HK1200717B - Abeta antibody formulation - Google Patents

Abstract

The present invention relates to a pharmaceutical formulation comprising about 50 mg/ml - 200 mg/ml of an Abeta antibody, about 0.01 % - 0.1% poloxamer, about 5 mM - 50 mM of a buffer, about 100 mM - 300 mM of a stabilizer at a pH of about 4.5 - 7.0.

Description

Abeta antibody formulations

The present invention relates to pharmaceutical formulations of antibody molecules, and/or mixtures of antibody molecules, directed against amyloid beta peptide (Abeta).

Antibody molecules, as part of the protein drug group, are highly susceptible to physical and chemical degradation (such as denaturation and aggregation, deamidation, oxidation and hydrolysis). Protein stability is affected by the properties of the protein itself (e.g., amino acid sequence), and by external influences (such as temperature, solvent pH, excipients, interfaces, or shear rate). Thus, it is important to specify optimal formulation conditions to protect the protein from degradation reactions during manufacturing, storage and administration (Manning, M.C., K.Patel, et al (1989). "Stability of protein pharmaceuticals", "Pharm Res6(11):903-18., Zheng, J.Y. and L.J.Janis (2005)." influx of pH, buffer speces, and storage temperature on physical Stability of a manipulated monomeric antigenic modification LAL 298. "_ Int.). Administration of antibodies by subcutaneous or intramuscular routes requires high protein concentrations in the final formulation due to the high doses and limited administration volumes that are often required. (Shire, s.j., z.shahrogh, et al (2004). "changing in The depth of high protein concentrations for purposes of formulation", "JPharm Sci93(6): 1390. quadrature.402", rossos, l.k., c.g. davis, et al (2004). "The clinical pharmacology of clinical monoclonal antibodies" Drug development research61(3): 108. quadrature.120.) large scale production of high protein concentrations can be achieved by ultrafiltration processes, drying processes (such as lyophilization or spray drying), and precipitation processes. (Shire, S.1., Z.Shahrokh, et al (2004). "changing inter-level of high protein concentration formulations", "J Pharm Sci93(6): 1390-.

It is an object of the present invention to provide a highly concentrated, stable formulation of an Abeta antibody or a mixture of such antibodies, which allows subcutaneous administration of the antibody to a patient.

The formulations of the invention show good stability after 8 months of storage at 2-8 ℃ and 25 ℃ without formation of visible particles. Shaking and multiple freeze-thaw steps are applied to the liquid formulation to simulate physical stress conditions that may occur during manufacture or transport of the drug product.

The pharmaceutical preparation of the present invention comprisesPoloxamersAs a surfactant to reduce aggregation and particle formation of the antibody. The term "poloxamer" as used herein includes the compounds referred to asPoloxamers188 polyoxyethylene-polyoxypropylene triblock copolymer, sold under the trade name BASF (Parsippany, N.J.)Sold as F68. Other poloxamers that may be used in the formulations of the present invention includePoloxamers403 (withSold by P123),poloxamers407 (to)Sold under the trade name P127),poloxamers402 (to)Sold by P122),poloxamers181 (to)Sold as L61),poloxamers401 (to)Sold as L121),poloxamers185 (to)Sold as P65), andpoloxamers338 (to)Sold as F108).

The present invention provides a stable liquid pharmaceutical antibody formulation comprising:

-about 50mg/ml to 200mg/ml of Abeta antibody,

-from about 0.01% to 0.1% of a poloxamer, preferably poloxamer 188,

-a buffer in the range of about 5mM to 50mM,

-a stabilizer in the range of about 100mM to 300mM,

a pH of about 4.5-7.0

In a particular embodiment of the invention, the concentration of the Abeta antibody is about 100mg/ml to 200mg/ml, preferably about 150 mg/ml.

In a particular embodiment of the invention, the poloxamer is present in a concentration of about 0.02% to 0.06%, preferably about 0.04%.

In a particular embodiment of the invention, the buffer is a sodium acetate buffer or a histidine buffer, preferably a histidine/histidine-HCl buffer.

