Stable isotonic lyophilized protein composition

 

Stable isotonic composition includes a protein and a suitable solvent that dilutes mobilisierung mixture of protein and bioprotector to obtain the current composition with a high concentration of protein without substantial loss of stability of the protein. The concentration of protein in the composition is at least 50 mg/ml Molar ratio bioprotector to the protein in dried mixture is (100-600):1, respectively, the concentration of protein in dilute existing songs in the 20-40 times higher than the concentration of protein in the mixture before lyophilization. The compositions contain, for example, anti-IgE and anti-HER2 antibody and is produced by lyophilization of these antibodies in the presence of bioprotector. Bioprotection is sucrose or trehalose. The composition is included in the composition of the medicinal product for subcutaneous administration in the treatment of mammals, which are disorders characterized by the overexpression of HR2 receptor, including cancer. Lyophilized protein composition is stable during storage and administration. 9 C. and 38 C.p. f-crystals, 19 ill., 10 table.

Background of the invention

The scope of the invention

This invention relates to liveu composition, which can be diluted with a solvent, forming a stable current composition suitable for subcutaneous injection.

An overview of well-known technical solutions

Over the past ten years, advances in biotechnology have made it possible to produce a number of proteins for pharmaceutical applications using genetic engineering. Because proteins are larger and more complex than traditional organic and inorganic drugs (i.e., have numerous functional groups in addition to the complex three-dimensional structures), the composition of these proteins pose particular challenges. To the protein remained biologically active composition should retain the original conformational integrity of at least the Central (main) amino acid sequence of the protein and at the same time to protect the many functional groups of the protein from destruction. Path of destruction (degradation) of proteins can include chemical instability (i.e., any process that involves the modification of the protein by formation or cleavage of communication, resulting in a new chemical entity) or physical instability (i.e., changes in structure, hydrolysis, oxidation,-elimination or disulfide exchange. Physical instability can result from denaturation, aggregation, precipitation or adsorption. The three most common destinations (routes) degradation are aggregation, deliciousa and oxidation of protein (Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993)).

Drying by freezing is a commonly used way to save protein, which helps to remove water from the protein of interest of the drug. Drying by freezing or lyophilization is a way by which the substance should be dried first frozen and then the ice or frozen solvent is removed by sublimation in vacuum. To increase stability during the freeze-drying and/or improve the stability of the lyophilized product upon storage of the composition prior to lyophilization may include a filler (Pikal, M., Biopharm. 3(9): 26-30 (1990) and Arakawa et al. Pharm. Res 8(3): 285-291 (1991)).

The aim of the present invention is to provide a lyophilized protein composition, which is stable during storage and administration. In addition, the purpose of this izobretenij variants goal is to create a composition of reusable which is stable for at least the time when it is administered to the patient.

The invention

This invention is based on the fact that stable lyophilized protein composition can be obtained using bioprotector (preferably carbohydrates, such as sucrose or trehalose), freeze-dried composition can be mixed with stable current composition with the concentration of the protein, which is significantly higher (e.g., 2-40 times greater, preferably 3-10 times higher, and most preferably 3-6 times higher) than the concentration of protein in the composition prior to lyophilization. In particular, while the concentration of protein in the composition prior to lyophilization may be 5 mg/ml or less, the concentration of protein in the present composition usually 50 mg/ml or more. Believe that such a high concentration of protein in the current composition will be particularly necessary when the composition is intended for subcutaneous administration. Found that the current composition is stable, despite the very high concentration of protein (i.e., does not detect major or minor chemical or physical instability of the protein), at 2-8

If the composition is diluted with a solvent containing a preservative (e.g., bacteriostatic water for injection, BWFI-bacteriostatic water for injection), this current composition can be used as a composition of multiple use. This composition is suitable when, for example, the patient requires frequent subcutaneous injection of protein for the treatment of chronic diseases. The advantage of the composition of multiple use is that it facilitates its use by the patient, reduces losses, contributing to full use of the contents of the vial (vial), and leads to significant savings for manufacturers, as multiple doses contained in one vial (vial) (below the cost of filling and transportation).

Based on the results of scientific observations described in this invention, one option provides stable istoricheskaya composition obtained from freeze-dried mixture of protein and bioprotector, where the concentration of protein in the diluted composition in ~2-40 times greater than the concentration of protein in the mixture before lyophilization.

In another embodiment, the invention provides a stable current composition containing the antibody in the amount of at least ~50 mg/ml and the solvent, when the current composition obtained from freeze-dried mixture of antibodies and bioprotector, where the concentration of antibodies in the present composition in ~2-40 times greater than the concentration of the antibodies in the mixture before lyophilization.

The ratio of bioprotector to the protein in lyophilized compositions, described in the previous paragraph, depends, for example, as from a choice of protein, and bioprotector, and also depends on the concentration of the selected protein and isotonicity of the current song. In the case of full length antibodies (protein) and trehalose or sucrose (as bioprotector) to obtain the current isotonic composition with a high concentration of protein, this ratio may, for example, be about 100-1500 moles trehalose or sucrose per 1 mol of antibody.

Typically, the composition of the protein and bioprotector before lyophilization further includes a buffer that provides compositions suitable pH hung in prodemonstrirovano below, his-tag buffer, because he, apparently, has bioprotective properties.

As noted in this invention, the composition may further include a surfactant (e.g. Polysorbate), which can reduce the aggregation of the protein and/or reduce the formation of particles in the current (diluted) compositions. Surfactant can optionally be added to the composition prior to lyophilization, freeze-dried composition and/or a diluted composition (but preferably to the composition prior to lyophilization).

In addition, we offer a method of obtaining a stable isotonic current composition, including the dilution of the lyophilized mixture of protein and bioprotector solvent so that the concentration of protein in the diluted composition would be at least 50 mg/ml, where the concentration of protein in the diluted composition in ~2-40 times greater than the concentration of protein in the mixture before lyophilization.

Another option provides for a method of obtaining a composition comprising the stage of: (a) freeze-drying the mixture of protein and bioprotector and (b) the dilution of the dried mixture from stage (a) with a solvent so that the diluted compositions the concentration of protein in the diluted composition can be from about 80 mg/ml to about 300 mg/ml Typically, the concentration of protein in dilute (current) of the composition is about 2-40 times greater than the concentration of protein in the mixture before lyophilization.

The product described in the invention, also includes:

a) a container (bottle, vial, which contains a lyophilized mixture of protein and bioprotector, and

b) instructions for diluting the lyophilized mixture solvent to the concentration of protein in the current composition of at least ~50 mg/ml product may further include a second container (bottle, vial, which contains the solvent (e.g., bacteriostatic water for injection, BWFI containing aromatic alcohol).

Moreover, it provides a method of treating mammals, including the appointment of a therapeutically effective amount of the current composition described in this invention, mammals, if mammals have the disorder for which treatment is required proteincoding composition. For example, the composition (composition) may be injected subcutaneously.

It was found that one useful pre-dried composition anti-R2 antibodies, as detected in the experiments described below, contains anmer, 40-80 mm), buffer (e.g., his-tag with a pH of 6 or succintly with pH 5) and surfactant (e.g. Polysorbate). It was found that freeze-dried composition is stable at 40With at least within 3 months and stable under 30With at least within 6 months. This anti-R2 composition can be diluted with solvent to obtain a composition suitable for intravenous injection containing anti-R2 in the amount of from 10 to ~30 mg/ml, which is stable at 2-8C for at least 30 days. If you require a higher concentration of anti-R2 antibodies (for example, if a subcutaneous injection of antibodies is the assumed form of the introduction of the patient), the freeze-dried composition can be diluted with stable current composition with a protein concentration of 50 mg/ml or more.

One suitable pre-dried composition of anti-IgE antibodies described in this invention, contains anti-IgE in the range from ~5 to 40 mg/ml (e.g., 20-30 mg/ml) and sucrose or trehalose from ~60 to 300 mM (e.g., 80-170 mM), buffer (preferably, a his-tag with pH 6) and surfactant (for example, is her least one year. This composition can be mixed to obtain compositions containing anti-IgE in the range from ~15 to ~45 mg/ml (e.g., 15-25 mg/ml), suitable for intravenous injection, which is stable at 2-8C for at least one year. On the contrary, if you require higher concentrations of anti-IgE in the composition, a freeze-dried composition can be mixed with stable compositions, the concentration of anti-IgE which is50 mg/ml

Brief description of drawings

In Fig.1 shows the influence of the diluted volume on the stability of lyophilized rhu HER2. The freeze-dried composition obtained from the composition prior to lyophilization contained 25 mg/ml protein, 60 mM trehalose, 5 mM sodium succinate pH 5.0 and 0.01% tween-20TM(Tween 20TM). Dried mass was kept at 40C and then diluted to 4.0 (on) and 20 ml (•) BWFI. The proportion of intact protein in the diluted composition was measured using gel chromatography and was identified as the peak area of the native protein to the total area of the peaks, including the units.

In Fig.2 shows the effect of the concentration of trehalose on the stability of lyophilized rhu HER2. P is warraty) and the concentration of trehalose in the range of 60 mM (molar ratio 360) to 200 mM (molar ratio 1200). Lyophilized protein was kept at 40From within or 30 days (filled symbols), or within 91 days (unfilled symbols).

The number of intact protein was determined after dilution of the lyophilized protein in 20 ml of BWFI.

In Fig.3 illustrates the effect of the concentration of trehalose on prolonged stability of lyophilized rhu HER2 put into storage at 40C. Protein liofilizirovanny or at a concentration of 25 mg/ml in 5 mM sodium succinate with pH 5.0, 0.01% of the tween (Tween 20TM) and 60 mM trehalose () or 5 mM histidine, with a pH of 6.0, 0,01% tween (Tween 20TM) and 60 mM trehalose () or at a concentration of 21 mg/ml in 10 mM sodium succinate at pH 5,0 0,2% tween-20TM(Tween 20TM) and 250 mM trehalose (•). Lyophilized protein was kept at 40C and then diluted with 20 ml of BWFI. The number of intact protein was determined after dilution.

