PEG-hmFGF-21 injection and preparation method and application thereof

Abstract

The present application relates to a kind of PEG-hmFGF-21 injection and its preparation method and application, the composition of the PEG-hmFGF-21 injection is as follows: PEG-hmFGF-21 15-30 mg / ml, pharmaceutically acceptable buffer system 0.5-50mM, at least one surfactant 0.01%-1% w / v, at least one stabilizer 5mg / ml-20mg / ml, pH 4.0-9.0.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a PEG-hmFGF-21 injection solution, its preparation method, and its application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD), also known as non-alcoholic fatty liver, is caused by a variety of factors. The lesions are mainly located in the liver lobules and are characterized by steatosis of hepatocytes and triglyceride (TG) accumulation (the lipid content of liver tissue exceeds 5% of the liver wet mass, or more than 1 / 3 of the hepatocytes are fatty in histological examination). The pathological changes are similar to those of alcoholic liver disease (ALD), but there is no history of excessive alcohol consumption (equivalent to ethanol intake of <140g / week for men and <70g / week for women) or other clear hepatotoxic factors.

[0003] The spectrum of non-alcoholic fatty liver disease includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), non-alcoholic cirrhosis (NAC), and hepatocellular carcinoma.

[0004] If non-alcoholic fatty liver disease (NAFLD) is not controlled in time, the continued excessive accumulation and degeneration of fat can easily progress to non-alcoholic steatohepatitis (NASH). The difference between NASH and NASH lies in the fact that, in addition to excessive fat accumulation, NASH also involves inflammatory cell infiltration, varying degrees of fibrosis, and hepatocyte damage in the liver. Furthermore, if NASH is not effectively controlled and continues to progress, it can easily lead to liver fibrosis, cirrhosis, and even liver cancer.

[0005] Since NASH was first identified as a disease in 1980, drug development for it has been ongoing for decades. However, it wasn't until March 15, 2024, that the first new drug for treating metabolic disorder-associated steatohepatitis (NASH), Resmetirom (trade name: Rezdiffra), was approved by the FDA. While Resmetirom offers an effective treatment strategy for NASH patients, like many drugs, it has some adverse reactions. The most common adverse reactions include diarrhea, nausea, itching, vomiting, constipation, abdominal pain, and dizziness.

[0006] Fibroblast growth factor-21 (FGF-21) is a recently discovered metabolic regulator in vivo. It belongs to the fibroblast growth factor family. Its ability to specifically act on liver, adipose tissue, and pancreatic islet cells and effectively and safely regulate blood glucose and lipids without relying on insulin has attracted the attention of researchers. There are also reports that fibroblast growth factor-21 (FGF-21) can effectively prevent and treat in vitro induced NAFLD (Liu Min et al., Effects of fibroblast growth factor-21 on lipid metabolism in in vitro induced non-alcoholic fatty liver cell model, Journal of Jilin University, May 2012, Vol. 38, No. 3, 477-481).

[0007] Chinese patent CN201310115210.0 (publication number CN103193878A) discloses a novel long-acting mutated human fibroblast growth factor (as shown in sequence 3 of the sequence listing) and a cross-linked product of polyethylene glycol (called PEG-hmFGF-21, also known as lipoprotein), and discloses its use in treating diabetes. Chinese patent CN201880013016.2 (publication number CN110520146A) discloses the use of PEG-hmFGF-21 in treating non-alcoholic steatohepatitis, and Example 1 discloses the composition of the aqueous injection: PEG-hmFGF-21 (concentration 10 mg / mL), histidine (pharmaceutical grade, concentration 10 mg / mL), and citrate-sodium citrate buffer (20 mM sodium citrate-citric acid, 100 mM NaCl, pH 5.5 ± 0.1).

[0008] The existing PEG-hmFGF-21 aqueous injection formulation has a concentration of 10 mg / mL, which is considered a low-concentration injectable. This leads to problems such as frequent and repeated administration, risks and inconvenience, and poor patient compliance. Therefore, there is an urgent need to develop a high-concentration, stable, sterile liquid formulation of PEG-hmFGF-21 suitable for subcutaneous injection. A high-concentration formulation can significantly reduce the dosage and frequency of administration, thus improving patient convenience and compliance. Furthermore, a high-concentration formulation offers many advantages, such as enabling subcutaneous administration of doses <2 ml, supporting self-administration, treating chronic diseases requiring long-term medication while ensuring patient compliance, and saving on production and logistics costs.

