. briefly, m3D6 light and heavy chain variable regions (V.sub.V.sub.3 D.3) can be isolated by reverse transcription-polymerase chain reaction (RT-PCR)_LAnd V_H) The DNA sequence of (a) to humanize a murine monoclonal antibody against the A β peptide (designated m3D6) according to the established m3D6V_LAnd V_HTo ensure that the humanized antibody retains the ability to interact with the A β peptide antigen, the critical murine V in the humanized 3D6 sequence_LAnd V_HFramework region residues were retained, thus maintaining the overall structure of the Constant Domain Regions (CDRs) in the human kappa light chain and IgG1 heavy chain sequences. The encoded humanized 3D6V identified by this procedure was generated by annealing synthetic overlapping DNA oligonucleotides followed by a DNA polymerase fill-in reaction_LAnd V_HThe DNA sequence of the sequence (including the 5 'signal peptide sequence and the 3' intron splice donor sequence), the integrity of each humanized variable region sequence was verified by DNA sequencing A schematic of the predicted structure of an exemplary humanized anti-A β peptide 3D6 antibody, designated h3D6v2, is depicted in FIG. 1, FIG. 2 identifies the full amino acid sequence of the h3D6v2 light and heavy chains.

The humanized 3D6 antibody can be expressed by transfecting a Chinese Hamster Ovary (CHO) host cell line with expression plasmids encoding the anti-a β antibody light and heavy chain genes. CHO cells expressing the antibody can be isolated using standard methotrexate-based drug selection/gene amplification methods. Clonal CHO cell lines exhibiting the desired yield and growth phenotype can be selected and chemically defined media free of animal or human derived components used to construct antibody expressing cell lines.

EXAMPLE II preparation of humanized anti-A β antibody drug substance

The polypeptide preparation method is started by thawing a starting culture of clonal cells stably expressing anti-a β antibodies. Cells can be cultured using chemically defined media that do not contain proteins of animal or human origin. The culture is then expanded and used to inoculate a seed bioreactor, which is then used in turn to inoculate multiple preparation reactor cycles. The preparation reactor was operated in batch mode. At the end of the production cycle, the conditioned media product in the produced product may be clarified by microfiltration for further downstream processing.

The purification process consisted of standard chromatography steps followed by filtration. The purified antibody can be concentrated by ultrafiltration and diafiltered into formulation buffer without polysorbate 80. Optionally, polysorbate 80 (plant derived) may be added to the ultrafiltration/diafiltration retentate pool, followed by the addition of the bacterial retentate filtrate. The drug substance can be stored frozen at-80 ℃ and maintained for further processing into pharmaceutical products, including the stable liquid formulations described in this specification.

EXAMPLE III preparation of antibody formulation and placebo

Two batches of antibody drug were prepared. The first batch was prepared by combining the drug substance into a formulation containing 20mg anti-a β antibody active per ml, 10mM histidine, 10mM methionine, 4% mannitol, 0.005% polysorbate 80, ph6.0, free of animal and human protein. The drug, 100mg anti a β antibody active substance per vial, was aseptically filled into the vial. The finished pharmaceutical vial contains no preservatives and may be intended for single use only.

A second batch of drug may be prepared by a similar method using a formulation buffer that does not contain polysorbate 80.

Example IV stability analysis of formulations with and without polysorbate-80

The stability, in particular, the physicochemical integrity (e.g., aggregation, deamidation, hydrolysis, and/or disulfide bond rearrangement) of the formulation can be assessed by the following methods well known in the art: appearance; the pH value; protein concentration (a 280); ELISA, in part, as a test for biological activity; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in part, as a test for aggregation; size exclusion high performance liquid chromatography (SEC-HPLC), in part, as a test for aggregation and stability generality; cation exchange high performance liquid chromatography (CEX-HPLC), in part, as a general test for deamidation and stability; and polypeptide mapping. These methods allow the recovery and integrity of the protein to be assessed under assay conditions at a variety of temperatures.

