NGF-Fc fusion protein preparation and application thereof

Abstract

The NGF-Fc fusion protein preparation is high in stability under the conditions of high temperature, illumination, oscillation, freeze thawing, acceleration and long term, good stability of the preparation in the preparation, transportation and storage processes can be guaranteed, and clinical medication safety and quality controllability are guaranteed.

Description

NGF-Fc fusion protein preparation and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an NGF-Fc fusion protein preparation and application thereof. Background Art

[0002] Nerve growth factor (NGF) is the first member of the neurotrophic factor family to be discovered. It was first discovered by Italian scientist Levi-Montlcini in mouse sarcoma cells in 1953. NGF plays an important regulatory role in the development, differentiation, growth, regeneration and functional expression of central and peripheral neurons. NGF has been used to treat abnormal development of the nervous system, including amblyopia, neuroma, various nerve injuries and nervous system diseases. However, its side effects such as causing pain, short half-life in vivo, low dose restrictions to avoid pain sensitivity and frequent administration limit the widespread application of NGF. Fusion of protein drugs with parts with longer half-life and / or larger molecular weight is a strategy to give certain protein drugs long-lasting activity.

[0003] However, there are currently few Fc fusion protein pharmaceutical preparations on the market, and no NGF-Fc fusion protein preparations have been disclosed. The present invention, through rational design, formulation screening, and testing, ultimately produces a highly stable NGF-Fc fusion protein preparation that can be used to prepare both injections and eye drops. The fusion protein preparation maintains excellent stability during preparation, transportation, and storage, ensuring quality controllability and clinical safety.

[0004] Summary of the Invention

[0005] In one aspect, the present invention provides an NGF-Fc fusion protein preparation, which comprises NGF-Fc fusion protein, a stabilizer, a surfactant, an antioxidant, and a buffer, wherein the stabilizer is trehalose or sucrose, the antioxidant is methionine, the surfactant is poloxamer, and the stabilizer is histidine buffer.

[0006] In some embodiments, the concentration of the fusion protein is 0.05 mg / ml-5.0 mg / ml, preferably 0.05 mg / ml-2.0 mg / ml, more preferably 0.1 mg / ml-0.5 mg / ml. In some specific embodiments, the concentration of the fusion protein is 0.05 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.10 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.14 mg / ml, 0.16 mg / ml, 0.18 mg / ml or 2.0 mg / ml.

[0007] In some embodiments, the concentration of the histidine buffer is 8 mM-50 mM, preferably 10 mM-30 mM. In some specific embodiments, the concentration of the histidine buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM.

[0008] In some embodiments, the concentration of the sucrose or trehalose is 6%-12%, preferably 7%-10%. In some specific embodiments, the concentration of the sucrose or trehalose is 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.

[0009] In some embodiments, the concentration of methionine is 0.05 mM-7 mM, preferably 0.1 mM-5 mM, more preferably 1 mM-3 mM. In some specific embodiments, the concentration of methionine is 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM.

[0010] In some embodiments, the concentration of the poloxamer is 0.005%-0.3%, preferably 0.01%-0.2%, more preferably 0.01%-0.03%. In some specific embodiments, the concentration of the poloxamer is 0.01%, 0.02%, 0.03%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%.

[0011] In some embodiments, the pH of the formulation is 5.0-6.5, preferably 5.0-6.2, more preferably 5.0-5.4. In some specific embodiments, the pH of the formulation is 5.0, 5.1, 5.2, 5.3, 5.4, 5.6, 5.8, 6.0 or 6.2.

[0012] In some embodiments, the concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0-6.2.

[0013] In some embodiments, the formulation further comprises cyclodextrin, preferably hydroxypropyl beta-cyclodextrin.

[0014] In some embodiments, the concentration of the cyclodextrin is 0.05 mM-8 mM, preferably 1 mM-6 mM, more preferably 5 mM. In some specific embodiments, the concentration of the cyclodextrin is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM or 6 mM.