[0009] However, high-concentration protein formulations are often accompanied by low solubility, protein aggregation, and high viscosity. The higher the product concentration, the more likely protein-protein interactions will occur. In some cases, these interactions can increase product instability and aggregation. Higher protein concentrations lead to higher protein aggregation rates because closer proximity between constituent substances results in stronger intermolecular attraction and a higher incidence of self-association events. This not only poses a significant challenge to developing drug formulations and dosage forms suitable for clinical use but also places higher demands on the production of both the drug substance and the finished product. Summary of the Invention

[0010] This invention develops a new, high-concentration PEG-hmFGF-21 injection based on the existing low-concentration (10 mg / mL) PEG-hmFGF-21 injection.

[0011] The high-concentration PEG-hmFGF-21 injection solution of the present invention has the following composition:

[0012] PEG-hmFGF-21 15-30 mg / ml

[0013] Pharmaceutically acceptable buffer systems are 0.5-50 mM.

[0014] At least one surfactant, 0.01%-1% w / v,

[0015] At least one stabilizer 5 mg / ml-20 mg / ml,

[0016] pH 4.0-9.0.

[0017] The above components were mixed to prepare an injection solution containing PEG-hmFGF-21 at a concentration of 15-30 mg / ml.

[0018] In one specific embodiment of the present invention, the concentration of PEG-hmFGF-21 is preferably 25 mg / ml.

[0019] In one specific embodiment of the present invention, the pharmaceutically acceptable buffer system is preferably a 20mM citric acid / sodium citrate-100mM sodium chloride buffer system (hereinafter referred to as the Citric B buffer system) of 15-25mM.

[0020] In one specific embodiment of the present invention, the surfactant is preferably polysorbate 80 0.01-0.05% w / v;

[0021] In one specific embodiment of the present invention, the stabilizer is preferably arginine hydrochloride, proline, or histidine at a concentration of 5 mg / ml to 15 mg / ml.

[0022] In one specific embodiment of the present invention, the pH is preferably 5.0-6.0.

[0023] In a specific embodiment of the present invention, the most preferred embodiment of the high-concentration PEG-hmFGF-21 injection solution of the present invention comprises:

[0024] PEG-hmFGF-21 25mg / ml

[0025] Lemon B buffer system 20mM,

[0026] Polysorbate 80 0.04% w / v

[0027] Arginine hydrochloride 10mg / ml

[0028] pH 5.5.

[0029] The present invention further includes the use of the PEG-hmFGF-21 injection solution described herein in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0030] The development strategy of the high-concentration PEG-hmFGF-21 injection of the present invention is as follows: (1) Preliminary screening of excipient combinations (2) Interaction experiment: design interaction to screen the best combination of stabilizer and surfactant (3) Stability confirmation: conduct influencing factors, accelerated and long-term stability studies on several formulation combinations that were better screened in the previous stage, and finally determine the best formulation for the protective effect of high-concentration PEG-hmFGF21 protein.

[0031] Preliminary screening of excipient combinations:

[0032] Using a 20mM citrate B buffer system at pH 5.5 as the base buffer system, trehalose, proline, arginine, and histidine were selected as stabilizers, and polysorbate 80 was selected as the surfactant. Five groups of samples were prepared, and the experimental design is shown in Table 1. The stability of each group of samples was measured under different conditions, and the stability scheme is shown in Table 2. The test results are shown in Tables 3-14. It was preliminarily determined that the proline / polysorbate 80 group and the arginine hydrochloride / polysorbate 80 group had better protective effects on higher concentrations of PEG-hmFGF21 protein.

[0033] Table 1 Experimental group design for primary screening of excipients

[0034]

[0035] Table 2 Experimental Scheme for Initial Screening Stability of Auxiliary Materials

[0036]

[0037] (1) Visual inspection results

[0038] Table 3 Results of Initial Screening / Accelerated / Long-Term Visual Inspection of Excipients

[0039]

[0040] Table 4. Visual Inspection Results of Factors Affecting Initial Screening of Auxiliary Materials

[0041]

[0042] (2) Results of the insoluble particulate test

[0043] Table 5 Results of accelerated / long-term insoluble microparticle screening of excipients

[0044]

[0045] Note: Due to the small number of samples filled in group 5, data at 25±2℃ in February were not monitored. However, based on the overall trend of the 5 groups, it can be determined that these 5 groups were eventually eliminated, thus not affecting the final result.

[0046] Table 6. Results of the test on insoluble microparticles in the initial screening of excipients.

