MXPA98000684A - Formulation of isotonic protocols isotonic s - Google Patents

Abstract

A stable lyophilized protein formulation is described which can be reconstituted with an appropriate diluent to generate a reconstituted formulation of high protein concentration, which is suitable for subcutaneous administration. For example, the anti-IgE and anti-HER2 formulations have been prepared by lyophilization of these antibodies in the presence of a lyoprotectant. The lyophilized mixture thus formed is reconstituted to a high concentration of protein without apparent loss of protein stability.

Description

FORMULATION OF STABILIZED ISOTONIC PROTEIN PROTEINS Field of the Invention This invention is directed to a lyophilized formulation of proteins. In particular it relates to a stable lyophilized formulation of proteins, which can be reconstituted with a diluent to generate a stable reconstituted formulation suitable for subcutaneous administration.

Description of Previous Art In the past ten years, advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA techniques. Because proteins are larger and more complex than traditional organic and inorganic drugs (that is, they have multiple functional groups in addition to complex three-dimensional structures), the formulation of such proteins presents special problems. For a protein to remain biologically active, the formulation must maintain intact the conformational integrity of at least REF: 26676 a core sequence of the amino acids of the protein, while at the same time protecting the multiple functional groups of the protein from degradation. The pathways of degradation for proteins can involve chemical instability (that is, any process that involves the modification of the protein through the formation of bonds or cleavages that result in a new chemical species) or physical instability (ie, changes in the higher order structure of the protein). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result from denaturation, aggregation, precipitation or adsorption, for example. The three most common protein degradation pathways are protein aggregation, deamidation and oxidation. Cleland et al. Critical Reviews in Therapeutic Systems Drug Carrier 10 (4): 307-377 (1993).

Drying-freezing is a technique commonly used to preserve proteins that serve to remove water from the protein preparation of interest. Drying-freezing, or lyophilization, is a process by which the material to be dried is frozen first and then the frozen or frozen solvent is removed by sublimation in a vacuum medium. An excipient may be included in the pre-lyophilized formulations to increase the stability during the drying-freezing process and / or to improve the stability of the lyophilized product during storage. Pikal, M. Biopharm. 3 (9) 26-30 (1990) and Arakawa et al. Pharm. Res. 8 (3): 285-291 (1991).

It is an object of the present invention to provide a lyophilized formulation of proteins that is stable during storage and delivery. It is a further object to provide a stable reconstituted formulation of proteins that is suitable for subcutaneous administration. In certain embodiments, it is an object to provide a multi-use formulation that is stable for at least the time during which it will be administered to the patient.

Brief Description of the Invention The invention is based on the discovery that a lyophilized stable protein formulation can be prepared using a lyoprotectant (preferably a sugar such as sucrose or trehalose), a lyophilized formulation that can be reconstituted to generate a stable reconstituted formulation having a concentration of protein that is significantly higher (eg, from about 2-40 times higher, preferably 3-10 times higher and more preferably 3-6 times higher) than the protein concentration in the pre-lyophilized formulation. In particular, since the concentration of protein in the pre-lyophilized formulation can be 5 mg / mL or less, the concentration of protein in the reconstituted formulation is generally 50 mg / mL or more. Such high concentrations of protein in the reconstituted formulation are considered particularly useful where the formulation is intended for subcutaneous administration.

Regardless of the very high protein concentration in the reconstituted formulation, it has been found that the reconstituted formulation is stable (that is, it fails to show unacceptable or significant levels of physical or chemical instability in the protein) at 2-8 ° C during at least around 30 days. In certain embodiments, the reconstituted formulation is isotonic. Despite the use of lower concentrations of the lyoprotectant to achieve said isotonic formulations during reconstitution, it has been discovered here that the protein in the lyophilized formulation retains essentially its physical and chemical stability and integrity during lyophilization and storage.

When reconstituted with a diluent comprising a preservative (such as bacteriostatic water for injection, BWFI for its acronym in English), the reconstituted formulation can be used as a multipurpose formulation. Such a formulation is useful for example, wherein the patient requires frequent subcutaneous administrations of the protein, to treat a chronic medical condition. The advantage of a multi-purpose formulation is that it simplifies the ease of use for the patient, reduces waste by allowing full use of the contents of the vial, and results in significant cost savings for the manufacturer, since different doses are packaged in a simple vial (lower filling and shipping costs).

Based on the observations described herein, in one aspect the invention provides a stable reconstituted isotonic formulation comprising a protein in an amount of at least about 50 mg / mL and a diluent, reconstituted formulation that has been prepared from a mixture lyophilized of a protein and a lyoprotectant, wherein the concentration of protein in the reconstituted formulation is about 2-40 times greater than the concentration of protein in the mixture before lyophilization.

In another embodiment, the invention provides a stable reconstituted formulation comprising an antibody in an amount of at least about 50 mg / mL and a diluent, reconstituted formulation that has been prepared from a lyophilized mixture of an antibody and a lyoprotectant, wherein the concentration of the antibody in the reconstituted formulation is about 2-40 times greater than the concentration of antibody in the mixture before lyophilization.

The ratio of lyoprotectant: protein in the lyophilized formulation of the preceding paragraphs depends, for example, on the protein and lyoprotectant of choice, as well as on the desired concentration of protein and the isotonicity of the reconstituted formulation. In the case of a full-length antibody (such as the protein) and trehalose or sucrose (as the lyoprotectant) to generate an isotonic reconstituted formulation of high protein concentration, the ratio may be, for example, of about 100-1500 moles of trehalose or sucrose: 1 mole of antibody.

Generally, the pre-lyophilized formulation of the protein and the lyoprotectant will additionally include a buffer solution, which provides the formulation at an appropriate pH depending on the protein in the formulation. For this purpose, it has been found desirable to use a histidine buffer in which, as demonstrated below, it appears to have lyoprotective properties.

The formulation may also include a surfactant (e.g., a polysorbate) in what has been observed here that it can reduce the aggregation of the reconstituted protein and / or reduce the formation of particles in the reconstituted formulation. The surfactant can be added to the pre-lyophilized formulation, the lyophilized formulation and / or the reconstituted formulation (but preferably the pre-lyophilized formulation) as desired.

The invention further provides a method for preparing a stable isotonic reconstituted formulation comprising reconstituting a lyophilized mixture of a protein and a lyoprotectant in a diluent such that the concentration of protein in the reconstituted formulation is at least 50 mg / mL, wherein the Protein concentration in the reconstituted formulation is about 2-40 times higher than the concentration of protein in the mixture before lyophilization.

In still a further embodiment, the invention provides a method for the preparation of a formulation comprising the steps of: (a) lyophilizing a mixture of a protein and a lyoprotectant; and (b) reconstituting the lyophilized mixture of step (a) in a diluent such that the reconstituted formulation is isotonic and stable and has a protein concentration of at least about 50 mg / mL. For example, the protein concentration in the reconstituted formulation can be from about 80 mg / mL to about 300 mg / mL. Generally, the concentration of protein in the reconstituted formulation is about 2-40 times greater than the concentration of protein in the mixture before lyophilization.

Also provided herein is a manufacturing article comprising: (a) a container holding a lyophilized mixture of a protein and a lyoprotectant; and (b) instructions for reconstituting the lyophilized mixture with a diluent at a protein concentration in the reconstituted formulation of at least about 50 mg / mL. The article of manufacture may further comprise a second container that holds a diluent (e.g., bacteriostatic water for injection (BWFI) comprising an aromatic alcohol).

The invention also provides a method for the treatment of a mammal comprising administering a therapeutically effective amount of a reconstituted formulation described herein to a mammal, wherein the mammal has a disorder requiring treatment with the protein of the formulation. For example, the formulation can be administered subcutaneously.

A useful pre-lyophilized formulation for anti-HER2 antibody, as found in the experiments detailed below, was found to comprise anti-HER2 in amounts of about 5-40 mg / mL (e.g., 20-30 mg / mL) and sucrose or trehalose in an amount from about 10-100 mM (eg, 40-80 mM), a buffer solution (eg, histidine, pH 6 or succinate pH 5) and a surfactant (eg, a polysorbate). The lyophilized formulation was found to be stable at 40 ° C for at least 3 months and stable at 30 ° C for at least 6 months. This anti-HER2 formulation can be reconstituted with a diluent to generate a formulation suitable for intravenous administration comprising anti-HER2 in an amount from about 10-30 mg / mL which is stable at 2-8 ° C for at least about 30 days. Where higher concentrations of anti-HER2 antibody are desired (eg, where subcutaneous delivery of the antibody is the intended mode of administration to the patient), the lyophilized formulation can be reconstituted to produce a stable, reconstituted formulation having a protein concentration. of 50 mg / mL or more.

A pre-lyophilized formulation for anti-IgE antibody disclosed herein, has anti-IgE in amount from about 5-40 mg / mL (eg 20-30 mg / mL) and sucrose or trehalose in an amount from about 60-300 mM (eg, 80-170 mM), a buffer solution (preferably histidine, pH 6) and a surfactant (such as a polysorbate). The lyophilized anti-IgE formulation is stable at 30 ° C for at least 1 year. This formulation can be reconstituted to produce a formulation comprising anti-IgE in an amount from about 15-45 mg / mL (eg, 15-25 mg / mL) suitable for intravenous administration which is stable at 2-8 °. C for at least 1 year. Alternatively, where higher concentrations of anti-IgE are desired in the formulation, the lyophilized formulation can be reconstituted in order to generate a stable formulation having an anti-IgE concentration of = .50 mg / mL.

Brief Description of the Drawings Figure 1 shows the effect of the reconstitution volume on the stability of lyophilized rHMA2 HER2. The lyophilized formulation was prepared from a pre-lyophilized formulation comprising 25 mg / mL protein, 60 mM trehalose, 5 mM sodium succinate, pH 5.0, and 0.01% Tween 20 ™. The lyophilized cake was incubated at 40 ° C and then reconstituted with 4.0 (o) or 20.0 L (•) of BWFI. The intact protein fraction in the reconstituted formulation was measured by natural size exclusion chromatography and defined as the peak area of the natural protein relative to the total peak area including the aggregates.

Figure 2 illustrates the effect of trehalose concentration on the stability of lyophilized rHMAb HER2. The protein was lyophilized at 25 mg / mL in 5 mM sodium succinate, pH 5.0 (circles) or 5 mM histidine, pH 6.0 (squares) and trehalose concentrations in the range from 60 mM (360 mole ratio) to 200 mM (ratio molar 1200). The lyophilized protein was incubated at 40 ° C for 30 days (closed symbols) or 91 days (open symbols). The amount of intact protein was measured after reconstitution of the lyophilized protein with 20 mL of BWFI.

