MXPA06009001A - Highly concentrated liquid formulations of anti-egfr antibodies - Google Patents

Abstract

The invention relates to methods for producing, by ultrafiltration, highly concentrated liquid formulations containing at least one anti-EGFR antibody and/or one of its variants and/or fragments, particularly monoclonal antibodies against the EGF receptor, particularly preferred Mab C225 (cetuximab) and Mab h425 (EMD 72000). The invention also relates to highly concentrated liquid formulations of anti-EGFR antibodies, particularly monoclonal antibodies against the EGF receptor, particularly preferred Mab C225 (cetuximab) and Mab h425 (EMD 72000) and/or their variants and/or fragments. The invention is characterized in that the highly concentrated liquid formulations have a content of anti-EGFR antibodies ranging from 10 to 250, preferably from 50 to 180 mg/ml, particularly preferred from 100 to 150 mg/ml. Finally, the invention relates to the use of these formulations.

Description

LIQUID FORMULATIONS OF ANTI-EGFR ANTIBODIES HIGHLY CONCENTRATED FIELD OF THE INVENTION The invention relates to processes for preparing highly concentrated liquid formulations containing at least one anti-EGFR antibody and / or one of its variants and / or fragments, especially monoclonal antibodies against the EGF receptor, with special preference Mab C225 (Cetuximab) and Mab h425 (EMD 72000), by ultrafiltration. The invention also relates to liquid formulations of highly concentrated anti-EGFR antibodies, especially of monoclonal antibodies against the EGF receptor, with special preference of Mab C225 (Cetuximab) and Mab h425 (EMD 72000) and / or its variants and / or fragments, characterized in that the highly concentrated liquid formulations have an anti-EGFR antibody content of 10-250, preferably 50-180 mg / ml, with special preference of 100-150 mg / ml, and their use. BACKGROUND OF THE INVENTION Advances in the area of biotechnology in the last 10 years have allowed the preparation of a series of proteins for pharmaceutical application by recombinant DNA techniques. For example, proteinaceous drugs, such as monoclonal antibodies, are used, for example in the therapy REF: 174336 of tumors, for example for specific immunotherapy or vaccines against tumors. The therapeutic proteins are larger and more complex than conventional organic and inorganic active ingredients, and present complex three-dimensional structures and numerous functional groups that condition the biological activity of the protein or, alternatively, may cause undesirable effects. During preparation, storage and transportation, protein drugs are exposed to numerous exogenous influences that may have a stability-reducing action on the active protein ingredient. Accordingly, it is necessary to study the causes and mechanisms of special degradation reactions accurately, in order to be able to stabilize the protein, for example by the addition of certain stabilizing aids (see, for example, Manning M. C, Patel K ., &Borchardt RT (1989) Stability of protein pharmaceuticals, Pharm. Res. 6, 903-918).

The literature describes numerous formulations of therapeutic proteins. However, the requirements of the composition of a pharmaceutical preparation of protein active ingredients can be very different and, in general, it is not possible to apply established protein formulations to new protein active ingredients, due to the specific physicochemical properties and the degradation reactions. of the different proteins. Consequently, it is still a major challenge to find suitable pharmaceutical formulations of these novel active ingredients. In the current literature, ultrafiltration is described as a standard method in downstream processing in the purification of recombinant proteins (Taylor and Francis (2000) Pharmaceutical Formulation Development of Peptides and Proteins, London, pgs. 1-212; McPherson A. (1989) Separation Methods, Preparation and Analysis of Protein Crystals: "New York, Robert E. Krieger Publishing Co., Inc., pp. 1-51), but in the downstream processing, nonetheless, high sales concentrations are not achieved. The dilution of the process solutions obtained can be carried out again by means of the following purification and chromatography steps: US 6,252,055 describes the preparation of highly concentrated antibody formulations by means of ultrafiltration, however, the antibody formulations prepared in this way they present directly after the preparation a large percentage of soluble aggregates of> 4% In addition, the formulations of antibodies obtained are not characterized in terms of their native structure and stability, which should be considered, for example, as very important with respect to the immunogenicity and efficacy of the antibody formulation.The disadvantageous influence of aggregates on the greater immunogenicity and lower efficacy, as well as the reduced bioavailability of protein formulations, is already known from the literature (SA Marshall, GA Lazar, A. J. Chirino, and JR Desjariais) Rational design and engineering of Drug Discovery Today 8 (5): 212-221, 2003, Schellekens H. Bioequivalence and the Imunogenicity of Biopharmaceuticals, Nat Rev Drug Discov 1 (6): 457-462, 2002). For the reasons mentioned above, it is obvious that the preparation of liquid formulations of highly concentrated antibodies that are stable for a sufficiently long time is extremely difficult for the person skilled in the art. Furthermore, the preparation of a highly concentrated liquid formulation was not attractive to the person skilled in the art, since the tendency to aggregation of the strongly labeled proteins and especially of the antibodies was already sufficiently known in low concentration ranges (SA Marshall , GA Lazar, AJ Chirino and JR Desjariais, Rational design and engineering of therapeutic proteins, Drug Discovery Today 8 (5): 212-221, 2003). Thus, protein aggregation is described in the literature as the physical reaction of instability that is most often present (W. Wang, Instability, stabilization, and formulation of liquid protein pharmaceuticals, Int. J. Pharm. ): 129-188, 1999).

Formulations containing Mab C225 (Cetuximab) or Mab h425 (EMD 72000) are known from WO03053465 and from WO03007988, but the known formulations of WO03053465 show a relatively low protein concentration and are not stable at room temperature in the long term . The known formulations of WO03007988 also have a relatively low protein concentration and the preparation (lyophilized) must be reconstituted before use. The lyophilization process for the stabilization of protein formulations is described, for example, in W093 / 00807 and W098 / 22136, but the significant disadvantages of lyophilized preparations are that the user must reconstitute the lyophilisate before use, which represents a considerable source of error in the preparation before use. Since another preparation process is added, compared to liquid formulations, the process is unfavorable with respect to the additional expense for the development of the process (to ensure the stability during lyophilization), the preparation (costs and duration of the preparation) and , for example, validation. In the case of formulations of low protein concentration known hitherto, high volumes of infusion are required with an intravenous administration. Therefore, the concentration of antibodies according to the invention is the object of the invention, so that, by reducing the volumes to be applied, a subcutaneous application can also be considered. The formulations to be applied subcutaneously should not exceed a volume of 1.0-1.5 ml and, in addition, should be euhydric (pH 7.2 or pH 4.0-9.0) and isotonic (approximately 290 mOsm). Another advantage of subcutaneous formulations lies in the possibility of self-administration by the patient. However, with the concentration, the stability of the protein should not be impaired, that is, the increase in decomposition and aggregation products should be acceptable within the specifications. In addition, these formulations should be free of toxicologically harmful substances or contain them only in physiologically harmless concentrations. Given that, due to the expected difficulties, the already established protein formulations can not be converted into new protein active principles, it was the object of the present invention to find new stable highly concentrated liquid formulations for therapeutic proteins., especially monoclonal antibodies against the EGF receptor, for example Mab C225 (Cetuximab) and Mab h425 (EMD 72000), which have greater stability against stress conditions, such as higher temperature, ambient humidity and / or shear forces , so that, in the case of preparation, storage, transport and application, their effectiveness is preserved and these formulations do not contain toxicologically harmful adjuvants. Detailed description of the invention Surprisingly, pharmaceutical preparations of highly concentrated