[0047]

[0048] Note: For 0D 2 (high temperature) and 1D 03 04, there was insufficient sample volume in one sample, which caused air bubbles to be drawn in during testing, resulting in higher values. However, this does not affect the overall trend judgment.

[0049] (3) SEC test results

[0050] Table 7 Results of Accelerated Long-Term Stability Test (SEC) for Primary Screening of Excipients

[0051]

[0052] Note: SEC (size exclusion chromatography) - a molecular size-based separation technique used to analyze the molecular weight distribution, polymer content, and purity of biopharmaceuticals.

[0053] Table 8. Results of SEC test on factors affecting the initial screening of excipients.

[0054]

[0055] (4) IEC test results

[0056] Table 9. IEC Results of Accelerated Long-Term Stability Test for Primary Screening of Additives

[0057]

[0058] Note: IEC (Ion Exchange Chromatography) - This method analyzes the distribution of charge isomers by utilizing the interaction between the ion exchange groups of the stationary phase and the charges of charged molecules in the sample.

[0059] Table 10. IEC Test Results of Factors Affecting Initial Screening of Auxiliary Materials

[0060]

[0061] (5) CE-SDS test results

[0062] Table 11 Results of Accelerated Long-Term Stability Test (CE-SDS) for Excipient B1344

[0063]

[0064] Note: (Capillary electrophoresis) - Under the influence of an electric field, samples are separated in a capillary based on differences in molecular charge, size, and shape to analyze the purity, charge isomers, and impurities of biopharmaceuticals.

[0065] Table 12 Results of CE-SDS test on factors affecting the initial screening of excipients.

[0066]

[0067] (6) Viscosity test results

[0068] Table 13 Results of viscosity determination of excipients during initial screening

[0069]

[0070] (7) Results of activity test

[0071] Table 14 Results of Preliminary Screening Activity Determination of Excipients

[0072]

[0073] Designing interactions to screen for the optimal combination of stabilizers and surfactants:

[0074] Using a 20mM pH 5.5 citrate B buffer system as the base buffer system, different concentrations of proline, arginine, and histidine were selected as stabilizers, and different concentrations of Tween 80 and Tween 20 were selected as surfactants. Ten groups of samples were prepared, and the experimental design is shown in Table 15. The stability of each group of samples was measured under different conditions, and the stability scheme is shown in Table 16. The test results are shown in Tables 17-23. It was determined that the combination of 10mg / ml arginine hydrochloride / 0.04% polysorbate 80 and the combination of 10mg / ml proline / 0.04% polysorbate 80 had better protective effects on higher concentrations (20-30mg / ml) of PEG-hmFGF21 protein.

[0075] Table 15 Interaction Experiment Design

[0076]

[0077] Table 16 Experimental Scheme for Interaction Stability

[0078]

[0079] (1) Visual inspection results

[0080] Table 17 Visual Inspection Results of Interactions

[0081]

[0082] (2) Results of insoluble microparticles

[0083] Table 18 Results of Interactions with Insoluble Particles

[0084]

[0085] (3) SEC results

[0086] Table 19 Interaction Results (SEC)

[0087]

[0088] (4) IEC results

[0089] Table 20 IEC Results of Interactions

[0090]

[0091] (5) CEC-SDS results

[0092] Table 21 Results of CE-SDS Interaction (%)

[0093]

[0094] (6) Results of biological activity

[0095] Table 22 Results of biological activity (%) of the interaction

[0096]

[0097] (7) Osmotic pressure results

[0098] Table 23 Results of Interaction Osmolarity (mOsmol / kg)

[0099]

[0100] Stability confirmed:

[0101] The best formulation combinations selected through interaction screening were studied for influencing factors, acceleration, and long-term stability. The optimal formulation for protecting high concentrations (15-30 mg / ml) of PEG-hmFGF21 protein was ultimately determined. Using a 20 mM citrate B buffer system (pH 5.5) as the base buffer, 0.4 mg / ml polysorbate 80 as the surfactant, and 10 mg / ml proline and arginine as stabilizers, four formulations with protein concentrations of 25 mg / ml and 30 mg / ml were prepared. A low-concentration original formulation was also prepared as a reference. The experimental design is shown in Table 24. The stability of each prepared sample under different conditions was measured. The stability scheme is shown in Table 25, and the test results are shown in Tables 26-50. Under conditions of high temperature, shaking, light exposure, and freeze-thaw cycles, the stability of the high-concentration formulation was basically the same as that of the low-concentration formulation, and the IEC purity and the number of insoluble particles were significantly improved. The high-concentration formulation was comparable to the low-concentration formulation in terms of long-term stability. The optimal formulation for the high-concentration (15-30 mg / ml) was determined to be: 10 mg / ml arginine hydrochloride, 0.04% w / v polysorbate 80, pH 5.5, and a 20 mM citric acid B buffer system. The formulation samples should be stored in the dark and protected from repeated freeze-thaw cycles and high temperatures.