[0015] In some embodiments, the formulation is any one of the following formulations:

[0016] (1) The concentration of the fusion protein is 0.05 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.10 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.14 mg / ml, 0.16 mg / ml, 0.18 mg / ml or 2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0-6.2;

[0017] (2) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the formulation is 5.0-6.2;

[0018] (3) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the formulation is 5.0-6.2;

[0019] (4) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the formulation is 5.0-6.2;

[0020] (5) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%, 0.02%, 0.03%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, and the pH of the formulation is 5.0-6.2;

[0021] (6) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0, 5.1, 5.2, 5.3, 5.4, 5.6, 5.8, 6.0 or 6.2.

[0022] In some embodiments, the formulation is any one of the following formulations:

[0023] (1) The concentration of the fusion protein is 0.1 mg / ml, the concentration of the histidine buffer is 10 mM, the concentration of sucrose or trehalose is 7%, the concentration of methionine is 1.5 mM, the concentration of poloxamer is 0.01%, and the pH of the preparation is 5.4;

[0024] (2) The concentration of the fusion protein is 0.1 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 9%, the concentration of methionine is 1.0 mM, the concentration of poloxamer is 0.03%, and the pH of the preparation is 5.2;

[0025] (3) The concentration of the fusion protein is 0.5 mg / ml, the concentration of the histidine buffer is 30 mM, the concentration of sucrose or trehalose is 10%, the concentration of methionine is 3 mM, the concentration of poloxamer is 0.02%, and the pH of the preparation is 5.0;

[0026] (4) The concentration of the fusion protein is 2.0 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 8%, the concentration of methionine is 2.0 mM, the concentration of poloxamer is 0.1%, and the pH of the preparation is 5.8;

[0027] (5) The concentration of the fusion protein is 0.5 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 10%, the concentration of methionine is 0.1 mM, the concentration of poloxamer is 0.2%, and the pH of the preparation is 6.2;

[0028] (6) The concentration of the fusion protein is 0.05 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 8.5%, the concentration of methionine is 5.0 mM, the concentration of poloxamer is 0.04%, and the pH of the preparation is 5.2.

[0029] In some embodiments, the cyclodextrin concentration is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM.

[0030] In some embodiments, the antibody formulation may further comprise a preservative, including but not limited to octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parahydroxybenzoate, and the like.

[0031] In some embodiments, the preparation is a liquid preparation, preferably, the preparation is an injection preparation or an eye drop preparation.

[0032] In some embodiments, the preparation is an eye drop preparation, further comprising a viscosity enhancer; preferably, the viscosity enhancer is hypromellose; more preferably, the viscosity enhancer is hypromellose K4M.

[0033] In some embodiments, the concentration of the viscosity increasing agent is 0.4%-0.5%.

[0034] In some embodiments, the eye drop formulation comprises the following components: the concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, the concentration of cyclodextrin is 1 mM-6 Mm, the concentration of hypromellose is 0.4%-0.5%, and the pH of the formulation is 5.0-6.2.

[0035] In some embodiments, the viscosity of the eye drop preparation is 25-50 cp, preferably 30-45 cp.

[0036] In some embodiments, the eye drop formulation comprises the following components: a fusion protein concentration of 0.1 mg / ml, a histidine buffer concentration of 20 mM, a trehalose concentration of 8.5%, a methionine concentration of 1.0 mM, a poloxamer concentration of 0.03%, a cyclodextrin concentration of 5 mM, and a hypromellose concentration of 0.4%, 0.45% or 0.5%, and the pH of the formulation is 5.2.

[0037] In some embodiments, the fusion protein in the preparation comprises an NGF portion and an Fc portion from N-terminus to C-terminus, wherein the Fc portion is derived from the Fc portion of IgG, or a mutant of the Fc portion of IgG, and the mutant of the Fc portion of IgG includes a mutation in a site associated with ADCC / CDC activity.

[0038] In some embodiments, the NGF portion is human nerve growth factor, preferably a human nerve growth factor mutant.

