EA044682B1 - PHARMACEUTICAL COMPOSITION CONTAINING ROMIPLOSTIM - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to improved lyophilized pharmaceutical compositions containing romiplostim, a buffer, an excipient, a stabilizer and a surfactant and a method for their preparation. The proposed compositions may find use in the production of a drug related to thrombopoietin receptor agonists used for the treatment of thrombocytopenia.

State of the art

Thrombopoietin (TPO) is a cytokine involved in the regulation of platelet synthesis (thrombocytopoiesis) by activating the growth and differentiation of megakaryocytes, platelet precursor cells, in the bone marrow.

TPO is synthesized as a 353 amino acid precursor protein. Most of the TPO is formed in the liver, while the rate of synthesis is maintained at the same level. As a result of processing, a mature protein is formed, consisting of two domains: receptor-binding and highly glycosylated C-terminal [1].

The level of TPO in the blood is normally inversely proportional to the number of platelets. The feedback mechanism is realized through receptor-mediated destruction of the cytokine upon binding to thrombopoietin receptors (CD110 or c-Mpl), which are present in large numbers on the surface of platelets. As thrombocytopenia develops, the total number of receptors decreases, so receptor-mediated destruction also decreases.

Romiplostim is a thrombopoietin receptor agonist. In its structure, it is an Fc-conjugated peptide (peptide antibody), which, like TPO, induces thrombocytopoiesis. The peptide antibody molecule consists of the Fc fragment of human immunoglobulin IgG1, in which each single-chain subunit of the Fc fragment is covalently linked at the C-terminus to a CD110 agonist peptide containing two receptor-binding domains. Romiplostim is produced by recombinant DNA technology using the producer strain of Escherichia coli (E.coli). The amino acid sequence of romiplostim is not homologous to the amino acid sequence of endogenous TPO. Preclinical and clinical studies did not reveal the formation of cross-reacting antibodies to romiplostim and endogenous TPO [2, 3].

Romiplostim is indicated for the treatment of chronic idiopathic (immune) thrombocytopenic purpura in adult splenectomy patients refractory to other treatments (eg, corticosteroids, immunoglobulins). The drug can be used as second-line therapy in patients who are contraindicated for splenectomy [2].

As a rule, peptides in an aqueous environment are physically and chemically unstable, which can lead to a loss of their biological activity during production and storage. Physical instability of the peptide may result in denaturation, aggregation, precipitation or adsorption. Chemical changes occur due to oxidation, deamidation, proteolysis, etc. All these processes pose a serious problem for therapeutically active peptides, the dosage of which depends on the biological activity [4, 5].

To solve the problem of physicochemical instability of therapeutic peptides in aqueous compositions, lyophilization (freeze drying) is widely used [5, 6]. Freeze drying is based on removing moisture from frozen material by sublimation (sublimation) of ice, which turns into steam, bypassing the liquid phase. The practical implementation of the method includes three stages: freezing, main drying (ice sublimation) and post-drying (removal of residual moisture at temperatures above 0°C).

When developing medicinal products containing peptides or proteins in the form of lyophilisates, the properties of both active and introduced excipients are taken into account, which should ensure the effectiveness of the drug, its chemical and physical stability. In this case, the issue of the composition of the drug and the concentration of substances is resolved for each therapeutically active peptide individually, with physical-chemical, technological and biopharmaceutical studies of the created compositions. Determining the optimal ratio between the main and excipients is a necessary condition for obtaining a high-quality medicinal product.

Lyophilized preparations of therapeutic peptide antibodies usually contain a buffer, excipient, and stabilizer. In the event that aggregation occurs during the lyophilization stage or during the recovery stage, a surfactant (surfactant) is additionally included in the composition.