anti-EGFR antibodies can be obtained with the aid of ultrafiltration methods, which allow in a liquid formulation protein concentrations of 10-250 mg / ml, with special preference of 50. - 180 mg / ml, with special preference of 100-150 mg / ml. The formulations obtained by means of the ultrafiltration process are preferably stable for a prolonged period or can be mixed if necessary with appropriate stabilizing adjuvants or by subsequent lyophilization. The formulations according to the invention are physiologically tolerable, can be prepared easily, can be precisely metered and are stable during the storage period, mechanically charged and, for example, in the case of multiple freezing and thawing processes. Surprisingly it was found that the highly concentrated anti-EGFR antibody formulations prepared according to the method according to the invention contain a part of >monomers; 99% The highly concentrated liquid formulations obtained according to the invention with a concentration of 10-250 mg / ml, with special preference of 50-180 mg / ml, with special preference of 100-150 mg / ml are physically and chemically stable, i.e. no modification of the monomer content occurs with "a simultaneous increase of soluble aggregates, which would be seen as very critical in the sense of efficacy and immunogenic side effects (Schellekens H. (2002) Bioequivalence and the immunogenicity of biopharmaceuticals: Nat. Rev. Drug Discov., V. 1, pp. 457-462) Furthermore, with the applied ultrafiltration procedure, there is no change in the primary structure of the protein, on the other hand, in terms of mechanical stability and the thermal stability, there are no disadvantages compared to the formulations of proteins of "low concentration. In particular, the characterized aggregation products are also in the range of specifications established for highly concentrated liquid antibody formulations. This was not to be expected, since, in the case of highly concentrated protein formulations, the tendency to instability is much greater than in the case of diluted protein formulations (Fields, G., Alonso, D., Stiger, D ., Dill, K. (1992) "Theory for the aggregation to proteins and copolymers", J. Phys. Chem. 96, 3974-3981). In the case of a high concentration of proteins, the "packing density" of the protein molecules is high. Accordingly, an increased number of collisions can be assumed, and occasional protein associations can occur. This process is usually carried out by nucleation and growth mechanisms, where the critical nuclei often represent soluble associated proteins, but which can be rapidly converted into precipitates of insoluble proteins (denatured protein) (Reithel, JF ( 1962) "The dissociation and association of protein structures", Adv. Protein Chem. 18, 123). The size of the protein aggregates increases with a higher protein concentration, as could already be shown for the β-lactoglobulin (Roefs, SPFM, De Kruif, KG (1994) "A model for the denaturation and aggregation of β + lactoglobulin "Eur. J. Biochem. 226, 883-889). The formulations of anti-EGFR antibodies according to the invention described below are surprisingly characterized by one or several advantages, selected from: high concentration of proteins, high stability, low tendency to aggregation, low viscosity, high purity, absence of pharmaceutically harmful agents and, with it, great security, good tolerance and possibility of direct application. The preparation methods according to the invention described below are surprisingly characterized by one or several advantages, selected from: simplicity, saving in time and costs, use of pharmaceutically innocuous agents, high yields. In this way, the methods according to the invention are preferably substantially simpler in the embodiment, save more time and are more cost-effective than the techniques described in the literature, since liquid formulations of anti-bodies are surprisingly obtained. -EGFR highly concentrated stable by ultrafiltration that present the advantages mentioned above.

Therefore, methods for preparing highly concentrated liquid formulations containing at least one anti-EGFR antibody and / or one of its variants and / or fragments by ultrafiltration are subject of the invention. The processes according to the invention are characterized in particular by the highly concentrated liquid formulations obtained having a content of at least one anti-EGFR antibody of 10-250 mg / ml, preferably 50-180 mg / ml, with special preference of 100-100 mg / ml. 150 mg / ml. In addition, the methods according to the invention are characterized in that the anti-EGFR antibodies are monoclonal and have murine or human origin, preferably murine origin, and are chimeric or humanized. Especially preferred are the anti-EGFR antibodies Mab C225 (Cetuximab) or Mab h425 (EMD72000) and / or its variants and / or fragments. Ultrafiltration processes according to the invention are ultrafiltration processes such as ultrafiltration under agitation and tangential flow filtration (TFF).

The ultrafiltration of the antibodies according to the invention is preferably carried out in an appropriate buffer system, that is to say that stabilization of the reaction solutions such as, for example, by detergents is not necessary. The use of detergents in preparations for parenteral application should be avoided or minimized in general, since a considerable toxic and immunogenic potential arises (Sweetana S. &Akers MJ (1996) Solubility principies and practices for parenteral drug dosage form development. PDA J. Pharm. Sci. Technol. 50, 330-342) and may also lead to the modification of the secondary structure of proteins (Vermeer AWP &Norde W. (2000) The influence of the binding of low molecular weight surfactants on the thermal stability and secondary structure of igG Colloids and Surfaces A: Physicochemical and Engineering Aspeets 161, 139-150). Furthermore, carrying out the process of ultrafiltration of formulations containing detergents becomes difficult, since, due to possible micelle formation of the detergent, a disadvantageous and uncontrolled accumulation of the detergent in the product can occur. With respect to the anti-EGFR antibodies according to the invention and within the framework of the present invention, by "biologically active", "native" and "effective" it is understood that the anti-EGFR antibodies according to the invention can also exert their biological action after of becoming formulations according to the invention, especially EGFR binding, inhibition of ligand binding, especially EGF, with EGFR, modulation, in particular inhibition of EGFR-mediated signal transduction and prevention or therapy of mediated diseases. EGFR. Anti-EGFR antibodies: Preferably, the anti-EGFR antibodies according to the invention are monoclonal and of murine or human origin; with particular preference they are of murine origin and are chimeric or humanized. In the case of the antibody directed against the epidermal growth factor receptor (EGFR), it is particularly preferred to treat Mab C225 (cetuximab) or Mab h.425 (EMD 72000) and / or its variants or fragments. Other antibodies directed against EGFR are. describe, for example, in EP 0586002, as well as in J. Nati. Cancer Inst. 1993, 85: 27-33 (Mab 528). Mab C225 (Cetuximab, Erbitux ™): Mab C225 (Cetuximab) is a clinically proven antibody that binds to the EGF receptor. Mab C225 (Cetuximab) is a chimeric antibody whose variable regions are of murine origin and whose constant regions are of human origin. It was first described by Naramura et al., Cancer Immunol. Immunotherapy 1993, 37: 343-349, as well as in WO 96/40210 Al.

Mab h425 (EMD 72000): Mab h425 (EMD 72000) is a humanized monoclonal antibody (Mab) obtained from murine anti-EGFR antibody 425 (Mab 425) (EP 0531472). The murine monoclonal antibody Mab 425 was developed in the human carcinoma cell line A431, since it bihere with an extracellular epitope of the epidermal growth factor receptor (EGFR). It was found to inhibit EGF binding (Murthy et al., 1987). Increased expression of EGFR was observed in malignant tissues from various sources and, consequently, Mab 425 is a possible active ingredient for the diagnosis and therapeutic treatment of human tumors. As a result, Mab 425 was found to mediate tumor cytotoxicity in vitro and suppress tumor growth of epidermoid and colorectal carcinoma cell lines in vi tro (Rodeck et al., 1987). In addition, it was shown that Mab 425 bito xenografts of human malignant gliomas in mice (Takahashi et al., 1987). Their humanized and chimeric forms are described, for example, in EP 0531472 Kettleborough et al., Protein Engineering 1991, 4: 773-783 Bier et al., Cancer Chemother. Pharmacol. 2001, 47: 519-524 Bier et al., Cancer Immunol. Immunother 1998, 46: 167-173. In this case, Mab h425 (EMD 72000) is a humanized antibody (h.425) in clinical phase I / II, whose constant region consists of a K chain and a human? -1 chain (EP 0531472).