In a particular embodiment of the invention, the buffer has a concentration of about 10 to 30mM, preferably about 20 mM.

In a particular embodiment of the invention, the pH of the formulation is about 5-6, preferably about 5.5.

In a particular embodiment of the invention, the stabilizer is selected from the group consisting of a sugar and an amino acid.

In a particular embodiment of the invention, the stabilizer is selected from trehalose and arginine.

In a particular embodiment of the invention, the stabilizing agent has a concentration of about 100mM to 300 mM.

In a particular embodiment of the invention, the stabilizer is trehalose and has a concentration of about 150mM to 250mM, preferably about 200 mM.

In a particular embodiment of the invention, the stabilizer is arginine and has a concentration of about 100mM to 150mM, preferably about 135 mM.

In a particular embodiment of the invention, the Abeta antibody is a monoclonal antibody comprising a heavy chain and a light chain.

In a particular embodiment of the invention, the heavy chain of the Abeta antibody comprises a VH domain comprising:

-a CDR1 comprising the amino acid sequence of seq. Id.No.4,

-a CDR2 comprising the amino acid sequence of seq. ID.No.5,

-a CDR3 sequence comprising the amino acid sequence of seq.

In a particular embodiment of the invention, the light chain of the Abeta antibody comprises a VL domain comprising:

-a CDR1 comprising the amino acid sequence of seq. Id.No.7,

-a CDR2 comprising the amino acid sequence of seq. Id.No.8,

-a CDR3 sequence comprising the amino acid sequence of seq.

In a particular embodiment of the invention, the VH domain of the Abeta antibody comprises the amino acid sequence of seq.id No.2 and the VL domain of the Abeta antibody comprises the amino acid sequence of seq.id No. 3.

In a particular embodiment of the invention, the heavy chain of the Abeta antibody comprises the amino acid sequence of seq.

In a particular embodiment of the invention, the light chain of the Abeta antibody comprises the amino acid sequence of seq.

In a particular embodiment of the invention, the monoclonal Abeta antibody is a mixture of mono-and di-glycosylated Abeta antibodies, wherein the mono-glycosylated antibody comprises glycosylated asparagine (Asn) at position 52 of seq.id.no.2 in the VH domain of one antibody binding site and wherein the di-glycosylated antibody comprises glycosylated asparagine (Asn) at position 52 of seq.id.no.2 in the VH domains of both antibody binding sites, and whereby the mixture comprises less than 5% of antibodies that are non-glycosylated at position 52 of seq.id.no.2 in the VH domain.

In a particular embodiment, the present invention provides the use of a pharmaceutical formulation of the present invention for subcutaneous administration of an Abeta antibody.

The terms "Abeta antibody" and "antibody that binds to Abeta" refer to an antibody that is capable of binding to a β peptide with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting a β peptide.

Notably, a β has some naturally occurring forms, of which the human forms are referred to as a β 39, a β 40, a β 41, a β 42, and a β 43 mentioned above. The most prominent form, a β 42, has the amino acid sequence (starting from the N-terminus):

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (seq. id. No. 1). In A β 41, A β 40, A β 39, the C-terminal amino acids A, IA and VIA, respectively, were deleted. In the a β 43 form, an additional threonine residue is included at the C-terminus of the sequence described above (seq. id No. 1).

The term "monoglycosylated Abeta antibody" refers to an antibody molecule comprising an N-glycosylation at position 52 of seq.id.no.2 in one (VH) -region of the individual antibody molecule; see also fig. 1. The term "bisglycosylated Abeta antibody" defines an antibody molecule that is N-glycosylated at position 52 of seq. id. No.2 on both variable regions of the heavy chain (fig. 1). Antibody molecules lacking N-glycosylation on both heavy chain (VH) -domains are referred to as "aglycosylated antibodies" (fig. 1). The mono-, di-and non-glycosylated antibodies may comprise the same amino acid sequence or different amino acid sequences. Mono-glycosylated antibodies and di-glycosylated antibodies are referred to herein as "glycosylated antibody isoforms". A purified antibody molecule characterized in that at least one antigen-binding site comprises glycosylation in the variable region of the heavy chain (VH) is a mono-glycosylated antibody, i.e. "purified mono-glycosylated antibody", which is not associated or associated to a very low extent with an isotype selected from the group consisting of di-glycosylated antibodies and non-glycosylated antibodies. Within the scope of the present invention, a doubly glycosylated antibody is not associated or associated to a very low extent with an isotype selected from mono-glycosylated antibodies and non-glycosylated antibodies, i.e. "purified doubly glycosylated antibodies".