Patent EA17085 describes lyophilized compositions of therapeutic peptide antibodies, including romiplostim, containing a buffer, excipient, stabilizing agent and, optionally, a surfactant. Patent EA22424 protects an invention isolated from patent EA17085, which is the closest analogue of the present invention - a stable composition of romiplostim (Fc-TMP), corresponding to the composition/technology for producing the original drug Enplate, containing romiplostim at a concentration of 0.5 mg/ml, 10 mM histidine buffer (pH 5), 4% w/v, mannitol as excipient

- 1,044,682 calves, 2% w/v, sucrose as a stabilizer and 0.004% w/v, polysorbate-20 as a surfactant. The indicated concentrations represent the concentrations in the liquid composition before lyophilization, which are equal to the concentrations in the lyophilisate reconstituted in accordance with the instructions for medical use [2]. Our study of the stability of the original drug showed that during storage of the lyophilisate, impurities of oxidation, deamidation, and proteolysis of romiplostim may appear. The presence of such impurities is undesirable due to the possible effect on the biological activity of the drug. The stability of romiplostim in the compositions according to the description of the invention decreases as the concentration of protein in the solution before lyophilization decreases. For example, it is possible to increase the rate of aggregation and oxidation at concentrations less than 0.5 mg/ml.

Application WO2015150968 describes lyophilized pharmaceutical compositions containing romiplostim at a concentration of 0.5 mg/ml, a buffer selected from citrate, phosphate, alanine, glycine, arginine or a combination thereof, polysorbate-20 as a sulfactant and an excipient selected from sucrose, trehalose or combinations thereof. The lyophilized compositions described in the application have stability (storage in closed vials, 15 days or 21 days at 40°C) comparable to the stability of the composition reproducing the composition of the original drug. However, the application does not contain data on studying stability during long-term storage (2-8°C) and during accelerated storage ((25±2)°C; (60±5)%).

A common disadvantage of the known lyophilized compositions of romiplostim is the use of polysorbate-20 (polyoxyethylene (20) sorbitan monolaurate) as a surfactant, which is a chemically unstable compound.

Polysorbates contain ester bonds, as well as unsaturated alkyl and polyoxyethylene chains, which easily hydrolyze and autoxidize in an aqueous environment, leading to the accumulation of highly reactive peroxides and aldehydes. The resulting peroxides are capable of oxidizing the methionine and tryptophan residues of the polypeptide. Aldehydes react with the primary amino groups of the polypeptide, which can increase immunogenicity [5, 7-10]. Lauric acid, formed as a result of hydrolysis of polysorbate-20, can reduce the acidity of solutions. When filtering solutions of polypeptides, polysorbate-20 can be adsorbed on filter membranes [11].

Therefore, there is a need to develop a lyophilized pharmaceutical preparation of romiplostim that does not contain polysorbates as surfactants and is physically and chemically stable under various storage conditions.

The essence of the invention

The purpose of the present invention is to obtain new stable lyophilized compositions of romiplostim. This goal is achieved by choosing the concentration of the active substance in the solution for lyophilization, choosing the qualitative and quantitative composition of excipients, and lyophilization conditions.

The compositions according to the invention contain romiplostim in a concentration from 0.1 to 1.0 mg/ml, histidine buffer in a concentration from 5 to 25 mM (pH of compositions 4-6), mannitol in a concentration from 3 to 4.5% w/v ., trehalose in a concentration of 1.5 to 3% w/v, poloxamer 188 in a concentration of 0.004 to 1% w/v, where the concentration of mannitol and trehalose is in a ratio of 1:1 to 3:1.

Functional purpose of excipients: histidine - buffering agent, mannitol - filler, osmotic agent, trehalose - stabilizer, osmotic agent, poloxamer 188 - surfactant. Dilute hydrochloric acid is used if necessary to adjust the pH of the compositions to values of 4.0-6.0, preferably 5.0.

The technical result achieved using the proposed compositions is to increase their stability compared to a composition that reproduces the composition of the original drug.

The stability of the compositions, namely the tendency to form related impurities, was determined by the following methods: reverse-phase HPLC (RP-HPLC), cation exchange HPLC (CEX-HPLC), size exclusion HPLC (SE-HPLC) [12-14].

The SE-HPLC method is based on the separation of polypeptides by size and allows the determination of romiplostim aggregation impurities (dimers, trimers, etc.). Aggregation of therapeutic proteins affects their effectiveness and may cause immunogenicity. According to the requirements of the International Conference on Harmonization ICH (Q6B), aggregates must be separated from the main product and quantitatively characterized [14].

The CEX-HPLC method is based on the separation of polypeptides by their total charge and allows the detection of impurities of deamidation and methionine oxidation in romiplostim.

