RU2589691C2 - Stable composition of antibody specifically bound with her2 receptors and preparation method thereof - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and biotechnology, namely concerns new stable composition of antibody specifically bound with HER2 receptors in dried form, which can be recovered by solvent, and in form of concentrated solution, as well as method for preparing composition.

EFFECT: invention can be used for creation of finished dosage form of preparation, as well as for preparing solution for infusions used for treating HER2-positive tumours.

9 cl, 1 dwg, 3 tbl, 21 ex

Description

This invention relates to the field of pharmaceuticals and biotechnology, and in particular to a new stable composition of an antibody that specifically binds to HER2 receptors, both in lyophilized form, which can be reconstituted with a solvent, and in the form of a concentrated solution, as well as a method for preparing the composition.

The invention can be used to create a finished dosage form of the drug, as well as to prepare a solution for infusion, used to treat HER2-positive tumors.

The HER2 receptor (also known as HER2 / neu and ErbB2) is one of four members of the epidermal growth factor receptor (EGFR) family. These receptors are expressed on the surface of most normal and tumor epithelial cells and are involved in the regulation of the most important cellular processes, controlling cell proliferation, differentiation, migration, and apoptosis.

Overexpression and / or amplification of the HER2 gene by cells leads to the development of a number of aggressive cancers, in particular breast cancer, breast cancer, gastric cancer, ovarian cancer, uterine cancer, which are characterized by low patient survival, as well as low efficiency of cytostatic therapy. The frequency of occurrence of HEP2-positive breast cancer and gastric cancer, according to various estimates, varies within 15-30% of the total number of patients with breast cancer and gastric cancer.

In recent years, significant progress has been observed in the treatment of HER2-positive cancers, in particular breast cancer, due to the introduction of targeted drugs into clinical practice, primarily monoclonal antibodies - trastuzumab (Herceptin ™) and recently approved for the use of pertuzumab (Perreta ™) .

Trastuzumab is a humanized recombinant monoclonal antibody to the epidermal growth factor receptor type 2 (HER2). The mechanisms of action of trastuzumab are still not fully understood. The antiproliferative and cytotoxic properties appear to be the result of a combination of inhibition of signaling cascades (mitogen-activated protein kinase (MAPK) and phosphatidylinositol triphosphate kinase (PI3K)) by binding to HER2, internalization and degradation of HER2 receptors, and the induction of p27, which leads to a decrease in the p27 cell cycle leading to a blocking of p27 cyclin-dependent kinase 2 (CDK2) activity, inhibition of DNA damage repair and antibody-dependent cellular cytotoxicity. In addition, trastuzumab makes tumor cells more sensitive to traditional chemotherapy.

Currently, trastuzumab is used to treat tumors with overexpression of HER2, namely metastatic breast cancer, breast cancer in the early stages, widespread adenocarcinoma of the stomach or esophageal-gastric transition. According to works published over the past 10-15 years, the effectiveness of combinations of trastuzumab and cytostatic agents in the treatment of metastatic breast cancer reaches 50-75%. Combinations of trastuzumab with paclitaxel and docetaxel have been approved in the United States and Europe as a first-line standard in the treatment of patients with metastatic breast cancer with HER2 overexpression. When trastuzumab is added to adjuvant chemotherapy in patients with early HER2-positive cancer, the risk of relapse and death is reduced by more than 30%. In this case, trastuzumab therapy is characterized by good tolerance.

Monoclonal antibodies, like other proteins used in clinical practice, undergo degradation over time. One of the challenges facing drug developers based on monoclonal antibodies is the creation of compositions that provide high stability, maintaining quality, maintaining the activity and structure of the active substance (monoclonal antibody) during storage. Monoclonal antibody-based preparations are made in the form of lyophilisates or concentrated solutions (concentrates). As a rule, a buffer with a certain pH, lyoprotectants, stabilizers, surfactants, and other components are additionally included in their composition. Moreover, for each monoclonal antibody, each of the components and their ratio are individually selected to ensure the greatest stability of the active component.