[0039] In some embodiments, the human nerve growth factor mutant, relative to the amino acid position of the wild-type human nerve growth factor mutant, has a mutation site including Phe12Glu; preferably, the human nerve growth factor mutant comprises the amino acid sequence of SEQ ID NO: 2.

[0040] In some embodiments, the NGF portion is fused to the Fc portion via a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 3.

[0041] In some embodiments, the Fc portion is derived from an IgG1 Fc comprising the amino acid sequence of SEQ ID NO:4.

[0042] In some embodiments, the NGF-Fc fusion protein comprises the amino acid sequence shown in SEQ ID No: 1.

[0043] In another aspect, the present invention discloses the use of the NGF-Fc fusion protein preparation in the preparation of drugs for treating NGF-related diseases.

[0044] In some embodiments, the NGF-related disease is a nervous system disease; preferably, the nervous system disease is selected from the group consisting of neonatal hypoxic-ischemic encephalopathy, cerebral palsy, critical illness myopathy, sensorineural hearing loss, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down syndrome, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, diffuse brain injury, thymic dysplasia, optic nerve contusion, follicular dysplasia, spinal cord injury, glaucoma, neurotrophic keratitis, optic nerve injury, neuromyelitis optica, retinal-related diseases, urinary incontinence, Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, hypertensive cerebral hemorrhage neurological dysfunction, cerebral small vessel disease, acute ischemic stroke, corneal endothelial dystrophy, diabetic neuropathy, diabetic foot ulcer, neurogenic skin ulcer, pressure sore, neurotrophic corneal ulcer, diabetic corneal ulcer and macular hole.

[0045] In some embodiments, the NGF-related disease is a non-neurological disease; preferably, the non-neurological disease is selected from the group consisting of splenic atrophy, splenic contusion, decreased ovarian reserve function, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle hypoplasia, spermatogenesis disorders (such as oligospermia, asthenozoospermia, oligoasthenozoospermia), ischemic ulcers, stress ulcers, rheumatoid ulcers, liver fibrosis, corneal ulcers, burns, oral ulcers and leg venous ulcers.

[0046] The NGF-Fc fusion protein preparation provided by the present invention has the following excellent effects:

[0047] 1. The present invention screened out an optimal formulation of NGF-Fc fusion protein. Based on this formulation, it can be used to prepare both injections and eye drops. The formulation of the present invention can significantly reduce the formation of protein aggregates in the formulation, slow down the change in charge heterogeneity, and maintain good stability of the fusion protein formulation.

[0048] 2. Hydroxypropyl methylcellulose is added as a thickener to the NGF-Fc fusion protein preparation of the present invention, which can be used to prepare eye drops to ensure that the eye drops have a viscosity suitable for intraocular administration and can also maintain the osmotic pressure of the eye drop preparation at around 300mOsm / kg, which is close to the osmotic pressure of the internal environment of the human eye.

[0049] 3. The injection preparation and eye drop preparation of the present invention have strong stability, and are stable under oscillation, freeze-thaw, accelerated and long-term conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, ensuring clinical drug safety and quality controllability. DETAILED DESCRIPTION

[0050] The specific embodiments of the present invention will be further described below in conjunction with the examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these examples are merely illustrative and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements fall within the scope of protection of the present invention.

[0051] If no specific conditions or methods are specified in the examples, all experiments were carried out according to conventional conditions or methods. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0052] The NGF-Fc fusion protein used in the embodiments of the present invention is disclosed in international patent application WO2022 / 105847A1, the disclosure of which is incorporated herein by reference.

[0053] The structure of the 2-118-L3Fc10-M3-5 polypeptide used in the examples of the present invention is shown in Table 1:

[0054] Table 1 NGF-Fc fusion protein sequence

[0055] Example 1: Screening of formulation composition of NGF-Fc fusion protein preparations

[0056] (1) Screening of surfactants and antioxidants

[0057] 1. Prescription design and preparation of preparations

[0058] The NGF-Fc fusion protein 2-118-L3Fc10-M3-5 was used to prepare samples of various formulations according to Table 2, wherein the concentration of the fusion protein was 0.1 mg / ml.