The RP-HPLC method is based on the separation of polypeptides with different hydrophobicity and allows the isolation of deamidated and N-terminally truncated forms of romiplostim.

In order to establish the effect of the concentration of histidine buffer in the compositions on their stability, compositions 1-4 were prepared (Table 1). The composition of composition 1 (control) reproduces the composition of the original drug before lyophilization. The concentration of histidine buffer in it

- 2 044682 mM. The qualitative and quantitative composition of compositions 2-4 is similar to the composition of composition 1, with the exception of the molar concentration of histidine buffer, which varies from 5 to 25 mM.

Table 1

Qualitative and quantitative composition of compositions 1-4

Song number 1 2 3 4 Romiplostim 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml L-histidine 10 mM 5 mM 15 mM 25 mM Mannitol 4% w/v 4% w/v 4% w/v 4% w/v Sucrose 2% w/v 2% w/v 2% w/v 2% w/v Polysorbate-20 0.004% w/v 0.004% w/v 0.004% w/v 0.004% w/v Dilute HC1 solution (10%) up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0

Preparation of compositions according to examples 1-4.

Compositions according to examples 1-4 were prepared in a volume of 0.5 l.

Preparation of buffer.

Pre-prepare 0.6 l of histidine buffer with a concentration of 5, 10, 15 or 25 mM, depending on the concentration given in the table. 1. Measure the pH and, if necessary, adjust the pH of the solution to 4.8-5.3 with a 10% solution of hydrochloric acid.

Preparation of placebo solution.

Using histidine buffer, prepare 0.15 l of solution containing 10 g of sucrose and 20 g of mannitol. In a separate container, 20 mg of polysorbate-20 is dissolved in histidine buffer, and the volume is adjusted to 0.007 l. The resulting solutions in the entire prepared volume are quantitatively transferred into a 0.2 l volumetric flask. The pH is measured, if necessary, the pH is adjusted with a 10% solution of hydrochloric acid to pH 4.8-5.3, then the volume is adjusted to 0.2 l with histidine buffer.

Preparation of compositions with a concentration of romiplostim of 0.5 mg/ml.

In a 0.5 liter volumetric flask with a 0.2500 g portion of romiplostim, add 0.26 liters of histidine buffer and stir until romiplostim is completely dissolved. Measure the pH and, if necessary, adjust the pH to 4.8-5.3 with a 10% solution of hydrochloric acid. Add the placebo solution in the entire prepared volume. The volume is adjusted to 0.5 l with histidine buffer.

The resulting solutions are filtered through a 0.22 μm polyethersulfone filter, poured under sterile conditions into 0.75 ml (for a delivered dose of 250 μg) or 1.5 ml (for a delivered dose of 500 μg) into 3 ml vials, sealed with stoppers and subjected to lyophilization . At the end of lyophilization, the vials are sealed with an aluminum cap.

The stability of the resulting lyophilisates was studied under the following conditions: for 28 days at a temperature (40±2)°C and relative humidity (75±5)%, for 6 months at a temperature (25±2)°C and relative humidity (60± 5)%.

The results of stability studies in terms of purity (% of the main peak) by RP-HPLC, CEX-HPLC and SE-HPLC showed that the concentration of histidine buffer, varying from 5 to 25 mM, did not affect the stability. For further studies, 25 mM histidine buffer with the highest buffer capacity was chosen to ensure maximum buffer stability.

In order to study the effect of replacing the surfactant polysorbate-20 with poloxamer 188, compositions were prepared according to examples 6-10 (Table 2) with a qualitative and quantitative composition similar to the composition of composition 4, except that poloxamer 188 was used as a surfactant concentration from 0.004% w/v to 1% w/v. As a control, composition 5 was prepared, reproducing the composition of composition 4.

The preparation of composition 5 is carried out similarly to composition 4. The preparation of compositions 6-10 is carried out similarly to composition 4, except that instead of polysorbate-20, poloxamer 188 is used in the quantities given in table. 2.