The degradation of monoclonal antibodies can be associated with the chemical destruction of certain amino acid residues (oxidation of methionine, tryptophan, histidine, deamidation of asparagine and glutamine, rearrangement of disulfide bonds between cysteines, β-elimination with rearrangement of aspartic acid, hydrolysis of aspartic acid, crosslinking, crosslinking , glutamic acid) with a violation of the tertiary structure of the protein or in connection with covalent or non-covalent aggregation of monomers of monoclonal antibodies in solution.

Due to the importance of preserving the properties of the drug during storage, there are a number of patents for stable drug compositions based on monoclonal antibodies to HER2. RF patent 2426554 describes a stable pharmaceutical composition comprising pertuzumab in histidine acetate buffer with a pH of 5.5-6.5. In US patents 6685940 and 7682609, as well as RF 2229288 described lyophilized antibody compositions, including compositions of antibodies against HER2 and IgE. US Pat. No. 7,074,404 describes an anti-HER2 antibody composition comprising a mixture of an anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant (s) is approximately less than 25%. An example of an anti-HER2 antibody is trastuzumab.

As the closest analogue, Herceptin may be indicated by F. Hoffmann-La Roche Ltd., Switzerland, in the form of a lyophilisate for the preparation of a solution for infusion, which contains:

Active substance: trastuzumab - 440 mg;

Excipients: L-histidine hydrochloride - 9.9 mg, L-histidine - 6.4 mg, α, α-trehalose dihydrate - 400.0 mg, polysorbate 20 - 1.8 mg.

Solvent (20 ml bacteriostatic water for injection containing 1.1% benzyl alcohol as an antimicrobial preservative): benzyl alcohol 229.9 mg, water for injection 20.9 mg.

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The objective of the invention is the development of a new stable antibody composition that specifically binds to HER2 receptors.

The problem is solved by a pharmaceutical composition comprising trastuzumab as an active substance, a pharmaceutically acceptable solvent, a lyoprotector selected from the group: trehalose, maltose, a mixture of sucrose with mannitol or glycine, a buffer selected from L-histidine buffer or sodium phosphate buffer, and a pharmaceutically acceptable solvent with the following components:

trastuzumab - 10-23 mg / ml

lyoprotector - 110-800 mm

buffer pH 5-6.5 - 5-10 mm

A surfactant such as polysorbates, polyoxyethylene-polyoxypropylene polymers and the like can be further added. at a concentration of 0.01-0.03%, methionine, EDTA, glycine, arginine, proline, as well as mixtures thereof, to provide additional stability.

The composition may be in the form of a liquid, its lyophilized form, or as a concentrated solution.

The solvent is preferably a known solvent suitable for the antibody, such as water for injection or sterile saline. An additional preservative may be added to the solvent, which is water for injection.

The problem is also solved by the production method, including dilution of trastuzumab with a solvent, dialysis into a buffer solution containing, if necessary, a surfactant, adding a lyoprotectant and, if necessary, other additional components, mixing until complete dissolution, sterilizing filtration, pouring into vials or ampoules. Before capping the bottles or sealing the ampoules, the solution can be concentrated or lyophilized.

The technical result of this invention is the creation of a stable trastuzumab composition both in lyophilized form, which can be reconstituted with a solvent, or in the form of a concentrated solution. When reconstituted with a solvent, stability is maintained before use.

The invention may be illustrated by the following embodiments.

Preparation of dosage forms for stability testing

The active pharmaceutical substance trastuzumab after dilution to the desired protein concentration was transferred using dialysis in 5 mM L-histidine buffer (examples 1-12, 14-21 in tables 2 and 3) or 10 mM sodium phosphate buffer, pH 6.0, ( example 13 in table 2) containing, if necessary, polysorbate 20 0.02% (vol./vol.). The concentration of additional components: polysorbate 20, trehalose, L-methionine, EDTA, sucrose, mannitol, maltose, glycine, arginine and proline was equalized by adding sterile concentrated solutions. After making all the working solutions, mixing was carried out until the components were completely dissolved. Sterilization of the components of the final mixture was carried out by sterilizing filtration through a membrane filter with a pore size of 0.22 μm. Ready sterile solution was poured into 0.5 ml into bottles with a nominal volume of 2 ml. In the case of concentrated solutions (examples 14 in table 2 and 17-21 in table 3), immediately after filling, the bottles were corked with rubber stoppers, crimped with aluminum caps and laid for storage. In the case of lyophilisates (examples 1-13 in table 2, examples 15, 16 in table 3) before corking the bottles, lyophilized drying of the obtained solutions was carried out according to the procedure described below. The compositions of all tested compositions are shown in tables 2 and 3.