Human anti-EGFR antibodies can be prepared by XenoMouse technology, as described in WO 9110741, WO 9402602 and WO 96/33735. An antibody subjected to clinical studies and prepared by this technology is, for example, ABX-EGF (Abgenix, Crit., Rev. Oncol, Hematol, 2001, 38: 17-23, Cancer Research, 1999, 59: 1236-43). Antibody: Antibody or immunoglobulin is used in the broadest sense, within the framework of the present invention, and relates, in particular, to polyclonal antibodies and multispecific antibodies (for example, bispecific antibodies) and, with particular preference, to monoclonal antibodies intact (Mab) that are biologically active, and their variants and fragments. The term also encompasses heteroantibodies that consist of two or more antibodies or their fragments and / or have different binding specificities, and bind to each other. Depending on the amino acid sequence of their constant regions, the antibodies can be assigned to different classes of antibody (immunoglobulin): IgA, IgD, IgE, IgG and IgM Several of them can be subdivided into subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

In general, the antibodies have a molecular weight of approximately 150 kDa and consist of two identical light chains (L) and two identical heavy chains (H). Monoclonal antibodies are obtained from a population of homogeneous cells. They are very specific and are directed against a single epitope, while polyclonal antibodies cover different "antibodies, directed against various epitopes." Methods for the preparation of monoclonal antibodies include, for example, the hybridoma method described by Kohier and Milstein (Nature 256, 495 (1975)) and in Burdon et al., (1985) "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas", eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers , Amsterdam can be prepared, in particular, by known recombinant DNA techniques (see, for example, US 4816567) Monoclonal antibodies can also be isolated from antibody phage libraries, for example with the aid of the techniques described in Clackson et al. (Nature, 352: 624-628 (1991)) and Marks et al. (J. Mol. Biol, 222: 58, 1-597 (1991)). Variants and fragments : In the case of the variants (muteins) of the antibodies, they are structurally related proteins, for example, those that can be obtained by modification of the primary sequence (amino acid sequence), by glucoengineering (variants of the sites or structures of glycosylation, also deglycosylated proteins), by PEGylation, by preparation in modified host cells or by other techniques. The variants according to the invention are not restricted in this case to the previous examples, but instead include all the variants of the antibodies according to the invention which are known to the person skilled in the art. In the case of fragments (partial segments) of antibodies, these are cleavage products of the antibodies obtained, for example, by limited enzymatic digestion with the aid of papain, pepsin and plasmin or by preparation of the partial segments by genetic engineering. Typical partial segments are, for example, the bivalent fragment F (ab ') 2 / the monovalent fragment Fab and the Fc fragment (Lottspeich F., H. Zorbas (ed.). Bioanalytik, Heidelberg, Berlin: Spektrum Akademischer Verlag GmbH, (1998) p.1035). The fragments according to the invention are not restricted to the previous examples, but instead include all fragments of antibodies according to the invention, known to the person skilled in the art. Pharmaceutical preparation: The terms "pharmaceutical formulation" and "pharmaceutical preparation" are used synonymously within the framework of the present invention. As used in the present"pharmaceutically tolerable" refers to medicaments, excipients, adjuvants, stabilizers, solvents and other agents that facilitate the administration of the pharmaceutical preparations thus obtained to a mammal, without undesired physiological side effects, such as nausea, dizziness, digestive problems or the like . In pharmaceutical preparations for parenteral administration, isotonicity, euhydria, as well as tolerability and safety of the formulation (low toxicity), of the used adjuvants and of the primary package are required. Surprisingly, liquid formulations of anti-EGFR antibodies according to the invention preferably have. the advantage that direct use is possible, since physiologically acceptable agents are used. Thus, the preparation of liquid formulations of highly concentrated anti-EGFR antibodies according to the invention preferably with a high simultaneous yield of native and pharmaceutically innocuous high purity protein is preferably simple, saves time and is inexpensive. In the case of ultrafiltration, it is a semi-permeable membrane process operated under pressure to separate dissolved and suspended materials. The principle of separation is based on the size and dimension of the molecule, that is, substances that are smaller than the size of the pores reach the filtrate (permeate), while substances that are larger than the pore size they remain in the retentate (concentrated). The force necessary to carry out the separation can be applied, for example, by centrifugal forces, a source of gas pressure (for example, nitrogen) or a membrane pump. Preferably, liquid formulations of highly concentrated anti-EGFR antibodies according to the invention can be prepared by concentrating a solution containing anti-EGFR antibodies by means of an ultrafiltration method. For this, a solution with a defined concentration of anti-EGFR antibodies according to the invention is conveniently poured (for example for C225: 0.01 to 150 mg / ml, preferably 2 to 100 mg / ml, with special preference approximately 20 mg / ml, for EMD 72000: 0.01 to 150 mg / ml, preferably 5 to 100 mg / ml, with special preference approximately 20 mg / ml), as obtained in its preparation, in an ultrafiltration unit and submits, under controlled and defined pressure conditions, to a concentration procedure. If the antibody is present as a solid substance, for example as a lyophilisate, the highly concentrated liquid formulation according to. The invention can be prepared by first dissolving anti-EGFR antibodies according to the invention in water or an aqueous solution containing one or more other adjuvants and then exposing it to an ultrafiltration process. The product obtained by means of the ultrafiltration process can then be stabilized by adding the adjuvants detailed below. The solution thus obtained containing the corresponding antibody is adjusted to a pH value of 4 to 10, preferably a pH value of 5 to 9, it is filtered under sterility and, if necessary, it is converted into a solid form through a lyophilization stage. later for its stabilization. The order of the addition of the different adjuvants or the antibody according to the invention is largely independent of the preparation process and is at the discretion of the expert. The anti-EGFR antibodies are present in the highly concentrated liquid formulations according to the invention, preferably in a biologically active form and, in the methods according to the invention, preferably no denaturation of the antibodies occurs. In this way, the biological efficacy of the protein is preferably preserved. In the compounds according to the invention, for example, polyethersulfone (PES) or regenerated cellulose can be used as ultrafiltration membranes: the theoretically imaginable cut-off is in the range of 5 to 500 kDa, preferably 10 a 100 kDa, with special preference of 30 to 50 kDa. Centrifugal forces applied to the Ultrafree centrifuge tubes (Millipore) vary in the range of 1 - 20,000 * g, preferably in the range of 1000 - 12,000 * g, with special preference in 2000 * g. The gas pressure applied in the case of the Amicon agitation cell (Millipore) varies in the range of 0.1 to 5 psi, preferably is 4 psi. The initial pressure applied in the Labscale-TFF installation (Millipore) varies in the range of 0.1 to 85 psi, preferably in the range of 10 to 30 psi, with special preference being 20 psi. The final pressure applied in the Labscale-TFF installation (Millipore) ranges from 0.1 to 85 psi, preferably in the range from 5 to 20 psi, with special preference being 10 psi. In the process according to the invention, the following buffers can be used, for example: phosphate buffer: Na (or K) phosphate; possible pH about 6.0 - 8.2; citrate buffer: Na citrate or citric acid, possible pH about 2.2 - 6.5, succinate buffer, pH about 4.8 - 6.3, acetate buffer, eg sodium acetate, pH about 2.5-6.0; histidine buffer, pH about 6.0-7.8; glutamic acid pH 8.0 to 10.2; glycine (N, N-bis (2-hydroxyethyl) glycine), pH about 8.6 to 10.6; glycinate buffer, pH about 6.5-7.5; imidazole, pH from 6.2 to 7.8; potassium chloride, pH about 1.0 to 2.2; lactate buffer, pH about 3.0-6.0; Maleate buffer, pH about 2.5-5.0; tartrate buffer, pH about 3.0-5.0; Tris: pH about 6.8-7.7; phosphate-citrate buffer. It is also possible to consider the addition of isotonic agents for isotonization (for example, Cl of Na (or K) or also other salts). In the methods according to the invention, the buffers mentioned for example in the following concentrations can be used: 1 mM to 200 mM, preferably 2-20 mM, with special preference about 10 mM. Preferably, the following pH ranges can be used: pH 4-10, pH = IEP +/- 2 pH units (2 pH units near the isoelectric point of the protein) are preferred. Preferably, the following isotonic agents (usual concentrations) can be used: sodium chloride approximately 5 mM - 305 mM; potassium chloride; glucose; glycerin; 4-5.5 mM dextrose; Sodium sulfate 1- 1.6 mM. Preferably, the following substances can be used to reduce the viscosity: sodium chloride, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate. Preferably, the following stabilizers can be used: 1) amino acids (about 1-100 mg / ml, with particular preference 3-10 mg / ml, such as hydrochloride) arginine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine , alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline. 2) Sugar and sugar alcohols (about 1-200 mg / ml, with particular preference 30-65 mg / ml) sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, trehalose, glucosamine, N- methylglucosamine, galactosamine, neuramic acid. 3) Antioxidants 0.2% sodium acetone bisulfite, 0.01% ascorbic acid, 0.015% ascorbic acid ester, 0.02% butylhydroxyanisole (BHA), 0.02% butylhydroxytoluene (BHT), 0.5% cysteine, nordihydroguayaretic acid (NDGA) 0.01%, monothioglycerol 0.5%, sodium bisulfite 0.15%, sodium metabisulfite 0.2%, tocopherol 0.5%, glutathione 0.1%. 