In Fig.4 shows the stability rhuMA HER2, dried in 38,4 mM mannitol (7 mg/ml), 20,4 mM sucrose (7 mg/ml), 5 mM histidine, with a pH of 6.0, 0.01% tween-20TM(Tween 20TM). Lyophilized protein was kept at 40And ZAT is p>

In Fig.5 illustrates the stability of the current rhu HER2, lyophilized in 5 mM succinate sodium pH 5.0, 60 mM trehalose, 0.01% of the tween (Tween 20TM). The samples were diluted to 4.0 ml (squares) or of 20.0 ml (circles) BWFI (20 ml: 0.9% of benzyl alcohol; 4,0 ml: 1.1% benzyl alcohol) and then kept at 5With (filled symbols) or 25With (empty symbols). The percentage of native protein was determined as the peak area of the native (not destroyed) protein to the total area of the peaks, as measured by cation-exchange chromatography.

In Fig.6 shows the stability of the current composition containing rhu R2 and dried in 5 mM histidine, with a pH of 6.0, 60 mM trehalose, 0.01% tween (Tween 20). The samples were diluted to 4.0 ml (squares) or 20 ml (circles) BWFI (20 ml: 0.9% of benzyl alcohol; 4,0 ml: 1.1% benzyl alcohol) and then kept at 5With (filled symbols) or 25With (empty symbols). The contents of the native protein (%) was determined as the peak area of the native (not destroyed) protein to the total area of the peaks, as measured by cation-exchange chromatography.

In Fig.7 shows the stability of the current composition containing rhu HER2 and lyophil the ATA) or 20 ml (circles) BWFI (20 ml: 0.9% of benzyl alcohol; 4,0 ml: 1.1% benzyl alcohol) and then kept at 5With (filled symbols) or 25With (empty symbols). The contents of the native protein (%) was determined as the peak area of the native (not destroyed) protein to the total area of the peaks, as measured by cation-exchange chromatography.

In Fig.8 shows the stability of the current composition containing rhu HER2 and lyophilized in 10 mM sodium succinate with pH 5.0, 250 mM trehalose, 0.2% tween-20 (Tween 20). The samples were diluted to 20.0 ml of BWFI (0.9% of benzyl alcohol) and then kept at 5With (•) or 25With (about). The contents of the native protein (%) was determined as the peak area of the native (not destroyed) protein to the total area of the peaks, as measured by cation-exchange chromatography.

In Fig.9 shows the aggregation rhu E25 taking place in the manufacture of the composition in the buffers in the range of pH from 5 to 7 at a concentration of buffer 10 mM and the antibody concentration 5 mg/ml Samples liofilizirovanny and analyzed at zero time and after 4 weeks, 8 weeks and 52 weeks after storage at 2-8C. the Buffers were: potassium phosphate pH 7.0 (o); sodium phosphate with a pH of 7.0 (); sodium succinate with a pH of 5.5 () and sodium succinate with a pH of 5.0 ().

In Fig.10 depicts the aggregation rhu E25, dried 5 mM his-tag buffer as at pH 6 and at pH 7, and analyzed after storage in the following way. A buffer with a pH of 6.0 was stored at 2-8C (o); pH 6 was kept at 25With (); with a pH of 6 was kept at 40With (); pH 7 was stored at 2-8With (•); with pH 7 was kept at 25With (and with a pH of 7 was kept at 40With ().

In Fig.11 shows the aggregation rhu E25 at a concentration of 5 mg/ml, occurring in the manufacture of a composition in 10 mM sodium succinate at pH 5.0 with bioprotection added at a concentration of 275 mM (isotonic). Bioprotector were: control sample without bioprotector (); mannitol); lactose (); maltose (•); trehalose () and sucrose (). Samples liofilizirovanny and analyzed at initial time and after 4 weeks, 8 weeks and 52 weeks is taking place in the manufacture of a composition in 10 mM sodium succinate at pH 5.0 with bioprotection, added in a concentration of 275 mM (isotonic). Bioprotector were: control sample without bioprotector (); mannitol); lactose (); maltose (•); trehalose () and sucrose (). Samples liofilizirovanny and analyzed during the initial (zero time) and after 4 weeks, 8 weeks and 52 weeks of storage at 40C.

In Fig.13 presents chromatography hydrophobic interaction rhu E25 with a concentration of 20 mg/ml, lyophilized his-tag in buffer at pH 6 with isotonic concentration (i.e., 275 mM) lactose left in storage for 24 weeks at 2-8, 25, or 40C and diluted to 20 mg/ml

In Fig.14 presents chromatography hydrophobic interaction rhu E25 with a concentration of 20 mg/ml, lyophilized his-tag in buffer at pH 6, stored for 24 weeks at 2-8, 25, or 40C and diluted to 20 mg/ml

In Fig.15 presents chromatography hydrophobic interaction rhu E25 with a concentration of 20 mg/ml, lyophilized his-tag in buffer at pH 6 isotonic concentration (i.e., 275 mM) sucrose and left n the effect of the concentration of carbohydrate in rhu E25, taking place at a concentration of 20 mg/ml in 5 mM histidine, with a pH of 6.0. To the composition was added sucrose (•) and trehalose () in molar ratios varying from 0 to 2010 (isotonic) (see table below. 1). Samples liofilizirovanny and analyzed after 12 weeks when stored at 50C.

In Fig.17 shows the aggregation rhuMA E25, occurring at a concentration of 25 mg/ml in 5 mM histidine at pH 6 with 85 mm sucrose (Oh); 85 mm trehalose (); 161 mM sucrose (or 161 mM trehalose (). Samples liofilizirovanny and kept at 2-8C, followed by dilution with 0.9% benzyl alcohol to 100 mg/ml antibody in 20 mM histidine at pH 6 with isotonic (340 mM) and hypertensive (644 mM) concentration of carbohydrate.

In Fig.18 shows the aggregation rhu E25, occurring at a concentration of 25 mg/ml in 5 mM histidine at pH 6 with 85 mm sucrose (Oh); 85 mm trehalose (); 161 mM sucrose (or 161 mM trehalose (). Samples liofilizirovanny and kept at 30C, followed by dilution with 0.9% benzylamine carbohydrate.

In Fig.19 shows the aggregation rhu E25, taking place at 25 mg/ml in 5 mM histidine at pH 6 with 85 mm sucrose (Oh); 85 mm trehalose (); 161 mM sucrose (or 161 mM trehalose (). Samples liofilizirovanny and kept at 50C, followed by dilution with 0.9% benzyl alcohol to 100 mg/ml antibody in 20 mM histidine at pH 6 with isotonic (340 mM) and hypertensive (644 mM) concentration of carbohydrate.

A detailed description of the preferred options

1. Definition

By "protein" refers to amino acid sequence, chain length which is sufficient for producing higher levels of tertiary and/or Quaternary structure. This differs from the "peptides" or other low molecular weight drugs, which do not have such structure. Usually the molecular weight of the protein described in this invention should be at least about 15-20 kD, preferably about 20 kD.

Examples of proteins covered by this definition include mammalian proteins, such as growth hormone, including human growth hormone and bovine growth hormone; factor secretion of growth hormone; parathyroid hormone; dash is proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors of the blood system, such as factor VIIIC, factor IX, tissue factor and von Willebrand factor (Willebrands); factors that prevent blood clotting, such as Protein C; atrial naturethese factor; lung surfactant; a plasminogen activator; for example, urokinase or tissue plasminogen activator (t-PA); bombesin; thrombin;andfactors that cause necrosis of tumor cells; enkephalinase; RANTES (regulates on activation normally T-cell expressed and secreted regulating activation of expression and secretion of normal T-cells); human phagocytic inflammatory protein (WORLD-1-); serum albumin, for example, serum albumin person; müller inhibiting substance; And-chain relaxin; In-circuit relaxin; prolactin; mouse gonadotropin-binding peptide; DNA-ASE; inhibin; activin; vascular endothelial growth factor (vascular endothelial growh factor, VEGF); receptors for hormones or growth factors; integrin; A - or D-protein; rheumatoid factors; a neurotrophic factor, for example, neurotrophic factor bone origin (bone-derived neurotrophic factor, BDNF), near the RNA growth factor (platelet-derived growth factor, PDGF); fibroblast growth factor (fibroblast growth factor, FGF), such as aFGF and bFGF; epidermal growth factor (epidermal growth factor, EGF); transforming growth factor (transforming growth factor, TGF) such as TGF-and TGF-including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-like growth factor-I and-II (insulin-like growth factor, IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-1); binding proteins insulin-like growth factor; CD proteins, for example, CD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; a protein involved in osteogenesis (bone morphogenetic protein, BMP); an interferon, for example,-,and-interferon; colony stimulating factors (colony-stimulating factors, CSFs); for example, M-CSF, GM-CSF and G-CSF; interleukins (ILS) such as IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; complementability stimulator of hemolysis (DAF-factor, decay accelerating factor; viral antigen, for example, some HIV-shell; transport proteins; homing"receptors; adressen; regulatory proteins; immunoadhesins; Anna, which is used to produce the compositions should preferably be sufficiently clean and preferably almost homogeneous (i.e., without contaminating proteins and so on). "Enough (essentially) pure protein means a composition containing at least 90% (mass) of protein from the total weight of the composition, preferably about 95% (mass). "Almost homogeneous" protein means a composition containing at least 99% (mass) of protein from the total weight of the composition.

In some embodiments, the protein is an antibody. The antibody may, for example, to contact any of the above molecules. Typical molecular targets for antibodies that are included in the present invention, are CD proteins, for example, CD3, CD4, CD8, CD19, CD20 and CD34; members of the family of HER receptor, for example, EGF receptor, HER2, HER3, or HER4 receptor; cell adhesion molecules, e.g. LFA-1, Mo1, p150,95, VLA-4, ICAM-1, VCAM andv/3 integrin, including itsor-subunit (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors, e.g., VEGF; IgE; antigens of blood groups; flk2/flt3 receptor; receptor obesity (obesity, OB); With protein, etc.

The term "antibody is Lina, having the Fc region of immunoglobulin), the compositions of antibodies with polyepitopic specificity, bespecifically antibodies, Diethelm and molecules with a single chain, as well as fragments of antibodies (e.g., Fab, F(ab’)2and Fv).

The term "monoclonal antibody" used in this invention refers to an antibody obtained from a population almost monoclonal antibodies, i.e., the individual antibodies comprising identical populations, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies of vysokospetsifichnymi, and directed towards one region determinants. In addition, in contrast to conventional formulations (polyclonal) antibodies, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage that they are synthesized using the hybridoma culture, uncontaminated by other immunoglobulins. The definition of "monoclonal" indicates the character of the antibody obtained from almost homogeneous population of antibodies, and not on the performance communications antibodies used in accordance with the present invention, can be obtained by the hybrid method, first described by Kohler et al., Nature, 256: 495 (1975), or can be obtained by the methods of recombinant DNA (genetic engineering) (see, for example, U. S. Patent No. 4816567). "Monoclonal antibodies" may also select from a library of phage antibodies using the methods described, for example, in the publications of Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991).