Figure 3 demonstrates the effect of trehalose concentration on the long-term stability of rhuMab HER2 stored at 40 ° C. The protein was lyophilized at 25 mg / mL, in 5mM sodium succinate, pH 5.0, 0.01% Tween 20 ™, and 60- mM trehalose (B) or 5 mM histidine, pH 6.0, 0.01% Tween 20 ™. , and 60mM of trehalose (D), or 21mg / mL in 10mM of sodium succinate, pH 5.0, 0.2% of Tween 20 ™ and 250mM of trehalose (•). The lyophilized protein was incubated at 40 ° C and then reconstituted with 20 mL of BWFI. The amount of intact protein was measured after reconstitution.

Figure 4 shows the stability of freeze-dried rhuMAb HER2 in 38.4 mM mannitol (7 mg / mL), 20.4 mM sucrose (7mg / mL), 5 mM histidine, pH 6.0, 0.01% Tween 20 ™ The lyophilized protein was incubated at 40 ° C and then reconstituted with either 4.0 ml (o) or 20 ml (#) of BWFI. The amount of intact protein was measured after reconstitution.

Figure 5 demonstrates the stability of the lyophilized reconstituted rHMAb HER2 in 5mM sodium succinate, pH 5.0, 60mM trehalose, 0.01% Tween 20 ™. Samples were reconstituted with either 4.0 mL (square) or 20.0 mL (circles) of BWFI (20 mL: 0.9% benzyl alcohol, 4 mL: 11% benzyl alcohol) and then stored at 5 ° C (solid symbols) or 25 ° C (open symbols). The percentage of natural protein was defined as the peak area of the natural protein (not degraded) relative to the total peak area as measured by cation exchange chromatography.

Figure 6 shows the stability of freeze-dried reconstituted rhuMAb HER2 in 5 mM histidine, pH 6.0, 60 mM trehalose, 0.01% Tween 20. Samples were reconstituted with either 4.0 mL (squared) or 20.0 mL (circles) of B FI (20 mL: 0.9% benzyl alcohol, 4 mL: ll% benzyl alcohol) and then stored at 5 ° C (solid symbols) or 25 ° C (open symbols). The percentage of natural protein was defined as the peak area of the natural protein (not degraded) relative to the total peak area as measured by cation exchange chromatography.

Figure 7 reveals the stability of freeze-dried reconstituted rhuMAb HER2 in 5 mM histidine, pH 6.0, 38.4 mM mannitol, 20.4 mM sucrose, 0.01% Tween 20. Samples were reconstituted with either 4.0 ml (square) or 20.0 ml (circles) of BWFI (20 mL: 0.9% benzyl alcohol, 4 mL: ll% benzylic alochol) and then stored at 5 ° C (solid symbols) or 25 ° C (open symbols). The percentage of natural protein was defined as the peak area of the natural protein (not degraded) relative to the total peak area as measured by cation exchange chromatography.

Figure 8 shows the stability of freeze-dried reconstituted rhuMAb HER2 in lOmM of sodium succinate, pH 5.0, 250 mM of trehalose, 0.2% of Tween 20. The samples were reconstituted with 20.0 mL of BWFI (0.9% benzyl alcohol) and then stored at 5 ° C (•) or 25 ° C (o). The percentage of natural protein was defined as the peak area of the natural protein (not degraded) relative to the total peak area as measured by cation exchange chromatography.

Figure 9 shows the aggregation of rhuMAb E25 formulated in buffer solutions in the range from pH 5 to pH 7 at a buffer concentration of 10 mM and 5 mg / mL antibody concentration. The samples were lyophilized and assayed at zero time and after 4 weeks, 8 weeks and 52 weeks of storage at 2-8 ° C. Buffer solutions are: potassium phosphate pH 7.0 (o); sodium phosphate pH 7.0 (D); histidine pH 7.0 (o); sodium succinate pH 6.5 (•), - sodium succinate pH 6.0 (•); sodium succinate pH 5.5 (), \ and sodium succinate pH 5.0 (?).

Figure 10 details the aggregation of freeze-dried E25 rhuMAb in a 5 mM histidine buffer at pH 6 and 7 and assayed following storage as follows. The buffer solution was at: pH 6.0 stored at 2-8 ° C (o); pH 6 stored at 25 ° C (D); pH 6 stored at 40 ° C «*); pH 7 stored at 2-8 ° C (•); pH 7 stored at 25 ° C (B); and pH 7 stored at 40 ° C ().

Figure 11 illustrates the aggregation of 5 mg / mL of rhuMAb E25 formulated into 10 mM sodium succinate at pH 5.0 with a lyoprotectant added at a concentration of 275 mM (isotonic). The lyoprotectants were: control, without lyoprotectant (o), - mannitol (D); lactose (* >); maltose (•); trehalose (•); and sucrose (). The samples were lyophilized and assayed at a zero time and after 4 weeks, 8 weeks and 52 weeks of storage at 2-8 ° C.

Figure 12 shows the aggregation of 5 mg / mL of rhuMAb E25 formulated into 10mM sodium succinate at pH 5.0 with a lyoprotectant added at a concentration of 275 mM (isotonic). The lyoprotectants were: control, without lyoprotectant (o); mannitol (D); lactose (»>); maltose (*), - trehalose (B); and sucrose (^). The samples were lyophilized and assayed at zero time and after 4 weeks, 8 weeks and 52 weeks of storage at 40 ° C.

Figure 13 details the hydrophobic interaction chromatography of 20 mg / mL of freeze-dried E25 rhuMAb in a histidine buffer at pH 6 with an isotonic concentration (ie, 275 mM) of lactose stored for 24 weeks at 2-8 , 25 or 40 ° C and reconstituted at 20 mg / mL.

Figure 14 shows the hydrophobic interaction chromatography of 20 mg / mL of rhuMAb E25 lyophilized in a histidine buffer at pH 6, stored for 24 weeks at 2-8, 25 or 40 ° C and reconstituted at 20 mg / mL -.

Figure 15 illustrates the hydrophobic interaction chromatography of 20 mg / mL of rhuMAb E25 lyophilized in a histidine buffer at pH 6 with an isotonic concentration (ie, 275 mM) of sucrose and stored for 24 weeks at 2-8 , 25 or 40 ° C and reconstituted at 20 mg / mL.

Figure 16 illustrates the effect of sugar concentration on rhuMAb E25 formulated at 20 mg / mL in 5mM histidine at a pH of 6.0. Sucrose (•) and trehalose (D) were added to the formulation at molar ratios ranging from 0 to 2010 (isotonic) (see Table 1 below). The samples were lyophilized and assayed after 12 weeks of storage at 50 ° C.

Figure 17 reveals the aggregation of rhuMAb E25 formulated at 25 mg / mL within 5 mM histidine at pH 6 with 85 mM sucrose (o); 85 mM trehalose (D), - 161 mM sucrose () or 161 mM trehalose (?). Samples were lyophilized and stored at 2-8 ° C followed by reconstitution with 0.9% benzyl alcohol to 100 mg / mL of antibodies in 20 mM histidine at pH 6 with an isotonic (340 mM) and hypertonic sugar concentration (644 mM).

Figure 18 shows the aggregation of rhuMAb E25 formulated at 25 mg / mL within 5 mM histidine at pH 6 with 85 mM sucrose (o); 85 mM trehalose (G); 161 mM sucrose () or 161 mM trehalose (?). The samples were lyophilized and stored at 30 ° C followed by reconstitution with 0.9% benzyl alcohol up to 100 mg / mL of antibodies in 20 mM histidine at pH 6 with an isotonic (340 mM) and hypertonic sugar concentration (644 mM).

Figure 19 illustrates the aggregation of rhuMAb E25 formulated at 25 mg / mL within 5 mM histidine at pH 6 with 85 mM sucrose (o); 85 mM trehalose (Q); 161 mM sucrose (^) or 161 mM trehalose (?). The samples were lyophilized and stored at 50 ° C followed by reconstitution with 0.9% benzyl alcohol up to 100 mg / mL of antibodies in 20 mM histidine at a pH of 6 with an isotonic (340 mM) and hypertonic sugar concentration (644 mM).

Detailed Description of the Preferred Modalities I. Definitions By "protein", it is meant a sequence of amino acids for which the chain length is sufficient to produce the highest levels of tertiary and / or quaternary structure. This is to distinguish from "peptides" or other small > low molecular weight drugs that do not have such structure. Typically, the protein here will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD.

Examples of proteins within the definition herein include mammalian proteins, such as, eg, growth hormone, including human growth hormone and human hormone from bovine; growth hormone releasing factor; parathyroid hormone, thyroid stimulating hormone; lipoproteins, - a-1-antitrypsin; Chain A insulin; B chain insulin; proinsulin; follicle stimulating hormone; Calciotonin; luteinizing hormone, - glucagon; coagulant factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-coagulant factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; a- and ß- factors of tumor necrosis; enkephalinase; RANTES (expressed and secreted T cells normally regulated in their activation); inflammatory protein of the human macrophage (MIP-1-a); serum albumin such as human serum albumin; inhibitory substance muleriana; chain relaxin A; B-chain relaxin; prorelaxin; associated peptide of mouse gonadotropin; Dnasa; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; an integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin -3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or a growth factor of nerves such as NGF-b, platelet-derived growth factor (PDGF); fibroblast growth factor such as FGF and bFGF, epidermal growth factor (EGF), transforming growth factor (TGF) such as TGF-α and TGF-β including TGF-β1, TGFβ2, TGF-β3, TGF-β4 TGF-β5; factor I- and II-insulin-like growth (IGF-I and IGF-II); des (1-3) -IGF-I (cerebral IGF-I); Insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO), - osteoinductive factors, -immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -β and - ?; colony-defining factors (CSFs), eg, M-CSF, GM-CSF; and G-CSF; interleukins (lys), e.g., IL-1 to IL-10; superoxide dismutase; T cell receptors; Surface membrane proteins; decline accelerating factor (DAF), - a viral antigen such as, for example, a portion of the envelope of AIDS; transport proteins; receptors at rest; adresinas; regulatory proteins; immunoadhesins; antibodies; and fragments or biologically active variants of any of the polypeptides listed above.

The protein that is formulated is preferably essentially pure and desirably essentially homogeneous (ie, free of contaminating proteins, etc.). "Essentially pure" protein means a composition comprising at least about 90% by weight of the protein, based on the total weight of the composition, preferably at least about 95% by weight. "Essentially homogeneous" protein means a composition comprising at least about 99% by weight of protein, based on the total weight of the composition.