An appearance analysis of the formulation can be performed to determine the quality of the formulation at different time points. The analysis was based on visual inspection of the clarity, color and presence of particulates. For example, the degree of transparency is analyzed with reference to a reference suspension. Analysis of the appearance of the formulations with and without polysorbate 80 according to the invention showed that when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ at the following time points: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months.

The pH analysis was performed to determine that the pH of the formulation was maintained within an acceptable range of about 5.5 to about 6.5. pH analysis of the formulations with and without polysorbate 80 according to the invention showed that when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ at the following time points: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. Generally, the pH must not be below 5.8 or above 6.2.

The protein concentration analysis of the A280 test was performed to determine that the protein concentration of the formulation remained within an acceptable range of about 17mg/ml to about 23 mg/ml. Analysis of protein concentrations as described in the present invention with and without the polysorbate 80 formulation showed that when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ at the following time points: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. Protein concentrations were maintained within acceptable ranges except that the protein concentration without the polysorbate 80 formulation at 5 ℃, 25 ℃ and 40 ℃ at the three month time point was slightly above about 23 mg/ml. Thus, protein concentration analysis showed that no detectable protein loss occurred even under harsh conditions, especially for formulations with polysorbate 80. In addition to this, the general decrease in protein concentration indicates a significant time or temperature dependent change after the initial time point.

Maintenance of biological activity was, in part, assayed by ELISA techniques. Biological activity was assayed by the acceptable active Binding Units (BU)/mg ≧ 2500BU/mg or 50% (i.e., 5000BU/mg corresponds to 100%). Analysis of the ELISA with and without the polysorbate 80 formulation as described in the present invention showed that the following time points when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ were observed: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. The biological activities of both formulations remained within acceptable limits except for the range of slightly less than 50% of the biological activity of both formulations at the 12 month time point when stored at 40 ℃.

SEC-HPLC analysis was performed as a general test for aggregation, purity and stability. SEC-HPLC performed under mobile phase chromatographic conditions using a dibasic buffer with sodium phosphate indicates that the formulation is acceptable if SEC-HPLC analysis identifies > 90% IgG monomer compared to the percentage of high molecular weight product and low molecular weight product. SEC-HPLC analysis of the formulations with and without polysorbate 80 as described herein showed that when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ at the following time points: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. Except that the percentage monomer range for both formulations was below 90% when stored at 40 ℃ at each time point and after 6 months (where the analysis identified a low molecular weight product above at least 10% for both formulations at each time point), the other percentage monomers remained within acceptable ranges. SEC-HPLC analysis can demonstrate that although the distribution of high and low molecular weight products varies over time in samples with and without polysorbate, the monomeric form of the antibody can generally remain constant, for example, at the 12 month time point when the formulation is stored at 5 ℃.

The CEX-HPLC analysis was performed as a general measure of amination and stability. CEX-HPLC procedures performed under conditions using mobile phase chromatography, NaCl buffer, produced an elution profile and retention time of the main peak with comparable or non-comparable analysis to the reference standard profile. Analysis of CEX-HPLC with and without the polysorbate 80 formulation as described herein showed that when stored at-80 ℃,5 ℃, 25 ℃ and 40 ℃ at the following time points: initially, both formulations were acceptable under conditions of 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. The other main peaks were comparable to the reference peak, except that the elution profile and retention time of the main peaks of both formulations were not comparable at each time point and after 3 months when stored at 40 ℃.

In general, analysis of formulations with polysorbate 80 stored at 5 ℃ leads to the following important conclusions: 1) clarity, pH, ELISA, CEX-HPLC, SEC-HPLC and SDS-PAGE analysis all showed only minor changes in the formulation in 9 months; 2) the formulation stored at 5 ℃ after 9 months is more like the reference sample than the sample under harsh conditions; 3) peptide mapping showed changes at 5 ℃; and 4) SEC-HPLC trend data at 5 ℃ predicts stability for at least 17.2 months (see FIG. 6), which then predicts stability for greater than 30 months after the chromatographic column, instrument and buffer variation are removed (see FIG. 7). In addition, samples stored under harsh conditions with polysorbate 80 at 25 ℃ also passed all indicators at 9 months (fig. 4).