The term "antibody" includes different forms of antibody structures, including but not limited to whole antibodies and antibody fragments. The antibody according to the invention is preferably a humanized antibody, a chimeric antibody, or a further genetically engineered antibody, as long as the characteristic properties according to the invention are retained.

An "antibody fragment" comprises a portion of a full-length antibody, preferably a variable domain thereof, or at least an antigen-binding site thereof. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, for example, described in Houston, J.S., Methods in enzymol.203(1991) 46-96). In addition, antibody fragments include those having V_HThe nature of the domains, i.e. being able to interact with V_LThe domains being assembled together, or having V's in combination with A β_LThe nature of the domains, i.e. being able to interact with V_HThe domains are assembled together into a single chain polypeptide that functions as an antigen binding site and thereby provides the property.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single amino acid composition.

The term "chimeric antibody" refers to an antibody comprising a variable region (i.e., a binding region) from one source or species and at least a portion of a constant region derived from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region is modified or altered from that of the original antibody to produce properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also known as "class-switched (class-switched) antibodies". Chimeric antibodies are the product of an immunoglobulin gene comprising the expression of a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art. See, e.g., Morrison, S.L., et al, Proc.Natl.Acad.Sci.USA 81(1984) 6851-6855; U.S. patent nos. 5,202,238 and 5,204,244.

The term "humanized antibody" refers to an antibody in which the framework regions or "complementarity determining regions" (CDRs) are modified to comprise the CDRs of an immunoglobulin of different specificity compared to the CDRs of the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to make a "humanized antibody. See, e.g., Riechmann, L., et al, Nature332(1988) 323-327; and Neuberger, M.S., et al, Nature 314(1985) 268-. Particularly preferred CDRs correspond to those representing sequences that recognize the antigens indicated above for the chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region is additionally modified or altered from that of the original antibody to produce properties according to the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, m.a., and van de Winkel, j.g., curr. opin. chem. biol.5(2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire or selected human antibody following immunization in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA90(1993) 2551-2555; Jakobovits, A., et al, Nature362(1993) 255-258; Bruggemann, M., et al, Yeast Immunol.7(1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J.Mol.biol.227(1992) 381-. The techniques of Cole et al and Boerner et al can also be used to prepare human monoclonal antibodies (Cole et al, monoclonal antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); and Boerner, P.et al, J.Immunol.147(1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes such antibodies which have been modified in the constant region to produce properties according to the invention (in particular with respect to C1q binding and/or FcR binding), for example by "class switching", i.e. by altering or mutating the Fc part (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutations).

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groups (groups) of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural properties, and or specific charge properties. An epitope is a region of an antigen that is bound by an antibody.

"variable domain" (light chain (V) as used herein_L) Of (2), the heavy chain (V)_H) The variable domains of (a) represent each of a pair of light and heavy chain domains directly involved in binding an antibody to an antigen, the variable light and heavy chain domains have the same general structure and each domain comprises four Framework (FR) regions of generally conserved sequence, connected by three "hypervariable regions" (or complementarity determining regions, CDRs).

The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The antigen-binding portion of an antibody includes amino acid residues from a "complementarity determining region" or "CDR". "framework regions" or "FR" regions are those variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. In particular, the CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. The CDR and FR regions are determined according to the standard definition of Sequences of proteins of Immunological Interest, 5 th edition, Public Health Service, national institutes of Health, Bethesda, MD (1991) and/or those residues from "hypervariable loops".