- 3 044682

table 2

Qualitative and quantitative composition of compositions 5-10

Song number 5 (control) 6 7 8 9 10 Romiplostim 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml L-histidine 25 mM 25 mM 25 mM 25 mM 25 mM 25 mM Mannitol 4% w/v 4% w/v 4% w/v 4% w/v 4% w/v 4% w/v Sucrose 2% w/v 2% w/v 2% w/v 2% w/v 2% w/v 2% w/v Polysorbate 20 0.004% w/v — — — — — Poloxamer 188 — 0.004% w/v 0.05% w/v 0.07% w/v 0.1% w/v 1% w/v Dilute HC1 solution (10%) up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0

The stability of the resulting lyophilisates was studied under the following conditions: for 28 days at a temperature (40±2)°C and relative humidity (75±5)%, for 6 months at a temperature (25±2)°C and relative humidity (60± 5)%. The results of studying the stability in terms of purity (% of the main peak) using the RP-HPLC, CEX-HPLC and SE-HPLC methods (Table 3, Fig. 1-3) showed that when using poloxamer 188 in any of the concentrations (from 0.004 wt. /vol., up to 1 wt./vol.) the stability of the compositions is better than when using polysorbate 20.

Table 3

Results of studying the stability of compositions 5 - 10 in terms of Purity,% of the main substance using RP-HPLC, CEX-HPLC, SE-HPLC___________

Song number (40±2)°С; (75±5)% (25±2)°; (60±5)% RP-HPLC 0 days 7 days 14 days 28 days 3 months 6 months 5 98.4 98.2 98.0 97.6 98.0 97.5 6 98.3 98.4 98.3 97.8 98.0 97.5 7 98.4 98.4 98.2 98.1 98.2 98.0 8 98.5 98.4 98.3 98.3 98.4 98.3 9 98.4 98.3 98.3 98.2 98.4 98.2 10 98.5 98.4 98.3 98.2 98.4 98.3 CEX-HPLC 5 98.4 98.2 98.1 97.6 97.5 97.5 6 98.5 98.4 98.2 97.9 98.3 98.0 7 98.5 98.4 98.3 98.1 98.3 98.1 8 98.6 98.4 98.3 98.4 98.6 98.5 9 98.5 98.2 98.3 98.3 98.5 98.3 10 98.4 98.4 98.3 98.3 98.2 98.2 SE-HPLC 5 99.4 99.2 99.1 98.6 99.0 98.5 6 99.5 99.4 99.2 98.9 99.3 99.0 7 99.5 99.3 99.3 99.1 99.3 99.1 8 99.5 99.4 99.3 99.4 99.5 99.4 9 99.4 99.3 99.2 99.2 99.4 99.3 10 99.4 99.4 99.3 99.1 99.3 99.1

The increase in stability, namely the decrease in the chemical degradation products of romiplostim when replacing polysorbate-20 with poloxamer 188, can be explained by the fact that poloxamer 188, due to the absence of an ester bond and an unsaturated alkyl chain in its chemical structure, is less susceptible to oxidation and hydrolysis compared to polysorbate 20, as a result of which in the compositions, fewer reactive surfactant decomposition products that interact with romiplostim accumulate. The decrease in romiplostim aggregation when using poloxamer 188 is due to both the better stability of poloxamer 188 compared to polysorbate 20 and an increase in its concentration. Thus, replacing surfactants and increasing the concentration of surfactants in the compositions are appropriate. The optimal concentration of poloxamer 188 is 0.05 to 1% w/v, preferably 0.07% w/v.

The next stage of development was to study the effect of replacing sucrose with trehalose, as well as determining the optimal mannitol:trehalose ratio at the optimal concentration of poloxamer 188.

Formulations 11-15 are prepared with different ratios of mannitol:trehalose, varying the amount of trehalose and adjusting the level of mannitol so that the osmolarity of the finished compositions is in the physiological range (consensus range for subcutaneous administration 269-360 mOsm/kg). Blood osmolarity is about 300 mOsm/kg. According to literature sources, for a noticeable painful effect from the injection, the osmolarity must be more than 600 mOsm/kg. It is accepted that drugs for injection should be prepared in the form of isotonic solutions (osmolality about 300 mOsm/kg) [15,16].