4) Preservatives m-cresol about 0.1 - 0.3%, chlorocresol about 0.1 - 0.3%, phenol about 0.5%, benzyl alcohol about 1.0 - 2.0%, methylparaben about 0, 2%, propylparaben approximately 0.02%, butylparaben approximately 0, 015%, chlorobutanol about 0.25-0.5%, phenyl mercurinitrate about 0.002%, phenyl mercury acetate about 0.002%, thimeral about 0.01-0.02%, benzalkonium chloride about 0.01%, chloride of benzethonium approximately 0.01%. 5) Cyclodextrins eg hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin. 6) Albumins human serum albumin (HSA), bovine serum albumin (BSA): "7) polyhydric alcohols glycerol, ethanol, mannitol 8) salts salts of acetate (eg, sodium acetate), magnesium chloride, chloride of calcium, tromethamine, EDTA (for example, sodium EDTA) The invention also comprises all the hydrates, salts and derivatives of the aforementioned agents, which are known and imaginable by the person skilled in the art. Highly concentrated liquid pools containing at least one anti-EGFR antibody and / or one of its variants and / or fragments These liquid formulations of highly concentrated anti-EGFR antibodies can be prepared by means of the ultrafiltration procedures described above. conceivable for concentration are chromatographic procedures such as, for example, size exclusion chromatography (e.g. gel filtration), c affinity romatography (e.g., protein A chromatography) or ion exchange chromatography, membrane separation method such as, for example, dialysis, electrodialysis, microfiltration, reverse osmosis, electrophoretic methods or drying procedures such as, for example, nitrogen gas drying, vacuum oven drying, lyophilization, washing in organic solvents and subsequent air drying, liquid bed drying, fluid bed drying, spray drying, roller drying, layer drying, air drying at room temperature environment and subsequent reconstitution in a small volume of solvents. Liquid formulations of highly concentrated anti-EGFR antibodies are characterized in particular by having a content of at least one anti-EGFR antibody of 10-250 mg / ml, preferably 50-180 mg / ml, with special preference of 100-150. mg / ml. The highly concentrated liquid formulations according to the invention are characterized in that the anti-EGFR antibodies are monoclonal and have murine or human origin, preferably murine origin, and are chimeric or humanized. Mab C225 anti-EGFR antibodies are especially preferred (Cetuximab) or Mab h425 (EMD72000) and / or its variants and / or fragments. Another object of the invention are highly concentrated liquid formulations containing at least one anti-EGFR antibody and / or one of its variants and / or fragments that can be obtained by means of processes according to the invention, ie by ultrafiltration methods described previously. In addition, liquid formulations of highly concentrated anti-EGFR antibodies according to the invention as storage stable medicaments are subject of the invention. The liquid formulations of highly concentrated anti-EGFR antibodies according to the invention may contain, in addition to the antibodies according to the invention, optionally excipients and / or adjuvants and / or other pharmaceutical active ingredients. Preferably, the processes according to the invention make it possible to prepare highly concentrated formulations without causing aggregations of the undesired and unfavorable antibodies according to the invention. In this way, ready-to-apply solutions can be prepared by means of the processes according to the invention with a high content of active ingredients. Highly concentrated formulations of protein active ingredients have recently had a large increase. Most antibodies used for therapy are used in doses in the mg / kg region. A high dose and the small volumes that must be administered (for example approximately 1 to 1.5 ml for subcutaneous administration) demonstrate the demand for highly concentrated protein preparations, with concentrations higher than 100 mg / ml. In addition, highly concentrated protein formulations have considerable advantages in preclinical trials for the investigation of in vitro and in vivo acceptability and efficacy (in an animal model), in clinical trials for the investigation of acceptability and efficacy in humans. and in the clinical use of the product (in particular for subcutaneous administration). Its advantages consist, in particular, in that a small volume of the preparation is used. In contrast to the infusion or injection of proteinaceous drugs with a relatively low concentration, this allows, for example, subcutaneous administration of proteinaceous drugs for the patient. Subcutaneous administration of proteic drugs can be due to several causes. For example, the address can be desired to a specific target in connection with a "therapeutic window". In addition, subcutaneous administration has the advantage that the patient can take charge of the administration by himself, without relying on medical personnel. The example of insulin clearly shows these advantages. However, since injections for subcutaneous administration have a maximum of 1-1.5 ml, highly concentrated protein formulations containing more than 100 mg / ml are often required. Surprisingly, liquid formulations of highly concentrated anti-EGFR antibodies can be obtained with the aid of processes according to the invention, which do not have the aforementioned disadvantages with protein concentrations of 10-250 mg / ml, preferably 50-180 mg / ml, with special preference of 100-150 mg / ml. The limit of highly concentrated immunoglobulin formulations is usually 2-50 mg / ml (Humira®) in liquid formulations of ready-to-use antibodies. With the methods according to the invention, however, clearly more concentrated and still stable formulations can be prepared, which was unexpected. Accordingly, highly concentrated, stable antibody formulations having lower viscosity and tendency to aggregation compared to known high-concentrated liquid antibody formulations can be obtained by the methods according to the invention., which simplifies the management in the case of parenteral administration. Advantageously, solutions containing antibodies with a pH value of 4 to 10, preferably with a pH value of 5 to 9, and an osmolality of 250 to 350 mOsmol can be prepared from the formulations according to the invention. / kg. The formulations according to the invention can then be administered directly, substantially painlessly, intravenously, intraarterially and also subcutaneously. In addition, the preparation can also be added to solutions for infusion, such as, for example, glucose solution, isotonic physiological solution or Ringer's solution, which may also contain other active ingredients, which allows the administration of greater amounts of active ingredients. . The formulations according to the invention are physiologically well tolerated, easy to prepare, can be administered accurately and are preferably stable with respect to the content, decomposition products and aggregates during storage and transport and during multiple freezing and thawing processes. Preferably, they can be stored stably for a prolonged period at refrigerator temperature (2-8 ° C) and at room temperature (23-27 ° C) and 60% relative humidity (RH). Preferably, the formulations according to the invention are comparatively stable with high ambient temperatures and humidity. The term "effective amount" denotes the amount of a medicament or active pharmaceutical ingredient that causes a biological or medical response in a tissue, system, animal or human that seeks or is of interest, for example, to a researcher or physician. In addition, the term "therapeutically effective amount" denotes an amount which, "compared to a corresponding subject who did not receive said amount, has the following consequences: better treatment, cure, prevention or elimination of a disease, syndrome, pathological condition, discomfort, disorder or prevention of side effects or also the reduction of the progress of a disease, discomfort or disorder.The term "therapeutically effective amount" also encompasses the amounts that are effective in increasing normal physiological function.Drugs can be administered as a dose units comprising a predetermined quantity of active ingredient per dosage unit A unit of this type may comprise, for example, 0.5 mg to 1 g, preferably 1 mg to 800 mg, of an active ingredient according to invention, according to the pathological condition treated, the route of administration and the age, weight and health of the The unit formulations preferably are those comprising a daily dose or sub-dose, as indicated above, or one of its fractions of an active ingredient. In addition, this type of medication can be prepared by means of one of the processes generally known in the pharmaceutical sector. The medicaments can be adapted for administration by any appropriate route, for example orally (including buccal or sublingual), rectal, pulmonary, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal). Medications of this type can be prepared by all processes known in the pharmaceutical sector, for example, by combining the active ingredient with the excipient (s) or adjuvants. Parenteral administration is preferably suitable for the administration of the medicaments according to the invention. In the case of parenteral administration, intravenous, subcutaneous or intradermal administrations are particularly preferred. In the case of intravenous administration, the injection can be applied directly or also as an addition to solutions by infusion. The medicaments according to the invention for