The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. The protein degradation pathway "introduction" 203(1-2):1-60. and E.F. Powell, et al (1993) "The degradation of stable protein formulations: a close hook at protein aggregation, degradation, and oxidation," Crit Rev Ther Drug System 10(4):307-77, Wang, W. (1999) "stability, stabilization, and modification of lipid protein pharmaceuticals" Int J Pharm185(2):129-88, Wang, W. (2000) "degradation and modification of lipid protein pharmaceuticals" introduction "203 (1-2):1-60. and E.Y. 1325. protein degradation, et al (9) protein degradation pathway. Stabilizers include, but are not limited to, sugars, amino acids, polyols, surfactants, antioxidants, preservatives, cyclodextrins, polyethylene glycols, such as PEG 3000,3350,4000,6000, albumins, such as Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), salts, such as sodium chloride, magnesium chloride, calcium chloride, chelating agents, such as EDTA, as defined below. As mentioned above, the stabilizing agent may be present in the formulation in an amount of about 10 to about 500mM, preferably in an amount of about 10 to about 300mM and more preferably in an amount of about 100mM to about 300 mM.

A "stable liquid pharmaceutical antibody formulation" is a liquid antibody formulation that does not observe significant changes at refrigeration temperatures (2-8 ℃) for at least 12 months, particularly 2 years, and more particularly 3 years. The stability is normalized in that not more than 10%, in particular 5%, of the antibody monomer is degraded when measured by size exclusion chromatography (SEC-HPLC). Furthermore, the solution was colorless or clear to slightly milky by visual analysis. The protein concentration of the formulation has a variation of no more than +/-10%. Not more than 10%, in particular 5%, of aggregates are formed. Stability is measured by methods known in the art such as UV spectroscopy, size exclusion chromatography (SEC-HPLC), ion exchange chromatography (IE-HPLC), turbidimetry and visual inspection.

Recombinant methods and compositions

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

For recombinant production of anti-Abeta antibodies, nucleic acids encoding, for example, antibodies as described above are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specific for the genes encoding the heavy and light chains of the antibody).

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

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells (in particular for transfection of Spodoptera frugiperda cells).

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

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (as, for example, in Graham et alHuman, 293 or 293 cells described in J.Gen Virol.36:59 (1977); baby hamster kidney cells (BHK); mouse support cells (sertoli cells) (such as, for example, TM4 cells described in Mather, biol. Reprod.23:243- & 251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine Kidney cells (MDCK; Buffalo) rat hepatocytes (BRL3A), human Lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells as described, for example, in Mather et al, AnnalsN.Y.Acad.Sci.383:44-68(1982), MRC 5 cells, and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ), pp.255-268 (2003).

Examples

The liquid pharmaceutical formulation for subcutaneous administration according to the present invention was developed as follows.

Example 1 preparation of liquid formulation

The following Abeta liquid formulations were prepared at a protein concentration of 150 mg/ml:

abeta antibodies prepared and obtained as described in WO2007/068429 are provided at a concentration of about 50-60mg/mL in 10mM histidine buffer at a pH of about 5.5. The Abeta antibodies used in the examples comprise the CDRs, VH domains, VL domains, heavy and light chains specifically defined in the sequence listing (seq. id. No. 2-11) of the present application.

To prepare a liquid formulation, Abeta was buffer exchanged against diafiltration buffer containing the expected buffer composition and concentrated by ultrafiltration to an antibody concentration of about 200 mg/mL. After the ultrafiltration operation is completed, an excipient (e.g., trehalose) is added to the antibody solution as a storage solution. The surfactant is then added in 50 to 125-fold stock solution. The protein concentration was finally adjusted with buffer to a final Abeta concentration of about 150 mg/mL.