Table 4

Qualitative and quantitative composition of compositions 11-15

Song number AND 12 13 14 15 Romiplostim 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml 0.50 mg/ml L-histidine 25 mM 25 mM 25 mM 25 mM 25 mM Mannitol 4% w/v 4.5% w/v 3% w/v 1.5 % w/v 2% w/v Trehalose 2% w/v 1.5% w/v 3% w/v 4.5 % w/v 4% w/v Poloxamer 188 0.07% w/v 0.07% w/v 0.07% w/v 0.07% w/v 0.07% w/v Dilute solution of HC1 (10%) up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0

Compositions 11-13 have better stability compared to the stability of compositions 110. According to the data presented in table. 5, and as can be seen in FIG. 4-6, the most preferred ratios of mannitol:trehalose are 2:1, 3:1 and 1:1 (compositions 11, 12 and 13). With an increase in the content of trehalose in the composition (compositions 14 and 15), the purity of the lyophilisate, determined during accelerated storage ((25±2)°C; (60±5)%) and storage under stress conditions ((40±2)°C , (75±5)%) falls, the compositions acquire an unsatisfactory appearance.

- 5 044682

Table 5

Results of studying the stability of compositions 11-15 in terms of Purity,% of the main substance __methods RP-HPLC, CEX-HPLC, SE-HPLC___________

Song number (40±2)°С; (75±5)% (25±2)°; (60±5)% RP-HPLC 0 days 7 days 14 days 28 days 3 months 6 months eleven 98.9 98.7 98.7 98.6 98.3 98.5 12 98.8 98.7 98.7 98.4 98.6 98.4 13 98.9 98.7 98.6 98.5 98.7 98.5 14 98.7 98.6 98.4 98.1 97.7 97.8 15 98.8 98.5 98.4 98.0 98.5 98.0 CEX-HPLC eleven 98.9 98.8 98.7 98.7 98.5 98.6 12 98.8 98.7 98.6 98.6 98.7 98.4 13 98.9 98.7 98.7 98.5 98.8 98.5 14 98.8 98.7 98.5 98.00 98.5 97.9 15 98.7 98.6 98.4 98.1 98.3 97.8 SE-HPLC AND 99.6 99.6 99.4 99.3 99.5 99.3 12 99.5 99.4 99.5 99.3 99.3 99.1 13 99.5 99.3 99.2 99.2 99.3 99.2 14 99.6 99.3 99.00 98.6 99.0 98.8 15 99.5 99.2 98.9 98.7 98.3 98.4

In order to evaluate the effect of minor changes in the concentration of romiplostim in solution before lyophilization on stability, compositions 16-19 were prepared in the romiplostim concentration range from 0.45 to 0.55 mg/ml. Compositions 16-19 were prepared as described above with the qualitative and quantitative composition given in table. 6. Due to the change in the concentration of the active substance in the compositions, the volume of the solution placed in the bottle for subsequent lyophilization was changed so that the content of the active substance in all bottles (for compositions 16-19) was equal to 375 mcg.

The results of studying the stability in terms of purity (% of the main peak) using the RP-HPLC, CEX-HPLC, SE-HPLC methods did not show statistically significant differences between compositions 16-19.

Table 6

Qualitative and quantitative composition of compositions 16-19

Song number 16 17 18 19 Romiplostim 0.45 mg/ml 0.5 mg/ml 0.521 mg/ml 0.55 mg/ml L-histidine 25 mM 25 mM 25 mM 25 mM Mannitol 3.6% w/v 4% w/v 4.2% w/v 4.4% w/v Trehalose 1.8 2 wt. %/about.% 2.1 wt.%/vol.% 2.2 wt.%/vol.% Poloxamer 188 0.07% w/v 0.07% w/v 0.07% w/v 0.07% w/v Dilute solution of HC1 (10%) up to pH 5.0 up to pH 5.0 up to pH 5.0 up to pH 5.0 The volume of the composition in a bottle with delivered dose 250 mcg* 0.833 ml 0.75 ml 0.72 ml 0.682 ml

* Actual content of romiplostim per vial with a delivered dose of 250-375 mcg.

Compositions 11, 12, 13, 16-19 are stable during long-term storage in the refrigerator (2-8°C) (in

- 6 044682 for at least 2 years), and with accelerated storage.