subcutaneous or intradermal administration are particularly suitable, since, with the help of highly concentrated liquid formulations according to the invention, small volumes that can be administered, required for subcutaneous administration, can be obtained. Subcutaneous administration has the advantage that the patient can administer the medication himself, without the need for expert medical help. The solid forms of the anti-EGFR antibodies according to the invention are also suitable for the preparation of medicaments for parenteral administration with controlled, sustained and / or delayed release of the active ingredient, for example also for the preparation of release formulations delayed that are advantageous for the patient, since an application is necessary only in large intervals. The pharmaceutical preparations according to the invention can also be injected directly into the tumor and, thus, develop their effect directly at the site of action as intended. Medications adapted for parenteral administration include sterile aqueous and non-aqueous injection solutions comprising antioxidants, buffers, bacteriostats and solutes, through which the formulation is isotonic with the blood of the recipient to be treated.; as well as sterile aqueous and non-aqueous suspensions, which may comprise suspension media and thickeners. The formulations can be administered in single or multiple dose containers, for example sealed ampoules and vials, and stored in the lyophilized state, whereby it is only necessary to add the sterile liquid carrier, for example water for injections, immediately before use. The solutions and suspensions for injection prepared according to the formulation can be produced from sterile powders, granules and tablets. The formulations of anti-EGFR antibodies according to the invention can also be administered in the form of liposome delivery systems, such as, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from various phospholipids such as, for example, cholesterol, stearylamine or phosphatidylcholines. Medicaments adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils, and incorporated into the formulations according to the invention. For the treatment of the eye or other external tissues, for example the mouth and the skin, the formulations are preferably applied as topical ointment or cream. In the case of the formulation as an ointment, the formulations according to the invention can be incorporated in a cream base with paraffin or miscible with water. Alternatively, a formulation according to the invention can be formulated to give a cream with an oil-in-water cream base or a water-in-oil base. Medications adapted for topical administration of the eye include ophthalmic drops. Medications adapted for rectal administration can be given in the form of suppositories or enemas. Medicaments adapted for administration by inhalation include powders or finely particulate mists that can be produced by various types of pressurized dispensers with aerosols, sprays or insufflators. Medications adapted for vaginal administration can be supplied as pessaries, tampons, creams, gels, pastes, foams or aerosol formulations. It is obvious that, in addition to the constituents particularly mentioned above, the medicaments according to the invention may also comprise other agents customary in the specialized sector in relation to the particular type of pharmaceutical formulation. Another object of the invention are kits composed of separate packages of a) a formulation according to the invention containing an effective amount of an anti-EGFR antibody, preferably a monoclonal anti-EGFR antibody, with particular preference of Mab C225 (Cetuximab) or Mab h425 (EMD 72000) and / or its variants or fragments and b) a formulation containing an effective amount of another medicament active ingredient. The kit contains suitable containers, such as boxes or cartons, bottles, bags or individual ampoules. The kit may contain, for example, separate ampoules, where there is an effective formulation of an anti-EGFR antibody according to the invention and a formulation of another medicament active ingredient in dissolved or lyophilized form. A therapeutically effective amount of an anti-EGFR antibody according to the invention depends on several factors, such as eg the age and weight of the patient, the precise pathological condition requiring treatment and its severity, the nature of the formulation and the route of administration, and ultimately determined by the attending physician or veterinarian. However, an effective amount of an anti-EGFR antibody according to the invention for the treatment of a neoplastic growth, for example intestinal or breast cancer, generally ranges in the range of 0.1 to 100 mg / kg of body weight. of the receiver (mammal) per day and, in particular, typically in the range of 1 to 10 mg / kg of body weight per day. Therefore, the actual amount per day for an adult mammal weighing 70 kg would usually be between 70 and 700 mg, where this amount can be given as a single dose per day or usually in a series of sub-doses (such as, for example, , two, three, four, five or six) per day, so that the total daily dose is the same. The appropriate antibody titer is determined by methods known to the person skilled in the art. The expected dose for administration is usually sufficient to achieve the inhibitory action of the tumor sought. However, the lowest possible dose should also be chosen so as not to have side effects, such as cross-reactions, undesired anaphylactic reactions, or the like. The medicaments according to the invention can be used, in particular, for the prevention and / or treatment of diseases and pathological conditions. Another object of the invention consists, therefore, in the use of liquid formulations of highly concentrated anti-EGFR antibodies according to the invention for preparing a medicament for the treatment and / or prevention of tumors and / or tumor metastasis, wherein the tumor is selected from the group consisting of brain tumor, urogenital tract tumor, tumor of the lymphatic system, stomach tumor, laryngeal tumor, monocytic leukemia, lung adenocarcinoma, small cell lung carcinoma, pancreatic cancer, glioblastoma and breast carcinoma . It could be demonstrated in various in vi tro and in vivo studies that blocking EGFR by antibodies acts against tumors at various levels, for example by inhibiting the proliferation of cancer cells, reducing tumor-mediated angiogenesis, inducing apoptosis of cancer cells and increased toxic effects of radiotherapy and conventional chemotherapy. Medicaments comprising formulations according to the invention are capable of regulating, modulating or effectively inhibiting EGFR and, therefore, can be used for the prevention and / or treatment of diseases connected with unregulated or altered EGFR activity. In particular, the formulations of anti-EGFR antibodies according to the invention can be used, consequently, in the treatment of certain forms of cancer and diseases caused by pathological angiogenesis, such as diabetic retinopathy or inflammations. Accordingly, the invention also relates to the use of formulations according to the invention for the preparation of a medicament for the treatment and / or prevention of diseases caused, mediated and / or propagated by EGFR and / or by signal transduction. mediated by EGFR. The medicaments according to the invention are particularly suitable for the treatment and / or prevention of cancer, including solid carcinomas such as for example, carcinomas (for example of the lung, pancreas, thyroid, bladder or colon), myeloid diseases (e.g. myeloid leukemia) or adenomas (for example hairy colonic adenoma), pathological angiogenesis and migration of metastatic cells. The drugs are also useful for the treatment of chronic inflammation dependent on complement activation (Niculescu et al (2002) Immunol Res., 24: 191-199) and HIV-1-induced immunodeficiency (human immunodeficiency virus type 1) (Popik et al. (1998) J Virol, 72: 6406-6413). In addition, the present medicaments are suitable as pharmaceutical active ingredients for mammals, in particular humans, for the treatment of EGFR-induced diseases. The term "EGFR-induced diseases" refers to disease states dependent on EGFR activity. EGFR intervenes directly or indirectly in the signal transduction pathways of various cellular activities, for example proliferation, adhesion and migration, as well as differentiation. Diseases associated with EGFR activity include the proliferation of tumor cells, pathological neovascularization that favors the growth of solid tumors, neovascularization in the eye (diabetic retinopathy, macular degeneration associated with age and the like) and inflammation (psoriasis, rheumatoid arthritis and the like). The diseases analyzed here are usually divided into two groups: hyperproliferative and non-hyperproliferative diseases. In this context, non-cancerous diseases are considered: psoriasis, arthritis, inflammations, endometriosis, scarring, benign prostatic hyperplasia, immunological diseases, autoimmune diseases and immunodeficiency diseases, of which arthritis, inflammation, immunological diseases, autoimmune diseases and Immunodeficiency are usually considered non-hyperproliferative diseases. In this context, cancerous diseases are considered brain cancer, lung cancer, squamous epithelium cancer, bladder cancer, stomach cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, breast cancer, cancer of head, neck cancer, esophageal cancer, gynecological cancer, thyroid cancer, lymphoma, chronic leukemia and acute leukemia, and all are usually included in the group of hyperproliferative diseases. In particular, the growth of cancer cells and in particular the growth of cancer cells mediated directly or indirectly by EGFR is a disease that represents an object of the present invention. It can be shown that the medicaments according to the invention have antiproliferative action in vivo in a xenotransplant tumor model. The medicaments according to the invention are administered to a patient