[0102] Table 24 Stability Confirmation Experimental Group Design

[0103]

[0104] Table 25 Stability Confirmation Experimental Protocol

[0105]

[0106] (1) Results of visual inspection experiment

[0107] Table 26. Experimental Visual Inspection Results of Factors Affecting Stability Confirmation

[0108]

[0109] Table 27 Visual Inspection Results of Accelerated Stability Test for Stability Confirmation

[0110]

[0111] Table 28 Visual Inspection Results of Long-Term Stability Confirmation Experiments

[0112]

[0113] (2) Results of the insoluble particulate experiment

[0114] Table 29. Factors affecting stability confirmation: Results of experiments on insoluble microparticles.

[0115]

[0116] Table 30 Results of Accelerated Stability Test for Insoluble Particles to Confirm Stability

[0117]

[0118] Table 31 Results of Long-Term Stability Testing for Insoluble Microparticles in Stability Confirmation

[0119]

[0120] (3) Results of the SEC experiment

[0121] Table 32 Stability Confirmation Factors and Experimental Results (SEC)

[0122]

[0123] Table 33. Results of Accelerated Stability Test (SEC) for Stability Confirmation.

[0124]

[0125] Table 34. Results of the Long-Term Stability Experiment (SEC) for Stability Confirmation.

[0126]

[0127] (4) IEC test results

[0128] Table 35 Stability Confirmation Influencing Factors Experimental IEC Results

[0129]

[0130] Table 36 Stability Confirmation: IEC Results of Accelerated Stability Test

[0131]

[0132] Table 37 Stability Confirmation: IEC Results of Long-Term Stability Tests

[0133]

[0134] (5) RP Experiment Results

[0135] Table 38 Results of Experimental RP on Stability Confirmation Factors

[0136]

[0137] Table 39 Results of Accelerated Stability Test (RP) for Stability Confirmation

[0138]

[0139] Table 40. Results of the Long-Term Stability Test (RP) for Stability Confirmation

[0140]

[0141] (6) CE-SDS Experimental Results

[0142] Table 41. Experimental CE-SDS Results of Factors Affecting Stability Confirmation

[0143]

[0144] Table 42 Results of Accelerated Stability Test (CE-SDS) for Stability Confirmation

[0145]

[0146] Table 43. Results of the Long-Term Stability Test (CE-SDS) for Stability Confirmation

[0147]

[0148] (7) Results of activity experiment

[0149] Table 44. Factors affecting stability confirmation and experimental activity results.

[0150]

[0151] Table 45. Results of accelerated stability testing for stability confirmation.

[0152]

[0153] Table 46. Results of long-term stability tests for stability confirmation.

[0154]

[0155] (8) Free PEG

[0156] Table 47. Factors affecting stability confirmation: Experimental results of free PEG.

[0157]

[0158] Table 48. Stability Confirmation Results of Accelerated Experiments on Free PEG

[0159]

[0160] Table 49. Results of long-term experiments confirming the stability of free PEG.

[0161]

[0162] (9) Osmotic pressure

[0163] Table 50 Stability Confirmation Osmotic Pressure Results

[0164] Attached Figure Description

[0165] Figure 1 Long-term stability (upright orientation) of the SEC main peak of high-concentration formulation at 5°C ± 3°C (6M trend graph)

[0166] Figure 2 Long-term stability (inverted) of the SEC main peak of high-concentration formulation at 5°C ± 3°C (6M trend chart)

[0167] Figure 3 Long-term stability (upright orientation) of high-concentration formulation SEC polymer at 5°C ± 3°C (6M trend chart)

[0168] Figure 4 Long-term stability (inverted) of high-concentration formulation SEC polymer at 5°C ± 3°C (6M trend chart)

[0169] Figure 5 Long-term stability (upright orientation) of high-concentration formulation SEC low molecular weight 6M at 5°C ± 3°C: trend chart

[0170] Figure 6 Long-term stability (inverted) of high-concentration formulation SEC low molecular weight at 5°C ± 3°C (6M trend chart)

[0171] Figure 7 Long-term stability (upright orientation) of the main peak of high-concentration formulation at 5°C ± 3°C for 6M trend graph