During storage of compositions 11, 12, 13, 16-19, specific biological activity was also determined. The determination was carried out in an in vitro test using a validated method. The 32D Clone 3 cell line transfected with the human thrombopoietin receptor was used. In response to the addition of romiplostim, the rate of cell proliferation, which was detected by fluorescence, changed in a dose-dependent manner.

There are no statistically significant changes in biological activity compared to the original during storage (see Tables 7 and 8).

Table 7 Results of determination of biological activity during accelerated storage ((25±2)°C; (60±5)%)

Song number % standard activity (from 80 to 125%) 0 months 3 months 6 months eleven 108 98 119 12 103 86 114 13 99 101 109 16 102 99 111 17 98 101 117 18 118 109 116 19 92 FROM 99

Table 8

Results of determination of biological activity during long-term storage at 2-8°C

Song number % standard activity (from 80 to 125%) 0 months 3 months 6 months 12 months 18 months 24 months AND 99 95 100 98 119 105 12 100 97 105 101 99 113 13 95 105 110 105 96 111 16 103 95 111 98 100 99 17 95 103 115 96 110 106 18 105 98 113 103 101 102 19 91 102 95 112 101 96

The stability of the compositions proposed in the invention was assessed in the concentration range of romiplostim from 0.1 to 1 mg/ml under accelerated storage and stress conditions. Poloxamer 188, at concentrations ranging from 0.004 to 1% w/v, has been shown to inhibit aggregation and chemical degradation over the entire concentration range of romiplostim studied. Increasing romiplostim concentrations above 1 mg/mL is not of interest due to the low dosage of romiplostim in the clinic. The use of formulations with romiplostim concentrations below 0.5 mg/mL may result in more accurate dosing during vial filling because volume measurement error decreases with increasing measured volume.

To evaluate the effect of pH on stability, compositions with qualitative and quantitative compositions corresponding to compositions 17 and 18 were prepared using a histidine buffer, the pH of which was varied from 4 to 6. Changing the pH of the compositions in the range from 4 to 6 does not affect the stability of the compositions.

- 7 044682

Bibliography.

1. Kuter D. J., Begley C. G. Recombinant human thrombopoietin: Basic biology and evaluation of clinical studies. Blood. 2002; 100(10):3457-3469. doi: 10.1182/blood.V 100.10.3457

2. Instructions for use of the medicinal product for medical use Enplate (NPLATE). URL: http://grls.rosminzdrav.ru (access date 05/31/21).

3. Mytych D. T., Park J. K., Kim J. et al. Assessment of romiplostim immunogenicity in adult patients in clinical trials and in a global postmarketing registry. British Journal of Haematology, 2020; 190 (6), 923 - 932. https://doi.org/10.1182/blood-2018-99-113484

4. Grassi L., Cabrele C. Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions. Amino Acids. 2019; 51: 1409-1431. https://doi.org/10.1007/s00726-019-02787-2

5. Zapadka K. L., Becher F. J., Gomes dos Santos A. L., Jackson S. E. Factors affecting the physical stability (aggregation) of peptide therapeutics. Interface Focus. 2017; 7(6):1-16 doi: 10.1098/rsfs.2017.0030

6. Arshinova O. Yu., Oborotova N. A., Sanarova E. V. Excipients in the technology of lyophilization of drugs. Development and registration of medicines. 2013; 1(2):20 - 25.

7. Edward T. Maggio. Polysorbates, peroxides, protein aggregation, and immunogenicity are a growing concern. J. Excipients and Food Chern. 2012; 3 (2): 45 - 53.

8. Kerwin B. A. Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways. Journal of Pharmaceutical Sciences. 2008; 97(8), 2924-2935. doi:10.1002/jps.21190

9. Frison-Norrie S., Spoms, P. Investigating the Molecular Heterogeneity of Polysorbate Emulsifiers by MALDI-TOF MS. Journal of Agricultural and Food Chemistry. 2001; 49(7): 3335-3340. doi:10.1021/jf010096w

10. Donbrow M., Azaz E., Pillersdorf A. Autoxidation of Polysorbates. Journal of Pharmaceutical Sciences. 1978; 67(12), 1676-1681. doi: 10.1002/jps.2600671211

11. Zhou J., Qiu J., Jiang G., Zhou C., Bingham N., Yeung H., Tressel T. Non-specific binding and saturation of Polysorbate-20 with aseptic filter membranes for drug substance and drug product during mAb production. Journal of Membrane Science. 2008; 325(2): 735-741. doi:10.1016/j.memsci.2008.08.046

12. ICH Topic QI A Stability testing of new drug substances and products.

13. ICH Topic Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products.

14. ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. CPMP/ICH/365/96 September 1999.