Freeze drying

Freeze drying was performed on a Martin Christ BETA 1-8KS freeze dryer (table 1). Vials were placed on a pre-chilled lyophilized drying shelf; the freezing temperature was in the range of -30 ÷ -40 ° С. Vials with the finished dosage form underwent freezing for 2 hours, after which the drying switched to sublimation (primary drying). Sublimation was carried out under a pressure of 0.09 mTorr at a shelf temperature of -20 ° C for 28 hours. After the time of sublimation, the drying was switched to the stage of desorption (secondary drying). The transition to desorption was carried out stepwise, for 1 hour, so that every 15 minutes the temperature of the shelf increased by 10 ° C. Upon completion of the transition to desorption, the vials were dried for two hours at a temperature of 20 ° C and a pressure of 0.09 mTorr. At the end of the drying, the bottles with the preparation were corked with rubber stoppers and crimped with aluminum caps.

At least two vials were used for each example, one of which, at the end of drying, was used to determine the residual moisture by coulometric titration using a standard method (Karl Fischer method). The required humidity value was less than 2%.

Stability study

In order to accelerate the study of stability, vials with the finished dosage form were incubated at a temperature of + 50 ° C for 40 days or at temperatures of + 37 ° C and + 50 ° C for a longer time (up to 92 days). The temperature of accelerated storage and its duration were pre-selected and it was found that the changes are sufficient to study the stability of the drug during storage. At the end of the accelerated storage period, as well as at intermediate points, vials were taken for analysis. To assess the stability of the protein during storage, we used the quality indicator “purity”, determined by gel filtration high performance liquid chromatography (GF-HPLC), and “activity” using the method for determining the activity of trastuzumab on BT-474 cells. A composition identical to that of the commercially available Herceptin preparation (F. Hoffmann-la Roche Ltd.) containing 21 mg / ml trastuzumab, 50 mM trehalose, 5 mM histidine buffer, polysorbate 20 0.01% (v / v) was used as a comparison composition. about.).

HF-HPLC

Before analysis, the column was balanced with the mobile phase (10 mM TRIS, 0.15 M NaCl, 0.15 mM EDTA, pH 7.0) at a flow rate of 0.5 ml / min. 20 μl of sample solution was injected into the gel filtration column using a flow rate of 0.5 ml / min. Separation was carried out in isocratic mode. Peaks eluting from the column were determined in the UV spectrum at 214 nm. The purity of the test sample was calculated as a percentage relative to the peak content of the immunoglobulin monomer.

Activity determination

A suspension of BT-474 cells (100 μl / well, 7500 cells / well) in RPMI 1640 medium with glutamine, gentamicin and 10% fetal bovine serum was added to the wells of a 96-well plate. 200 μl of cell-free medium was added to the marginal wells. The plate was incubated at a temperature of 37 ° C and 5% carbon dioxide for 1-3 hours. 100 μl of a dilution of a standard sample (example 1 in table 2) and a test sample (examples 2-14) in various dilutions (0.0001-10 μg / ml, three wells for each dilution) were added to the cell wells, the contents of the tablets were mixed for 5 minutes on an orbital shaker, incubated for 145 hours at a temperature of 37 ° C and 5% carbon dioxide. At the end of the incubation, the number of living cells was evaluated using WST-1 stain (Roche). WST-1 was added 20 μl to all wells of a 96-well plate. Stirred for 5 min and incubated for 2 to 4 hours at a temperature of 37 ° C and 5% carbon dioxide. Readings in relative fluorescence units at an excitation wavelength of 450 nm were read. The EC50 was calculated using a 4-parameter logistics program.

results

To study the effect of various components on the stability of the monoclonal antibody trastuzumab in the finished dosage form, compositions of various compositions were prepared and a comparative analysis of stability during accelerated storage was performed (Table 2). One antibody preparation with a purity of 99.9% (determined by HF-HPLC) was used to prepare the compositions shown in Table 2. After lyophilization and the end of the accelerated storage program, the samples were tested for critical values.