[0172] Figure 8 Long-term stability (inverted) of the main peak of high-concentration formulation at 5°C ± 3°C (6M trend chart)

[0173] Figure 9 Long-term stability (upright orientation) of RP residual F-related proteins in high-concentration formulations at 5°C ± 3°C (6M trend chart)

[0174] Figure 10 Long-term stability (inverted) of high-concentration formulation RP residual F-related protein at 5°C ± 3°C (6M trend chart)

[0175] Figure 11 Long-term stability (upright orientation) of the IEC main peak of high-concentration formulation at 5°C ± 3°C for 6M trend graph

[0176] Figure 12 Long-term stability (inverted) of high-concentration formulation at IEC main peak at 5°C ± 3°C (6M trend chart)

[0177] Figure 13 Long-term stability (upright orientation) of high-concentration formulation IEC acid peak at 5°C ± 3°C (6M trend graph)

[0178] Figure 14 Long-term stability (inverted) of IEC acid peak in high-concentration formulations at 5°C ± 3°C (6M trend chart)

[0179] Figure 15 Long-term stability (upright orientation) of high-concentration formulation IEC alkali peak at 5°C ± 3°C for 6M trend chart

[0180] Figure 16 Long-term stability (inverted) of high-concentration formulation IEC alkali peak at 5°C ± 3°C (6M trend chart)

[0181] Figure 17 Long-term stability (upright orientation) of the main peak of non-reducing CE-SDS in high-concentration formulations at 5°C ± 3°C (6M trend graph)

[0182] Figure 18 Long-term stability (inverted) of high-concentration formulation non-reducing CE-SDS main peak at 5°C ± 3°C (6M trend chart)

[0183] Figure 19 Biological activity of high-concentration formulations, long-term stability at 5°C ± 3°C (upright orientation), 6M trend chart

[0184] Figure 20 Biological activity of high-concentration formulations, long-term stability at 5°C ± 3°C (inverted), 6M trend chart

[0185] Figure 21 High-concentration formulation SEC main peak at 25°C±2°C, 60%±5%RH accelerated stability 6M trend graph

[0186] Figure 22 High-concentration formulation SEC polymer accelerated stability 6M trend chart at 25°C±2°C and 60%±5%RH

[0187] Figure 23 High-concentration formulation SEC low molecular weight 25°C±2°C, 60%±5%RH accelerated stability 6M trend graph

[0188] Figure 24 High-concentration formulation RP main peak at 25°C±2°C, 6M accelerated stability trend at 60%±5%RH

[0189] Figure 25 High-concentration formulation RP residual F-related protein accelerated stability 6M trend graph at 25°C±2°C and 60%±5%RH

[0190] Figure 26 High-concentration formulation IEC main peak at 25°C±2°C, 60%±5%RH accelerated stability 6M trend graph

[0191] Figure 27 High-concentration formulation IEC acid peak at 25°C±2°C, accelerated stability at 60%±5%RH (6M trend graph)

[0192] Figure 28 High-concentration formulation IEC alkali peak at 25°C±2°C, 60%±5%RH accelerated stability 6M trend graph

[0193] Figure 29 High-concentration formulation non-reducing CE-SDS main peak at 25°C±2°C, 60%±5%RH accelerated stability 6M trend graph

[0194] Figure 30 Biological activity of high-concentration formulations at 25°C±2°C and 60%±5%RH accelerated stability (6M trend chart) Detailed Implementation

[0195] Experimental Example 1

[0196] 1. Composition

[0197] PEG-hmFGF-21 25mg / ml

[0198] Lemon B buffer system 20mM,

[0199] Polysorbate 80 0.04% w / v

[0200] Arginine hydrochloride 10mg / ml

[0201] pH 5.5.

[0202] 2. Preparation method:

[0203] (1) PEG-hmFGF-21 protein solution with a concentration of 27.8 mg / ml, the buffer system is citrate-sodium citrate buffer (20 mM citrate-sodium citrate, 100 mM NaCl, pH 5.5 ± 0.1), for later use;

[0204] (2) 10X excipient stock solution (0.4w / v% polysorbate 80, 100mg / ml arginine hydrochloride), the buffer system is citrate-sodium citrate buffer (20mM citrate-sodium citrate, 100mM NaCl, pH5.5±0.1), for later use;

[0205] (3) Mix the PEG-FGF21 protein solution from step (1) and the 10X excipient stock solution from step (2) at a volume ratio of 9:1, and fill them into 2ml vials at a specification of 0.6ml / vial.

