Liquid preparation for injection of recombinant human granulocyte macrophage stimulating factor

Abstract

The invention relates to the field of biological agents, and particularly provides a liquid preparation for injection of a recombinant human granulocyte macrophage stimulating factor (GM-CSF). The preparation provided by the invention does not contain human serum albumin, a preservative, an EDTA chelating agent and the like, solves the potential safety risk problem of the human serum albumin and the preservative in the prior art, and keeps the GM-CSF stable for a long time in a liquid state by selecting a proper stabilizer, a proper antioxidant and a proper pH value range; the water injection with the preferable formula has excellent stability under the condition that the long-term time is 30 months at the temperature of 2-8 DEG C.

Description

Technical Field

[0001] The present invention relates to the field of biological agents. Specifically, the present invention relates to a liquid preparation for injection of recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF). Background Art

[0002] Human granulocyte macrophage colony-stimulating factor is a cytokine with multiple potentials. It can not only act on hematopoietic stem and progenitor cells to promote bone marrow hematopoiesis, but also act on dendritic cells, which are currently known as the most powerful antigen-presenting cells in the body, to promote immune responses, regulate immune reactions, and also act on keratinocytes, fibroblasts, mucosal cells, etc. Currently, recombinant human granulocyte macrophage colony-stimulating factor has been successfully used clinically to treat leukopenia caused by radiotherapy and chemotherapy of malignant tumors, as well as the treatment of bone marrow transplantation, aplastic anemia, and certain immunodeficiency diseases with leukopenia. In addition, the research on recombinant human granulocyte macrophage colony-stimulating factor in new fields such as promoting angiogenesis, promoting immune responses, and anti-infection has also received increasing attention.

[0003] Currently, the commercially available GM-CSF preparations at home and abroad include various dosage forms such as injections, inhalants, gels or ointments, etc., but all the injections are lyophilized preparations and there is no liquid preparation. The patent research data of WO02 / 069999 shows that IMMUNEX COPRORATION developed an early GM freeze-dried powder injection (LEUKINE lyophilized formulation) and a GM aqueous injection (LEUKINE Liquid formulation) containing EDTA. However, in January 2008, the US FDA found that due to the presence of EDTA in the aqueous injection leukine, the adverse reaction of syncope showed an increasing trend, while there was no upward trend in the adverse reaction of syncope in the EDTA-free lyophilized formulation leukine. Therefore, the leukine aqueous injection preparation on the US market has been withdrawn from the market.

[0004] Currently, the original research drug leukine and domestic and foreign biosimilars mainly exist in the form of powder injections. The lyophilized formulation leukine is equipped with a diluent containing benzyl alcohol as a reconstituting solvent. The liquid medicine containing benzyl alcohol will cause pain at the injection site during injection, is irritating to the human body, and will also affect the activity of GM-CSF. Moreover, the toxic and side effects of benzyl alcohol limit the applicable population of the drug.

[0005] Most domestic biosimilars contain human albumin. As a commonly used excipient in biopharmaceuticals, human albumin is mainly extracted from human plasma. Using blood-derived products is prone to infection with other diseases in the blood, which will bring certain risks, such as carrying infectious sources, including some pathogenic microorganisms and unknown viruses, etc. Potential adverse reactions of human albumin include local skin swelling, rash, asthma, vomiting, abdominal pain, etc.

[0006] Lyophilized preparations need to be reconstituted before use, which is inconvenient to operate and may cause secondary contamination; if the reconstitution is not good, it may also affect the therapeutic effect. At present, there is no approved injectable liquid preparation of GM-CSF at home and abroad. Compared with the lyophilized dosage form, the liquid preparation has obvious advantages, such as no need to dissolve, convenient administration, simple manufacturing process, significantly reduced cost, easy to be accepted by doctors and patients, and less quality risk, etc. There is no aqueous injection form of GM-CSF on the market at present. Since the N-terminus of GM-CSF is easily degraded in the liquid solution state and the product is easily oxidized, it may affect the stability of the product. Summary of the Invention