As a critical indicator, purity was used, estimated as the percentage of antibody monomer. This indicator is widely used to assess the stability of antibodies, since a change in various properties of a protein molecule, as a rule, leads to a change in the purity index. Moreover, even a slight decrease in purity allows us to judge the stability of the antibody. As an additional indicator of stability, the activity of trastuzumab was used on a VT474 cell model. The values of this indicator allow you to directly judge the biological activity of the antibody. Thus, the data obtained allow us to fully carry out a comparative assessment of the stability of various lyophilisate compositions (Table 2).

From the results shown in the table, it can be seen that both purity and biological activity significantly changed after the expiration of the accelerated storage period. So, with an initial purity of 99.9% for the composition of the comparison (example 1 in table 2) after an incubation of 40 days at 50 ° C, the value dropped to less than 95%, which is lower than the acceptable purity value for the final control for most monoclonal antibody preparations. The value of biological activity also varies significantly, but in most cases it is higher for compositions having a higher degree of purity compared with the composition of the comparison at the end of accelerated storage.

From the results it is seen that 11 compositions (examples 2-13, table 2) showed stability greater than or equal to that of the composition of the comparison in purity. 9 formulations showed greater stability in activity (examples 2, 3, 5-12, table 2) of the antibody. Nevertheless, two examples 4 and 13, which showed significantly greater stability in purity, have an activity value slightly lower, but comparable with the composition of the comparison, and therefore deviations from the composition of the comparison can be considered promising.

Thus, by changing the concentration or composition of the components relative to the composition of the comparison (example 1 in table 2), it is possible to increase the stability of the antibody. The increased content of trehalose (examples 2-5 in table 2) has a positive effect on storage stability. The appearance of additional components in the compositions can also positively affect the stability of a monoclonal antibody. So, L-methionine at a concentration of 5 mm has a stabilizing effect (comparison of examples 4 and 6 in table 2). The addition of 5 mM L-methionine along with 0.03% ethylenediaminetetraacetic acid or its disodium salt also allows for greater storage stability compared to the comparison composition (comparison of examples 4 and 7 in table 2), although only with L-methionine has a positive effect was slightly larger (comparison of examples 6 and 7 in table 2). The addition of other amino acids, namely arginine and proline, at concentrations of 25 mM had an unambiguous effect on the stability of the antibody (comparison of formulations 4 and 11). On the one hand, the purity was slightly lower (99.0% versus 99.4%), but the antibody was more active after accelerated storage when these amino acids were present in the composition.

Replacement of the lyoprotector with respect to the composition of comparison (trehalose, 50 mM) with sucrose with mannitol at concentrations of 40 mg / ml (example 8), with maltose at a concentration of 50 mg / ml (example 9) or sucrose with glycine at concentrations of 30 mg / ml (example 10) also significantly increased stability both in terms of purity and in the final activity of trastuzumab.

A composition with a high content of monoclonal antibody trastuzumab showed greater stability with respect to the composition of the comparison (comparison of examples 12 and 1 in table 2), however, it is impossible to unequivocally conclude the effect of an increased concentration of trastuzumab during storage, since the composition also increased trehalose content.

The replacement of histidine buffer with Na-phosphate did not cause a significant decrease in the stability of the lyophilized composition (comparison of examples 13 and 1 in table 2). Thus, sodium phosphate buffer may also be considered to provide a stable trastuzumab composition.