15. Usach I., Martinez R., Festini T., Peris J.-E. Subcutaneous Injection of Drugs: Literature Review of Factors Influencing Pain Sensation at the Injection Site. Advances in Therapy 2019; 36: 2986 - 2996. doi:10.1007/sl2325-019-01101-6

16. Wang W. Tolerance of hypertonic injectables. International Journal of Pharmaceutics.

2015; 490: 308 - 315. https://doi.Org/10.1016/j.ijpharm.2015.05.069

List of figures, drawings and other materials

Fig. 1. Study of the stability of compositions 5-10 using the RP-HPLC method (storage conditions: (40±2)°C; (75±5)%).

Fig. 2. Study of the stability of compositions 5-10 using the CEX-HPLC method (storage conditions: (40±2)°C; (75±5)%).

Fig. 3. Study of the stability of compositions 5-10 by SE-HPLC (storage conditions: (40±2)°C; (75±5)%).

Fig. 4. Study of the stability of compositions 11-15 by RP-HPLC method (storage conditions: (40±2)°C; (75±5)%).

- 8 044682

Fig. 5. Study of the stability of compositions 11-15 by CEX-HPLC (storage conditions: (40±2)°C; (75±5)%).

Fig. 6. Study of the stability of compositions 11-15 by SE-HPLC (storage conditions: (40±2)°C; (75±5)%).

Claims (5)

CLAIM 1. A pharmaceutical composition containing romiplostim, in the form of a solution for intravenous or subcutaneous administration, characterized in that it contains histidine buffer, mannitol, trehalose and poloxamer 188 as excipients, where a) concentration of romiplostim from 0.1 to 1.0 mg/ml; b) concentration of histidine buffer from 5 to 25 mM, pH 4-6; c) mannitol concentration from 3 to 4.5% w/v; d) trehalose concentration from 1.5 to 3% w/v; e) the concentration of poloxamer 188 is from 0.004 to 1% w/v, where the concentration of mannitol and trehalose is in a ratio of 1:1 to 3:1. 2. A pharmaceutical composition containing romiplostim in the form of a solution for lyophilization, characterized in that it contains histidine buffer, mannitol, trehalose, poloxamer 188 as excipients, where a) concentration of romiplostim from 0.1 to 1.0 mg/ml; b) concentration of histidine buffer from 5 to 25 mM, pH 4-6; c) mannitol concentration from 3 to 4.5% w/v; d) trehalose concentration from 1.5 to 3% w/v; e) the concentration of poloxamer 188 is from 0.004 to 1% w/v, where the concentration of mannitol and trehalose is in a ratio of 1:1 to 3:1. 3. Pharmaceutical composition according to claim 2, characterized in that a) the concentration of romiplostim is 0.5 mg/ml; b) the concentration of histidine buffer is 25 mM, pH 4.8-5.3; c) the concentration of mannitol is 4% w/v; d) trehalose concentration is 2% w/v; e) the concentration of poloxamer 188 is 0.07% w/v, where the concentrations indicated are the concentrations in solution before lyophilization. 4. Pharmaceutical composition according to claim 2, characterized in that a) the concentration of romiplostim is 0.521 mg/ml; b) the concentration of histidine buffer is 25 mM, pH 4.8-5.3; c) the concentration of mannitol is 4.2% w/v; d) trehalose concentration is 2.1% w/v; e) the concentration of poloxamer 188 is 0.07% w/v, where the concentrations indicated are the concentrations in solution before lyophilization. 5. Lyophilisate for preparing a solution of romiplostim for intravenous or subcutaneous administration, characterized in that it is obtained from the compositions according to claims 2-4.