[0059] 2. Stability testing method

[0060] The sample immediately after preparation was designated as the time 0 sample. Subsequently, each sample was stored at -70°C ± 10°C, 25°C ± 2°C, 5°C ± 3°C for one month, and at 40°C ± 3°C for two weeks. After storage, the stability of each sample was tested using the following method.

[0061] The detection methods are: visual observation of the solution appearance; detection of protein concentration by Titer assay; determination of protein aggregation characteristics by SEC-HPLC; and determination of charge heterogeneity by WCX.

[0062] Table 2. Formulation design for surfactant and antioxidant screening

[0063] 3. Stability test results

[0064] (1) Appearance and protein concentration

[0065] After the samples of the above-mentioned preparations were placed at -70℃±10℃, -20℃±10℃, and 5℃±3℃ for one month, the concentration of the fusion protein did not decrease significantly and was basically consistent among the groups.

[0066] However, in terms of appearance, after being placed at -70℃±10℃, -20℃±10℃, and 5℃±3℃ for one month, insoluble particles visible to the naked eye appeared in prescription groups 2, 3, and 5, but no visible foreign matter appeared in the other groups.

[0067] After being placed at a high temperature of 40℃±3℃ for 2 weeks, only prescription groups 7 and 10 were free of visible foreign matter. Insoluble particles visible to the naked eye appeared in the samples of other groups, mainly white sand-like, short fiber-like or flaky protein aggregates. This may be because the structure of the NGF-Fc fusion protein was destroyed, resulting in insoluble particles and even precipitation.

[0068] The above results preliminarily indicate that high temperature has a destructive effect on NGF-Fc fusion protein, but compared with other prescriptions, prescriptions 7 and 10 can provide better protection for the fusion protein.

[0069] (2) Aggregate detection

[0070] The aggregation of each formulation was detected by SEC-HPLC, and the results are listed in Table 3.

[0071] The results showed that at 0h, the proportion of the SEC-HPLC main peak of prescription groups 2, 3 and 5 was significantly lower, at 77.81%, 91.26% and 92.36% respectively. This indicates that the protein structure of prescription groups 2, 3 and 5 had been damaged to a certain extent after being prepared into liquid preparations. In comparison, the main peak proportions of the samples in the other groups were all above 98%.

[0072] As previously mentioned, after one month of storage at -70°C, -20°C, and 5°C, samples from Formulations 2, 3, and 5 showed visible insoluble particles and clear precipitation, and therefore were not subjected to SEC-HPLC analysis. The main protein peak in samples from the remaining groups remained above 97%, with a decrease of only 0.05%-2.37% compared to the zero-time level.

[0073] After 2 weeks at 40°C, formulations 2, 3, and 5 also failed SEC-HPLC analysis due to the presence of significant precipitation. The main peak percentages for the remaining formulations decreased. For formulations 4, 6-8, and 10, the main peak percentage remained above 95% across all test conditions, with a decrease of only 2.4%-3.2%. The main peak percentage for formulation 9 decreased to 90.25% by SEC-HPLC, a significant decrease of 9.27%.

[0074] The above results indicate that the addition of polysorbate surfactants to the preparation will cause the NGF-Fc fusion protein to aggregate. In comparison, the addition of surfactant P188 has a better protective effect on the NGF-Fc fusion protein.

[0075] Table 3. SEC-HPLC test results of NGF-Fc fusion protein preparation

[0076] (3) Charge heterogeneity detection

[0077] The charge heterogeneity of each formulation was detected by WCX, and the results are listed in Tables 4-6.

[0078] The results showed that at 0h, the proportion of the main WCX peak in each group was in the range of 54.19%-59.25%, and the proportions of the acidic peak and alkaline peak were in the range of 13.39%-14.63% and 26.87%-32.42%, respectively, which were basically consistent among the groups.

[0079] After one month at 70°C, -20°C, and 5°C, and two weeks at 40°C, samples from recipes 2, 3, and 5 were not tested due to precipitation as mentioned above.