Furthermore, analysis of the formulations without polysorbate 80 stored at 5 ℃ leads to the following important conclusions: 1) the transparency, pH and ELISA analyses all showed only minor changes in the formulation over 9 months; 2) CEX-HPLC and SDS-PAGE results demonstrated comparable findings to the reference sample or control at-80 ℃ at 9 months; 3) SEC-HPLC analysis showed only minor changes after 9 months and more pronounced changes at elevated temperatures; and 4) SEC-HPLC trend data predicts stability for at least 18 months, consistent with analytical variability (see FIG. 8).

FIGS. 3-5 are graphical depictions of shelf life predictions for formulations prepared according to the present invention (with and without PS80) stored at 5 deg.C, 25 deg.C and 40 deg.C, respectively. In general, fig. 3-5 show that storage of the formulations of the present invention at higher temperatures reduces the expected shelf life. Figure 3, in particular, shows that the expected shelf life of the formulation is at least 18 months when the formulation is stored at 5 ℃. Figure 4 illustrates that storage of the formulation at room temperature (25 ℃) reduces the expected shelf life to about 12 months. Figure 5 further demonstrates that storage of the formulation at 40 ℃ reduces the expected shelf life to about 4 months.

Example V stability Studies Using methionine as an antioxidant

Studies were conducted to determine the effect of methionine on maintaining the stability of the antibodies of the antibody formulations. At different temperatures, inFour antibody samples (using anti-CD 22 IgG) within 6 months₄Antibody): antibody preparation with 20mM succinate at pH 6.0; an antibody formulation having 20mM succinate and 10mM methionine; an antibody preparation with 20mM succinate and 0.01% PS 80; and an antibody formulation with 20mM succinate, 10mM methionine and 0.01% PS80 was analyzed by SEC-HPLC. The results indicate that methionine can reduce the formation of high molecular weight (HMV) as expected. Furthermore, methionine reduced the temperature-dependent increase in percentage HMW (see figure 10).

In addition, the following four antibodies (anti-B7.2IgG) were treated at different temperatures (5 ℃ and 40 ℃) within 6 weeks₂Antibody) sample: (1) samples containing antibody, 10mM histidine and 150mM nacl; (2) samples containing antibody, 10mM histidine, 150mM nacl and 0.01% PS 80; (3) a sample containing antibody, 10mM histidine, 150mM nacl and 10mM methionine; and (4) samples containing antibody, 10mM histidine, 150mM NaCl, 10mM methionine and 0.01% PS80 were subjected to pH stability studies (at pH5.8, pH6.0 and pH 6.2). SEC-HPLC analysis was performed. The results show that methionine reduces the increase in temperature-dependent percent by-product formation (e.g., HMV by-product) over a specified pH range, e.g., from about pH5.8 to about pH6.2 (see fig. 11). As shown in FIG. 11, the methionine-containing samples showed a small amount of aggregation when kept at 40 ℃ for 6 weeks, which is similar to the case of keeping at 5 ℃ for 6 weeks.

Example VI excipients for IgG1 antibodies by differential scanning calorimetry Analysis of

The main purpose of a protein pharmaceutical formulation is to stabilize the protein in its native, biologically active form. Generally, this can be done by screening various excipients in a base formulation and monitoring their effect on the molecular weight and activity of the molecule. These parameters may account for stability. Other stability measurements are thermal denaturation, which can be monitored using a variety of biophysical techniques. In general, protein stabilityThe elevated level of (a) is due to high dissolution, denaturation or decomposition temperatures. Thus, the thermal properties of a representative IgG1 monoclonal antibody can be monitored using a VP-capillary differential scanning calorimetry meter in the presence of various excipients. In particular, for surface T containing 10mM histidine (pH6.0) formulations with various excipients_ms was measured. Several excipients have been shown to enhance or reduce thermal stability. Since the increased level of protein stability is due to the higher solubilization temperature, so, compared to control T_m2/T_m3 values (74.9 ℃ and 83.4 ℃ respectively) can have an increase in T in the sample_m2 or T_mExcipient 3 is considered to be a particularly desirable excipient (see table 1 below).