In certain embodiments, the protein is an antibody. The antibody can be linked to any of the above-mentioned molecules, for example. Exemplary molecular targets for antibodies, covered by the present invention, include CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family such as the EGF receptor, HER2 receptor, HER3 or HER4; cell adhesion molecules such as LFA-1, Mol, pl50-95, VLA4, ICAM-1, VCAM and av / β3 integrin including either α or β subunits thereof (eg, anti-CDlla antibodies, anti-CD18 or anti-CDII); growth factors such as VEGF; IgE; blood group antigens; flk2 / flt3 receiver; obesity receptor (OB), - protein C, etc.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length antibodies having an immunoglobulin Fe region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single chain molecules, as well as antibody fragments (e.g., Fab, F (ab ') 2, and Fv).

The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, except for possible naturally occurring mutations, which can be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a simple antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the culture of the hybridoma, not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not interpreted to require the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. , Nature, 256: 495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries, using the techniques described in Clackson et al. , Nature, 352: 624-628 (1991) and Marks et al. , J. Mol. Biol. , 222: 581-597 (1991), for example.

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of antibody in particular, while the remaining chain (s) is identical with or homologous to the corresponding sequences of antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, provided they show the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Nati, Acad. Sci. USA, 81: 6851-6855 (1984)).

The "humanized" forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other binding subsequences of antibody antigens) containing a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the residues of a determinant complementarity region (CDR) of the container are replaced by residues of a CDR of a non-human species (donor antibody) as a mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the residues of the region of the Fv (FR) framework of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported framework or CDR sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will substantially comprise all of at least one, and typically two, variable domains, in which all or substantially all regions of FR are those of a human immunoglobulin sequence. The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For more details, see Jones et al. , Nature, 321: 522-525 (1986); Reichman et al. , Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. , 2: 593-596 (1992). The humanized antibody includes a Primatized ™ antibody wherein the antigen binding region of the antibody is derived from an antibody produced by immunization of macaque monkeys with the antigen of interest.

A "stable" formulation is one in which the protein in it retains essentially its physical and chemical stability and its storage integrity. Various analytical techniques for the measurement of protein stability are available in the art and are reviewed in Peptides and Supply of Protein Medications, 247-301, Vincent Lee De., Marcel Dekker, Inc., New York, New York, Pubs (1991) and Jones, A. Adv. rug. Delivery Rev. 10: 29-90 (1993). The stability can be measured at a selected temperature for a selected period of time. For rapid screening, the formulation can be maintained at 40 ° C for 2 weeks up to 1 month, at which time the stability is measured. Where the formulation is to be stored at 2-8 ° C, the formulation should generally be stable at 30 ° C or 40 ° C for at least 1 month and / or stable at 2-8 ° C for at least 2 years. Where the formulation is to be stored at 30 ° C, generally the formulation must be stable for at least 2 years at 30 ° C and / or stable at 40 ° C for at least 6 months. For example, the degree of aggregation that follows lyophilization and storage should be used as an indicator of the stability of the protein (see examples here). For example, a "stable" formulation may be one in which less than about 10% and preferably less than about 5% of the protein is present as an aggregate in the formulation. In other embodiments, any increase in aggregate formation following lyophilization and storage of the lyophilized formulation can be determined. For example, a "stable" lyophilized formulation may be one in which the increase in aggregate in the lyophilized formulation is less than about 5% and preferably less than about 3%, when the lyophilized formulation is stored at 2-8 °. C for at least one year. In other embodiments, the stability of the protein formulation can be measured using a biological activity assay (see, eg, Example 2 below).

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation suitable for administration (e.g., parenteral administration) to a patient to be treated with the protein of interest and, in certain embodiments of the invention, may be one that is suitable for subcutaneous administration.

By "isotonic", it is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. The isotonicity can be measured using an osmometer of the ice-freezing or vapor pressure type, for example.

A "lyoprotectant" is a molecule that, when combined with a protein of interest, prevents or significantly reduces the chemical and / or physical instability of the protein with lyophilization and subsequent storage. Exemplary lyoprotectants include sugars such as sucrose or trehalose; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as sugar or trihydric higher alcohols, pej. , glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; Pluronic and combinations thereof. The preferred lyoprotectant is a non-reducing sugar, such as trehalose or sucrose.

The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotective amount" which means that, following lyophilization of the protein in the presence of a lyoprotectant amount of the lyoprotectant, the protein essentially retains its stability and physical and chemical integrity with lyophilization and storage.

The "diluent" of interest here is one that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (B FI), a solution buffered in its pH (eg, phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.

A "preservative" is a compound that can be added to the diluent to essentially reduce the bacterial action in the reconstituted formulation, thereby facilitating the production of a multi-use reconstituted formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative here is benzyl alcohol.

A "bulking agent" is a compound that adds dough to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake that maintains an open pore structure. ). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and sorbitol.

The "treatment" refers to the therapeutic and prophylactic treatment or preventive measures. Those in need of treatment, include those who already have the disorder as well as those in which the disorder is to be avoided.

"Mammal" for treatment purposes, refers to any animal classified as a mammal, including humans, domestic and farm animals, and animals for sport, zoo and pets, such as dogs, horses, cats, cows, etc. Preferably the mammal is a human being.

A "disorder" is any condition that would benefit from treatment with the protein. This includes chronic or acute diseases or disorders, including those pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of the disorders to be treated here include carcinomas and allergies.

II. Ways to Carry Out the Invention A. Preparation of the Protein. The protein to be formulated is prepared using techniques that are well established in the art, including synthetic techniques (such as recombinant techniques and peptide synthesis or a combination of these techniques) or can be isolated from an endogenous source of the protein. In certain embodiments of the invention, the protein of choice is an antibody. They follow the techniques for the production of antibodies. (i) Polyclonal antibodies Polyclonal antibodies are generally produced in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, eg, hemocyanin from limpets of the genus Fissurella (keyhole limpet), serum albumin, bovine triglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl ester of sulfosuccinimide (conjugation through cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, S0Cl2, or R1N = C = NR, wherein R and R1 are different alkyl groups.

The animals are immunized against the antigen, immunogenic conjugates or derivatives, by combining 1 mg or 1 μg of the peptide or conjugate (for rabbits and mice, respectively) with 3 volumes of Freund's complete adjuvant. One month later, the animals are increased with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody concentration. The animals are increased until the concentration has no change in the variation of the count. Preferably, the animal is increased with the conjugate of the same antigen, but conjugated to a different protein and / or through a different reagent reagent. The conjugates can also be made in recombinant cell cultures as protein fusions. Also, aggregating agents such as alum are appropriately used to increase the immune response. (ii) Monoclonal Antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Thus, the "monoclonal" odifier indicates the character of the antibody as being not a mixture of discrete antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al. , Nature, 256: 495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to evoke lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used for immunization. Alternatively, lymphocytes can be immunized in vi tro. The lymphocytes are then fused with myeloma cells using an appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium which preferably contains one or more substances that inhibit the growth or survival of the myeloma-generating cells without melting. For example, if the myeloma generating cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), whose substances prevent the growth of the cells deficient in HGPRT.

Preferred myeloma cells are those that efficiently fuse, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as the HAT medium. Among these, the preferred myeloma cell lines are murine myeloma lines, such as those derived from the mouse tumors MOPC-21 and MPC-11 available from the Cell Distributor Center of the Salk Institute, San Diego, California USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and human-mouse heteromyeloma cell lines have also been described for the production of monoclonal human antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Techniques and Production Applications of Monoclonal Antibodies, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells grow is tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al. , Anal. Biochem. , 107: 220 (1980).

After the hybridoma cells which produce antibodies of the specificity, affinity and / or desired activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, p. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can grow in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subcloned ones are appropriately separated from the culture medium, ascites fluids or serum, by conventional methods of immunoglobulin purification such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or chromatography. of affinity.

The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes which are capable of specifically binding to the genes encoding the light and heavy chains of murine antibodies). . Hybridoma cells serve as the preferred source of said DNA. Once isolated, the DNA can be placed into expression vectors, which are transfected (cell infection) into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary cells (CHO) , or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on expression of recombinants in DNA bacteria encoding the antibodies include Skerra et al. , Curr, Opinion in Immunol. , 5: 256-262 (1993) and Plückthun, Immunol. , Revs. , 130: 151-188 (1992).

In a further embodiment, the antibodies can be isolated from phage antibody libraries generated using the techniques described in McCafferty et al. , Nature, 348: 552-554 (1990). Clackson et al. , Na ture, 352: 624-628 (1991) and Marks et al. , J. Mol. Biol. , 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM range) by chain change (Marks et al., Bio / Technology, 10: 779: 783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy to build very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)): Thus, these techniques are viable alternatives to the traditional hybridoma techniques of monoclonal antibodies for the isolation of monoclonal antibodies.

DNA can also be modified, for example, by substituting the sequence coding sequence for heavy or light chain human constant domains, instead of murine homologous sequences (US Patent No. 4,816,567; Morrison, et al., Proc. Nati, Acad. Sci. USA, 81: 6851 (1984)), or by covalently binding to the immunoglobulin coding sequence, in whole or in part of the coding sequence for a non-immunoglobulin polypeptide.

Typically, said non-immunoglobulin polypeptides are replaced by the constant domains of an antibody, or are substituted by the variable domains of an antigen-combining site of an antibody, to create a bivalent chimeric antibody comprising a combining antigen site that has specificity for an antigen and another antigen-combining site that has the specificity for a different antigen.

Hybrid or chimeric antibodies can also be prepared in vitro, using methods known in synthetic protein chemistry, including those involving entanglement agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. (iii) Humanized and humanized antibodies Methods for humanization of non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced therein, from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "Imported" variable domain. Humanization can essentially be carried out following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Recih ann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. In this way, said "humanized" antibodies are chimeric antibodies (U.S. Patent 4,816,567); wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies.

The choice of human, light and heavy variable domains to be used in making humanized antibodies is very important to reduce antigenicity. In accordance with the so-called "best fit" method, the variable domain sequence of a rodent antibody is screened against the entire sequence library of known human variable domains. The human sequence that is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Si s et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light and heavy chains. The same framework can be used for various humanized antibodies (Cárter et al., Proc Nati Acad Sci USA, 89: 4285 (1992), Presta et al., J. Immunol., 151: 2623 (1993)) .