[0007] In order to solve the above-mentioned technical problems, the present invention creatively develops a recombinant human granulocyte macrophage colony-stimulating factor injectable liquid preparation without human albumin, preservatives and EDTA, and this preparation has excellent stability and safety. In order to achieve the above technical effects, the present invention provides the following technical solutions:

[0008] First of all, in the first aspect of the present invention, there is provided a recombinant human granulocyte macrophage colony-stimulating factor injectable liquid preparation, which includes recombinant human granulocyte macrophage colony-stimulating factor, a stabilizer and an antioxidant, and does not contain human albumin and preservatives; the pH value of the preparation is 6-8.

[0009] In one embodiment, the stabilizer is selected from one or more of methionine, glycine, mannitol, sucrose, and trehalose.

[0010] In a preferred embodiment, the antioxidant is selected from one of methionine, sodium metabisulfite, and sodium bisulfite.

[0011] In a preferred embodiment, the concentration of methionine in the preparation is 10-100 mM.

[0012] In one embodiment, the concentration of methionine in the preparation is 20 mM.

[0013] In one embodiment, the preparation further includes other pharmaceutical excipients, including one or several of non-ionic surfactants, isotonic agents, and buffers.

[0014] In a preferred embodiment, the non-ionic surfactant includes one or more of polysorbate and poloxamer, the isotonic agent includes one or more of sodium chloride, potassium chloride, sodium sulfate, sorbitol, and potassium sulfate, and the buffer includes phosphate buffer solution.

[0015] In a preferred embodiment, the concentration of sodium chloride is 20 - 100 mM.

[0016] In a most preferred embodiment, the formulation of the preparation is 10 mM PB (pH 7.0) + 5% mannitol + 20 mM methionine + 0.15 mg / ml GM-CSF; or 10 mM PB (pH 7.0) + 20 mM methionine + 100 mM NaCl + 0.15 mg / ml GM-CSF.

[0017] In a most preferred embodiment, the concentration range of GM-CSF in the preparation is 0.05 - 10 mg / ml.

[0018] Compared with the prior art, the present invention has obtained the following remarkable progress:

[0019] 1. The stable GM-CSF aqueous solution preparation provided by the present invention does not contain human serum albumin, preservatives, EDTA chelating agents, etc., solving the potential safety risk problems of human serum albumin and preservatives in the prior art;

[0020] 2. The present invention also discovers that appropriate stabilizers and pH value ranges are important factors for GM-CSF to maintain stability in solution state, and adding suitable antioxidants plays an important role in maintaining the stability of GM-CSF aqueous solution, providing a basis for the development of GM-CSF liquid preparations;

[0021] 3. Through post-translational modification analysis of peptide segments, the present invention first discovers the influence of the M1 / M37 / M47 / M80 / W123 (M is methionine, W is tryptophan) sites on the GM-CSF protein on the stability of the preparation, as well as the mechanism of methionine improving the stability of the GM-CSF aqueous solution preparation;

[0022] 4. Compared with the same type of products, such as the liquid preparation of GM-CSF disclosed in the publication number CN115297844A, it can only be stored for 18 months at 5°C, while the water injection dosage forms of the preferred formulations 18 and 8 of the present invention have excellent stability under the conditions of 2 - 8°C for 30 months in the long term.

[0023] 5. Under the preferred formulation of the present invention, the GM-CSF protein concentration range can cover 0.05 - 10 mg / ml. Brief Description of the Drawings

[0024] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the accompanying drawings:

[0025] Figure 1 It is the amino acid sequence diagram of rhGM-CSF

[0026] Figure 2 It is the long-term stability trend diagram of rhGM-CSF injection at 2-8°C - purity (HPLC-SEC method) - formulation 18 (left) and formulation 8 (right);

[0027] Figure 3 It is the long-term stability trend diagram of rhGM-CSF injection at 2-8°C - purity (HPLC-RP method) - formulation 18 (left) and formulation 8 (right);

[0028] Figure 4 It is the long-term stability trend diagram of rhGM-CSF injection at 2-8°C - activity change - formulation 18 (left) and formulation 8 (right);

[0029] Figure 5 It is the overlay diagram (left) and enlarged diagram (right) of the results of the influence of methionine and EDTA on the GM-CSF aqueous solution - related proteins (HPLC-RP method).