Monoclonal antibodies definitely include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a single species or belonging to a separate class or subclass of antibody, while the remainder of the chain (chain) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies, but also fragments of these antibodies, as long as they exhibit the desired biological activity (U. S Patrent No. 4816567; Morrison et al., Proc Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

"Refined" forms antibodies that do not belong to the human race (for example, related to mice and rats) are chimeric immunoglobulins, CE the activity of antibodies), which contain minimal sequence derived from the immunoglobulin, not belonging to the human race. Mainly, "refined" antibodies are human immunoglobulins (recipient antibody) in which hypervariable residues plot (complementarity determining region CDR) of the recipient are replaced by residues of the CDR of non-human genus species (donor antibody) such as mouse, rat or rabbit, which possess the desired specificity, affinity and capacity. In some cases the remains of Fv spanning region (framework region FR) of a human immunoglobulin are replaced by corresponding residues not belonging to the human race. In addition, the purified antibodies can include residues which are not found in the recipient antibody nor in the input (imported) CDR or spanning sequences. These modifications are forced to conduct additional cleanup and optimize the characteristics of the antibody. Typically, purified antibody must include substantially all or at least one, typically two variable regions, in which all or substantially all of the CDR areas correspond to areas of immunoglobulin that does not belong to the human race, and all or osnove antibody should also include, at least part of a constant region of immunoglobulin (Fc), typically a constant region of human immunoglobulin. More detail is described in Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988) and Presta, Curr. Op. Struct. il., 2: 593-596 (1992). Purified antibody includes the antibody is PrimatizedTMwhere antigennegative region of antibodies derived from antibodies produced by immunization of monkeys (macaques) the desired antigen.

"Stable" composition is one in which the protein is essentially retains its physical and chemical stability and integrity upon storage. There are various analytical methods to determine the stability of the protein, which are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be defined at the selected temperature for a selected period of time. For rapid screening, which determines the time stability, the composition can be maintained at 40With 2 weeks to one month. If the composition should be stored at 2-8Since, then, usually, it must be stable under 30With 40 orWith thecapacity should be stored at 30With, it generally must be stable at least for two years when 30C and/or stable at 40With in 6 months. For example, as an indicator of the stability of the protein (see examples in the invention) may be the degree of aggregation after lyophilization and storage. For example, a stable composition can be considered as such, which is less than 10% and preferably less than 5% of the protein present in the composition in the form of the unit. In other embodiments, may be determined by any increase in the formation of aggregates after lyophilization and storage of the lyophilized composition. For example, stable lyophilized composition can be considered as such, in which an increase in units less than 5% and preferably less than 3%, if the freeze-dried composition is stored at 2-8C for at least one year. In other embodiments, the stability of the protein composition can be assessed using the test of biological activity (see below, e.g., example 2).

"The current (diluted)" the song is called such if it is prepared by dissolving lyophilized protein composition in a solvent so that the protein emer, injecting) the patient should be treated by a given protein, in some embodiments, the composition may be suitable for subcutaneous injection.

Under "isotonic" understand such a composition that has almost the same osmotic pressure as human blood. Isotonic compositions typically should have an osmotic pressure of from 250 to 350 mOsm (milliosmoles). Isotonicity can be measured, for example, by using steam or vygorajivayuschii of osmometry.

"Bioprotector" is a molecule which, when mixed with necessary protein, significantly prevents or reduces chemical and/or physical instability of this protein during lyophilization and subsequent storage. Typical bioprotector include carbohydrates, such as sucrose or trehalose; amino acids, e.g., one-deputizing monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol, for example, a trivalent or more vysokodetalnye alcohols carbohydrates, for example, glycerin, aritra, Arabic, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronics (Pluronics), and combinations thereof. The preferred bioprotection is neostriatal in "bioprotection number", that means that after lyophilization of the protein in the presence bioprotectors number of bioprotector, protein, essentially retains its physical and chemical stability and identity during lyophilization and storage.

Selected "solvent" is as if he pharmaceutically acceptable (safe and non-toxic when administered to a human) and are suitable for the preparation of the current (diluted) compositions. Typical solvents include sterile water, bacteriostatic water for injection (BWFI), a buffer solution with a specific pH (e.g., phosphate buffered saline, sterile saline solution, ringer's solution (Ringer''s solution or dextrose.

"Preservative" means a compound that can be added to the solvent to substantially reduce bacterial impact on the diluted composition, thus facilitating, for example, to obtain the current composition of multiple use. Examples of potential preservatives are octadecyltrimethylammonium chloride, hexamethonium chloride, benzalconi chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are groups with long chain) is benzyl alcohol, alkylarene, for example, methyl - or propylparaben, catechin, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative in the present invention is benzyl alcohol.

"Filler" is a compound that increases the weight of the dried mixture and leads to the physical structure of the dried mass (for example, contributes to obtaining essentially homogeneous dried mass, which retains an open porous structure). Typical excipients include mannitol, glycine, polyethylene glycol and sorbitol.

"Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have the disorder, and those with these disorders should be warned.

Being treated with "mammal" is any animal classified as a mammal, including humans, domestic and farm animals, zoo animals, sports or animals, e.g. dogs, horses, cats, cows, etc. Preferred mammal is a human.

"Disorder" is any condition that will improve with treatment which predispose the mammal to the disorder, in question. Unlimited examples of disorders that are treated, are cancer and allergies.

II. Ways of carrying out the invention

A. Obtaining protein

The protein used in the composition, is obtained using methods that are well known in the art, including synthetic methods (e.g., recombinant methods of genetic engineering) and peptide synthesis, or a combination of these methods), or can stand out from the endogenous protein source. In some embodiments of the invention the protein is selected from antibodies. The following are methods of producing antibodies.

(a) polyclonal antibodies

Polyclonal antibodies usually occur in animals at multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. This can be useful for linking the relevant antigen to a protein that is immunogenic in the immunized species, for example, hemocyanin lymph snails, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, ester of maleimidophenylmethacrylates (conjugation through SOCl2or R N=C=NR, where R and R’ are different alkyl groups.

Animals subjected to immunization with the antigen, immunogenic conjugates, or derivatives by mixing 1 mg or 1 μg of peptide or conjugate (for rabbits or mice, respectively) with three volumes complete adjuvant's adjuvant. A month later the animals revaccinated from 1/5 to 1/10 part of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days the animals take blood and serum will be analyzed by the titer of antibodies. Animals revaccinated up until the title keeps changing. Preferably, animals revaccinated conjugate of the same antigen, but is associated with a different protein and/or through a different cross-linking reagent. The conjugates can also be prepared in recombinant cell culture by merging the protein. Also the agents that cause aggregation, such as alum, is successfully used to enhance the immune response.

b) Monoclonal antibodies

Monoclonal antibodies are obtained from populations mainly monoclonal antibodies, i.e., the individual antibodies comprising identical populations, except perhaps in the nature of the meetings is clonal" indicates the nature of the antibodies, which is not a discrete mixture of antibodies.

For example, monoclonal antibodies can be obtained using the hybrid method first described by Kohler et al., Nature, 256:495 (1975), or can be obtained by means of genetic engineering (recombinant DNA) (U. S. Patent No. 4816567).

In the hybrid method, a mouse or other suitable animal host, such as hamster, subjected to immunization, as described above, to identify lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Or lymphocytes can undergo immunization in vitro. The lymphocytes are then fused with myeloma cells using a suitable agent that causes cell fusion, as for example, polyethylene glycol, with the formation of cell hybridoma (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The thus obtained cells are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or natural selection nalivshihsya parental myeloma cells. For example, if the parental myeloma cells lack the enzyme - hypoxanthineguanine (HGPRT G), substances which prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that are easy to blend, support stable high-level production of antibodies selected antibody-producing cells, and are sensitive to the environment, as for example, to GAT environment. Among them, preferred myeloma cell lines are related to mice or rats myeloma lines, such as, for example, obtained from the MORSE-21 and MPC-11 tumors of mice available from the center Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available from the American culture collections (American Type Culture Collection, Rockville, Maryland USA). For production of monoclonal human antibodies have been described myeloma cell lines of human and heteromyinae cell line mouse-human (Kozbor, J. Immunol. 133: 3001 (1984): Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

The cultural environment in which cultured cells are hybridomas were tested for the production of monoclonal antibodies directed to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybrid cells, determined using the thus or reaction binding iay, ELISA).

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

After cell hybridoma that produce antibodies of the desired specificity, affinity and/or activity identified, the clones can be subclinical using technology limited dilution and to cultivate by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose is, for example, D-MEM or RPMI-1640 medium. In addition, cell hybridomas can be grown in vivo as ascitic tumors in animals.

Monoclonal antibodies are excreted (secreted) by the subclones can easily be separated from the culture medium, ascites fluid, or serum by conventional means of purification of immunoglobulin, such as protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is easily detected, and sequeiros using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to a coding genes, caselotti DNA. Once the selected DNA may be placed in expressing vector, which is then transfections in the host cell, such as E. coli cells, the cells COS monkey cells of the ovary of the Chinese hamster (Chinese hamster ovary, CHO) or myeloma cells that otherwise do not produce the protein of the immunoglobulin by applying the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expressii in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion In Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

In another embodiment, antibodies can be isolated from libraries of phage antibodies generated using the techniques described in the publication McCafferty et al., Nature, 348: 552-554 (1990). In the works of Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) described the selection of antibodies specific to mice or rats and humans, respectively, using phage libraries. In subsequent publications describe the production of antibodies of high affinity (within PM) by moving (move) chain (Marks et al., Bio/Technology, 10: 779-783 (1992)), as well as combinatorial infection and recombination in vivo as a strategy for creating very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Thus, these IU the ate for selection of monoclonal antibodies.

DNA also can be modified, for example, by substituting the coding sequence for the constant region of the heavy and light chain instead of a homologous sequences related to mice or rats (U. S. Patent No. 4816567); Morrison, et al., Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)), or covalent connection of the coding sequence of the immunoglobulin with the entire coding sequence nimmanahaeminda polypeptide or a part of it.