[0080] For the remaining formulations, after one month of storage at -70°C, -20°C, and 5°C, the proportions of the main peak, acidic peak, and basic peak for formulations 4, 6-8, and 10 remained unchanged, indicating that under these conditions, formulations 4, 6-8, and 10 also exhibited excellent stability in terms of charge heterogeneity. In contrast, the proportion of the WCX main peak in formulations 1 and 9 showed a downward trend, while the proportions of the acidic and basic peaks also increased.

[0081] After 2 weeks at 40°C, the main peak ratios of the remaining formulations decreased, particularly formulation 9, which saw a 28.11% decrease relative to time 0. While formulations 4, 6-8, and 10 saw decreases of only approximately 19-20%, formulation 7 exhibited the best stability performance of all groups, with a decrease of only 14.73%. The proportions of both the acidic and alkaline peaks increased in each formulation, with formulation 7 performing the best, with an 11.82% increase in its acidic peak (relative to time 0) and a 2.91% increase in its alkaline peak (relative to time 0).

[0082] Table 4. Detection results of the main peak of NGF-Fc fusion protein preparation WCX

[0083] Table 5. Results of WCX acidic peak detection of NGF-Fc fusion protein preparation

[0084] Table 6. Results of basic peak detection of NGF-Fc fusion protein preparation WCX

[0085] Based on these results, the addition of polysorbate-based surfactants to NGF-Fc fusion protein formulations tends to cause aggregation, whereas P188 and / or methionine exhibit excellent protective effects. Therefore, the NGF-Fc fusion protein of the present invention uses P188 as a surfactant and methionine as an antioxidant as a basis for the next stage of formulation screening.

[0086] (2) Screening of buffers and stabilizers

[0087] 1. Prescription design and preparation of preparations

[0088] The NGF-Fc fusion protein 2-118-L3Fc10-M3-5 was used to prepare samples of various formulations according to Table 7, wherein the concentration of the fusion protein was 0.1 mg / ml.

[0089] 2. Stability testing method

[0090] The sample immediately after preparation was designated as the sample at time 0. Subsequently, each sample was placed under different influencing conditions (accelerated condition: 25°C ± 2°C, high temperature condition: 40°C ± 3°C) for 2 weeks and then taken out to test the stability of each sample.

[0091] The detection method is as follows: observe the appearance of the solution with the naked eye; detect the osmotic pressure by freezing point osmometer and the protein concentration by titer method; determine the protein aggregation characteristics by SEC-HPLC and the charge heterogeneity by WCX.

[0092] Table 7. Formulation design for buffer and stabilizer screening

[0093] 3. Stability test results

[0094] (1) Osmotic pressure, appearance, and protein concentration

[0095] At 0h, the osmotic pressure of each preparation prescription sample was 285mOsm / kg-299mOsm / kg; the pH of prescriptions 1-4 and 9-12 was 5.15-5.38, and the pH of prescriptions 5-8 was 6.84-6.86.

[0096] After 2 weeks of storage under accelerated and high temperature conditions, the protein concentration of each formulation group did not change significantly.

[0097] After 2 weeks of storage under accelerated and high temperature conditions, the samples were observed with the naked eye and found that only small white granular protein aggregates appeared in prescriptions 1, 2 and 4; although prescription 3 had a slight opalescence, there were no particles visible to the naked eye; however, prescriptions 5-12 all produced large flaky insoluble matter.

[0098] The above results preliminarily indicate that histidine buffer is more suitable for the stability preparation of NGF-Fc fusion protein than phosphate buffer and acetate buffer.

[0099] (2) Aggregate detection

[0100] Aggregation of each formulation was tested by SEC-HPLC, and the results are listed in Table 8. Similarly, after 2 weeks of storage under accelerated and high temperature conditions, due to severe precipitation, aggregation testing was no longer performed for formulations 5, 6, and 8.

[0101] From the test results in Table 8, it can be seen that at 0h, the main peak proportion of each group is above 98%, which is basically consistent among the groups.