[0030] Figure 6 It is the overlay diagram (left) and enlarged diagram (right) of the results of the influence of different protein concentrations on the GM-CSF aqueous solution - related proteins (HPLC-RP method).

[0031] Figure 7 It is the overlay diagram (left) and enlarged diagram (right) of the results of the influence of different methionine concentrations on the high-concentration GM-CSF aqueous solution - related proteins (HPLC-RP method). Detailed implementation manners

[0032] The following is an illustration of the preferred embodiments of the present invention with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0033] Example 1 Influence of pH value on the stability of GM-CSF solution

[0034] When considering the influence of the pH value, the influence of other stabilizers on the GM-CSF liquid preparation for injection is not considered temporarily. GM-CSF formulations with different pH values are filled and placed in an incubator at 37°C for 7 days. The results are shown in the following table:

[0035] Table 1 Influence of different pH values on the GM-CSF aqueous solution

[0036]

[0037] As can be seen from the results in Table 1, relatively more related proteins are likely to be produced when the pH value of the GM-CSF solution is in the range of 4.0 - 5.0. At the same time, it is found that when the pH value exceeds 8.0, it is also unstable and the related proteins also increase. When the pH value is 7.0 in the phosphate buffer system, the content of related proteins is relatively low. It can be seen that the quality of the GM-CSF solution is relatively stable under this condition.

[0038] Example 2: Influence of Different Amino Acids on Aqueous Solution of GM-CSF

[0039] When considering the influence of different amino acids (such as glycine, lysine, aspartic acid, methionine, etc.) on the aqueous solution of GM-CSF and comparing with the formula without amino acids, from Example 1, it is known that the GM-CSF solution is relatively stable when the pH is 7.0 in the phosphate buffer system. Therefore, in this example, the pH and buffer system are fixed, and after preparing the following solutions, the samples are examined in an incubator at 37°C for 7 days:

[0040] Table 2: Influence of Different Amino Acids on Aqueous Solution of GM-CSF

[0041]

[0042]

[0043] As can be seen from the results in Table 2: Compared with the control group (formula 3), after adding glycine, aspartic acid, and lysine to the aqueous solution of GM-CSF, the content of related proteins is relatively high, and after adding methionine, the content of related proteins is relatively low. It can be seen that adding methionine can improve the stability of the GM-CSF solution.

[0044] Example 3: Influence of Different Antioxidants on Aqueous Solution of GM-CSF

[0045] When considering the influence of different antioxidants (such as methionine, sodium metabisulfite, sodium bisulfite) on the aqueous solution of GM-CSF, from Example 1, it is known that the GM-CSF solution is relatively stable when the pH is 7.0 in the phosphate buffer system. Therefore, in this example, the pH and buffer system are fixed, and the solution samples with the following formulas are examined in an incubator at 37°C for 7 days:

[0046] Table 3: Influence of Different Antioxidants on Aqueous Solution of GM-CSF

[0047]

[0048] As can be seen from the results in Table 3: Compared with the control group (formula 3), after adding methionine, the content of related proteins is relatively low, and after adding sodium metabisulfite and sodium bisulfite, impurity bands are detected. It can be seen that adding methionine can improve the stability of the GM-CSF solution.

[0049] Example 4: Influence of Different Methionine Concentrations on the Aqueous Solution of GM-CSF

[0050] When considering the influence of different methionine concentrations on the aqueous solution of GM-CSF, it can be seen from Example 1 that the GM-CSF solution is relatively stable at pH 7.0 under the phosphate buffer system. Therefore, in this example, the pH and buffer system are fixed, and a suitable methionine concentration is screened. Solution samples with the following formulations are examined in an incubator at 37°C for 60 days:

[0051] Table 4: Influence of Different Methionine Concentrations on the Aqueous Solution of GM-CSF

[0052]

[0053] It can be seen from the results in Table 4 that compared with the control group (Formulation 3), the content of the relevant protein is lower after adding 20 mM methionine. It can be seen that a methionine concentration of 20 mM is relatively optimal.