Typically, such nimmanahaeminda polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable regions of one antigennegative centre of antibody to create a chimeric bivalent antibody comprising one antigennegative centre antibody, having specificity for the antigen, and the other antigennegative centre antibody, having specificity for a different antigen.

Chimeric or hybrid antibodies can also be obtained in vitro using known chemistry of synthetic proteins of ways, including methods involving cross-linking agents. For example, immunotoxins can be created using the reaction of disulfide exchange or the formation of thioester linkages. Examples suitable for these purposes reagent>the means of refining antibodies that do not belong to the human race, are well known. Usually purified antibody contains one or more amino acid residues introduced into the antibody from a source that does not belong to the human race. These amino acid residues that do not belong to the human race, often referred to as "entered" ("import") residues, which are typically taken from an "import" variable domain. Improvement can be done, mainly following the method of winter with TCS. (Winter) (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing the CDRs or CDR sequences of rodents on the corresponding sequences of human antibodies. Accordingly, such "refined" antibodies are chimeric antibodies (U.S. Patent No. 4816567), in which a much smaller proportion undamaged (intact) variable regions of the person replaced by the corresponding sequence from a species not belonging to the human race. In practice, purified antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced remnants of similar centers in antibodies grisonni antibodies very important to reduce antigenicity. According to the so-called method of "best match", conduct the screening sequence variable regions of the antibodies of the rodent relatively full (all) library of known sequences of variable regions of human rights. The sequence of the person that is closest to the sequence of the rodent, then accepted as spanning region (FR) of a person for purified antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a separate spanning region, obtained from the synthesis of a typical sequence of all human antibodies of a separate subgroup of light or heavy chains. The same frame can be used for some other purified antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

In addition, it is important that antibodies are refined with retention of high affinity for the antigen and preserving other important biological properties. In accordance with the preferred option to achieve this goal purified antibodies produced by the method of analysis of the original (parent) sequences and various speculative refined products, using tree available and known in the art. There are computer programs that illustrate and demonstrate probable three-dimensional conformational structures of selected sequences of immunoglobulin. Analysis of these images allows to make a conclusion about the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., concludes residues that influence the ability of the candidate immunoglobulin to bind antigen. This way, FR residues can be selected and separated from the recipient and import sequences so as to achieve the desired characteristics of the antibodies, such as increased affinity for antigen(new) target. Typically, the CDR residues are directly and most severely affect binding to the antigen.

On the contrary, at the present time it is possible to produce transgenic animals (e.g. mice) that when immunization is able to produce a full range of human antibodies in the absence of producing endogenous immunoglobulin, for Example, has been described that the homozygous deletion of the heavy chain region of the connection (JH) gene antibodies in chimeric and line-embryo mutant mice results in complete inhibition of the production of endogenous antibodies. Transfer Rangiroa is odwzorowania of human antibodies with the antigenic stimulus (see for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Measurement., 7: 33 (1993). Human antibodies can be obtained from libraries of phage - display (Hoogenboom et al., J. Mol. il. 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991)).

g) Bespecifically antibodies

Bespecifically antibodies (BsAbs) are antibodies that have binding specificity for at least two different epitopes. Such antibodies can be obtained from the full length antibodies or fragments of antibodies (e.g., F(ab’)2bespecifically antibodies).

Known methods of obtaining bespecifically antibodies. Traditional production bespecifically antibodies full length based on the co-expression of two pairs of heavy - light chain immunoglobulin, where the two chains have different specificnosti (Millstein et al., Nature, 305: 537-539 (1983)). Because of the random selection of heavy and light chains of immunoglobulin, these hybridoma (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the corresponding molecule, which is usually carried out using affinity chromatography, a rather time-consuming and the yield of the product when it is low. Similar to what comes from the fusion of the variable regions of antibodies, characterized by specific binding specificnosti (antibody-antigennegative center) with the sequences of the constant region of the immunoglobulin. Mainly the merger takes place with a constant region of a heavy chain immunoglobulin comprising at least the hinge CH2 and CH3 region. Preferably, the first constant region of the heavy chain (SN) containing the center needed to bind light chain, was attended by at least one merge. DNA-are you encoding the fusion heavy chain immunoglobulin and, if required, the light chain of the immunoglobulin, are inserted into separate expressing vector and cotransfected in a suitable host organism. This ensures a high rigidity in the regulation of the mutual proportions of the three polypeptide fragments in the case when unequal ratios of the three polypeptide chains used in the construction provide the optimum outputs. However, it is possible to insert the coding sequences for two or all three polypeptide chains in one expressing vector when the expression of at least two polypeptide chains, taken in equal ratios results in high outputs or when the ratio of persons do not have the heavy chain of the immunoglobulin with the initial binding specificity and a second shoulder - from hybrid pair of heavy - light chain immunoglobulin (providing secondary binding specificity). It was found that this asymmetric structure facilitates the separation of the corresponding especifismo connection from unnecessary combinations of chains of immunoglobulin, as the presence of light chain immunoglobulin only in one part of bespecifically molecules provides a convenient way of separating. This approach is proposed in WO 94/04690 published March 3, 1994. A more detailed description of the receiving bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210(1986).

Bespecifically antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate can connect with Avidya, and the other with Biotin. Such antibodies, for example, been proposed to target cells of the immune system to unnecessary (unnecessary) cells (U. S. Patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373). Heteroconjugate antibodies can be obtained using any convenient cross-linking methods. Suitable cross-linking agents, along with methods of cross-stitching are well known and described in US Patent No. 467980.

Methods of obtaining bespecifically antibodies of the fragment is of Rahmanov bivalent antibodies, which are not necessarily bespecifically. For example, Fab’ fragments derived from E. coli, can chemically bind in vitro with the formation of bivalent antibodies (see Shalaby et al., J. Exp. Med., 175: 217-225(1992)).

There were also describes various methods of acquisition and allocation of fragments, bivalent antibodies directly from recombinant cell culture. For example, the divalent heterodimer were obtained using binding-leucine (Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Latinlatin peptides from the Fos and Jun proteins were added to the Fab’ portions of two different antibodies at the confluence of the gene. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. Technology "diately" described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), provides an alternative mechanism for obtaining fragments especifismo/bivalent antibodies. The fragments comprise variable region heavy chain (Vn) associated with the variable region of the light chain (VL), by using a linker that is too small to allow the connection of two regions on the same circuit. Thus, VHand VLthe scope of a single fragment of Prta, thereby forming two antigenspecific center. It was also reported other strategies of obtaining fragments especifismo/bivalent antibodies using Fv(sFv) dimers of the same chain (see Gruber et al., J. Immunol., 152: 5368(1994)).

B. obtaining a lyophilized composition

After obtaining the desired protein, as described above, receive "reliabilityand composition". The amount of protein present in the composition prior to lyophilization, determine, taking into account the volume of the required dose, method(s) of introduction and so on, If the selected protein is intact (undamaged) antibody (such as anti-IgE or anti-HER2 antibody), a typical initial concentration protein concentration of ~2 mg/ml to about 50 mg/ml, preferably from about 5 mg/ml to 40 mg/ml and most preferably from ~20-30 mg/ml Protein normally present in the solution. For example, the protein may be present in the buffer solution with a specific pH - pH~4 to 8, preferably pH 5 to 7. Typical buffers are his-tag, phosphate buffer, Tris, citrate, succinate buffer and the buffers containing other organic acids. The concentration of the buffer may range from about 1 mM to about 20 mm, or from 3 mM to 15 mm, depending, for example, from the buffer and ner is the his-tag buffer since, as will be shown below, it has bioprotective properties, it is Shown that other suitable buffer is succinate buffer.

Bioprotector added to the composition prior to lyophilization. In preferred embodiments, bioprotector is a non carbohydrate, such as sucrose or trehalose. The number of bioprotector in the composition prior to lyophilization is usually such that upon dilution of the obtained composition would be isotonic. However, there may be also suitable hypertensive existing compositions. In addition, the number of bioprotector should not be too small, because an insufficient number of it leads to degradation or aggregation of the protein during lyophilization. If bioprotection is carbohydrates (e.g. sucrose or trehalose), and protein - antibody, the typical concentration of bioprotector in the composition prior to lyophilization ranges from ~10 mm ~400 mm and preferably from about 30 mm to about 300 mm, and most preferably from about 50 mM to about 100 mm.

The ratio of protein to bioprotector choose for each combination of protein and bioprotector. If the protein is selected from antibodies and bioprotector for isotonic current song with the e bioprotector to the antibody can range from ~100 to ~1500 moles of bioprotector on 1 mol of antibody and preferably from ~200 to ~1000 moles of bioprotector 1 mol antibodies for example, from ~200 to ~600 moles of bioprotector on 1 mol of antibody.

In preferred embodiments of the invention to the composition prior to lyophilization is proposed to add a surfactant. Conversely, or in addition, the surfactant can be added to the freeze-dried composition and/or to the current composition. Typical surfactants are nonionic surfactants, such as Polysorbate (for example, Polysorbate 20 or 80); poloxamer (for example, poloxamer 188); Triton; sodium dodecyl sulphate (sodium dodecyl sulfate, SDS); sodium lauryl sulfate; octylglucoside sodium lauryl-, myristyl-, linoleyl or saarilahti; lauryl; myristyl; linoleyl or sterilisation; linoleyl-, myristyl or zeilboten; lauramidopropyl, cocamidopropyl, linoleamide, myristamide-; alminoprofen or isostearamide (for example, lauramidopropyl); myristamide, alminoprofen or easternmediterranean; metalcolor sodium and disodium salt of metalreinforced and MONAQUATTMseries (MOP Industries, Inc., Paterson, New Jersey), polyethylene glycol, polypropyleneglycol and copolymers of ethylene and propylene (e.g., pluronic (Pluronics, PF68 and totein in the current composition and brings to a minimum the formation of particles after receipt of the current song (after dilution). For example, the surfactant may be present in the composition prior to lyophilization from ~0.001 to 0.5% and preferably 0.005 to 0.05%.