All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 6mL glass vials sealed with ETFE (copolymer of ethylene and tetrafluoroethylene) coated stoppers and aluminum crimp caps (alucrimcap). The packing volume was about 2.4 mL. These formulations were stored for different periods of time in different climatic conditions (5 ℃,25 ℃ and 40 ℃) and stressed by shaking (1 week at 5 ℃ and 25 ℃ at a frequency of 200 min-1) and by the freeze-thaw stress method (five cycles at-80 ℃/+5 ℃). Samples were analyzed before and after applying the stress test and after storage by the following analytical methods:

UV spectroscopy

Size Exclusion Chromatography (SEC)

Ion Exchange Chromatography (IEC)

Transparency and opalescence of the solution

Visual inspection of

UV spectroscopy for determining protein content was performed on a Perkin Elmer λ 35 UV spectrophotometer at a wavelength range from 240nm to 400 nm. Pure protein samples were diluted to about 0.5mg/mL with the corresponding formulation buffer. Protein concentration was calculated according to equation 1.

Equation 1:

the UV absorbance at 280nm was corrected for light scattering at 320nm and multiplied by the dilution factor (which was determined from the weighing and density of the pure sample and dilution buffer). The molecule is divided by the product of the optical path length d and the extinction coefficient of the cuvette.

Size Exclusion Chromatography (SEC) was used to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysis products (LMW) in the formulation. The method was performed on a Waters Alliance 2695HPLC instrument with a Waters W2487 double absorbance Detector (DualaAbsorbance Detector) and a TosoHaas TSK-Gel G3000SWXL column. Intact monomers, aggregates and hydrolysates were separated by isocratic elution mode using 0.2M K2HPO4/0.25M KCL, pH 7.0 as mobile phase and detected at a wavelength of 280 nm.

Ion Exchange Chromatography (IEC) was performed to detect chemical degradation products that alter the net charge of Abeta in the formulation. The method uses a Waters Alliance 2695HPLC instrument with a Waters W2487 double Absorbance Detector (Dual Absorbance Detector) equipped with a (detection wavelength 280nm) and Mono S TM 5/50GL column (Amersham Biosciences). As mobile phases A and B, 50mM malonic acid/malonate pH 5.3 and 1M sodium acetate (in mobile phase A) pH 5.3 were used, respectively, at a flow rate of 1.0 mL/min.

Gradient program:

minute (min)	Mobile phase A	Mobile phase B
			0	100	0
1	100	0
			20	48	52
22	48	52
			24	0	100
25	0	100
			26	100	0
30	100	0

The transparency and opalescence were measured by nephelometry as Formalin Turbidity Units (FTU). The pure samples were transferred to a clear glass tube of 11mm diameter and placed in a HACH 2100AN turbidimeter.

The samples were examined for the presence of visible particles by using a Seideneader V90-T visual inspection apparatus.

The stability data provided above show that all formulations comprising polysorbate 20 and polysorbate 80 form visible granules upon storage at 5 ℃,25 ℃ or 40 ℃ for 8 months. On the other hand, the formulations containing poloxamer had almost no visible particles after 8 months of storage at 5 ℃,25 ℃ and 40 ℃. Thus poloxamers are able to prevent the formation of visible particles in Abeta antibody formulations.

Amino acid sequences disclosed in the present application

Amino acid sequence	Seq.Id.No.
		Abeta peptide A β	1
VH domains of Abeta antibodies	2
		VL domains of Abeta antibodies	3
CDR1 of the VH domain of Abeta antibodies	4
		CDR2 of the VH domain of Abeta antibodies	5
CDR3 of the VH domain of Abeta antibodies	6
		CDR1 of the VL domain of Abeta antibodies	7
CDR2 of the VL domain of Abeta antibodies	8
		CDR3 of the VL domain of Abeta antibodies	9
Heavy chain Abeta antibodies	10
		Light chain Abeta antibodies	11

Claims (23)

1. A stable liquid pharmaceutical antibody formulation, said formulation comprising:

-50mg/ml to 200mg/ml of Abeta antibody,

-0.01% -0.1% poloxamer, 5mM-50mM buffer,

-100mM to 300mM of a stabilizer,

the pH value is 4.5-7.0,

wherein the Abeta antibody is a monoclonal antibody comprising a heavy chain and a light chain,

wherein the heavy chain of the Abeta antibody comprises a VH domain comprising:

-a CDR1 consisting of the amino acid sequence of seq. Id.No.4,

-a CDR2 consisting of the amino acid sequence of seq. Id.No.5,

a CDR3 sequence consisting of the amino acid sequence of seq. Id.No.6,

wherein the light chain of the Abeta antibody comprises a VL domain comprising:

-a CDR1 consisting of the amino acid sequence of seq. Id.No.7,

-a CDR2 consisting of the amino acid sequence of seq. Id.No.8,

CDR3 sequence consisting of the amino acid sequence of seq.

2. The pharmaceutical formulation of claim 1, wherein the poloxamer is poloxamer 188.

3. The pharmaceutical formulation of claim 1, wherein the concentration of the Abeta antibody is 100mg/ml to 200 mg/ml.

4. The pharmaceutical formulation of claim 1, wherein the Abeta antibody concentration is 150 mg/ml.

5. The pharmaceutical formulation of any one of claims 1-4, wherein the poloxamer is present at a concentration of 0.02% -0.06%.

6. The pharmaceutical formulation of any one of claims 1-4, wherein the poloxamer is present at a concentration of 0.04%.

7. The pharmaceutical formulation of any one of claims 1-4, wherein the buffer is a sodium acetate buffer or a histidine buffer.

8. The pharmaceutical formulation of any one of claims 1-4, wherein the buffer is a histidine/histidine-HCl buffer.

9. The pharmaceutical formulation of any one of claims 1-4, wherein the buffer has a concentration of 10 to 30 mM.

10. The pharmaceutical formulation of any one of claims 1-4, wherein the buffer has a concentration of 20 mM.

11. The pharmaceutical formulation of any one of claims 1-4, wherein the formulation has a pH of 5-6.

12. The pharmaceutical formulation of any one of claims 1 to 4, wherein the pH of the formulation is 5.5.

13. The pharmaceutical formulation of any one of claims 1 to 4, wherein the stabilizer is selected from the group consisting of a sugar and an amino acid.

14. The pharmaceutical formulation of claim 13, wherein the stabilizer is selected from trehalose and arginine.

15. The pharmaceutical formulation of claim 14, wherein the stabilizer is trehalose and has a concentration of 150mM to 250 mM.

16. The pharmaceutical formulation of claim 14, wherein the stabilizer is trehalose and has a concentration of 200 mM.

17. The pharmaceutical formulation of claim 14, wherein the stabilizer is arginine and has a concentration of 100mM to 150 mM.

18. The pharmaceutical formulation of claim 14, wherein the stabilizer is arginine and has a concentration of 135 mM.

19. The pharmaceutical formulation of any one of claims 1-4, wherein the VH domain of the Abeta antibody comprises the amino acid sequence of seq.Id.No.2 and the VL domain of the Abeta antibody comprises the amino acid sequence of seq.Id.No. 3.

20. The pharmaceutical formulation of any one of claims 1-4, wherein the heavy chain of the Abeta antibody comprises the amino acid sequence of seq.

21. The pharmaceutical formulation of any one of claims 1-4, wherein the light chain of the Abeta antibody comprises the amino acid sequence of seq.

22. The pharmaceutical formulation of any one of claims 1-4, wherein the monoclonal Abeta antibody is a mixture of a monoglycosylated Abeta antibody and a bisglycosylated Abeta antibody, wherein the monoglycosylated antibody comprises a glycosylated asparagine (Asn) at position 52 of seq.Id.No.2 in the VH domain of one antibody binding site, and wherein the bisglycosylated antibody comprises a glycosylated asparagine (Asn) at position 52 of seq.Id.No.2 in the VH domain of both antibody binding sites, and whereby the mixture comprises less than 5% of antibodies that are non-glycosylated at position 52 of seq.Id.No.2 in the VH domain.

23. Use of a pharmaceutical formulation according to any one of claims 1 to 22 in the manufacture of a medicament for subcutaneous administration of the Abeta antibody.