with a hyperproliferative disease, for example to inhibit the development of the tumor, to reduce the inflammation associated with a lymphoproliferative disease, to inhibit the rejection of a transplant or the neurological damage due to tissue repair, etc. The present medications are useful for preventive or therapeutic purposes. As used herein, the term "treat" is used as a reference both for the prevention of diseases and for the treatment of existing discomforts. The prevention of proliferation is achieved by the administration of the drugs according to the invention before the development of the apparent disease, for example, to prevent tumor growth, prevent metastatic growth, reduction of restenosis associated with cardiovascular surgery, etc. Alternatively, the medications are used for the treatment of continuing illnesses by stabilizing or improving the clinical symptoms of the patient. The host or patient can belong to any species of mammal, for example, a species of primate, and in particular human; rodents, for example mice, rats and hamsters; rabbits horses, cows, dogs, cats, etc. Animal models are of interest for experimental studies, since they provide a model for the treatment of human disease. The receptivity of a certain cell to treatment with the medicaments according to the invention can be determined by in vitro tests. Typically, a culture of the cell is incubated with a medicament according to the invention at different concentrations for a period sufficient to allow the active ingredients to induce cell death or inhibit migration, usually between about one hour and one week. In vi tro tests can be carried out by cultured cells from a biopsy sample. Then the remaining vital cells are counted after treatment. The dose varies according to the specific medications used, the specific disease, the patient's condition, etc. Typically, a therapeutic dose is sufficient to substantially reduce the unwanted cell population in the target tissue, while maintaining the vitality of the patient. Generally the treatment is continued until a considerable reduction is obtained, for example a reduction of at least about 50% of the specific cell count, and it can be continued until essentially no unwanted cells are detected in the organism. There are several test systems for the identification of EGFR inhibitors. In the proximity scintillation assay (Sorg et al., J. of Biomolecular Screening, 2002, 7, 11-19) and the flash plate assay, the radioactive phosphorylation of a protein or peptide as a substrate is measured by? ATP. In the presence of an inhibitor compound, a reduced or no radioactive signal can be detected. In addition, homogeneous fluorescence resonance energy transfer technologies by time resolution (HTR-FRET) and fluorescence polarization (FP) are useful as assay methods (Sills et al., J. of Biomolecular Screening, 2002, 191 - 214). Other non-radioactive assay methods by ELISA use specific phospho-antibodies (phospho-AC). Phospho-AC only binds to the phosphorylated substrate. This binding can be detected by a second peroxidase conjugated anti-peroxidase antibody by chemiluminescence (Ross et al., 2002, Biochem J., in press, manuscript BJ20020786). There are many diseases and pathological conditions associated with the deregulation of cell proliferation and cell death (apoptosis). Diseases and disease states that can be treated, prevented or alleviated by medicaments according to the invention include the diseases and conditions listed below, but without limitations. The medicaments according to the invention are useful for the treatment and / or prevention of various diseases and various pathological conditions, which affect the proliferation and / or migration of smooth muscle cells and / or inflammatory cells in the intimal layer of a vessel, which results in a restriction of blood flow through said vessel, for example in occlusive lesions of the neointima. Occlusive diseases of vessels by transplantation include atherosclerosis, coronary vascular disease after transplantation, transplanted vein stenosis, restenosis of perianastomotic prosthesis, restenosis after angioplasty or stenting and the like. The present invention relates to the use of medicaments according to the invention for the treatment or prevention of cancer. Accordingly, the invention particularly preferably relates to the use of solid forms of anti-EGFR antibodies according to the invention for the preparation of a medicament for the. treatment and / or prevention of tumors and / or tumor metastasis, where the tumor is selected with particular preference from the group consisting of brain tumor, tumor of the urogenital tract, tumor of the lymphatic system, stomach tumor, laryngeal tumor, monocytic leukemia, lung adenocarcinoma, small cell lung carcinoma, pancreatic cancer, glioblastoma and breast carcinoma, without restrictions. The invention also relates to the use of medicaments according to the invention for the preparation of a medicament for the treatment of diseases selected from the group consisting of cancer diseases consisting of cancer of the squamous epithelium, bladder cancer, stomach cancer, cancer of the liver, kidney cancer, colorectal cancer, breast cancer, head cancer, neck cancer, esophageal cancer, gynecological cancer, pancreatic cancer, lymphoma, chronic leukemia and acute leukemia. The medicaments according to the invention can be administered to patients for the treatment of cancer. The present medicaments inhibit tumor angiogenesis and, thus, influence the development of tumors (J. Rak et al., Cancer Research, 55: 4575-4580, 1995). The inhibitory properties of angiogenesis of the medicaments according to the invention are also suitable for the treatment of certain forms of blindness associated with neovascularization of the retina. Accordingly, the invention also relates to the use of formulations of anti-EGFR antibodies according to the invention for the preparation of a medicament for the treatment and / or prevention of diseases caused, mediated and / or propagated by angiogenesis. A disease of this type including angiogenesis is an ocular disease, such as retinal vascularization, diabetic retinopathy, macular degeneration associated with age and the like. Accordingly, the invention also relates to the use of formulations of anti-EGFR antibodies according to the invention for the preparation of a medicament for the treatment and / or prevention of diseases selected from the group consisting of retinal vascularization, diabetic retinopathy, degeneration macular disease associated with age and / or inflammatory diseases. The invention also relates to the use of formulations of anti-EGFR antibodies according to the invention for the treatment and / or prevention of diseases selected from the group consisting of psoriasis, rheumatoid arthritis, contact dermatitis, type hypersensitivity reaction late, inflammations, endometriosis, scarring, benign prostatic hyperplasia, immunological diseases, autoimmune diseases and immunodeficiency diseases.

The invention also relates to the use of formulations of anti-EGFR antibodies according to the invention for the treatment and / or prevention of osteopathies selected from the group consisting of osteosarcoma, osteoarthritis and rickets. The medicaments according to the invention can also be used to provide additive or synergistic effects in certain chemotherapies and existing irradiation against cancer, and / or can be used to restore the efficacy of certain chemotherapies and existing irradiations against cancer. Accordingly, the invention also relates to the use of anti-EGFR antibody formulations according to the invention for the preparation of a medicament for the treatment and / or prevention of diseases wherein a therapeutically effective amount of an anti-human antibody is administered. -EGFR according to the invention in combination with a compound of the group consisting of 1) estrogen receptor modulator, 2) androgen receptor modulator, 3) retinoid receptor modulator, 4) cytotoxic agent, 5) antiproliferative agent, 6) prenyl protein transferase inhibitors, 7) HMG-CoA reductase inhibitors, 8) HIV protease inhibitors, 9) reverse transcriptase inhibitors , 10) inhibitors of growth factor receptor and 11) angiogenesis inhibitors. Accordingly, the invention also relates to the use of formulations of anti-EGFR antibodies according to the invention for the preparation of a medicament for the treatment and / or prevention of diseases in which a therapeutically effective amount of an anti-EGFR antibody according to the invention in combination with radiotherapy and a compound from the group consisting of 1) estrogen receptor modulator, 2) androgen receptor modulator, 3) retinoid receptor modulator 4) cytotoxic agent, 5) antiproliferative agent, 6) prenyl protein transferase inhibitors, 7) inhibitors of HMG-CoA reductase, 8) HIV protease inhibitors, 9) reverse transcriptase inhibitors, 10) growth factor receptor inhibitors and 11) angiogenesis inhibitors. Accordingly, the medicaments according to the invention can also be administered together with other well-known therapeutic agents that are selected for their particular utility against the condition being treated. For example, in the case of osteopathies, combinations that would be favorable include those containing antiresorptive bisphosphonates, such as alendronate and risedronate, integrin blockers (as defined below), such as vß3 antagonists, conjugated estrogens used in therapy of hormone replacement, such as Prempro®, Premarin® and Endometrion®; Selective estrogen receptor modulators (SERMs), such as raloxifene, droloxifene, CP-336,156 (Pfizer) and lasofoxifene, cathepsin K inhibitors and ATP proton pump inhibitors. The present medicaments are also suitable for combination with known anticancer agents. These known anticancer agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors , reverse transcriptase inhibitors, inhibitors of growth factor and inhibitors of angiogenesis. The present compounds are particularly suitable for administration at the same time as radiotherapy.