In some embodiments of the invention upon receipt of a composition prior to lyophilization is used a mixture of bioprotector (e.g., sucrose or trehalose) and filler (e.g., mannitol or glycine). The filler allows dried to obtain a homogeneous mass without excessive costs to do this and so on

In the composition prior to lyophilization (or freeze-dried and/or existing (diluted) composition) may include other pharmaceutically acceptable carriers, excipients or stabilizers, such as, for example, described in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), provided that they have no harmful effects on the desired properties of the composition. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations and include additional, creating the buffer agents; preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegration polymers, which eat the invention, may also contain more than one protein, when such a composition is required when a specific indication for the treatment, preferably with complementary activities that do not impact negatively on another protein. For example, it may be desirable to use two or more antibodies, which in a single composition connected with HER2 receptor, or IgE. In addition, in one composition may be combined anti-R2 and anti-VEGF antibodies. Such proteins, respectively, are present in the composition in amounts effective for their intended purpose.

Compositions are provided for administration in vivo, must be sterile. This is easily achieved by filtration through sterile filtration membranes, prior to or following lyophilization and dilution (the creation of the current song). In addition, the sterility of the whole mixture can be achieved by autoclaving components, with the exception of protein, for example, at a temperature of ~120C for 30 minutes

After mixing the protein, bioprotector and other possible components, the composition liofilizirovanny. For these purposes there are many different capable of drying at low temperatures agents, for example, Hull50TM(Provided that the product remains frozen during the primary drying) with appropriate pressure, varying usually from 50 to 250 mtorr. The composition, size and type of container containing the sample (for example, glass bubble), and the volume of liquid mainly determine the time required for drying, which can vary from several hours to several days (e.g., 40-60 hours). Secondary drying can be carried out in the temperature range 0-40With, primarily depending on the type and size of container and the type of protein. However, it is found that the secondary drying is optional. For example, the temperature-keeping for complete removal of water by lyophilization may be 15-30(For example, about 20C). The time and pressure required for secondary drying, should be such that you could get appropriate freeze-dried weight, depending, for example, temperature and drugiego required at least 5 h (for example, 10-15 h). Pressure may be the same as during primary drying. The lyophilization conditions can vary depending on the composition and size of the bubble.

In some cases it is desirable to liofilizirovanny protein composition in the container in which you spend getting the current (diluted) composition in order to avoid phase transfer. The container in this case can be, for example, a bubble with a volume of 3, 5, 10, 20, 50 or 100 cm3.

As a rule, lyophilization will lead to a freeze-dried composition in which the moisture content is less than 5% and preferably less than 3%.

C. the Dilution of the lyophilized composition

(getting the current song)

In a certain period, usually, when it comes time for the introduction of the protein to the patient, the freeze-dried composition can be diluted with a solvent so that the concentration of protein in the current composition amounted to at least 50 mg/ml, for example, from ~50 mg/ml to ~400 mg/ml, more preferably from 80 mg/ml to 300 mg/ml and most preferably from 90 mg/ml to 150 mg/ml. Believe that such a high concentration of protein in the present composition is particularly necessary, when the current composition of p which may require a lower concentration of protein in the current composition (for example, from ~5 to 50 mg/ml, or from 10 to 40 mg/ml of protein in the current song). In some embodiments, the concentration of protein in the current composition is significantly higher than the concentration of protein in the composition prior to lyophilization. For example, the concentration of protein in the current (diluted) of the composition may be in 2-40 times, preferably from 3 to 10 times and most preferably 3-6 times higher (for example, at least three-fold or four-fold), compared to the composition prior to lyophilization.

The creation of an active composition is at a temperature of approximately 25With providing full hydration, although they may suitable be used and other temperature. The time required to create a valid (for dilution) of the composition will depend, for example, the type of solvent, the amount of filler(s) and protein. Typical solvents are sterile water, bacteriostatic water for injection (BWFI), buffer solution with a specific pH (e.g., phosphate buffered saline), sterile saline solution, ringer's solution or dextrose. The solvent typically contains a preservative. Typical preservatives, reported above, answereing preservative determined by testing different concentrations of preservatives for compatibility with the protein and test the effectiveness of preservatives. For example, if the preservative is an aromatic alcohol (e.g., benzyl alcohol), it can be present in an amount of from ~0.1 to 2.0% and preferably from 0.5 to 1.5%, but most preferably from 1.0 to 1.2%.

Preferably, the current composition has less than 6000 particles whose size is10 μm, in the bubble.

Was the Introduction of the current composition

The current composition is assigned a mammal, which should be treated protein, preferably it is assigned to a person in accordance with known methods, for example, intravenous administration, the introduction of a bolus or by continuous infusion over a certain period of time, intramuscularly, intraperitoneally, vnutrizonovoy introduction, subcutaneously, intraarticularly intra-articular, podvoloshino (vnutriobolochechnoe), oral, local or inhalation methods.

In preferred embodiments, the current composition of introduced mammal subcutaneously (i.e. under the skin). For these purposes, the composition can be entered using a syringe. However, acceptable and other provisions for the introduction of the composition, for example, the injection device (e.g., devices Inject-easeTMand BiojectorTMand subcutaneous "Ochakovo" delivery system.

The appropriate dosage ("therapeutically effective amount") of the protein will depend, e.g., conditions of treatment, the severity of the disease and course of treatment, whether appointed protein for preventive or therapeutic purposes, previous treatment, clinical condition of the person and the sensitivity to the protein, the type of protein and the care of the attending physician. The protein can be assigned to patients once or over the course of treatment and can be entered at any time after diagnosis. The protein can be assigned as the sole treatment or in combination with other drugs or therapies that are necessary for treatment of the condition.

If the protein is an antibody, an initial test dose for administration to a patient is between 0.1 to 20 mg/kg, or for example, one or more separate injections. However, it may be suitable and other doses. The progress of this therapy is easily monitored by conventional methods.

In the case of anti-R2 antibodies therapeutically effective amount of the antibody can be administered in the treatment or prevention of cancer, characterized by sverkhsinteza HER2 receptacles, ovarian, stomach, endometrial cancer, cancer of the salivary glands, lung, kidney, large intestine and/or bladder. For example, anti-R2 antibody can be used for the treatment of flowing carcinoma in situ (ductal carcinoma in situ, DCIS). Typical doses of anti-R2 antibodies are in the range from 1 to 10 mg/kg in one or more separate injections.

Purpose anti-IgE compositions used in the treatment or prevention of, for example, allergic diseases caused by IgE, parasitic infections, intermediate cystitis and asthma. Depending on the disease or disorder, which is subjected to treatment, patient a therapeutically effective amount (for example, from 1 to 15 mg/kg) of anti-IgE antibodies.

D. the Finished product

Another variant of the invention provides a finished product which contains the freeze-dried composition and provides instructions for obtaining and/or application. The finished product includes a container. Suitable containers include, for example, a vial, a vial (e.g., double bubbles), syringes (e.g., dual-chamber syringes and test tubes. The containers can be manufactured from various materials, such as glass or plastic. The container contains leofiles who obtain the current composition and/or use. For example, on the label (sticker) may indicate that the freeze-dried composition is diluted to concentrations of protein, reported above. In addition, the label may indicate that the composition is suitable or intended for subcutaneous injection. The container containing the composition, may be a bubble reusable, which allows re-introduction (for example, from 2 to 6 injections) diluted (current) composition. The finished product can also include a second container containing a suitable solvent (for example, BWFI). When mixing of the solvent and freeze-dried compositions, the final concentration of protein in dilute compositions should generally be at least 50 mg/ml of the Finished product can also include other materials required from a commercial and user perspective, including other buffer solutions, solvents, filters, needles, syringes and packaging tabbed sheets with application instructions.

The invention can be more fully discerned by reference to the following examples. They, however, cannot be interpreted as limiting the scope of invention. All cited literature are included as links.

Prima is safe malignant neoplasms. Related to the mouse or rat monoclonal antibodies, known as muMAb4D5, directed to the extracellular region (extra-cellular domain, ECD) p185HER2. Molecule muMAb4D5 was refined to improve its clinical efficacy by reducing immunogenicity and provide support for the effector functions of man (see WO 92/22653). This example describes the creation of a freeze-dried composition that includes a purified antibody huMAb4D5-8 full length, described in WO 92/22653.

To create a freeze-dried composition initially organized the screening of fillers and buffer solutions, evaluating the stability of the protein after lyophilization and obtain the current (diluted) compositions. Lyophilized protein in each composition are also exposed to research on accelerated aging to determine the potential ability of protein to sustain its shelf life. These studies on accelerated aging is usually carried out at temperatures higher than proposed for storage, and data is then used to calculate the activation energies of degradation (decay), using the kinetic regularities of the Arrhenius equation (Cleland et al., Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993)). The activation energy ispolzuetsya.

In earlier works on screening studied the stability of several of lyophilised compositions of recombinant purified anti-R2 antibodies (rhuMAbHER2) after keeping them at 5(The proposed storage condition) and 40With (accelerated aging). It has been observed that in the liquid state rhuMAbHER2 destroyed by deliciouse (30 Asn light chain) and education isoaspartate through cyclic emenee derivative - succinimide (Asp 102 of the heavy chain). Deliciousa minimum at pH 5.0, leading to decay mainly to succinimide. At pH 6.0 in a liquid protein composition was slightly stronger diaminononane. Therefore, the lyophilized compositions were studied in

a) 5 or 10 mM succinate buffer solution with a pH of 5.0 or

b) 5 or 10 mM his-tag buffer solution with a pH of 6.0.

Both the buffer solution contained surfactant is Polysorbate 20 (tween-20TMTween 20TM), which was used to reduce aggregation of the current (diluted) protein composition and to minimize the formation of particles upon dilution of the composition. These buffer solutions used with various carbohydrates and Bdale liofilizirovanny and checked the protein stability after two weeks of incubation at 5With 40C. installation for lyophilization, the vials were cooled at a temperature-keeping -55C for about 5 h, followed by primary drying at a temperature of cure 5With or 5 mtorr for 30 hours; and drying to 1-2% residual moisture was achieved by secondary drying at a temperature keeping 20With over 10 hours Main path decomposition of this protein during lyophilization was in aggregation, and therefore the stability of the protein was checked by gel-chromatography to determine the output (content) of intact native protein (% of intact protein are shown below in table. 2).

The stabilizing influence of different bioprotector carbohydrates on the lyophilized protein was determined in 10 mM sodium succinate with pH 5.0 (table. 2). At high concentrations of carbohydrates (250-275 mM), low concentration of protein (5.0 mg/ml) trehalose and lactose stabilized the protein from aggregation during lyophilization of the protein, which was stored for 2 weeks at 40C. However, it was noted that lactose regenerating the carbon reacts with the protein for a longer period of storage at 40With in two weeks. At higher concentrations of protein (21,0 mg/ml), the composition containing mannitol or mannitol together with sorbitol or glycine, in both cases, contained aggregated protein after lyophilization and storage. On the contrary, in both cases, storage, trehalose and sucrose prevented aggregation.