It is further important that the antibodies are humanized with the retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in accordance with the preferred method, the humanized antibodies are prepared by a process of analysis of the parental sequences and of several humanized conceptual products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computational programs are available that illustrate and display the probable three-dimensional conformation structures of the selected immunoglobulin candidate sequences. The inspection of these deployments allows the analysis of the probable role of the residues in the functioning of the immunoglobulin candidate sequence, that is, the analysis of the residues that influence the ability of the candidate immunoglobulin to bind with its antigen. In this manner, the FR residues can be selected and combined, from the container and the import sequences so that the desired antibody characteristic such as increasing affinity for the target antigen (s) is achieved. In general, CDR residues are directly and more substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable of, by immunization, producing a broad repertoire of human antibodies in the absence of the production of endogenous immunoglobulin. For example, homozygous removal of the gene from the binding region of heavy chain antibody (JH) in chimeric and germ line mutant mice has been reported to result in the total inhibition of endogenous antibody production. The transfer of the arrangement of the human immunoglobulin gene in the germ line into said mouse, mutant in the germ line, will result in the production of human antibodies under the challenge of the antigen. See, e.g., Jakobovits et al. , Proc. Nati Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al. , Nature, 362: 255-258 (1993); Bruggermann et al. , Year in Immuno. , 7:33 (1993). Human antibodies can also be derived from phage display libraries (Hoogenboom et.al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581. -597 (1991)). (iv) Bispecific antibodies antibodies Bispecific antibodies (BsAbs) are antibodies that have binding specificities for at least two different epitopes. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., bispecific antibodies F (ab ') 2).

Methods for making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the coexpression of two pairs of light chain and heavy chain immunoglobulins, wherein the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random variety of heavy and light immunoglobulin chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is quite problematic, and product yields are low. Similar procedures are described in WO 93/08829 and in Traunecker et al. , EMBO J., 10: 3655-3659 (1991).

In accordance with a different approach, variable domains of antibodies with the desired binding specificities (antibody-antigen combining sites) are fused to constant domain immunoglobulin sequences. The fusion is preferably with a heavy chain immunoglobulin constant domain, comprising at least part of the joint of the CH2 and CH3 regions. It is preferred to have. the first heavy chain constant region (CH1) containing the sites necessary for the light chain linkage present in at least one of the fusions. The DNAs encoding the heavy chain immunoglobulin fusions and, if desired, the light chain immunoglobulin, are inserted into separate expression vectors, and counter-transfected into an appropriate host organism. This provides a great flexibility to adjust the mutual proportions of the three polypeptide fragments into embodiments when the unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is however possible to insert the coding sequences for the two or all three polypeptide chains into an expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratios are not particular importance.

In a preferred embodiment of this approach, the specific antibodies are composed of a heavy immunoglobulin hybrid chain with a first binding specificity in one arm, and a light chain-heavy chain immunoglobulin hybrid pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from undesirable immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy form of separation. This approach is described in the patent application WO 94/04690 published on March 3, 1994. For further details on the generation of bispecific antibodies, see for example, Suresh et al. , Methods in Enzymology, 121: 210 (1986).

Bispecific antibodies include entangled or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled with avidin, the other with biotin. Such antibodies have been proposed, for example, to signal cells from immune systems to undesirable cells (US Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373). Heteroconjugate antibodies can be made using any convenient interlacing method. Suitable interlacing agents are well known in the art, and are described in US Patent No. 4,676,980, along with various interlacing techniques.

The techniques for generating specific antibodies from antibody fragments have also been described in the literature. The following techniques can also be used for the production of bivalent antibody fragments, which are not necessarily bispecific. For example, Fab 'fragments recovered from E. coli can be chemically coupled in vi tro to form bivalent antibodies. See, Shalaby et al. , J. Exp. Med., 175: 217-225 (1992).

Various techniques for making and isolating bivalent antibody fragments directly from the culture of recombinant cells have also been described. For example, bivalent heterodimers have been produced using a leucine lock. Kostelny et al. , J. Immunol. , 148 (5): 1547-1553 (1992). Peptides from the leucine lock from the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the region of articulation, to form monomers and then re-oxidized to form the antibody heterodimers. The "diabody" technology described by Hollinger et al., Proc. Nat. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific / bivalent antibody fragments. The fragments comprise a heavy chain variable (VH) domain connected to a light chain variable (VL) domain by a linker that is too short to allow pairing between the two domains on the same chain. In this manner, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen binding sites. Another strategy for fragmenting bispecific / bivalent antibodies by the use of single chain dimers Fv (sFv) has also been reported. See Gruber et al. , J. Immunol. , 152: 5368 (1994).

B. Preparation of the Lyophilized Formulation After preparing the protein of interest as described above, a "pre-lyophilized" formulation is produced. The amount of protein present in the pre-lyophilized formulation is determined by taking into account the desired dose volumes, mode (s) of administration, etc. Where the protein of choice is an intact antibody (such as an anti-IgE or anti-HER2 antibody), from about 2 mg / mL to about 50 mg / mL, preferably about 5 mg / mL to about 40 mg / mL and more preferably from about 20-30 mg / mL, is an exemplary concentration of protein start.

The protein is usually present in solution. For example, the protein may be present in a buffered solution of pH at a pH from about 4-8, and preferably from about 5-7. Exemplary buffer solutions include histidine, phosphate, Tris, citrate, succinate or other organic acids. The concentration of the buffer solution can be from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending for example on the buffer solution and the desired isotonicity of the formulation (pej., of the reconstituted formulation). The preferred buffer is histidine since, as demonstrated below, it may have lyoprotective properties. The succinate is shown as another useful buffer.

The lyoprotectant is added to the pre-lyophilized formulation. In preferred embodiments, the lyoprotectant is a non-reducing sugar such as sucrose or trehalose. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that, upon reconstitution, the resulting formulation will be isotonic. However, hypertonic reconstituted formulations may also be appropriate. In addition, the amount of lyoprotectant should not be too low so that an unacceptable amount of protein degradation / aggregation occurs during lyophilization. Where the lyoprotectant is a sugar (such as sucrose or trehalose) and the protein is an antibody, the exemplary concentrations of lyoprotectant in the pre-lyophilized formulation are from about 10 mM to about 400 mM, and preferably from about 30 mM to about 300 mM, and more preferably from about 50 mM to about 100 M.

The ratio of protein to lyoprotectant is selected from each protein and combination of lyoprotectant. In the case of an antibody as the protein of choice and a sugar (eg, sucrose or trehalose) as the lyoprotectant to generate an isotonic reconstituted formulation with a high protein concentration, the molar ratio of lyoprotectant to antibody can be from about 100 to about 1500 moles of lyoprotectant per 1 mole of antibody, and preferably from about 200 to about 1000 moles of lyoprotectant to 1 mole of antibody, for example, about 200 to about 600 moles of lyoprotectant per mole of antibody.

In preferred embodiments of the invention, it has been found to add a surfactant to the pre-lyophilized formulation. Alternatively or in addition, the surfactant may be added to the lyophilized formulation and / or reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (eg, polysorbates 20 or 80); poloxamers (pe.j., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-. cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (eg, lauroamidopropyl), -myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT ™ series (Mona Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and ethylene and propylene glycol copolymers (eg, Pluronics, PF68 etc.). The aggregate amount of surfactant is such that it reduces the aggregation of the reconstituted protein and minimizes the formation of particles after reconstitution. For example, the surfactant may be present in the pre-lyophilized formulation in an amount from about 0.001-0.5% and preferably from about 0.005-0.05%.

In certain embodiments of the invention, a mixture of lyoprotectant (such as sucrose or trehalose) and a bulking agent (eg, mannitol or glycine) are used in the preparation of the pre-lyophilized formulation. The bulking agent can allow the production of a uniform lyophilized cake without excessive gaps in it, etc.

Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington 's Pharmaceutical Sciences, 16a. Ed., Osol, A. Ed. (1980) can be included in the pre-lyophilized formulation (and / or the pre-lyophilized formulation and / or the reconstituted formulation) as long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors at the concentrations and doses employed and include; additional buffering agents; conservatives; co-solvents, - antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes), biodegradable polymers such as polyesters; and / or salt forming counterions such as sodium.

The formulation herein may also contain more than one protein as necessary, for the particular indication being treated; preferably those with complementary activities that do not adversely affect the other protein. For example, it may be desirable to provide two or more antibodies that bind to the HER2 or IgE receptor in a single formulation. Additionally, anti-HER2 and anti-VEGF antibodies can be combined in a formulation. Said proteins are appropriately present in combination in amounts that are effective for the purpose sought.

Formulations for use in in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes, before, or followed by, lyophilization and reconstitution. Alternatively, sterility of the entire mixture can be achieved by autoclaving the ingredients, except for the protein, at about 120 ° C for about 30 minutes for example.

After the protein, lyoprotectant and other optional components are mixed together, the formulation is lyophilized. Many different freezer-dryers are available for this purpose such as the Hull50 ™ (Hull, USA) or GT20 ™ (Leybold-Heraeus, Germany) dryers-freezers. Drying-freezing is achieved by freezing the formulation and subsequently subliming the ice from the frozen contents at a temperature appropriate for primary drying. Under this condition, the temperature of the product is below the eutectic point or the collapse temperature of the formulation. Typically the shelf temperature for primary drying will be in the range of from about -30 to 25 ° C (provided the product remains frozen during primary drying) at an appropriate pressure, typically in the range of from about 50 up to 250 mTorr. The formulation, size and type of the container that holds the sample (eg, a glass vial) and the volume of liquid, will dictate mainly the required drying time, which can vary from a few hours to several days (eg. j., 40-60 hours). A secondary drying step can be carried out at around 0-40 ° C, depending mainly on the type and size of the container and the type of protein used. However, it has been found here that a secondary drying step may not be necessary. For example, the shelf temperature through the complete phase of lyophilization water removal may be from about 15-30 ° C (e.g., around 20 ° C). The time and pressure required for secondary drying will be that which produces an appropriate lyophilized cake, dependent, eg, on temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (eg, 10-15 hours). The pressure can be the same as that used during the primary drying stage. Freezing drying conditions may vary depending on the formulation and the size of the vial.

In some cases, it may be desirable to lyophilize the protein formulation in the container in which the reconstitution of the protein is carried out in order to avoid a transfer step. The container in this case may for example be a vial of 3, 5, 10, 20, 50 or 100 ce.

As a general proposition, lyophilization will result in a lyophilized formulation in which the moisture content thereof is less than about 5% and preferably less than about 3%.