[0054] Example 5: Influence of Whether to Add Polysorbate on the Aqueous Solution of GM-CSF

[0055] When considering whether adding polysorbate has an impact on the aqueous solution of GM-CSF, it can be seen from Example 4 that the GM-CSF solution is relatively stable under the phosphate buffer system, pH 7.0, and 20 mM methionine. Therefore, in this example, the pH, buffer system, and methionine concentration are fixed, and solution samples with the following formulations are examined in an incubator at 37°C for 60 days:

[0056] Table 5: Influence of Polysorbate on the Aqueous Solution of GM-CSF

[0057]

[0058]

[0059] It can be seen from the results in Table 5 that compared with Formulation 8 and Formulation 18, the addition of polysorbate does not significantly increase the content of the relevant protein. Considering that polysorbate has functions such as solubilization, emulsification, stabilization, and wetting, it can be added according to requirements.

[0060] Example 6: Influence of Different Salt Concentrations on the Aqueous Solution of GM-CSF

[0061] When examining the influence of different salt concentrations (such as NaCl) on the stability of the GM-CSF solution, it can be seen from Example 4 that the GM-CSF solution is relatively stable under the phosphate buffer system, pH 7.0, and 20 mM methionine. Therefore, in this example, the pH, buffer system, and methionine concentration are fixed, and solution samples with the following formulations are examined in an incubator at 37°C for 60 days:

[0062] Table 6 Effects of salts at different concentrations on GM-CSF aqueous solution

[0063]

[0064] As can be seen from the results in Table 6: Compared with Formulation 8, the content of related proteins decreased slightly after adding salts at different concentrations. It can be seen that adding a small amount of sodium chloride to the GM-CSF aqueous solution can effectively reduce the production of GM-CSF-related proteins and enhance the stability of the GM-CSF aqueous solution. The concentration of sodium chloride is 20 - 100 mM.

[0065] Example 7 Effects of sugars and polyols on GM-CSF aqueous solution

[0066] When considering the effects of sugars and polyols on the stability of the GM-CSF solution, it can be seen from Example 1 that the GM-CSF solution is relatively stable at pH 7.0 under the phosphate buffer system. Therefore, in this example, the pH and buffer system were fixed, and the following formulations were prepared:

[0067] Table 7 Effects of sugars and polyols on GM-CSF aqueous solution

[0068]

[0069]

[0070] As can be seen from the results in Table 7: The content of related proteins in Formulations 21, 22, and 23 is relatively low. Compared with Formulation 23, the content of related proteins in Formulation 8 is lower. It can be seen that mannitol has a certain stabilizing effect and is also an isotonic agent, and its applicable content is 0.5% - 5%.

[0071] Example 8 Stability study of the optimized formulation

[0072] Three batches of samples of the optimized Formulation 18 (Batch 1, Batch 2, Batch 3, specification: 100 μg / 0.5 ml / vial) and three batches of samples of Formulation 8 (Batch 4, Batch 5, Batch 6, specification: 200 μg / 0.5 ml / vial) were subjected to a 30-month long-term stability study at 2 - 8°C. Items such as appearance, visible foreign matters, insoluble particles, filling volume difference, pH value, protein content (HPLC-SEC method), purity (HPLC-SEC method), purity (HPLC-RP method), impurities (reduced and non-reduced SDS-PAGE methods), biological activity, sterility test, abnormal toxicity test, and bacterial endotoxin test were all investigated. The specific investigation item frequencies and evaluation criteria are shown in Table 8.