[0102] After two weeks under accelerated conditions, the main peak percentages of Prescriptions 1-4 remained almost unchanged compared to time 0, while the main peak percentages of Prescriptions 7 and 9-12 decreased slightly by 1.58%-2.20% by SEC-HPLC. This indicates that under these conditions, all of the above formulations exhibited good stability.

[0103] After two weeks under high temperature conditions, the main peak proportions in all groups decreased compared to time 0. The SEC-HPLC main peak proportions of Prescriptions 1-4 decreased by 1.2%-6.46%, and those of Prescriptions 7, 9-12 decreased by 4.42%-9.14%. Prescription 3 showed the best stability, with the main peak proportion decreasing by only 1.40%.

[0104] Table 8. SEC-HPLC test results of NGF-Fc fusion protein preparation

[0105] (3) Charge heterogeneity detection

[0106] The charge heterogeneity of each formulation was tested using WCX, and the results are listed in Tables 9-11. Similarly, after 2 weeks of storage under accelerated and high temperature conditions, due to severe precipitation, charge heterogeneity testing was no longer performed for formulations 5, 6, and 8.

[0107] From the test results in Tables 9-11, it can be seen that at 0h, the main peak proportion of the samples in each prescription group was in the range of 52.0%-58.0%, and the acidic peak and alkaline peak proportions were between 12.7%-15.5% and 27.1%-35.2%, respectively, which were basically consistent among the groups.

[0108] After 2 weeks under accelerated conditions, compared to time 0, the proportions of the main peak, acidic peak, and alkaline peak in prescription groups 1-4 remained almost unchanged, with the main peak decreasing by less than 4.0%, and the acidic peak and alkaline peak proportions increasing by less than 3.0%. In contrast, the main peak proportion of prescription groups 7 and 9-12 decreased by 6.1%-12.8%, the acidic peak proportion increased by 2.5%-6.8%, and the alkaline peak proportion increased by 3.5%-8.5%. Among them, the main peak proportion of prescription group 3 samples decreased by only 1.7%, and the acidic and alkaline peak proportions did not change significantly (≤1.0%), showing the best stability.

[0109] After two weeks under high temperature conditions, the proportion of the main peak in each group decreased. Compared to time 0, the main peak proportion of prescriptions 1-4 decreased by 9.4%-20.1%, and the main peak proportion of prescriptions 7 and 9-12 decreased by 25.8%-30.9%. Among them, the main peak proportion of prescription 3 decreased by only 9.4%, showing the best stability among all prescriptions. The proportion of acidic peaks and alkaline peaks increased in each prescription, but in comparison, prescription 3 still performed the best, with an increase of 9.1% in its acidic peak (relative to time 0) and a mere 0.3% in its alkaline peak (relative to time 0).

[0110] Table 9. Detection results of the main peak of NGF-Fc fusion protein preparation WCX

[0111] Table 10. Results of WCX acidic peak detection of NGF-Fc fusion protein preparation

[0112] Table 11. Results of basic peak detection of NGF-Fc fusion protein preparation WCX

[0113] From the above results, it can be seen that compared with other buffers and stabilizers, NGF-Fc fusion protein has a better stability effect in the histidine buffer / trehalose system. Therefore, it can be determined that the most suitable formulation for NGF-Fc fusion protein includes the following components: histidine buffer, trehalose, P188 and methionine.

[0114] Example 2: Determination of NGF-Fc fusion protein formulation prescription

[0115] In order to determine the formulation of the NGF-Fc fusion protein preparation and further verify the stability effect and advantages of the above formulation, the following experiments were conducted.

[0116] 1. Prescription design and preparation of preparations

[0117] The NGF-Fc fusion protein 2-118-L3Fc10-M3-5 was used to prepare samples of various formulations according to Table 12. The concentration of the fusion protein was 0.1 mg / ml.

[0118] 2. Stability testing method

[0119] The sample just prepared was taken as the sample at time 0. Subsequently, each sample was placed under different influencing factors (accelerated temperature 25℃±2℃, high temperature 40℃±3℃) for 2 weeks and then taken out. The stability of each sample was tested using the following method.