C. Reconstitution of the Lyophilized Formulation At the desired stage, typically when it is time to administer the protein to the patient, the lyophilized formulation can be reconstituted with a diluent such that the protein concentration in the reconstituted formulation is at least 50 mg / mL , for example, from about 50 mg / mL to about 400 mg / mL, more preferably from about 80 mg / mL, to about 300 mg / mL and more preferably from about 90 mg / mL to about 150 mg / mL. Said high concentrations of protein in the reconstituted formulation are considered particularly useful where the subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration such as intravenous administration, lower concentrations of the protein in the reconstituted formulation may be desirable (eg, from about 5-50 mg / mL, or from about 10-40 mg / mL, of protein in the reconstituted formulation). In certain embodiments, the concentration of protein in the reconstituted formulation is significantly higher than that in the pre-lyophilized formulation. For example, the concentration of orteine in the reconstituted formulation can be about 2-40 times, preferably 3-10 times and more preferably 3-6 times (eg, at least three times or at least four times) that of the pre-lyophilized formulation.

The reconstitution generally takes place at a temperature of around 25 ° C to re complete hydration, although other temperatures may also be employed as desired. The time required for reconstitution will depend, eg, on the type of diluent, the amount of excipient (s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a solution buffered at pH (eg, buffered saline-phosphate), sterile saline, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above with aromatic alcohols such as benzyl alcohol or phenol being the preferred preservatives. The amount of preservative employed is determined by evaluating different concentrations of preservatives for compatibility with the protein and for testing the efficacy of the preservative. For example, if the preservative is an aromatic alcohol (such as benzylic alchol), it may be present in an amount of from about 0.1-2.0% and preferably from about 0.5-1.5% but more preferably around 1.0-1.2%.

Preferably the reconstituted formulation has less than 6000 particles per vial which are > 10 μm in size.

D. Administration of the Reconstituted Formulation The reconstituted formulation is administered to a mammal in need of treatment with the protein, preferably a human, according to known methods, such as intravenous administration as a bolus, or by continuous infusion over a period of time. of time, by intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes.

In preferred embodiments, the reconstituted formulation is administered to the mammal by subcutaneous administration (ie, under the skin). For such purposes, the formulation can be injected using a syringe. However, other devices for administration of the formulation are available such as injection devices (e.g., the Inject-ease ™ and Genject ™ devices); injector p(such as the GenPen ™), needleless devices (eg MediJector ™ and BioJector ™), and - small exton subcutaneous delivery systems.

The appropriate dose ("therapeutically effective amount") of the protein will depend, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, prior therapy, the clinical history of the patient and the response to the protein, the type of protein used, and the doctor's discretion. The protein is appropriately administered to a patient, at a time or over a series of treatments, and can be administered to the patient at any time after the diagnosis. The protein can be administered as the sole treatment or in conjunction with other medications or therapies useful in the treatment of the condition in question.

Where the protein of choice is an antibody, from about 0.1-20 mg / kg is an initial dose candidate for administration to the patient, either for example, by one or more separate administrations. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

In the case of an anti-HER2 antibody, a therapeutically effective amount of the antibody can be administered to treat or prevent cancer, characterized by overexpression of the HER2 receptor. It is contemplated that a reconstituted formulation of the anti-HER2 antibody can be used to treat cancers of the breast, ovaries, stomach, endometrium, salivary glands, lung, kidney, colon and / or vegija. For example, the anti-HER2 antibody can be used to treat ductal carcinoma in situ (DCIS). Exemplary doses of the anti-HER2 antibody are in the range of 1-10 mg / kg for one or more separate administrations.

The use of an anti-IgE formulation includes the treatment or prophylaxis of IgE-mediated allergic disorders, parasite infections, intersitial cystitis and asthma, for example. Depending on the disease or disorder to be treated, a therapeutically effective amount (e.g., from about 1-15 mg / kg) of the anti-IgE antibody is administered to the patient.

E. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing the lyophilized formulation of the present invention is supplied and provides instructions for its reconstitution and / or use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (eg, dual chamber vials), syringes (such as dual chamber syringes), and test tubes. The container can be formed from a variety of materials such as plastic or glass. The container maintains the lyophilized formulation and the label on the container, or associated with it, can indicate the directions for its reconstitution and / or use. For example, the label may indicate that the lyophilized formulation is reconstituted at the protein concentrations as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration. The container holding the formulation can be a multi-use vial, which allows repeated administrations (eg, from 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g., BWFI). When mixing with the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg / mL. The article of manufacture can also include other desirable materials from a commercial and user's point of view. , including other shock absorbers, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

The invention will be more fully understood with reference to the following examples. However, they should not be interpreted as limiting the scope of the invention. All citations in the literature are incorporated as a reference.

EXAMPLE 1 FORMULATION ANTI-HER2 Overexpression of the proto-oncogene product HER2 HER2 (pl85) has been associated with a variety of aggressive human ills. The murine monoclonal antibody known as muMAb4D5 is directed against the extracellular domain (ECD) of pl85HER2. The muMAb4D5 molecule has been humanized in an effort to improve its clinical efficacy by reducing its immunogenicity and allowing it to support the functions of the human effector (see WO 92/22653). This example describes the development of a lyophilized formulation comprising a humanized full-length antibody huMAb4D5-8 described in WO 92/22653.

In the development of a lyophilized formulation, the excipients and buffer solutions are initially screened by measuring the stability of the protein after lyophilization and reconstitution. The lyophilized protein in each formulation is also subject to accelerated stability studies to determine the potential stability of the protein during its shelf life. These accelerated studies are usually carried out at temperatures above the proposed storage conditions and the data is then used to estimate the activation energy for the degradation reactions assuming Arrhenius kinetics (Celeland, et al., Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993)). The activation energy is then used to calculate the expected shelf life of the protein formulation at the proposed storage conditions.

In preliminary screening studies, the stability of various formulations of freeze-dried recombinant humanized anti-HER2 antibody (rhuMAb HER2) was investigated after incubation at 5 ° C (proposed storage condition) and 40 ° C (accelerated stability condition). In the liquid state, it was observed that rhuMAb HER2 is degraded by deamidation (30 Asn of light chain) and isoaspartate formation by means of a cyclic intermediate of imide, succinimide (102Asp of heavy chain). Deamidation was minimized to pH 5.0, resulting in degradation mainly in succinimide. At a pH of 6.0, a slightly higher deamidation was observed in the liquid protein formulation. The lyophilized formulations were therefore studied with: (a) 5 or 10 mM succinate buffer, pH 5.0 or (b) 5 or 10 mM histidine buffer, pH 6.0. Both buffer solutions containing the surfactant, polysorbate 20 (Tween 20 ™), which was used to reduce the aggregation potential of the reconstituted protein and minimize particle formation after reconstitution. These buffers are used with or without various sugars. The protein was formulated in the buffer at 5.0, 21.0 or 25.0 mg / mL. These formulations were then lyophilized and evaluated for protein stability after 2 weeks at 5 ° C and 40 ° C. In the lyophilizer, vials were frozen at shelf temperature of -55 ° C for approximately 5 hours, followed by primary drying at room temperature of 5 ° C and 150 mTorr for 30 hours, and 1-2% drying was achieved of residual moisture with secondary drying at shelf temperature of 20 ° C for 10 hours. The main degradation pathway for this protein with lyophilization was aggregation, and therefore the stability of the protein was evaluated naturally by exclusion chromatography, to measure the recovery of the intact natural protein (% of intact protein in Table 2 below).

The stabilizing effects of the various lyoprotectant sugars on the lyophilized protein were measured in 10 mM sodium succinate, pH 5.0 (Table 2). At higher concentrations of sugar (250-275 mM) and low protein concentration (5.0 mg / mL), trehalose and lactose stabilized the protein against aggregation, for the lyophilized protein stored for 2 weeks at 40 ° C. However, lactose, a reducing sugar, was found to react with the protein during a storage period greater than 40 ° C. Formulations at 5.0 mg / mL protein containing sorbitol or mannitol produced added protein after storage at 40 ° C for 2 weeks. At higher protein concentrations (21.0 mg / mL), formulations containing mannitol, or mannitol in combination with sorbitol or glycine, contained added protein after lyophilization and storage at both conditions. In contrast, trehalose and sucrose avoided aggregation at both operating conditions.

Formulations of 250 mM trehalose and 250 mM lactose were evaluated for long-term stability. After 9 months at 40 ° C or 12 months at 5 ° C, there was no change in the percentage of intact protein for the trehalose formulation. For the lactose formulation,% intact protein remained constant (the same as the initial one) after 3 months at 40 ° C or 6 months at 25 ° C. The trehalose formulation could be stored at controlled room temperature (15-30 ° C) for 2 years without a significant change in% intact protein. The 10 mM formulation of histidine, pH 6.0 with mannitol, contained less protein added after storage at 40 ° C for 2 weeks than the lOmM formulation of succinate, pH 5.0 with mannitol. This result may be related to some stabilizing effect contributed only by histidine. After storage at 40 ° C for 2 weeks, there was nevertheless no significant aggregation only for histidine or histidine / mannitol formulations. The addition of sucrose to an equal amount of mannitol (10 mg / mL of each) in the histidine formulation, stabilized the protein against aggregation for both storage conditions. The use of glycine with mannitol did not improve the stability of the protein, while the sucrose / glycine formulation provided the. same stability as the sucrose / mannitol formulation. These results further indicate that sucrose was useful in preventing aggregation of the lyophilized protein during storage.

TABLE 2 to. a fraction of intact protein was measured by natural size exclusion HPLC, and the peak area of the natural protein in relation to the total area of the peak including aggregates (column TSK3000 SW XL, TosoHaas, with a flow of 1.0 mL / min; elution with phosphate buffer saline, detection at 214 and 280 nm). The protein formulations were analyzed before lyophilization (liquid, 5 ° C) and after lyophilization and storage at 5 ° C or 40 ° C for 2 weeks. b. Formulations containing 5 mg / mL of protein were reconstituted with distilled water (20 mL, 5.0 mg / mL of protein), and formulations containing 21 mg / mL of protein were reconstituted with bacteriostatic water for injection (BWFI, 0.9 % benzyl alcohol, 20 mL, 20 mg / mL of protein).

The provision of a high protein concentration is often required for subcutaneous administration due to volume limitations (<1.5 mL) and dose requirements (> 100 mg). However, high protein concentrations (> 50 mg / mL) are often difficult to achieve in the manufacturing process because at high concentrations, the protein has a tendency to aggregate during processing and becomes difficult to manipulate ( eg, pump) and sterile filter. Alternatively, the lyophilization process can provide a method to allow the concentration of the protein. For example, the protein is filled in vials to a volume (Vf) and then lyophilized. The lyophilized protein is then reconstituted with a smaller volume (Vr) of water or conservative (eg, BWFI) than the original volume (eg, Vr = 0.25Vf) resulting in a higher concentration of protein in the reconstituted solution. This process also results in the concentration of buffer solutions and excipients. For administration. subcutaneously, the administration is desirably isotonic.