"Estrogen receptor modulators" refer to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of the mechanism. Examples of estrogen receptor modulators include, without limitation, tamoxifen, raloxifene, idoxifen, LY353381, LY 117081, toremifene, fulvestrant, 2, 2-dimethylpropanoate of 4- [7- (2, 2-dimethyl-l-oxo-propoxy-4-methyl-2- [4- [2- (1-piperidinyl) ethoxy] phenyl] -2H-1-benzopyran-3-yl] phenyl, 4,4'-dihydroxybenzophenone- 2,4-dinitrophenylhydrazone and SH646. "Androgen receptor modulators" refer to compounds that interfere with or inhibit the binding of androgens with the receptor, regardless of the mechanism. Examples of androgen receptor modulators include finasteride and other inhibitors of 5a-reductase, nilutamide, flutamide, bicalutamide, liarozole and abiraterone acetate. "Retinoid receptor modulators" refer to compounds that interfere with or inhibit the binding of retinoids to the receptor, regardless of the mechanism. Examples of retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, α-difluoromethylornithine, ILX23-7553, trans-N- (4'-hydroxyphenyl) retinamide and N-4- carboxyphenylretinamide. "Cytotoxic agents" refers to compounds that result in cell death, mainly by direct action on cell function, or inhibit or interfere with cellular myosis, and include alkylating agents, tumor necrosis factors, intercalators, microtubulin inhibitors and topoisomerase inhibitors. Examples of cytotoxic agents include, without limitation, tirapazimine, setain, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosylate, trophosphamide. , nimustine, dibrospide chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulvene, dexiphosphamide, cis-aminodichloro (2-methylpyridine) platinum, benzylguanine, glufosfamide, GPXIOO, tetrachloride (trans, trans, trans) -bis-mu - (hexan-1, 6-diamin) -mu- [diamin-platinum (II)] bis [diamin (chloro) platinum (II)], diazizinyl-spermine, arsenic trioxide, 1- (11-dodecylamino-10-) hydroxyundecyl) -3,7-dimethylxanthine, zorrubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, 'valrubicin, amrubicin, antineoplastone, 3'-de-samino-3' -morpholino-13-deoxo-10-hydroxycarinomycin, anamicina, gal rrubíciñ a, elinafide, MEN10755 and 4-demethoxy-3-deamino-3-azindinyl-4-methylsulfonyldaunorubicin (see WO 00/50032). Examples of microtubulin inhibitors include paclitaxel, vindesine sulfate, 3 ', 4' -dideshydro-4 '-deoxy-8' -norvincaleucoblastin, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotine, RPR109881, BMS184476, vinflunine, cryptophycin, 2, 3, 4, 5, 6-pentaf luoro-N- (3-fluoro-4-methoxyphenyl) benzenesulfonamide, anhydrovinblastine, N, N-dimethyl-L-valyl-L-valyl-N-methyl-L- valil-L-prolyl-L-prolynt-butylamide, 'TDX258 and BMS188797. Some examples of topoisomerase inhibitors are topotecan, hicaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3 ', 4'-O-exobenzylidenecartreusine, 9-methoxy-N, N-dimethyl-5-nitropyrazolo [3, 4,5-kl. ] acridin-2- (6H) -propanamine, l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH, 12H-benzo [de] -pyran [3 ' , 4 ': b, 7] -indolizino [1, 2b] -quinolin-10, 13- (9H, 15H) dione, lurtotecan, 7- [2- (N-isopropylamino) ethyl] - (2OS) camptothecin, B ? P1350, B? PI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'-dimethylamino-2'-deoxytetoside, GL331, N- [2- (dimethylamino) ethyl] -9-hydroxy-5, 6- dimethyl-6H-pyrido (4, 3-b) carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa, 9b) -9- [2- [N- [2- (dimethylamino) ethyl] -N-methylamino ] ethyl] -5- [4-hydroxy-3,5-dimethoxyphenyl] -5,5a, 6,8,8a, 9-hexohydrofuro (3 ', 4': 6,7) -naphtho- (2, 3- d) -1,3-dioxol-6-one, 2, 3- (methylenedioxy) -5-methyl-7-hydroxy-8-methoxybenzo [c] phenanthridinium, 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinolin-5,10-dione, 5- (3-aminopropylamino) -7,10-dihydroxy-2- (2-hydroxyethylaminomethyl) -6H-pyrazolo [4, 5, 1-de] acridin-6-one , N- [l- [2- (Diethylamino) ethylamino] -7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl] -formamide, N- (2- (dimethylamino) ethyl) acridine-4-carboxamide, 6- [[2- (dimethylamino) ethyl] amino] -3-hydroxy-7HT-indene [2, lc] quinolin-7-one and dimesna . "Anti-proliferative agents" include antisense RNA and DNA oligonucleotides, such as G3139, ODN698, RVASKRAS, GEM231 and INX3001, and antimetabolites such as enocythabin, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocphosphate, sodium phosteabine hydrate, raltitrexed, paltitrexid, emitefur, thiazofurine, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2 '-methylidenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N- [5- (2, 3-dihydrobenzofuryl) sulfonyl] -N '- (3,4-dichlorophenyl) urea, N6- (4-deoxy-4- [N2- [2 (E), 4 (E) -tetradecadienoyl] glycylamino] -L-glycero -BL-mannoheptopyranosyl] adenine, aplidine, ecteinascidin, troxacitabine, 4- [2-amino-4-oxo-4, 6, 7, 8-tetrahydro-3H-pyrimidino [5, 4-b] -1, 4- thiazin-6-yl- (S) -ethyl] -2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil, alanine, l-acetyl-8- (carbamoyloxymethyl) -4-formyl-6-acid ester methoxy-14-oxa-l, 11-diaza tetra-cycle (7.4.1.0.0) tetradeca-2, 4,6-trien-9-yl-acetic, swainsonin, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1- BD-arabinofuranosyl-cytosine and thiosemicarbazone 3-aminopyridine-2-carboxaldehyde. "Anti-proliferative agents" also include monoclonal antibodies against growth factors other than those listed above under "angiogenesis inhibitors", such as trastuzumab, and tumor suppressor genes, such as p53 that can be delivered through a gene transfer mediated by recombinant virus (see, for example, the US Na document). 6. 069,134). The medicaments according to the invention can also be administered in combination with all other therapeutic antibodies known to those skilled in the art or active pharmaceutical ingredients which are suitable in connection with the aforementioned diseases.