Compositions containing 250 TM trehalose and 250 mM lactose was tested for stability in the long term. After storage for 9 months at 40With or 12 months at 5For compositions with trehalose was not observed changes in % of intact protein. For compositions with lactose % intact protein remained constant (match start) after 3 months of storage at 40C or 6 months at 25C. Composition comprising trehalose, can be stored at a controlled room temperature (15-30(C) within 2 years without significant changes % of intact protein.

Composition with mannitol in the presence of 10 mM his-tag buffer with a pH of 6.0 contained less aggregated protein when stored at 40With in 2 weeks than composition with mA is m effect, due to histidine. When stored at 40With in 2 weeks was observed, however, significant aggregation of the compositions of the histidine or histidine/mannitol. The addition of an equal amount of sucrose to mannitol (concentration of each 10 mg/ml) in the his-tag composition stabilized the protein from aggregation in both cases storage. The use of glycine with mannitol did not improve the stability of the protein, while sucrose/glycine composition provided the same stability as the composition of the sucrose/mannitol. These results, moreover, meant that sucrose is suitable for preventing aggregation of lyophilized protein during storage.

Introduction high concentrations of protein is often required after subcutaneous administration due to the limitations of volumes (1.5 ml) and the required doses (100 mg). However, in the production process is often difficult to achieve high concentrations of protein (50 mg/ml), because at high concentrations of protein can aggregate processing and it becomes difficult to apply (for example, Perekatov the s concentration of the protein. For example, protein filled bubbles with a volume (Vfand then lyophilizers. Lyophilized protein is then diluted with a smaller volume (Vrwater or preservative (e.g., BWFI) than the original volume (e.g., Vr=0,25 Vf), leading to a higher concentration of protein obtained in (diluted) solution. This process also leads to the concentration of the buffer solutions and fillers. It is desirable that the solution for subcutaneous injection was isotonic.

The amount of trehalose in dried rhuMAbHER2 reduced to isotonic solution when diluted with a protein concentration of 100 mg/ml Stabilizing effect of trehalose was determined as a function of concentration of 5 mM sodium succinate pH 5.0 and 5 mM histidine at pH 6.0 with protein concentration of 25.0 mg/ml (table. 3). At concentrations of trehalose from 60 to 200 mM was not significant aggregation after keeping the lyophilized protein for 4 weeks at 40C. These compositions were diluted with 20 ml of bacteriostatic water for injection (BWFI, USP, 0.9% of benzyl alcohol). Dilution composition containing 50 mM trehalose (5 mM sodium succinate), 4 ml of BWFI (protein concentration of 100 mg/ml) promotion. Conserved current (diluted) composition had an advantage in the possibility of multiple extract the contents from the same bubble without breaking sterility. When using sterile needles, these compositions can be used to integrate multiple doses from the same vial.

Currently rhu HER2 is in the process of research as a therapeutic agent for the treatment of breast cancer. Protein is administered to patients on a weekly basis in the amount of 2 mg/kg as the average weight of these patients is ~65 kg, the average weekly dose of 130 mg rhuMA in HER2. When subcutaneous injection well tolerated injection volume of 1.5 ml or less, and therefore, the concentration of protein for weekly subcutaneous injection rhu HER2 may be approximately 100 mg/ml (130 mg average dose/1.5 ml). As mentioned above, such a high concentration of protein is hard to get and keep in a stable form. To achieve this high concentration of protein rhuMA in HER2 contained in: (a) 5 mM sodium succinate pH 5.0 or (b) 5 mM histidine, with a pH of 6.0, liofilizirovanny when protein concentration of 25 mg/ml in 60 mM trehalose, 0.01% tween-20C for about 5 h, followed by primary drying at a temperature of cure 5C and 150 mtorr for 30 h, and drying to 1-2% residual moisture was achieved by secondary drying at a temperature keeping 20With over 10 hours Thermocouple, placed in vials containing placebo (composition without protein), showed that the product in the vials was maintained at a temperature below -10With throughout primary drying. The application of the sealing device during lyophilization found that residual moisture after primary drying, usually less than 10%. Lyophilized protein was further diluted or 4 or 20 ml of BWFI (to 0.9 or 1.1% benzyl alcohol), receiving concentrated protein solutions:

a) 4 ml: 102 mg/ml rhuMA in HER2, 245 mM trehalose, 21 mM sodium succinate pH 5.0 or 21 mM histidine, with a pH of 6.0, and 0.04% tween-20TM;

b) 20 ml: 22 mg/ml rhuMA in HER2, 52 mM trehalose, 4 mM sodium succinate pH 5.0 or 4 mM histidine, with a pH of 6.0, 0,009% tween-20TM.

During storage of lyophilised compositions for 4 weeks at 40With and dilute to 22 mg/ml protein amount of aggregated protein, apparently, was slightly increased when clever As shown in Fig.1, the number of intact protein after keeping the lyophilized protein at 40With was the same as for the composition with 60 mM trehalose, 5 mM sodium succinate with pH 5.0, 0.01% tween-20TMdiluted 4 or 20 ml of BWFI.

The results are shown in table. 3, suggest that there may be a correlation between the concentration of trehalose and stability of the protein. To further assess this relationship compositions containing different concentrations of trehalose dissolved or sodium succinate, or his-tag in buffer maintained within 91 days at 40C. Stability was then determined as a function of molar ratio of trehalose to the protein for each concentration of trehalose. As shown in Fig.2, the stability of the protein undoubtedly decreases the concentration of trehalose for both compositions. Not there were obvious differences between the two buffer solutions - succinate and histidine; in these compositions, it was assumed that the primary stabilizer in these terms is trehalose. In addition, the observed decrease of intact protein in the two compositions will take place even at low concentrations of trehalose for composers stability at controlled room temperature (maximum temperature 30(C) required higher concentrations of trehalose (trehalose : protein600:1) depending on the requirements for stability of the product (i.e., the requirement for the number of intact protein remaining after storage for 2 years). Usually conditions store at controlled room temperature require stability for 6 months at 40S, which is equivalent to storage at 30With in 2 years.

As shown in Fig.3, the composition containing 250 mM trehalose did not change after 6 months of storage at 40With, while both compositions containing 60 mM trehalose, were less stable. Compositions containing 60 mM trehalose, should be stored in a cool place, to the end of the shelf life of the product the contents of the intact protein, according to the technical levels, constituted, for example, >98% according to gel-chromatography.

Preliminary screening showed that sucrose was also inhibited aggregation rhu HER2 after lyophilization and subsequent storage. To achieve isotonicity of the solution after dilution, intended for subcutaneous injection (approximately fourfold increase in content weight concentration of sucrose and mannitol (excipient), used for screening, prevented aggregation of the protein. For possible subcutaneous rhu HER2 composition chose a lower concentration of sucrose and mannitol (equal weight concentrations). The protein solution (25 mg/ml protein, 5 mM histidine, with a pH of 6.0, 38,4 mM (7 mg/ml mannitol, 20,4 mM (7 mg/ml) sucrose, 0.01% tween-20TM) liofilizirovanny in the same way as a composition containing 60 mM trehalose, except that the primary drying cycle was extended to 54 hours After 4 weeks of storage at 40With a slight increase in the number of units after dilution or 4,0 20,0 ml of BWFI (PL. 3). The amount of aggregated protein was the same as for the current (diluted) composition with the concentration of the protein 22 or 100 mg/ml (Fig.4). Similar compositions containing 60 mM trehalose, the composition containing mannitol/sucrose yielded less intact protein in a time when 40C. the Molar ratio of sucrose to protein for this composition was 120:1, showing that the combination of mannitol/sucrose may be more effective than one trehalose at the same molar ratio of stabilizing carbohydrate (Fig.2 and 4).

In the previous examples, the stability of leofiles the AI, containing trehalose and mannitol/sucrose prevented the destruction of the protein in the dried state at high temperature (40C). However, these experiments did not belong to the stability of the protein after dilution and storage. Once diluted with BWFI lyophilized rhu HER2 compositions can be used for multiple injections of the drug. In particular, the bubble containing 450 mg rhu HER2, was intended to provide three doses to an average patient (130 mg rhu HER2 for one dose). Because the drug is administered on a weekly basis, the bubble can be stored for at least three weeks after dilution. To make sure that rhuMA HER2 remained stable after dilution, studies on the stability of existing (diluted) rhu HER2 compositions was carried out at 5C and 25C.

For subcutaneous administration of the composition was diluted to a protein concentration of 100 mg/ml (4 ml of BWFI). At this high concentration of protein may be more susceptible to aggregation than the form for intravenous injection, which was diluted to a protein concentration of 22 mg/ml (20 ml of BWFI). Four rhu HER2 songs from the previous example were evaluated on the subject of the positions, diluted to a protein concentration of 22 and 100 mg/ml in Addition, these compositions retain the protein is fully intact up to 90 days of storage at 5C and 30 days at 25With, showing that the diluted protein composition can be stored in a cool place for at least 90 days. In contrast to the stability of lyophilized protein in the preceding example, the concentration of trehalose in the composition did not affect the stability of the protein (table. 7).

As mentioned above, the main route of degradation (destruction) rhu HER2 in aqueous solutions is deliciousa or education succinimide. Loss of native protein due to deliciouse or education succinimide was estimated for four operating rhu HER2 compositions.

Analysis of deliciouse rhu HER2 and education succinimide was performed using cation-exchange chromatography. Column Bakerbond Wide-Pore Carboxy Sulfon (CSX) (4,h mm) worked at a flow rate of 1 ml/min Buffer solutions for the mobile phase were: (A) 0.02 M sodium phosphate, pH 6 follows:

The results are given in table. 8.

Eluruumis peak was detected at 214 nm, for each analysis took 75 mg of protein.

Again there were no differences in the stability of the compositions, which are diluted to 22 and 100 mg/ml of protein (Fig.5-7). Degradation (destruction) of protein for each song flowed faster at 25With than when 5With, and the rate of degradation for all compositions stored at 5With, was comparable. Compositions containing histidine, was characterized by a slightly greater rate of degradation at 25With than compositions containing succinate. The amount of trehalose in the composition did not affect the rate of degradation at any temperature (Fig.5 and 8). These results showed that for the intended use period (30 days after dilution BWFI), these four songs are acceptable rate of degradation under conditions of storage in a cool place (5C).