[0206] Experimental Example 2

[0207] 1. Composition

[0208] PEG-hmFGF-21 30mg / ml

[0209] Lemon B buffer system 20mM,

[0210] Polysorbate 80 0.05% w / v

[0211] Arginine hydrochloride 15mg / ml

[0212] pH 6.0.

[0213] 2. Preparation method:

[0214] (1) PEG-hmFGF-21 protein solution with a concentration of 33.4 mg / ml, the buffer system is citrate-sodium citrate buffer (20 mM citrate-sodium citrate, 100 mM NaCl, pH 6.0 ± 0.1), for later use;

[0215] (2) 10X excipient stock solution (0.5w / v% polysorbate 80, 150mg / ml arginine hydrochloride), the buffer system is citrate-sodium citrate buffer (20mM citrate-sodium citrate, 100mM NaCl, pH6.0±0.1), for later use;

[0216] (3) Mix the PEG-FGF21 protein solution from step (1) and the 10X excipient stock solution from step (2) at a volume ratio of 9:1, and fill them into 2ml vials at a specification of 0.6ml / vial.

[0217] Experimental Example 3

[0218] 1. Composition

[0219] PEG-hmFGF-21 20mg / ml

[0220] Lemon B buffer system 25mM,

[0221] Polysorbate 80 0.02% w / v

[0222] Arginine hydrochloride 5mg / ml

[0223] pH 5.0.

[0224] 2. Preparation method:

[0225] (1) PEG-hmFGF-21 protein solution with a concentration of 22.3 mg / ml, the buffer system is citrate-sodium citrate buffer (25 mM citrate-sodium citrate, 100 mM NaCl, pH 5.0 ± 0.1), for later use;

[0226] (2) 10X excipient stock solution (0.2w / v% polysorbate 80, 50mg / ml arginine hydrochloride), the buffer system is citrate-sodium citrate buffer (25mM citrate-sodium citrate, 100mM NaCl, pH5.0±0.1), for later use;

[0227] (3) Mix the PEG-FGF21 protein solution from step (1) and the 10X excipient stock solution from step (2) at a volume ratio of 9:1, and fill them into 2ml vials at a specification of 0.6ml / vial.

[0228] Experiment Example 4

[0229] 1. Composition

[0230] PEG-hmFGF-21 15mg / ml

[0231] Lemon B buffer system 15mM,

[0232] Polysorbate 80 0.01% w / v

[0233] Proline 10mg / ml

[0234] pH 5.5.

[0235] 2. Preparation method:

[0236] (1) PEG-hmFGF-21 protein solution with a concentration of 16.7 mg / ml, the buffer system is citrate-sodium citrate buffer (15 mM citrate-sodium citrate, 100 mM NaCl, pH 5.5 ± 0.1), for later use;

[0237] (2) 10X excipient stock solution (0.1 w / v% polysorbate 80, 100 mg / ml proline), the buffer system is citrate-sodium citrate buffer (15 mM citrate-sodium citrate, 100 mM NaCl, pH 5.5 ± 0.1), for later use;

[0238] (3) Mix the PEG-FGF21 protein solution from step (1) and the 10X excipient stock solution from step (2) at a volume ratio of 9:1, and fill them into 2ml vials at a specification of 0.6ml / vial.

[0239] Experimental Example 5

[0240] 1. Composition

[0241] PEG-hmFGF-21 25mg / ml

[0242] Lemon B buffer system 20mM,

[0243] Polysorbate 80 0.04% w / v

[0244] Histidine 10mg / ml

[0245] pH 5.5.

[0246] 2. Preparation method:

[0247] (1) PEG-hmFGF-21 protein solution with a concentration of 31.25 mg / ml, the buffer system is citrate-sodium citrate buffer (20 mM citrate-sodium citrate, 100 mM NaCl, pH 5.5 ± 0.1), for later use;

[0248] (2) 5X excipient stock solution (0.2w / v% polysorbate 80, 50mg / ml histidine), the buffer system is citrate-sodium citrate buffer (20mM citrate-sodium citrate, 100mM NaCl, pH5.5±0.1), for later use;

[0249] (3) Mix the PEG-FGF21 protein solution from step (1) and the 5X excipient stock solution from step (2) at a volume ratio of 4:1, and fill them into 2ml vials at a specification of 0.6ml / vial.

[0250] Experimental Example 6

[0251] Stability comparison of the high-concentration PEG-hmFGF-21 injection of the present invention with that of the low-concentration PEG-hmFGF-21 injection in Example 1 of CN110520146A

[0252] The high-concentration PEG-hmFGF-21 injection solution of the present invention was prepared according to the method of Example 1.