Thus, it is believed that excipients such as glucose (formulated at 4% and 10% concentrations), sucrose (formulated at 4% and 10% concentrations), sorbitol (formulated at 4% and 10% concentrations) and mannitol (formulated at 4% and 10% concentrations) work particularly well to stabilize liquid polypeptide formulations, particularly antibody IgG formulations.

TABLE 1 analysis of excipients

Equivalent variations

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalent variations are also intended to be encompassed by the following claims.

Claims (55)

1. A stable formulation comprising:

(a) a humanized 3D6 monoclonal antibody at a concentration of 0.1mg/ml to 100mg/ml, wherein the humanized 3D6 antibody comprises: a light chain comprising a light chain constant region and the light chain variable region from SEQ ID NO:1, and a heavy chain comprising a heavy chain constant region and the heavy chain variable region from SEQ ID NO: 2;

(b) histidine at a concentration of 0.1mM to 25 mM;

(c) mannitol in an amount of 1% w/v to 10% w/v;

(d) methionine at a concentration of 0.1mM to 25 mM; and

(e) polysorbate in an amount of 0.001% w/v to 0.01% w/v;

wherein the pH of the formulation is between 5.5 and 6.5.

2. The formulation of claim 1, wherein histidine is present at a concentration of 10 mM.

3. The formulation of claim 2, wherein the polysorbate is present at a concentration of 0.005% w/v and the mannitol is present at a concentration of 4% w/v.

4. The formulation of claim 2, wherein the polysorbate is present at a concentration of 0.005% w/v and the mannitol is present at a concentration of 10% w/v.

5. The formulation of claim 3, wherein methionine is present at a concentration of 10 mM.

6. The formulation of claim 4, wherein methionine is present at a concentration of 10 mM.

7. The formulation of claim 1, wherein histidine is present at a concentration of 8mM to 12 mM.

8. The formulation of claim 7, wherein mannitol is present in an amount of 4% w/v to 6% w/v.

9. The formulation of claim 8, wherein methionine is present at a concentration of 8mM to 12 mM.

10. The formulation of claim 9, wherein the polysorbate is present in an amount of 0.003% w/v to 0.007% w/v.

11. The formulation of claim 10 wherein the pH is 5.8 to 6.2.

12. The formulation of claim 11, further comprising 2% w/v sucrose.

13. A stable unit dosage form comprising:

(a) a humanized 3D6 monoclonal antibody in an amount of 10mg to 250mg, wherein the humanized 3D6 antibody comprises: a light chain comprising a light chain constant region and the light chain variable region from SEQ ID NO:1, and a heavy chain comprising a heavy chain constant region and the heavy chain variable region from SEQ ID NO: 2;

(b) histidine at a concentration of 0.1mM to 25 mM;

(c) mannitol in an amount of 1% w/v to 10% w/v;

(d) methionine at a concentration of 0.1mM to 25 mM; and

(e) polysorbate in an amount of 0.001% w/v to 0.01% w/v;

wherein the pH of the unit dosage form is from 5.5 to 6.5.

14. The unit dosage form of claim 13, wherein histidine is present at a concentration of 10 mM.

15. The unit dosage form of claim 14, wherein the polysorbate is present at a concentration of 0.005% w/v and the mannitol is present at a concentration of 4% w/v.

16. The unit dosage form of claim 14, wherein the polysorbate is present at a concentration of 0.005% w/v and the mannitol is present at a concentration of 10% w/v.