[0073] Table 8 Investigation item frequencies and evaluation criteria for the optimized formulations (Formulation 8 and Formulation 18)

[0074]

[0075]

[0076] Note: " ★ " means that obvious visible foreign matters such as glass chips, metal chips, fibers, color spots, and color patches are recorded as Class A; white spots, fine protein flocs or protein particles are recorded as Class B; a small amount of flocs or protein particles, trace deposits, and precipitates that cannot be shaken off are recorded as Class C.

[0077] The inspection results show that the optimized prescription rhGM-CSF aqueous solution preparation was inspected at 2-8°C for 30 months. The results of appearance, visible foreign matters, insoluble particles, osmotic pressure, filling volume difference, pH value, biological activity, sterility inspection, abnormal toxicity inspection, bacterial endotoxin, impurities (reduced and non-reduced SDS-PAGE methods), purity (HPLC-RP method), protein content (HPLC-SEC method), and purity (HPLC-SEC method) all meet the standard requirements. Among them, the purity range of (HPLC-SEC method) is 99.8% - 100.0%, and the purity range of (HPLC-RP method) is 97.5% - 100.0%. This shows that under the condition of long-term 30 months at 2-8°C, the water injection dosage forms of the optimized formula 18 and formula 8 (positive control) both have excellent stability.

[0078] The trend analysis charts of purity (HPLC-SEC method), purity (HPLC-RP method), and biological activity are as Figures 1 to 3 shown.

[0079] Mechanism study on the stability of Example 9

[0080] The optimized formula 8 (containing methionine) and the control group formula 3 (without methionine) were used to compare and analyze the relevant proteins and post-translational modifications after 7 / 14 / 30 days of inspection at 40°C high temperature. The results show that the content of the relevant proteins (proteins with GM-CSF modification or degradation or aggregation) in the prescription containing methionine is slightly lower than that of the control group (see Figure 4 , Table 9). After adding methionine, the oxidation ratio of the sample is about 3% - 8% lower than that of the control group (Table 10), and the antioxidant effect of methionine becomes more obvious with the extension of the inspection time; according to the results of post-translational modification, methionine mainly plays an antioxidant protective role on the M1 / M37 / M47 / M80 / W123* (M represents methionine, W represents tryptophan) sites on the GM-CSF protein, thereby enhancing the stability of the rhGM-CSF aqueous solution preparation.

[0081] Note: The amino acid numbers of "M1 / M37 / M47 / M80 / W123*" are based on the amino acid sequence in "Human Granulocyte-Macrophage Colony-Stimulating Factor for Injection" in the Chinese Pharmacopoeia 2020 Edition (see Figure 1) Numbering starts from the 1st amino acid M1 at the N-terminus to the 128th amino acid E128 at the C-terminus.

[0082] Table 9 Summary Table of Results of Related Proteins in the Study on the Mechanism of Action of Antioxidant Methionine

[0083]

[0084] Table 10 Summary Table of Results of Post-Translational Modification Oxidation in the Study on the Mechanism of Action of Antioxidant Methionine

[0085]

[0086] Example 10 Comparing the Effects of Methionine and EDTA on Aqueous GM-CSF Solution

[0087] Comparing the effects of methionine and EDTA on aqueous GM-CSF solution, comparing Formulation 24 and Formulation 25 with Formulation 8 for research. Under the condition of 40 °C high temperature, after 7 / 14 / 30 days, comparative analysis was carried out through the situation of related proteins. Compared with Formulation 8 (containing methionine), as the inspection time extended, the content of related proteins in the formulation containing EDTA increased significantly (see Table 11, Figure 5 ) It can be seen that adding methionine to the aqueous GM-CSF solution has a better effect than EDTA, and adding methionine can make the aqueous GM-CSF solution more stable.