In addition, anti-EGFR antibody formulations according to the invention can be used for the isolation and investigation of EGFR activity or expression. In addition, they are particularly suitable for use in diagnostic methods of diseases related to the unregulated or altered activity of EGFR. For diagnostic purposes, the antibodies according to the invention can receive, for example, a radioactive label. A preferred labeling method is the yodogenic method (Fraker et al., 1978). For diagnostic purposes, the antibody used with particular preference is the F (ab ') 2 fragment. This results in excellent results, which means that it is not necessary to subtract the bottom. Fragments of this type can be prepared by known methods (for example, Herlyn et al., 1983). In general, digestion with pepsin is performed at acid pH, and the fragments are separated from undigested IgG and heavy chain fragments by protein A chromatography on Sepharose ™. The anti-EGFR antibodies preferably exhibit in the formulations according to the invention an advantageous biological activity, which is easily detected in the enzyme assays, as described in the examples. In enzyme-based assays of this type, the antibodies according to the invention exhibit and preferably cause an inhibitory effect, which is usually documented by IC50 values in a suitable range, preferably in the micromolar range, and more preferably in the range nanomolar The determination of the size of the protein, the structural integrity, the purity or the glycosylation pattern of the antibodies according to the invention in formulations according to the invention include, without restrictions, SE-HPLC, peptide mapping (digestion), sequence N-terminal, SDS-PAGE, TRIS / glycine gradient gel (non-reducing), FTIR (Fourier transformation infrared spectrum), CD (circular dichroism), RAMAN spectroscopy, carbohydrate staining (PAS method) , oligosaccharide profile, determination of monosaccharide composition or isoelectric focusing. The stability of the formulations according to the invention can be determined, for example, without restrictions, with the help of stabilization programs, for example storage at 25 ° C and 60% ambient relative humidity and at 40 ° C and 70% of ambient relative humidity over an extended period and determination of the stability or structural integrity of the protein at regular intervals, for example by the aforementioned determination methods (SE-HPLC, FT-IR, SDS-PAGE (reducing or non-reducing) ). The methods for determining the biological activity or the effectiveness of the antibodies according to the invention in formulations according to the invention, for example, without limitations, ELISA, cell biological assays, FTIR or CD. The determination of the reduction of the aggregation tendency of the highly concentrated formulations according to the invention include, for example, without limitations, visual inspection, analysis of sub-visible particles, nephelometry or turbidimetry or dynamic characterization of the dispersion. of the light . Example 1: Preparation of a liquid formulation of highly concentrated anti-EGFR antibodies by tangential flow filtration (TFF) 380 ml of protein (17 mg / ml in 10 mM phosphate + 145 mM NaCl are concentrated, pH 7.2) by means of a Labscale-TFF installation (Millipore) with a polyethersulfone ultrafiltration membrane incorporated with a 30 kDa cut for 226 min with an initial pressure of 20 psi and a final pressure of 10 psi. The retentate obtained has a protein concentration of approximately 132 mg / ml. The yield is 85%. Either 470 ml of protein (17 mg / ml in lOmM citrate) are concentrated by means of a Labscale TFF installation (Millipore) with a polyethersulfone ultrafiltration membrane incorporated with a 30 kDa cut-off for 226 min with an initial pressure of 20 psi and a final pressure of 10 psi. The retentate obtained has a protein concentration of approximately 123 mg / ml. The yield is 95%. Example 2: Preparation of a liquid formulation of highly concentrated anti-EGFR antibodies by ultrafiltration under stirring 25 ml of protein (10 mg / ml in 10 mM phosphate + 145 mM NaCl, pH 7.2) are concentrated by means of a Amicon agitation cell with a polyethersulfone ultrafiltration membrane incorporated with a 30 kDa cut for 144 min with a nitrogen gas pressure of 4 bar. The retentate obtained has a protein concentration of approximately 92 mg / ml. The yield is 95%. Either 25 ml of protein (10 mg / ml in 10 mM citrate, pH 5.5) are concentrated by means of an Amicon agitation cell with a polyethersulfone ultrafiltration membrane incorporated with a 30 kDa cut for 168 min with a nitrogen gas pressure of 4 bar. The retentate obtained has a protein concentration of approximately 82"" mg / ml. The yield is 95%. Example 3: Preparation of a liquid formulation of highly concentrated anti-EGFR antibodies by ultrafiltration under the action of centrifugal forces 15 ml of protein are centrifuged (2 mg / ml in 10 mM phosphate + 145 mM NaCl, pH 7.2 ) in an Ultrafree centrifuge tube (Millipore) with a polyethersulfone ultrafiltration membrane incorporated with a cut-off of 30 kDa at 2000 * g for 90 min.The retentate obtained has a protein concentration of approximately 116 mg / ml. yield is 95% Example 4: Investigation of soluble aggregates of the liquid formulation of highly concentrated anti-EGFR antibodies The retentates obtained in Examples 1 to 3 were tested by means of SE-HPLC for the content of soluble aggregates. In this case, the monomer content after concentration was >99% Example 5: Nativity test of the liquid formulation of highly concentrated anti-EGFR antibodies The retentates obtained in Example 1 were tested by FT-IR spectrophotometry. In this case, the spectra of the 2-derivatization of the amide I of the starting material were congruent before concentration by means of tangential flow filtration and the obtained retentate. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (20)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Method for preparing a highly concentrated liquid formulation characterized in that it contains at least one anti-EGFR antibody and / or one of its variants and / or fragments by ultrafiltration.
2. 2. Method according to claim 1, characterized in that the highly concentrated liquid formulation obtained has a content of an anti-EGFR antibody of 10-250 mg / ml.
3. 3. Method according to claim 1, characterized in that the highly concentrated liquid formulation obtained has a content of an anti-EGFR antibody of 50-180 mg / ml.
4. 4. Method according to claim 1, characterized in that the highly concentrated liquid formulation obtained has a content of an anti-EGFR antibody of 100-150 mg / ml.
5. Method according to one or more of claims 1 to 4, characterized in that the anti-EGFR antibody is monoclonal and has murine or human origin.
6. Method according to one or more of claims 1 to 5, characterized in that the anti-EGFR antibody is of murine origin and is chimeric or humanized.
7. Method according to one or more of claims 1 to 6, characterized in that the anti-EGFR antibody is Mab C225 (Cetuximab) or Mab h425 (EMD72000).
8. 8. Highly concentrated liquid formulation characterized in that it contains at least one anti-EGFR antibody and / or one of its variants and / or fragments.
9. 9. Highly concentrated liquid formulation according to claim 8, characterized in that the highly concentrated liquid formulation has a content of an anti-EGFR antibody of 10-250 mg / ml.
10. 10. Highly concentrated liquid formulation according to claim 8, characterized in that the highly concentrated liquid formulation has a content of an anti-EGFR antibody of 50-180 mg / ml.
11. 11. Highly concentrated liquid formulation according to claim 8, characterized in that the highly concentrated liquid formulation has a content of an anti-EGFR antibody of 100-150 mg / ml.
12. 12. Highly concentrated liquid formulation according to one or more of claims 8 to 11, characterized in that the anti-EGFR antibody is monoclonal and has murine or human origin.
13. 13. Highly concentrated liquid formulation according to one or more of claims 8 to 12, characterized in that the anti-EGFR antibody is of murine origin and is chimeric or humanized.
14. 14. Highly concentrated liquid formulation according to one or more of claims 8 to 13, characterized in that the anti-EGFR antibody is Mab C225 - (Cetuximab) or Mab h425 (EMD72000).
15. 15. Highly concentrated liquid formulation characterized in that it contains at least one anti-EGFR antibody and / or one of its variants and / or fragments that can be obtained by means of a method according to one or more of claims 1 to 7.
16. 16 Highly concentrated liquid formulation according to one or more of claims 8 to 15 as a storage stable medicament.
17. 17. Highly concentrated liquid formulation according to one or more of claims 8 to 16, characterized in that it optionally contains excipients and / or adjuvants and / or other pharmaceutical active ingredients.
18. 18. Use of a highly concentrated liquid formulation according to one or more of claims 8 to 17 for preparing a medicament.
19. 19. Use of a highly concentrated liquid formulation according to one or more of claims 8 to 17 for preparing a medicament for the treatment and / or prevention of tumors and / or tumor metastases.
20. 20. Use according to claim 19, wherein the tumor is selected from the group consisting of brain tumor, tumor of the urogenital tract, tumor of the lymphatic system, stomach tumor, laryngeal tumor, monocytic leukemia, lung adenocarcinoma, carcinoma of the small cell lung, pancreatic cancer, glioblastoma and breast carcinoma.