17. The unit dosage form of claim 15, wherein methionine is present at a concentration of 10 mM.

18. The unit dosage form of claim 16, wherein methionine is present at a concentration of 10 mM.

19. The unit dosage form of claim 13, wherein the humanized 3D6 antibody is present in an amount of 40mg to 60 mg.

20. The unit dosage form of claim 13, wherein the humanized 3D6 antibody is present in an amount of 60mg to 80 mg.

21. The unit dosage form of claim 13, wherein the humanized 3D6 antibody is present in an amount of 80mg to 120 mg.

22. The unit dosage form of claim 13, wherein the humanized 3D6 antibody is present in an amount of 120mg to 160 mg.

23. The unit dosage form of claim 13, wherein the humanized 3D6 antibody is present in an amount of 160mg to 240 mg.

24. The unit dosage form of claim 13, wherein histidine is present at a concentration of 8mM to 12 mM.

25. The unit dosage form of claim 24, wherein mannitol is present in an amount of 4% w/v to 6% w/v.

26. The unit dosage form of claim 25, wherein methionine is present at a concentration of 8mM to 12 mM.

27. The unit dosage form of claim 26 wherein the polysorbate is present in an amount of 0.003% w/v to 0.007% w/v.

28. The unit dosage form of claim 27 wherein the pH is from 5.8 to 6.2.

29. The unit dosage form of claim 28 further comprising 2% w/v sucrose.

30. A pharmaceutical product comprising:

(a) a glass vial comprising a stable antibody formulation, said formulation comprising:

(i) a humanized 3D6 monoclonal antibody in an amount of 10mg to 250mg, wherein the humanized 3D6 antibody comprises: a light chain comprising a light chain constant region and the light chain variable region from SEQ ID NO:1, and a heavy chain comprising a heavy chain constant region and the heavy chain variable region from SEQ ID NO: 2;

(ii) histidine at a concentration of 0.1mM to 25 mM;

(iii) mannitol in an amount of 1% w/v to 10% w/v;

(iv) methionine at a concentration of 0.1mM to 25 mM; and

(v) polysorbate in an amount of 0.001% w/v to 0.01% w/v;

wherein the pH of the formulation is from 5.5 to 6.5; and

(b) use of a label comprising an indication of the volume required to use an appropriate dose to reach 0.0001mg/kg to 100 mg/kg.

31. The product of claim 30 wherein the dosage is from 0.15mg/kg to 5 mg/kg.

32. The product of claim 31 wherein the dosage is from 0.5mg/kg to 3 mg/kg.

33. The product of claim 30, wherein the dose is 1mg/kg to 2 mg/kg.

34. The product of claim 30, wherein histidine is present at a concentration of 10 mM.

35. The product of claim 34 wherein the polysorbate is present at a concentration of 0.005% w/v and mannitol is present at a concentration of 4% w/v.

36. The product of claim 34 wherein the polysorbate is present at a concentration of 0.005% w/v and mannitol is present at a concentration of 10% w/v.

37. The product of claim 35, wherein methionine is present at a concentration of 10 mM.

38. The product of claim 36, wherein methionine is present at a concentration of 10 mM.

39. The product of claim 30, wherein the humanized 3D6 antibody is present in an amount of 40mg to 60 mg.

40. The product of claim 30, wherein the humanized 3D6 antibody is present in an amount of 60mg to 80 mg.

41. The product of claim 30, wherein the humanized 3D6 antibody is present in an amount of 80mg to 120 mg.

42. The product of claim 30, wherein the humanized 3D6 antibody is present in an amount of 120mg to 160 mg.

43. The product of claim 30, wherein the humanized 3D6 antibody is present in an amount of 160mg to 240 mg.

44. The product of claim 30, wherein histidine is present at a concentration of from 8mM to 12 mM.

45. The product of claim 44 wherein mannitol is present in an amount of 4% w/v to 6% w/v.

46. The product of claim 45, wherein methionine is present at a concentration of from 8mM to 12 mM.

47. A product according to claim 46 wherein the polysorbate is present in an amount of 0.003% w/v to 0.007% w/v.

48. The product of claim 47 wherein the pH is from 5.8 to 6.2.

49. The product of claim 48, further comprising 2% w/v sucrose.

50. The product of claim 30, wherein the label further comprises an indication of the subcutaneous dose.

51. The product of claim 30 wherein the label further comprises an indication of the dosage to be administered intravenously.

52. Use of a formulation according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment or prevention of a disease characterized by precipitation of a β peptide.

53. The use of claim 52, wherein the disease is Alzheimer's disease.

54. The use of claim 52, wherein the medicament is for intravenous administration to a patient.

55. The use of claim 52, wherein the medicament is for subcutaneous administration to a patient.