[0088] Table 11 Summary Table of Results of Related Proteins on the Effect of EDTA on Aqueous GM-CSF Solution

[0089]

[0090] Example 11 Comparing the Effects of Different Protein Concentrations on Aqueous GM-CSF Solution

[0091] Explore and study the concentration of rhGM-CSF in the optimized prescription. Compare Formulation 8, Formulation 26, Formulation 27, Formulation 28, and Formulation 29 for research. Under the condition of 40 °C high temperature, after 3 / 7 days, comparative analysis was carried out through the situation of related proteins (HPLC-RP) and post-translational modification (PTMs). It was found that the total sum of related proteins and the proportion of post-translational modification oxidation in rhGM-CSF aqueous solutions with different protein concentrations all increased slightly and the increase amounts were basically the same (see Table 12, Table 13, Figure 6 ) It shows that different protein concentrations (in the range of 0.05 mg / ml - 10.0 mg / ml) have basically the same effect on rhGM-CSF aqueous solution.

[0092] Table 12 Summary Table of Results of Related Proteins on the Effect of Different Protein Concentrations on Aqueous GM-CSF Solution

[0093]

[0094]

[0095] Table 13 Summary Table of the Effects of Different Protein Concentrations on GM-CSF Aqueous Solution - Results of Post-Translational Modification Oxidation

[0096]

[0097] Example 12 Comparing the Effects of Different Methionine Concentrations on High-Concentration GM-CSF Aqueous Solution

[0098] To compare the effects of different methionine concentrations on high-concentration GM-CSF aqueous solution, Formulation 28 was compared with Formulation 30. Under the high-temperature condition of 40°C, after 3 / 7 days, comparative analysis was carried out through relevant proteins (HPLC-RP) and post-translational modifications (PTMs). It was found that the total sum of the relevant proteins and the proportion of post-translational modification oxidation in high-concentration GM-CSF aqueous solutions with different methionine concentrations both increased slightly and the increase amounts were basically the same (see Table 14, Figure 7 ), indicating that the effects of different methionine concentrations (in the range of 20 mM - 100 mM) on high-concentration rhGM-CSF aqueous solution are basically the same.

[0099] Table 14 Summary Table of the Effects of Different Methionine Concentrations on High-Concentration GM-CSF Aqueous Solution - Results of Relevant Proteins

[0100]

[0101]

[0102] Table 15 Summary Table of the Effects of Different Methionine Concentrations on High-Concentration GM-CSF Aqueous Solution - Results of Post-Translational Modification Oxidation

[0103]

[0104] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A liquid preparation for injection of recombinant human granulocyte macrophage-stimulating factor, characterized in that, The preparation comprises recombinant human granulocyte macrophage-stimulating factor, a stabilizer and an antioxidant, and does not contain human serum albumin and preservatives; the pH value of the preparation is 6-8.

2. The preparation according to claim 1, characterized in that, The stabilizer is selected from one or more of methionine, glycine, mannitol, sucrose, and trehalose.

3. The preparation according to claim 1, characterized in that, The antioxidant is selected from one of methionine, sodium metabisulfite, and sodium bisulfite.

4. The preparation according to claim 2 or 3, characterized in that, The concentration of methionine in the preparation is 10-100 mM.

5. The preparation according to claim 4, characterized in that, The concentration of methionine in the preparation is 20 mM.

6. The preparation according to claim 1, characterized in that The preparation further comprises other pharmaceutical excipients, including one or more of non-ionic surfactants, isotonic agents, and buffers.

7. The preparation according to claim 6, characterized in that, The non-ionic surfactant includes one or more of polysorbate and poloxamer, the isotonic agent includes one or more of sodium chloride, potassium chloride, sodium sulfate, sorbitol, and potassium sulfate, and the buffer includes phosphate buffer solution.

8. The preparation according to claim 7, characterized in that, The concentration of sodium chloride is 20-100 mM.

9. The preparation according to claim 1, characterized in that, The concentration of GM-CSF in the preparation is 0.05-10 mg / ml.

10. The preparation according to any one of claims 1-3, 5-8, characterized in that, The formulation of the preparation is 10 mM PB (pH 7.0) + 5% mannitol + 20 mM methionine + 0.15 mg / ml GM-CSF; or 10 mM PB (pH 7.0) + 20 mM methionine + 100 mM NaCl + 0.15 mg / ml GM-CSF.

Images