The amount of trehalose in the freeze-dried rhuMAb HER2 was reduced to produce an isotonic solution upon reconstitution to produce 100 mg / mL of protein. The stabilizing effect of trehalose was determined as a function of the concentration of 5 mM sodium succinate, pH 5.0 and 5 mM histidine, pH 6.0 at 25.0 mg / mL of protein (Table 3). At trehalose concentrations from 60 to 200 mM, there was no significant aggregation after incubation of the lyophilized protein for 4 weeks at 40 ° C. These formulations were reconstituted with 20 mL of bacteriostatic water for injection (BWFI, USP, 0.9% benzyl alcohol). Reconstitution of the formulation of 50 mM trehalose (5 mM sodium succinate) with 4 ml of BWFI (100 mg / mL protein) after incubation for 4 weeks at 40 ° C produced a slight increase in aggregate formation . The preserved reconstituted formulations provided the advantage of multiple withdrawals from the same vial without concerns of sterility. When sterile needles are used, these formulations would then be allowed several doses from a single vial.

TABLE 3 to. to intact protein fraction it was measured by exclusion HPLC size, and defined as the peak area of the native protein relative to total peak area including aggregates (column TSK3000 SW XL, TosoHaas, co n a flow of 1.0 mL / min; elution with phosphate buffer saline; detection at 214 and 280 nm). The protein formulations were analyzed before lyophilization (liquid, 5 ° C) and after lyophilization and storage at 5 ° C or 40 ° C for 2 weeks. The formulations were reconstituted with bacteriostatic water for injection (BWFI, USP, 0.9% by weight of benzyl alcohol, 20 mL, 22 mg / mL of protein). b. Reconstituted with 4 ml of BWFI (0.9% benzyl alcohol) to produce 100 mg / mL c. Reconstituted with 4 ml of BWFI (1.1% benzyl alcohol) to produce 100 mg / mL d. Sample incubated for two weeks at 5 ° C and 40 ° C and then reconstituted with 20 mL of BWFI (0.9% benzylic alchol) to produce 22 mg / mL of protein.

Currently, the rhuMAb HER2 is under investigation as a therapeutic for breast cancer. The protein is dosed to patients at 2 mg / kg on a weekly basis. Since the average weight of these patients is 65 kg, the average weekly dose is 130 mg of rhuMAb HER2. For subcutaneous administration, injection volumes of 1.5 mL or less are well tolerated and, therefore, the protein concentration for a weekly subcutaneous administration of rhuMAb HER2 may be approximately 100 mg / mL (130 mg average dose / 1.5 mL ). As mentioned above, high protein concentration is difficult to manufacture and maintain in stable form. To achieve this high protein concentration, rhuMAb HER2 is formulated in: (a) 5 mM sodium succinate, pH 5.0 or (b) 5 mM histidine, pH 6.0, lyophilized at 25 mg / mL protein in 60 mM trehalose, 0.01% Tween 20 ™. Lyophilization was carried out by filling 18 mL of the protein formulation into 50-ce vials. In the lyophilizer, the vials were frozen at a shelf temperature of -55 ° C for about 5 hours, followed by a primary drying at a shelf temperature of 5 ° C and 150 mTorr for 30 hours, and drying at 1-2 % residual moisture was achieved with secondary drying at a shelf temperature of 20 ° C for 10 hours. The thermocouples placed in the vials containing the placebo (formulation without protein), indicated that the product in the vials was kept below -10 ° C through primary drying. Sequential puffing studies during lyophilization revealed that residual moisture after primary drying was usually less than 10%.

The lyophilized protein was then reconstituted with either 4 or 20 ml of BWFI (0.9 or 1.1% benzyl alcohol) to produce the concentrated protein solutions: (a) 4 mL: 102 mg / mL of rhuMAb HER2, 245 mM of trehalose, 21 mM of sodium succinate, pH 5.0 or 21 mM of histidine, pH of 6.0, 0.04% of Tween 20 ™; (b) 20 mL: 22 mg / mL rhuMAb HER2, 52 mM trehalose, 4 mM sodium succinate, pH 5.0 or 4 mM histidine, pH 6.0, 0.009% Tween 20 ™.

After storage of the lyophilized formulations for 4 weeks at 40 ° C and reconstitution at 22 mg / mL protein, the amount of added protein appeared to increase slightly with the decreasing concentration of trehalose. The stability of the lyophilized protein was not affected by the volume of reconstitution. As shown in Figure 1, the amount of intact protein after incubation of lyophilized protein at 40 ° C was the same for trehalose 60mM, 5 mM sodium succinate, pH 5.0, 0.01% Tween 20 ™ , formulation reconstituted with either 4 or 20 mL of BWFI.

The results shown in Table 3 suggest that there may be a relationship between trehalose concentration and protein stability. To further evaluate this relationship, formulations containing different concentrations of trehalose formulated in sodium succinate or histidine were incubated for 91 days at 40 ° C. The stability was then measured as a function of the molar ratio trehalose to protein for each concentration of trehalose. As shown in figure 2, the stability of the protein clearly decreased as the concentration of trehalose decreased for both formulations. There was no apparent difference between the two buffer solutions, succinate and histidine, in these formulations, suggesting that the primary stabilizer under these conditions is trehalose. In addition, the observed decrease in intact protein for both of these formulations would be acceptable even at the low concentration of trehalose for a formulation that is stored at 2-8 ° C throughout its shelf life. However, if stability is required at controlled room temperature (30 ° C maximum temperature), higher concentrations of trehalose (600: 1 trehalose: protein) may be necessary, depending on stability specifications for the product (ie, the specification for the amount of intact protein remaining after 2 years of storage). Typically, a storage condition at controlled room temperature would require stability for 6 months at 40 ° C which is equivalent to storage at 30 ° C for 2 years.

As shown in Figure 3, the 250mM formulation of trehalose remained unchanged after 6 months at 40 ° C, while loved 60mM trehalose formulations were less stable. The 60 mM trehalose formulations may then require refrigerated storage if the product specification at the end of its shelf life is for example, > 98% intact protein by size-exclusion chromatography.

In the study . Prior to sieving, it was also observed that sucrose prevented the aggregation of rhuMAb HER2 after lyophilization and subsequent storage. To achieve isotonic solutions after reconstitution for subcutaneous administration (approximately a four-fold concentration of the formulation and protein components), the sucrose concentration must be significantly reduced. The same concentration of mass of sucrose and mannitol (bulking agent) used in the sieving studies, prevented the aggregation of protein. A lower concentration of sucrose and mannitol (equal mass concentrations) was chosen as a potential subcutaneous formulation of rhuMAb HER2. The protein solution (25 mg / mL protein, 5 mM histidine, pH 6.0, 38.4 mM (7 mg / mL) mannitol, 20.4 mM (7 mg / mL) sucrose, 0.01% Tween 20 ™) lyophilized in the same manner as the 60 mM trehalose formulation, except that the primary drying cycle was extended for 54 hours. After 4 weeks at 40 ° C, there was a slight increase in the amount of aggregates after reconstitution with 4.0 or 20.0 mL of BWFI (Table 3). The amount of protein added was the same for reconstitution at 22 or 100 mg / mL of protein (Figure 4). Like the 60 mM trehalose formulations, the mannitol / sucrose formulation produced less intact protein over time at 40 ° C. The molar ratio of sucrose to protein for this formulation was 120 to 1, indicating that the mannitol / sucrose formulation may be more effective than trehalose only at the same molar ratio of stabilizing sugar (Figures 2 and 4).

In the previous examples, the stability of the lyophilized formulations of rhuMAb HER2 was determined as a function of temperature. These studies demonstrated that trehalose and mannitol / scarosa formulations prevented degradation of the protein in the lyophilized state at elevated temperatures (40 ° C). However, these experiments did not address the stability of the protein after reconstitution and storage. Once reconstituted with BWFI, the lyophilized formulations of rhuMAb HER2 can be used for various drug administrations. In particular, the vial configuration (450 mg rhuMAb HER2) was designed to provide 3 doses to the average patient (130 mg rhuMAb HER2 per dose). Since the medication is dosed weekly, the vial can be stored at least three weeks after reconstitution. To ensure that rhuMAb HER2 remained stable after reconstitution, stability studies on the reconstituted formulations of rhuMAb HER2 were carried out at 5 ° C and 25 ° C.

For subcutaneous administrations, the formulations were reconstituted at 100 mg / mL (4 mL of BWFI). At this high protein concentration, the protein may be more susceptible to aggregation than the intravenous dose form that was reconstituted at 22 mg / mL protein (20 mL BWFI). The four formulations of rhuMAb HER2 of the previous example were evaluated for aggregation (loss of intact protein). As shown in Tables 4 to 6, there was no difference in the stability of the reconstituted formulations at 22 and 100 mg / mL of protein. In addition, these formulations kept the protein completely intact for up to 90 days at 5 ° C and 30 days at 25 ° C, indicating that the reconstituted protein could remain refrigerated for at least 90 days. In contrast to the stability of the lyophilized protein in the previous example, the concentration of trehalose in the formulation did not affect the stability of the protein,. (Table 7).

TABLE 4 Stability of the reconstituted formulations of rhuMAb HER2 lyophilized at 25 mg / mL protein in 5 mM sodium succinate, pH 5.0, 60 mM trehalose, 0.01% Tween 20 ™ Samples were reconsti- tuted with 4.0 20.0 mL of BWFI (1.1% or 0.9% benzyl alcohol), and then stored at 5 ° C or 25 ° C. The% intact protein was defined as the fraction of the natural peak area as measured by size-exclusion chromatography. ND = Not determined.

TABLE 5 Stability of the reconstituted formulations of rhuMAb HER2 lyophilized at 25 mg / mL protein in 5 mM histidine, pH 6.0, 60 mM trehalose, 0.01% Tween 20 Samples were reconstituted with 4.0 or 20.0 mL of BWFI (1.1% or 0.9% benzyl alcohol), and were then stored at 5 ° C or 25 ° C% intact protein was defined as the fraction of the natural peak area as it is measured by size-exclusion chromatography. ND = Not determined.

TABLE 6 Stability of the reconstituted formulations of rhuMAb HER2 lyophilized at 25 mg / mL protein in 5 mM histidine, pH 6.0, 38.4 mM mannitol, 20.4 mM sucrose, 0.01% Tween 20 ™ Samples were reconsti- tuted with 4.020.0 mL of BWFI (1.1% or 0.9% benzyl alcohol), and then stored at 5 ° C or 25 ° C% intact protein was defined as the area fraction of natural peak as measured by size-exclusion chromatography. ND = Not determined.