[0253] Table 51 Batch Information of Formulations Used in Comparability Studies

[0254]

[0255] The preparation method of the aqueous injection in Example 1 of CN110520146A is as follows:

[0256] Composition: Lipoprotein (concentration 10 mg / mL), histidine (pharmaceutical grade, concentration 10 mg / mL), citrate-sodium citrate buffer (20 mM sodium citrate-citric acid, 100 mM NaCl, pH 5.5 ± 0.1).

[0257] Preparation method:

[0258] 1) A 12.5 mg / mL lipoprotein stock solution was prepared with a citrate-sodium citrate buffer system and the pH was adjusted to 5.5 ± 0.1.

[0259] 2) A stock solution of histidine with a concentration of 50 mg / mL, buffered in a citrate-sodium citrate buffer solution, with the pH adjusted to pH 5.5 ± 0.1, is ready for use.

[0260] 3) Mix the lipoprotein stock solution from step 1) and the histidine stock solution from step 2) at a volume ratio of 4:1, and fill 500μL / vial into 2mL vials.

[0261] 1. The long-term stability test temperature is 5°C ± 3°C, and the duration is tentatively set at 36 months. The accelerated stability test temperature and duration are set at 25°C ± 2°C, relative humidity 60% ± 5%, and 6 months. The long-term stability test protocol is shown in Table 52, and the accelerated stability test protocol is shown in Table 53.

[0262] Table 52 Test Protocol for Long-Term Stability Comparison Study

[0263]

[0264] Table 53 Accelerated Stability Comparison Test Protocol

[0265]

[0266] 2. The storage temperature for the long-term stability test was 5°C±3°C. The stability test has been completed for 6 months. The data of batches ZJ202404001 and ZJ202107002 are summarized and the results are shown in Tables 54 and 55.

[0267] Table 54 Results of long-term stability study (upright orientation) of ZJ202404001 and ZJ202107002 at 5°C±3°C

[0268]

[0269] *ND (Not determined): Not detected

[0270] **Since May 2022, the RP-HPLC method has replaced the SDS-PAGE iodine staining method as the release method for detecting the content of free polyethylene glycol.

[0271] Table 55 Results of long-term stability (inverted) studies of ZJ202404001 and ZJ202107002 at 5°C±3°C.

[0272]

[0273] 3. The storage temperature for accelerated stability testing is 25°C±2°C. The stability test has been completed for 6 months. The data of batches ZJ202404001 and ZJ202107002 are summarized and the results are shown in Table 56.

[0274] Table 56 Results of accelerated stability studies of ZJ202404001 and ZJ202107002 at 25°C±2°C

[0275]

[0276] *ND (Not determined): Not detected

[0277] **Since May 2022, the RP-HPLC method has replaced the SDS-PAGE iodine staining method as the release method for detecting the content of free polyethylene glycol.

[0278] 4. Results of long-term stability test

[0279] The completed 6-month long-term stability study results show that, under storage conditions of 5°C ± 3°C, all test results meet the quality standards. Under long-term 6M upright and inverted conditions, the number of insoluble particles and the content of free PEG in the high-concentration formulation are lower than those in the low-concentration formulation. Regarding purity, the long-term stability SEC peak purity of the high-concentration formulation ZJ202404001 shows a consistent trend with that of the low-concentration formulation ZJ202107002. Figures 1-2 As shown, the long-term stability of the SEC polymer content in the high-concentration formulation ZJ202404001 shows a consistent trend with that of the low-concentration formulation ZJ202107002, as indicated. Figures 3-4 As shown, the long-term stability of the high-concentration formulation ZJ202404001, with its low molecular weight SEC content, exhibits a consistent trend with that of the low-concentration formulation ZJ202107002. Figures 5-6 As shown.

[0280] The long-term stability, RP main peak purity, and residual F-related protein of the high-concentration formulation ZJ202404001 showed the same trend as those of the low-concentration formulation ZJ202107002. Figures 7-10 As shown.

[0281] The long-term stability of the high-concentration formulation ZJ202404001, particularly the purity of its main IEC peak, shows a consistent trend with that of the low-concentration formulation ZJ202107002. Figure 11-12 As shown. Due to fluctuations in the IEC method itself (around 5%), the 6M result of the acid peak in the high-concentration formulation ZJ202404001 showed a slight decrease compared to previous values. Figure 13-14As shown. Overall, the IEC acid peak should show an upward trend. We will continue to monitor the changes in the content of the IEC acid peak at long-term stability testing points such as 9M, 12M, and 24M. The long-term stability IEC alkali peak of the high-concentration formulation ZJ202404001 shows a consistent trend with that of the low-concentration formulation ZJ202107002, as shown. Figure 15-16 As shown.