Composition reusable when used in the United States should be tested for preservative efficacy according to United States Pharmacopeia (US Pharmacopeia, USP). Dried rhu HER2 kamilowaso alcohol, concentrations which ranged from 0.9 to 1.5% (wt.). At concentrations of 1.3% (wt.) or above diluted solution became turbid after standing over night at room temperature (~25C). Dilution standard BWFI solution (0.9% benzyl alcohol) led to the solution, which not always was not tested for preservative action. However, dilution of 1.0 or 1.1% benzyl alcohol was compatible with the composition and validation (test) on the preserving action. As the requirements of the manufacturer for solutions are ±10%, therefore, the freeze-dried composition was diluted to 1.1% benzyl alcohol (1,1±0,1%).

Developed single-stage cycle lyophilization rhu HER2 composition. In one cycle the lyophilization rhu HER2 at 25 mg/ml, 60 mM trehalose, 5 mM histidine, pH 6 and 0.01% Polysorbate 20 was carried out at a temperature keeping 20C and a pressure of 150 mtorr. After 47 h the content of residual moisture in dried weight was less than 5%. Believe that this cycle lyophilization is interesting because it simplifies the production process, excluding the stage of secondary drying.

Example 2

Anti-IgE composition

IgE antibodies bind with the specific the Yu mediators, for example, histamine, which produces the symptoms associated with allergies. Therefore, anti-IgE antibodies that block the binding of IgE to the high-affinity receptor, have potential therapeutic value in the treatment of allergies. These antibodies should not contact with IgE, as long as they are associated with the receptor, as this will stimulate the secretion (secretion) of histamine. This example describes the improvement of the freeze-dried composition that includes purified anti-IgE antibody Mae full length described in Presta et al., J. Immunology, 151: 2623-2632 (1993).

Materials. In the compositions described below, used highly refined rhuMAb E25 (recombinant purified anti-IgE antibody Mae), which did not contain tween-20TM. Membranes for dialysis - Spectra/Por 7 bought in Spectrum (Los Angeles, CA). All other reagents used in this study were obtained from commercial sources and were of analytical grade. Buffer solutions used for the composition, and the chromatographic mobile phase was prepared by mixing appropriate amounts of buffer and salt with Milli-Q water in a volumetric flask.

Composition. E25 pool S sepharose subjected, as is customary, the dialysis buffer solutions, which �/176.gif">C.

After dialysis to some songs, as required, was added bioprotector in isotonic concentration. The concentration of protein after dialysis was determined using UV spectroscopy using the molar absorption coefficient, equal to 1.60. Validirovannyj protein was diluted to a predetermined concentration in the composition of the corresponding buffer, sterilized using a 0.22 μm Millex-GV filter (Millipore), and placed in pre-cleaned and autoclaved glass vials (vials). Bubbles supplied siliconized (siliconized) Teflon tubes for lyophilization and liofilizirovanny using the following conditions: E25 composition was cooled to -55With a cooling rate of 80C/h and the contents of the vial was kept in a frozen state within 4 hours For the primary drying temperature of incubation was increased to 25With a speed of 10With/including Primary drying was carried out at 25C, 50a vacuum chamber for 39 h so that residual moisture in dried mass was 1-2%. After lyophilization, the bubble-containing composition is Torah, which was -70With, 2-8With 25C, 30With (controlled room temperature), 40With 50C.

The chromatography. Gel chromatography was performed on a Bio-Rad SIV-SelectTM, SEC 250-5 column (h,8 mm). The column was balanced and missed PBS (phosphate buffered saline) at a flow rate of 0.5 ml/min, using a Hewlett Packard 1090L HPLC chromatograph, equipped with a (supplied) diode matrix detector. For calibration of the column used the standards of a certain molecular weight (Bio-Rad, Inc.), containing thyroglobulin (670 kd),-globulin (158 kDa), ovalbumin (44 kd) and cyanocobalamin (1,35 kd). The sample was 25 µg and protein were detected by recording UV absorbance at 214 nm using Turbochrom software 3 (D Nelson, Inc.).

Chromatography hydrophobic interaction. F(ab’)2fragments E25 antibody were analyzed on a column (TosoHaas Butyl-NPR (3,5x4,6 mm) and chromatograph Hewlett Packard 1090L HPLC equipped with a diode array detector. Buffer And elution consisted of: 20 mM Tris, 2M ammonium sulfate, 20% (mass.) glycerol, pH 8.0, while buffer B with the eye of 1.0 ml/min for at least 20 minutes Load the sample was 5 µg and protein were detected by monitoring UV absorbance at 214 nm using Turbochrom software 3 for information collection (RE Nelson, Inc.). After the introduction of the sample the column was supported by a 10% buffer B for one minute, followed by an increase in the content of buffer B in a linear gradient from 10% to 62% during the 20 minutes Between successive input samples, the column was washed with 100% buffer B for 5 min and re-balanced 10% buffer B for 20 minutes

Antielastase activity: Analysis of inhibition of binding of IgE receptor (E:2) was performed as described in Presta et al., supra on samples diluted to 20 μg/ml and 30 μg/ml in the solvent in which carry out the analyses (phosphate buffered saline with 0.5% BSA, 0.05% Polysorbate 20, 0.01% thimerosal (Thimerosol)). Each diluted solution was further analyzed three times and the results were multiplied by the appropriate dilution factor, giving active concentration. The results of 6 tests were averaged. The analysis assesses the ability rhu E25 competitive contact with IgE and thereby inhibit the binding of IgE to its high-affinity receptor, which is immobilized on a plate of solid-phase immunoflu UV absorption spectroscopy, and get specific activity. Preliminary experiments showed that this analysis is an indicator of stability.

The test particles. The contents of the vials (bubbles) with diluted liofilizirovannam rhu E25 combined to a volume of approximately 7 ml was Determined by the number of particles ranging in size from 2 to 80 μm, present in 1 ml of sample using a counter Hiac/Royco model 1800. The counter is initially washed three times with 1 ml of the sample, followed by three times, measure 1 ml of the sample. The device determines the number of particles in 1 ml equal to or greater than 10 μm, and the number of particles equal to or greater than 25 microns.

The first stage in the creation (improvement) of the composition of anti-IgE antibodies consisted in finding a suitable buffer and pH for lyophilization and storage of the product. Antibodies at a concentration of 5.0 mg/ml was used to create a composition of 10 mM succinate buffer at a pH from 5.0 to pH 6.5 and nutrifaster buffer califorina and his-tag buffers at pH 7.0. In Fig.9 shows an enlarged aggregates of antibodies, which were observed in the compositions with higher pH, both before and after lyophilization. An exception was represented by a composition comprising a his-tag buffer with a pH of 7, which did not observe the line in 5 mM his-tag buffer as at pH 6, and at pH 7 and stored for one year at 2-8, 25, and 40 - C. Analysis that meets each point in time and the storage temperature of the composition with a pH of 6, showed that the composition contains less units than the composition with the antibody at pH 7.0. These results showed that the histidine at pH 6 is particularly suitable buffer system to prevent aggregation of the antibody.

To facilitate screening bioprotection to obtain compositions used anti-IgE antibody in sodium succinate buffer with pH 5 with bioprotection or without him. Potential bioprotector added in isotonic concentrations were divided into three categories:

a) non monosaccharides (i.e., mannitol);

b) reducing disaccharides (e.g. lactose, and maltose) and

C) non disaccharides (i.e., trehalose and sucrose).

Aggregation compositions after storage at 2-8 ° and 40With in one year is shown in Fig.11 and 12. When stored at 2-8With a composition comprising a monosaccharide (mannitol), gregorovius with a speed close to that of buffer solution (control), while the composition containing 6.gif">Were similar, except for the composition containing sucrose, which quickly aggregates (which correlates with obtaining dark dried mass (Fig.12). It was later shown that this is the cause of destruction (degradation) of sucrose during storage as in solutions with an acidic pH and at high temperature.

Chromatography hydrophobic interaction of the composition antibody his-tag in buffer at pH 6 with lactose shows that the antibody was changed after storage for 6 months at 40With (Fig.13).

Chromatographic peaks were usasales and retention time was decreased. These changes were not observed with control buffer and to compositions containing sucrose during storage under similar conditions, as shown in Fig.14 and 15, respectively. In addition, isoelectric focusing showed a shift in the acidic region l antibodies in the composition containing lactose, and when stored at 25 and 40C. This indicates that reducing carbohydrates is not suitable as bioprotectors for antibodies.

Aggregation of lyophilized compositions of anti-IgE at a concentration of 20 mg/ml in 5 mM his-tag buffer at pH 6 at different concentrations of sugar is and have the same protective effect against aggregation, if the concentration of carbon is greater than 500 moles of carbohydrate per 1 mol of antibody. According to these results isotonic and hypertonic composition containing both sucrose and trehalose were proposed for future applications. It was assumed that the composition will be filled prior to freeze-drying at a relatively low concentration of antibodies and dried product is mixed with a smaller volume than when filling bacteriostatic water for injection (BWFI), containing 0.9% benzyl alcohol. This allows the concentration of the antibody immediately before subcutaneous injection, and include a preservative for potential composition of multiple use, while avoiding interaction between the protein and preservative at the long-term storage.

Isotonic composition. Anti-IgE at a concentration of 25 mg/ml was used to create a composition of 5 mM histidinol buffer at pH 6 with 500 moles of carbohydrate per 1 mol of antibodies, which corresponds to the concentration of carbohydrate 85 mM. This composition is diluted with BWFI (containing 0.9% benzyl alcohol) to volume, which is four times less than the volume used in the filling. This leads to 100 mg/ml antibody in 20 mM his-tag buffer with a pH of 6 was isotonically to create a composition of 5 mM his-tag buffer at pH 6 with 1000 moles of carbohydrate per 1 mol of antibody which corresponds to the concentration of carbohydrate in 161 mM. This composition was diluted BWFI (containing 0.9% benzyl alcohol) to volume, which is four times less than when filling. This leads to 100 mg/ml antibody in 20 mM his-tag buffer with pH 6 and with hypertonic concentration of carbohydrate - 644 mM.