TABLE 7 Stability of the reconstituted formulations of rhuMAb HER2 lyophilized at 21 mg / mL protein in 10 mM sodium succinate, pH 5.0, 250 mM trehalose, 0.2% Tween 20 ™ Samples were reconstituted with 20.0 mL of BWFI (0.9% benzyl alcohol), and then stored at 5 ° C or 25 ° C% intact protein was defined as the fraction of the natural peak area as measured by life-size exclusion. ND = Not determined.

As previously mentioned, the major route of degradation for rhuMAb HER2 in aqueous solutions is the deamidation or formation of succinimide. The loss of natural protein due to deamidation or succinimide formation was evaluated for the four reconstituted formulations of rhuMAb HER2.

The analysis of the deamidation and succinimide formation of rhuMAb HER2 was carried out using a cation exchange cormatography. A Bakerbond column of Broad Pore Carboxy Sulphonate (CSX) was operated at a flow of 1 mL / min. The buffer solutions of the mobile phase were (A) 0.02 M sodium phosphate, pH 6.9, and (B) 0.02 M sodium phosphate, pH 6.9, 0.2 M NaCl. The chromatography was then run at 40 ° C as follows: TABLE 8 Peak elution was monitored at 214 nm and 75 μg of protein was loaded for each analysis.

Again, there were no differences in the stability of the reconstituted formulations at 22 and 100 mg / mL of protein (Figures 5 to 7). Protein degradation was faster at 25 ° C than at 5 ° C for each formulation, and the rate of degradation was comparable for all formulations stored at 5 ° C. The formulations containing histidine suffered a degradation rate slightly higher than 25 ° C than the succinate formulations. The amount of trehalose in the formulation did not affect the rate of degradation at any temperature (Figures 5 and 8). These results indicated that these four formulations provided an acceptable rate of degradation under refrigerated storage conditions (5 ° C) for the intended period of use (30 days after reconstitution with BWFI).

Multipurpose formulations must approve the conservative efficacy test as described in the US Pharmacopeia (USP) for use in the United States. The lyophilized formulation of rhuMAb HER2 consisting of 25 mg / mL protein, 5 mM histidine, pH 6.0, 60 mM trehalose, 0.01% Tween 20 ™, was reconstituted with 20 mL of benzyl alcohol at concentrations between 0.9 and 1.5% weight / weight For concentrations of or greater than 1.3% w / w the reconstituted solution became turbid after an overnight incubation at room temperature (around 25 ° C). Reconstitution with the normal BWFI solution (0.9% benzyl alcohol) resulted in a solution that did not consistently pass the challenge tests with conservative. However, reconstitution with 1.0 or 1.1% benzyl alcohol was compatible with the formulation and passed the challenge tests with preservative. The manufacturer's specifications for the solution required a range of ± 10%, and therefore, the lyophilized formulations were reconstituted with 1.1% benzyl alcohol (1.1 ± 0.1%).

A simple cycle of the lyophilization stage was developed for the formulation of rhuMAb HER2. In the simple cycle of the lyophilization step, rhuMAb HER2 was lyophilized at 25 mg / mL, 60 mM trehalose, 5 mM histidine pH 6 and 0.01% polysorbate 20, at a shelf temperature of 20 ° C and a pressure of 150 mTorr. After 47 hours, the residual moisture content of the lyophilized cake was less than 5%. This lyophilization cycle is considered to be useful since it simplifies the manufacturing process by eliminating the secondary drying stage.

EXAMPLE 2 ANTI-IgE FORMULATION IgE antibodies are linked to high affinity specific receptors on barley cells, leading to the degranulation of barley cells and the release of mediators such as histamine, which produces symptoms associated with allergy. Hence, anti-IgE antibodies that block the binding of IgE with its high affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies should also not bind to IgE once it binds to the receptor because this would trigger the release of histamine.

This example describes the development of a lyophilized formulation comprising full-length humanized anti-IgE antibody described in Presta et al. J. Immunology, 151: 2623-2632 (1993).

Materials: rhuMAb E25 highly purified (recombinant humanized anti-MaEll IgE antibody) not containing Tween 20 ™ was used in the formulations described below. Spectra / Por 7 dialysis membranes from Spectrum (Los Angeles, CA) were purchased. All the other reagents used in this study were obtained from commercial sources and were of analytical grade. Formulation buffers and the chromatographic mobile phase were prepared by mixing an appropriate amount of buffer solution and salt with Milli-Q water in a volumetric flask.

Formulation: Sepharose E25 S solution was dialyzed into the formulation buffer solutions as specified. Dialysis was carried out by means of a minimum of exchanges of 4 x 2L buffer solutions over a period of 48 hours at 2-8 ° C. After dialysis, lyoprotectant was added at an isotonic concentration to some of the formulations as required. The protein concentration following dialysis was determined by UV spectroscopy using a molar absorption of 1.60. The dialyzed protein was diluted to the predetermined concentration of the formulation with a suitable, sterile formulation buffer solution, filtered using a 0.22 μm Millex-GV filter (Millipore) and dosed into pre-washed and autoclaved glass vials. The vials were prepared with Teflon lyophilization plungers and lyophilized using the following conditions: Formulation E25 was frozen to -55 ° C to 80 ° C / hour and the contents of the vial kept frozen for 4 hours. The shelf temperature was tilted to 25 ° C at 10 ° C / hour for primary drying. The primary drying was carried out at 25 ° C, a vacuum chamber pressure of 50μ for 39 hours such that the residual moisture of the lyophilized cake was 1-2%. After lyophilization, one vial of each formulation was separated for a t = 0 analysis, and the remaining vials were maintained at various temperatures including -70 ° C, 2-8 ° C, 25 ° C, 30 ° C ( controlled temperature of the environment) 40 ° C and 50 ° C.

Chromatography: Life-size exclusion chromatography was performed on a Bio-Rad Bio-Select ™ SEC 250-5 column (300x7.8 mm). The column was equilibrated and operated in PBS at a flow rate of 0.5 mL / min using a Hewlett Packard 1090L HPLC equipped with a detector in diode array. Molecular weight standards were used (BioRad, Inc.) consisting of thyroglobulin (670 kd), gamma-globulin (158 kd), ovalbumin (44 kd) and cyanocobalamin (1.35 kd) to calibrate the column. The sample load was 25μg and the protein was detected by monitoring UV absorption at 214 nm using the Turbochrom 3 computer program (PE Nelson, Inc.) Hydrophobic Interaction Chromatography: F (ab ') 2 fragments of the E25 antibody were chromatographed using a Butyl-NPR TosoHaas column (3.5 x 4.6 mm) and a Hewlett Packad 1090L HPLC equipped with a diode array detector. The elution buffer solution A was 20 mM Tris, 2 M ammonium sulfate, 20% (v / v) glycerol, pH 8.0, while the elution buffer B was: 20 mM Tris, 20% (v / v) ) glycerol, pH 8.0. The column was equilibrated with a 10% B elution buffer at a flow of 1.0 mL / min for a minimum of 20 minutes. The sample loading was 5 μg and the protein was detected by monitoring UV absorption at 214 nm using the Turbochrom 3 data acquisition computer program (PE Nelson, Inc.). After injection of the sample, the column was maintained at 10% of the buffer B for 1 minute, followed by a linear gradient from 10% to 62% of the buffer B in 20 minutes. The column was washed with 100% buffer B for 5 minutes and re-equilibrated with 10% buffer B for a minimum of 20 minutes between successive sample injections.

Antibody Linkage Activity: The inhibition assay of the IgE receptor linker (IE25: 2) was carried out as described in Presta et al. , supra, on samples diluted to 20 μg / L and 30 μg / L in a test diluent (phosphate buffered saline, 0.5% BSA, 0.05% polysorbate 20, 0.01% Timerosol). Each dilution was then tested in triplicate and the results multiplied by an appropriate dilution factor to produce an active concentration. The results of 6 trials were averaged. The assay measures the ability of rhuMAb E25 to bind competitively to IgE and thereby prevent IgE from binding to its high affinity receptor that is immobilized with an ELISA plate. The results were divided by the concentration of antibodies as determined by UV absorption spectroscopy and reported as a specific activity. Previous experiments have shown that this test is an indicator of stability.

Particle Assay: The reconstituted vials of lyophilized rhuMAb E25 were poured to reach a volume of approximately 7 mL. The counting of various size particles in the range from 2 to 80 mm present in 1 mL of sample was determined using a Hiac / Royco model 8000 counter. The counter was first washed with 1 mL of sample three times followed by the measurement of 1 mL of sample in triplicate. The instrument determines the number of particles per mL that are equal to or greater than 10 μm and the number of particles per mL that are equal to or greater than 25 μm.

The first step in the development of a formulation for the anti-IgE antibody was to determine an appropriate buffer solution and the pH for lyophilization and storage of the product. Antibody was formulated at a concentration of 5.0 mg / mL within buffer solutions of lOmM succinate in a range from pH 5.0 to pH 6.5 and within sodium phosphate, potassium phosphate and histidine buffer at pH 7.0. Figure 9 shows an increasing aggregate of antibodies that was observed in the higher pH formulations before and after lyophilization. An exception was the histidine formulation at pH 7, where no increase in aggregate was observed during storage at 2-8 ° C. Figure 10 shows the lyophilized rhuMAb E25 in 5 mM histidine buffer at pH 6 and 7 and stored for 1 year at 2-8 ° C, 25 ° C, and 40 ° C. For each test time point and storage temperature, the pH 6 formulation had less aggregate than the antibody formulated at pH 7. These results show that histidine at pH 6 is a system of solutions. buffers particularly useful to prevent the aggregation of the antibody.

To facilitate screening of the lyoprotectants, the anti-IgE antibody was formulated in sodium succinate at pH 5. with or without a lyoprotectant. The potential lyoprotectants aggregated at isotonic concentrations were grouped into 3 categories: (a) non-reducing monosaccharide (ie, mannitol), - (b) reducing disaccharides (ie, lactose and maltose); and (c) non-reducing disaccharides (ie, trehalose and sucrose).

The aggregation of the formulations that followed storage at 2-8 ° C and 40 ° C for one year are shown in Figures 11 and 12. With storage at 2-8 ° C, the monosaccharide (mannitol) formulation was added at a rate similar to the control of the buffer solution, while the formulations containing the disaccharides were equally effective in controlling aggregation (Figure 11). The results that followed storage at 40 ° C were similar with the exception of the sucrose formulation that was added rapidly (which correlated with a darkening of the dried-frozen cake (Figure 12)). This was subsequently shown to be caused by the degradation of sucrose following storage at acid pH and high temperature.