[0282] The long-term stability and purity of the non-reducing CE-SDS main peak of the high-concentration formulation ZJ202404001 showed a consistent trend with that of the low-concentration formulation ZJ202107002. Figure 17-18 As shown.

[0283] The long-term stability biology of the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 both meet the quality standards, showing a consistent trend. Figures 19-20 As shown.

[0284] In summary, the long-term stability results of the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 over 6 months, both upright and inverted, are comparable. After the formulation change, the product quality of the high-concentration formulation did not show a significant change compared to the low-concentration formulation.

[0285] 5. Results of accelerated stability test

[0286] The completed 6-month accelerated stability study results showed that, under storage conditions of 25°C±2°C and 60%±5%RH, the number of insoluble particles and the content of free PEG in the high-concentration formulation were both lower than those in the low-concentration formulation. Regarding purity, the SEC purity of both the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 decreased, failing to meet quality standards at 6M, with a consistent trend. Figure 21 As shown, the 6M accelerated stability SEC polymer content of the high-concentration formulation ZJ202404001 is higher than that of the low-concentration formulation ZJ202404001, both within acceptable ranges. Figure 22 As shown, the increased protein content in high-concentration formulations leads to an increased polymer content, a trend consistent with expectations. The SEC low molecular weight content changes in high-concentration formulation ZJ202404001 and low-concentration formulation ZJ202107002 show the same trend. Figure 23 As shown.

[0287] The high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 both showed decreased purity of the RP main peak and increased residual F protein content during 6M accelerated stability testing, failing to meet the quasi-quality standards at 6M. The trends were consistent. Figure 24-25 As shown.

[0288] The 6M accelerated stability and IEC purity of both the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 decreased, and the accelerated 6M stability did not meet the quality standards. The trends were consistent. Figure 26 As shown, the IEC acid and alkali peaks of both the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 increased, showing a consistent trend. Figure 27-28 As shown.

[0289] The purity of non-reduced CE-SDS in both high-concentration formulation ZJ202404001 and low-concentration formulation ZJ202107002 decreased, failing to meet quality standards at accelerated 6M, with a consistent trend. Figure 29 As shown.

[0290] The long-term biological stability of the high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 both showed a decreasing trend in biological activity under accelerated conditions at 6M. Figure 30 As shown.

[0291] In summary, under accelerated conditions of 25°C ± 2°C and 60% ± 5% RH, the purity and activity of the high-concentration PEG-hmFGF-21 injection (ZJ202404001) and the low-concentration PEG-hmFGF-21 injection (ZJ202107002) in Example 1 of CN110520146A showed a decreasing trend, indicating that the formulations in this project are temperature-sensitive. The high-concentration formulation ZJ202404001 and the low-concentration formulation ZJ202107002 showed the same decreasing trend in purity and activity under accelerated conditions, and their accelerated stability results were comparable.

[0292] 6. Conclusion:

[0293] Based on the results of the 6-month long-term stability and 6-month accelerated stability tests, there was no significant difference between the high-concentration PEG-hmFGF-21 injection (ZJ202404001) of this invention and the low-concentration PEG-hmFGF-21 injection (ZJ202107002) in Example 1 of CN110520146A.

[0294] This invention maintains good stability while increasing the concentration of PEG-hmFGF-21 injection.

Claims

1. A high-concentration PEG-hmFGF-21 injection solution, the composition of which is: PEG-hmFGF-21 15-30 mg / ml 20mM citric acid / sodium citrate-100mM sodium chloride buffer system 15-25mM Polysorbate 80 0.01-0.04% w / v Arginine hydrochloride 5mg / ml-10mg / ml pH 5.0-6.

0.

2. The PEG-hmFGF-21 injection solution according to claim 1, wherein, The concentration of PEG-hmFGF-21 was 25 mg / ml.

3. The PEG-hmFGF-21 injection solution according to claim 1, wherein the composition is as follows: PEG-hmFGF-21 25mg / ml 20mM citric acid / sodium citrate-100mM sodium chloride buffer system 20mM Polysorbate 80 0.04% w / v Arginine hydrochloride 10mg / ml pH 5.

5.

4. The use of the PEG-hmFGF-21 injection solution according to claim 1 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

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