The absence of a lyophilization stage would simplify the process and is more convenient in treating patients, since there is no stage of dissolution of the lyophilized dried tablet. In this regard, in parallel to the lyophilized compositions, the preparation of a concentrated antibody solution of the same composition as the comparative preparation was investigated (examples 14 and 1 in table 2). After accelerated storage, the concentrated solution showed less purity and activity of the antibody (examples 1, 14, table 2).

Nevertheless, in view of the obvious technological and practical advantages of using concentrated solutions, a more detailed stability test was carried out during storage of concentrated solutions with different compositions. For this, compositions of various compositions were stored at two temperatures: 37 ° C and 50 ° C for a longer period and purity was measured at various time intervals (table 3, figure 1).

When stored both at 37 ° C and at 50 ° C, the results are comparable, although in the case of elevated temperatures are more indicative in view of the greater drop in purity during storage. The composition corresponding to the composition of the drug Herceptin was used as a comparison composition. For control, a lyophilized composition with an increased trehalose content was also tested, which once again demonstrated greater stability compared to the comparison composition (example 4 in table 2, as well as example 16 in table 3). Of the five tested compositions of concentrated solutions, four (examples 18-21, table 3), although they showed stability slightly below the lyophilized composition of the comparison (example 15 in table 3), can be considered as potentially promising due to the convenience in production and medical practice. One composition of the concentrated solution (example 17, table 3) showed greater stability compared to the freeze-dried composition of the comparison. Compared to the latter, the concentration of trehalose in this concentrated solution composition is significantly higher, which, most likely, makes a significant stabilizing effect.

The resulting forms can undergo numerous freezing-thawing processes.

Thus, in comparison with the composition of a commercially available and used in practice preparation with the main active ingredient trastuzumab, this patent describes the compositions of more stable lyophilisates, as well as a more stable concentrated solution. At the moment, there are a number of drugs based on a variety of monoclonal antibodies and a large number are in development. In this regard, these compositions are of great interest, since they can be considered to create stabilized drug compositions based on other monoclonal antibodies.

Claims (9)

1. A pharmaceutical composition comprising trastuzumab as an active substance, a pharmaceutically acceptable solvent, a lyoprotector, and a buffer, characterized in that the lyoprotector is selected from the group: trehalose, maltose, a mixture of sucrose with mannitol or glycine, a buffer selected from L-histidine buffer or sodium -phosphate buffer, with the following components:
trastuzumab 10-23 mg / ml lyoprotector 110-800 mm buffer pH 5-6.5 5-10 mm 2. The composition according to p. 1, characterized in that it further includes stabilizers selected from the group: polysorbate, methionine, EDTA, glycine, arginine, proline, as well as mixtures thereof. 3. The composition according to p. 2, characterized in that it includes 5 mm L-methionine together with 0.03% ethylenediaminetetraacetic acid or its disodium salt. 4. The composition according to p. 1, characterized in that it can be in the form of a lyophilized form or in the form of a concentrated solution. 5. The composition according to p. 1, characterized in that the solvent is selected from water for injection or sterile saline. 6. A method for producing a composition according to claim 1, characterized in that trastuzumab is diluted with a pharmaceutically acceptable solvent and transferred to a buffer solution, then a lyoprotectant is added, mixed until completely dissolved, sterilized filtration is carried out and the resulting solution is poured into vials or ampoules. 7. A method of obtaining a composition according to claim 6, comprising the stage of concentration or lyophilization after filling the solution. 8. The method according to p. 6, in which the stage of lyophilization is carried out by freezing at a temperature in the range of -30 ÷ -40 ° C for 2 hours, sublimation under a pressure of 0.09 mTorr at a temperature of -20 ° C for 28 hours, followed by transition to desorption stepwise, for 1 hour, so that every 15 minutes the temperature rises by 10 ° C, and drying for two hours at a temperature of 20 ° C and a pressure of 0.09 mTorr. 9. The method according to claim 6, in which stabilizers selected from the group: polysorbate, methionine, EDTA, glycine, arginine, proline, and also mixtures thereof are additionally introduced before sterilizing filtration.

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