Comparison of aggregation antibodies after storage isotonic or hypertonicity mix until 36 weeks is shown in Fig.17-19. There were no changes in aggregation neither hypertensive nor in isotonic compositions when stored at 2-8With (Fig.17). In hypertensive composition was not observed increasing aggregation when stored at controlled room temperature (30C), while there was an increase in aggregation from 1 to 2% in isotonic compositions (Fig.18). Finally, after storage at 50Since there was a minimal increase in aggregation for hypertensive compositions, a 4% increase in aggregation occurs with isotonic composition containing trehalose, and 12% increase in aggregation occurs with isotonic composition containing sucrose (Fig.19). These results show that isotonic composition contains minimalism src="https://img.russianpatents.com/chr/176.gif">C.

Binding activity of anti-IgE in isotonic or hypertonic compositions were evaluated using analysis of inhibition of IgE receptors. It was found that the binding activity of isotonic and hypertonic compositions containing sucrose and trehalose, does not change significantly after storage at - 70With, 2-8C, 30With 50With up to 36 weeks.

It is known that the lyophilized composition of proteins contain insoluble aggregates or particles (Cleland et al., Critical Reviews in Therapeutic Drug Carrier Systems, 10(4): 307-377 (1993)). Thus, the conducted analysis of the particle antibody, lyophilized at a concentration of 25 mg/ml in 5 mM his-tag buffer with pH 6 by addition of 85 mM and 161 mm sucrose and trehalose. To the composition was added Polysorbate 20 at a concentration of 0.005, 0.01 and 0.02%. Samples liofilizirovanny and analyzed after dilution to 100 mg/ml antibody in 20 mM his-tag buffer with pH 6 with 340 mM and 644 mM carbohydrate. The concentration of Polysorbate 20 after dilution were 0.02, and 0.04 and 0.08%.

Below in the table. 9 shows the number of particles of 10 μm or more and 25 μm or more in isotonic and hypertonic compositions containing Saha the ATA show the addition of tween (TweenTM) to the composition significantly reduces the number of particles for each of the scanned range of particle sizes. The USP (US Pharmacopeia, USP) requirement for small volume injections is not more than 6000 particles 10 microns, or no more than 600 particles of 25 μm or more in a container (bottle, vial) (Cleland et al., supra). With the addition of Polysorbate 20 as hypertonic, and isotonic composition satisfy this requirement.

One song anti-IgE antibodies (i.e., 143 mg isotonic composition rhu E25 in the bubble), which, when considered, can be used for subcutaneous injection of this antibody, shown below in table. 10. Bubble volume of 10 cm3fill composition containing 5,7 ml rhu E25 at a concentration of 25 mg/ml in 5 mM his-tag buffer with a pH of 6.0 with 0.01% Polysorbate 20. Sucrose was added as bioprotector at a concentration of 85 mM, which corresponds to a molar ratio of carbohydrate to the antibody as 500:1. Bubble liofilizirovanny and diluted with 0.9% benzyl alcohol to 1/4 of the filled volume of 1.2 ml Final concentration of components in the composition was increased four times to 100 mg/ml rhu E25 20 mM g the. The composition contains a his-tag buffer with a pH of 6, because it demonstrates a protective effect against aggregation of the antibody. Sucrose was added as bioprotector, as previously used in the pharmaceutical industry. The concentration of carbohydrate was chosen so that upon dilution to obtain an isotonic composition. Finally, Polysorbate 20 was added to prevent the formation of insoluble aggregates.

Claims

1. Stable isotonic current composition comprising a protein in an amount of at least 50 mg/ml and the solvent that dilutes the composition obtained from the freeze-dried mixture of protein and bioprotector, characterized in that the molar ratio of bioprotector and protein in the mixture is 100 to 600 mol bioprotector on 1 mol of the protein, and the concentration of protein in dilute existing compositions 2-40 times greater than the concentration of protein in the mixture before lyophilization.

2. The composition according to p. 1, characterized in that bioprotection is sucrose or trehalose.

3. Composition under item 1 or 2 further includes a buffer solution.

4. The composition according to p. 3, characterized in Nichelino contains a surfactant.

6. Stable current composition comprising the antibody in the amount of at least 50 mg/ml and the solvent that dilutes the composition obtained from the freeze-dried mixture of antibodies and bioprotector, characterized in that the molar ratio of bioprotector and protein in the mixture is 100 to 600 mol bioprotector on 1 mol of antibody, and the concentration of antibodies in the diluted current song 2-40 times greater than the concentration of the antibodies in the mixture before lyophilization.

7. The composition according to p. 6, characterized in that the antibody is an anti-IgE antibody or anti-R2-antibody.

8. Composition under item 6 or 7, which is isotonic.

9. The method of obtaining a stable isotonic current composition, including the dilution of the lyophilized mixture of protein and bioprotector, characterized in that the molar ratio of bioprotector and protein in the mixture do 100 to 600 mol bioprotector on 1 mol of the protein in the solvent, and the concentration of protein in dilute current composition is at least 50 mg/ml, and the concentration of protein in dilute existing compositions 2-40 times greater than the concentration of protein in the mixture before lyophilization.

10. The way of getting to what otector and protein in the mixture is 100 to 600 mol bioprotector on 1 mol of the protein, and b) diluting the lyophilized mixture of stage a) with a solvent, with the current composition of perform isotonic and stable, having a protein concentration of at least 50 mg/ml

11. The method according to p. 10, characterized in that the concentration of protein in the current composition of from 80 to 300 mg/ml.

12. The method according to p. 10 or 11, characterized in that the concentration of protein in the current composition 2-40 times greater than the concentration of protein in the mixture before lyophilization.

13. The method according to any of paragraphs.10-12, characterized in that the lyophilization is carried out at a holding temperature maintained 15-30With throughout the process of lyophilization.

14. The finished product, comprising a vial containing a lyophilized mixture of protein and bioprotector, and the molar ratio of bioprotector and protein in the mixture is 100 to 600 mol bioprotector on 1 mol of the protein, diluted with solvent to the concentration of protein in dilute current composition of at least 50 mg/ml

15. The finished product under item 14, further comprising a second vial, which contains a solvent.

16. The finished product under item 15, wherein the solvent is bacteriostatic water

17. Composition comprising a lyophilized mixture of bioprotector and antibodies, characterized in that the molar ratio of bioprotector and antibodies is 100 to 600 mol bioprotector on 1 mol of antibody.

18. Drug for the treatment of mammals, which are disorders characterized by the overexpression of the HER2 receptor containing composition under item 1.

19. Drug under item 18, characterized in that it is intended for subcutaneous administration.

20. The composition comprising the anti-R2-antibody in an amount of 5 to 40 mg/ml, sucrose or trehalose in an amount of from 10 to 100 mm buffer solution and surfactant.

21. The composition according to p. 20, further comprising a filler.

22. The composition according to p. 20 or 21, which is lyophilized.

23. Composition according to any one of paragraphs.20-22, which is diluted with a solvent so that the concentration of anti-R2-antibodies in the present composition is from 10 to 30 mg/ml

24. A composition comprising an anti-IgE antibody in an amount of 5 to 40 mg/ml, sucrose or trehalose in an amount of from 80 to 300 mm, buffer solution and surfactant.

25. The composition according to p. 24, which is lyophilized.

26. The composition according to p. 6, characterized in that odnaznachno for the treatment of cancer, characterized by overexpression of the HER2 receptor in mammals, by introducing to a mammal a therapeutically effective amount of the composition, the antibody in the composition binds the HER2 receptor.

28. The composition according to p. 27, characterized in that it is intended for subcutaneous injection.

29. The composition according to p. 6, characterized in that the molar ratio of bioprotector and antibodies is 200-600 mol bioprotector on 1 mol of antibody.

30. The composition according to p. 17, wherein the antibody is a monoclonal antibody.

31. The composition according to p. 17 or 30, characterized in that bioprotection is sucrose or trehalose.

32. The composition according to p. 26, characterized in that it includes a monoclonal antibody in an amount of from 50 to 400 mg/ml and the solvent, diluting the composition obtained from the freeze-dried mixture of monoclonal antibodies and sugar selected from the group consisting of sucrose and trehalose, with the molar ratio of sugar and monoclonal antibodies in the mixture is between 200 and 600 mol of sugar per 1 mol of monoclonal antibodies and the concentration of monoclonal antibodies diluted in the current composition 2-40 times greater than the concentration of monoclonal antibodies in the mixture to young Lida, and is intended for the treatment of cancer selected from the group consisting of endometrial cancer, lung cancer, colon and urinary bladder in humans, by introducing a therapeutically effective amount of the composition.

34. The composition according to p. 6, characterized in that it contains an antibody that binds the HER2 receptor in mammals, and is intended for the treatment of cancer characterized by overexpression of the HER2 receptor in a mammal by subcutaneous injection a therapeutically effective amount of the composition.

35. The composition according to p. 34, characterized in that the concentration of the antibody in the composition is from 50 to 400 mg/ml.

36. The composition according to p. 34 or 35, characterized in that it is obtained by dissolving the lyophilized antibody in the solvent.

37. Composition according to any one of paragraphs.34-36, wherein the mammal is man.

38. Composition according to any one of paragraphs.34-37, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, stomach cancer, endometrial cancer, cancer of the salivary gland, cancer of the lung, kidney, colon and bladder.

39. The composition according to p. 27, characterized in that the composition comprises an antibody that binds to the HER2 receptor, the number is, the ri, this concentration of antibody diluted in the current composition 2-40 times greater than the concentration of the antibodies in the mixture before lyophilization.

40. The composition according to p. 39, characterized in that it is intended for subcutaneous injection.

41. The composition according to p. 39 or 40, characterized in that the mammal is man.

42. The composition according to p. 6, characterized in that it contains an antibody that binds the HER2 receptor in humans, and is designed to treat a flow of carcinoma in situ in humans by introducing a therapeutically effective amount of the composition.

43. The composition according to p. 6, characterized in that is used to treat cancer characterized by the overexpression of the HER2 receptor, and antibody at the indicated composition binds the HER2 receptor.

44. The composition according to p. 43, characterized in that it is intended for the treatment of cancer selected from the group consisting of endometrial cancer, lung cancer, colon and bladder.

45. The composition according to p. 43, characterized in that it is intended for subcutaneous injection.

46. The composition according to p. 17, characterized in that it contains an antibody that binds IgE and is intended for the treatment of IgE-mediated allergic diseases, parasitic infections, interstitial Cys is Oia.

 

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