Hydrophobic interaction chromatography of the antibody formulated in the histidine buffer at a pH of 6 with lactose shows that the antibody is altered after storage for 6 months at 40 ° C (Figure 13). The peaks of cormatography expand and the retention time decreases. These changes are not observed with the control of the buffer solution and the sucrose formulations stored under similar conditions as shown in Figures 14 and 15 respectively. Additionally, the isoelectric focus showed an acidic change in the pl of the antibody formulated in lactose and stored at 25 ° C and 40 ° C. This indicates that the reducing sugars are not suitable as lyoprotectants for the antibody.

The aggregation of the lyophilized formulations of the anti - IgE at a concentration of 20 mg / mL in 5 mM histidine buffer at a pH of. 6 with various concentrations of scarosa and trehalose after storage for 12 weeks at 50 ° C, is shown in Figure 16. Both sugars have a similar protective effect on aggregation, when the sugar concentration is greater than 500 moles of sugar per mole of antibody. From these results, the isotonic and hypertonic formulations of sucrose and trehalose were identified for further development. The formulations were designed to be filled prior to lyophilization at a relatively low concentration of antibody and the lyophilized product was reconstituted with less volume than was filled with bacteriostatic water for injection (BWFI), comprising 0.9% benzyl alcohol. This allows the concentration of the antibody immediately prior to subcutaneous delivery and includes a preservative for a potential multi-use formulation while avoiding interactions between the protein and the preservative under long-term storage.

Isotonic formulation: Antibody at 25 mg / mL, formulated in 5 mM of histidine buffer at a pH of 6 with 500 moles of sugar per mole of antibody equivalent to sugar concentrations of 85 mM. This formulation is reconstituted with BWFI (0.9% benzyl alcohol) at a volume that is four times lower than that which was filled. This results in 100 mg / mL of antibody in 20 mM of histidine at a pH of 6 with an isotonic concentration of sugar of 340 mM.

Hypertonic formulation: Anti-IgE at 25 mg / mL formulated in 5 mM histidine buffer at pH 6 with 1000 moles of sugar per mole of antibody which corresponds to sugar concentrations of 161 M. This formulation is reconstituted with BWFI (0.9% benzyl alcohol) at a volume that is four times lower than it was filled. This results in 100 mg / mL of antibody in 20 mM of histidine at a pH of 6 with a hypertonic sugar concentration of 644 mM.

The comparison of antibody aggregation after storage of the isotonic and hypertonic formulations for up to 36 weeks is shown in Figures 17 and 19. No change in aggregation was observed in the hypertonic or isotonic formulations with storage at 2-8 °. C (Figure 17). With storage at controlled room temperature (# 0 ° C), no increased aggregation was observed in the hypertonic formulations while an increase in the aggregate from 1 to 2% occurs in the isotonic formulations (Figure 18). Finally, after storage at 50 ° C, there is a minimum increase in the aggregate with the hypertonic formulations, a 4% increase in the aggregate occurs with the isotonic trehalose formulation and 12% of the increase in the aggregate occurs with the formulation of isotonic sucrose (Figure 19). These results show that the isotonic formulation contains the minimum amount of sugar needed to maintain the stability of the antibody with storage at a temperature of up to 30 ° C.

The binding activity of the anti-IgE in the isotonic and hypertonic formulations was measured in an inhibition assay of the IgE receptor. It was found that the binding activity of the formulations of trehalose and hypertonic and isotonic sucrose was essentially unchanged after storage at -70 ° C, 2-8 ° C, 30 ° C and 50 ° C for up to 36 weeks.

Lyophilized protein formulations are known to contain aggregates or inso-lubles particulates (Cleland et al., Critique Reviews in Therapeutic Drug Carrier Systems, 10 (4) -201-311 (1993)). In this manner, a particular assay of lyophilized antibody at a concentration of 25 mg / mL in 5 mM histidine, pH of 6 with the addition of 85 mM and 161 mM of sucrose and trehalose was carried out. Polysorbate 20 was added to the formulations at a concentration of 0.005%, 0.01% and 0.02%. The samples were lyophilized and assayed following reconstitution at 100 mg / mL of antibody in 20 mM histidine, pH of 6 with 340 mM and 644 mM of sugar. The concentration of polysorbate 20 following reconstitution was 0.02%, 0.04% and 0.08%.

Table 9 below shows the number of particles of size equal to or greater than 10 mm and equal to or greater than 25 mm of the hypertonic trehalose and sucrose formulations. The polysorbate 20 was added to the formulations at concentrations of 0.005%, 0.01% and 0.02% prior to lyophilization. The results show that the addition of Tween ™ to the formulation significantly reduces the number of particles in each size range tested. The specification of the US Pharmacopeia (USP) for low volume injections is no more than 6,000 particles larger than or equal to • 10 μm and no more than 600 particles larger than or equal to 25 μm per container (Cleland et al. , supra). With the addition of polysorbate 20, the hypertonic and isotonic formulations pass this specification.

TABLE 9 A formulation developed for the anti-IgE antibody (ie, 143 mg of isotonic formulation of the rhuMab E25 vial) which is considered useful for subcutaneous delivery of this antibody, is shown in Table 10 below. Fill a 10-c vial with 5.7 mL of rhuMAb E25 at a concentration of 25 mg / mL formulated in 5 mM histidine at a pH of 6.0 with 0.01% polysorbate 20. Sucrose is added as a lyoprotectant at a concentration of 85 mM which corresponds to a molar ratio of sugar to antibody of 500 to 1. The vial is lyophilized and reconstituted with 0.9% benzyl alcohol to a quarter of the volume of the filling at 1.2 mL. The final concentration of the components in the formulation is increased by four to 100 mg / mL of rhuMAb E25 in 20 mM histidine at pH of 6 with 0.04% polysorbate 20 and 340 mM sucrose (isotonic) and 0.9% benzyl alcohol . The formulation contains histidine buffer at a pH of 6 due to its protective effect demonstrated on the aggregation of antibodies. Sucrose was added as a lyoprotectant due to its previous use in the pharmaceutical industry. The concentration of sugar was chosen to result in an isotonic formulation after reconstitution. Finally, the polysorbate 20 was added to avoid the formation of insoluble aggregates.

TABLE 10 It is noted that with respect to this date, the best method known to the applicant to carry out the invention is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (25)

1. A stable isotonic reconstituted formulation, characterized in that it comprises a protein in an amount of at least about 50 mg / mL and a diluent, reconstituted formulation that has been prepared from a lyophilized mixture of a protein and a lyoprotectant, wherein the Protein concentration in the reconstituted formulation is about 2-40 times higher than the concentration of protein in the mixture before lyophilization.

2. The formulation according to claim 1, characterized in that the lyoprotectant is sucrose or trehalose.

3. The formulation according to claim 1, characterized in that it also comprises a buffer solution.

4. The formulation according to claim 3, characterized in that the buffer is histidine or succinate.

5. The formulation according to claim 1, characterized in that it also comprises a surfactant.

6. A stable reconstituted formulation, characterized in that it comprises an antibody in an amount of at least about 50 mg / mL and a diluent, reconstituted formulation that has been prepared from a lyophilized mixture of an antibody and a lyoprotectant, wherein the concentration of The antibody in the reconstituted formulation is about 2-40 times greater than the concentration of antibody in the mixture before lyophilization.

7. The formulation according to claim 6, characterized in that the antibody is an anti-IgE or anti-HER2 antibody.

8. The formulation according to claim 6, characterized in that it is isotonic.

9. A method for the preparation of an isotonic reconstituted formulation, characterized in that it comprises the reconstitution of a lyophilized mixture of a protein and a lyoprotectant in a diluent such that the concentration of protein in the reconstituted formulation is at least 50 mg / mL, where the concentration in the reconstituted formulation is about 2-40 times greater than the concentration of protein in the mixture before lyophilization.

10. A method for the preparation of a formulation, characterized in that it comprises the steps of: (a) lyophilizing a mixture of a protein and a lyoprotectant; and (b) reconstituting the lyophilized mixture of step (a) in a diluent such that the reconstituted formulation is isotonic and stable and has a protein concentration of at least about 50 mg / mL.

11. The method according to claim 10, characterized in that the concentration of protein in the reconstituted formulation is from about 80 mg / mL to about 300 mg / mL.

12. The method according to claim 10, characterized in that the concentration of protein in the reconstituted formulation is about 2-40 times greater than the protein in the mixture before lyophilization.

13. The method according to claim 10, characterized in that the lyophilization is carried out at a shelf temperature maintained at about 15-30 ° C through the complete lyophilization process.

14. An article of manufacture characterized in that it comprises: (a) a container that maintains a lyophilized mixture of a protein and a lyoprotectant; and (b) instructions for reconstituting the lyophilized mixture with a diluent up to a protein concentration in the reconstituted formulation of at least about 50 mg / mL.

15. The article of manufacture according to claim 14, characterized in that it also comprises a second container that maintains a diluent.

16. The article of manufacture according to claim 15, characterized in that the diluent is bacteriostatic water for injection (BWFI) comprising an aromatic alcohol.

17. A formulation comprising a lyophilized mixture of a lyoprotectant and an antibody, characterized in that the molar ratio of lyoprotectant: antibody is about 100-1500 moles of lyoprotectant: 1 mole of antibody.

18. The use of the formulation of claim 1, in the preparation of a medicament for the treatment of a mammal having a disorder that requires treatment with the protein in the formulation.

19. The use according to claim 18, characterized in that the formulation is for subcutaneous administration.

20. A formulation characterized in that it comprises anti-HER2 antibody in an amount of about 5-40 mg / mL, sucrose or trehalose in an amount of about 10-100 mM, a buffer and a surfactant.

21. The formulation according to claim 20, characterized in that it also comprises a bulking agent.

22. The formulation according to claim 20, characterized in that it is lyophilized and stable at 30 ° C for at least 6 months.

23. The formulation according to claim 20, characterized in that it is reconstituted with a diluent such that the concentration of anti-HER2 antibody in the reconstituted formulation is from about 10-30 mg / mL, wherein the reconstituted formulation is stable at 2- 8 ° C for at least about 30 days.

24. A formulation comprising anti-IgE antibody in an amount of about 5-40 mg / mL, sucrose or trehalose in an amount of about 80-300 mM, a buffer and a surfactant.

25. The formulation according to claim 24, characterized in that it is lyophilized and stable at around 30 ° C for at least 1 year.