[0120] The detection method is as follows: observe the appearance of the solution with the naked eye; detect the osmotic pressure by freezing point osmometer and the protein concentration by titer method; determine the protein aggregation characteristics by SEC-HPLC and the charge heterogeneity by WCX.

[0121] Table 12. Formulation of NGF-Fc fusion protein

[0122] 3. Stability test results

[0123] (1) Osmotic pressure, appearance, and protein concentration

[0124] At 0h, the osmotic pressure of each sample was between 285mOsm / kg and 310mOsm / kg; the pH was between 5.15 and 6.28.

[0125] After 2 weeks of storage under accelerated and high temperature conditions, the samples were observed with the naked eye and found that the solutions of prescription groups 1-9 had a slight opalescence, but no particles visible to the naked eye. However, a large amount of precipitation appeared in prescription group 10, indicating that the preparation could not maintain the stability of the NGF-Fc fusion protein. Therefore, SEC-HPLC and WCX tests were no longer performed in subsequent experiments.

[0126] After 2 weeks of storage under accelerated and high temperature conditions, there was no significant change in the histone concentration of each formulation.

[0127] (2) Aggregate detection

[0128] The aggregation of each formulation was detected by SEC-HPLC, and the results are listed in Table 13.

[0129] The test results in Table 13 show that after 2 weeks under accelerated and high temperature conditions, the decrease in the proportion of the main peak by SEC-HPLC in Prescriptions 1-9 was minimal compared to time 0, indicating that Prescriptions 1-9 can effectively ensure the stability of the fusion protein. Specifically, the decrease in the proportion of the main peak in Prescriptions 4-6 was only 0.01%-0.06% after 2 weeks under accelerated conditions, and only 0.77%-0.93% after 2 weeks under high temperature conditions, demonstrating relatively superior stability.

[0130] Table 13. SEC-HPLC test results of NGF-Fc fusion protein preparation

[0131] (3) Charge heterogeneity detection

[0132] The charge heterogeneity of each formulation was detected by WCX, and the results are listed in Tables 14-16.

[0133] The test results in Tables 14-16 show that after two weeks under accelerated and high-temperature conditions, compared to time 0, the proportion of the WCX main peak in Prescriptions 1-9 decreased by less than 13%, and the proportion of the acidic and alkaline peaks increased by less than 10%, indicating that Prescriptions 1-9 can effectively ensure the stability of the fusion protein. Among them, the proportion of the main peak in Prescriptions 4-6 decreased by only approximately 8%, demonstrating relatively superior results.

[0134] Table 14. Main peak detection results of NGF-Fc fusion protein preparation

[0135] Table 15. Acidic peak detection results of NGF-Fc fusion protein preparations

[0136] Table 16. Basic peak detection results of NGF-Fc fusion protein preparations

[0137] The above results indicate that the formulations 1-9 of the present invention can well ensure the stability of the fusion protein, and in particular, the addition of cyclodextrin can further improve the stability of the fusion protein formulation.

[0138] Example 3: Stability Verification of the Formulation under Other Conditions

[0139] 1. Preparation of fusion protein preparation

[0140] Take NGF-Fc fusion protein and prepare fusion protein preparation sample according to the following formula:

[0141] Recipe 1: 0.1 mg / mL 2-118-L3Fc10-M3-5 fusion protein, 20 mM L-histidine, 9% trehalose, 1 mM methionine, 0.03% poloxamer 188, pH adjusted to 5.2 with acetic acid;

[0142] Formulation 2: 0.1 mg / mL 2-118-L3Fc10-M3-5 fusion protein, 20 mM L-histidine, 9% trehalose, 1 mM methionine, 5 mM hydroxypropyl beta-cyclodextrin, 0.03% poloxamer 188, pH adjusted to 5.2 with acetic acid;

[0143] The prepared sample is the 0h sample. After filtering through a 0.22μm filter membrane, the sample is divided into sterile 2ml vials, 1ml per vial, stoppered, and capped.

[0144] 2. Stability testing method

[0145] After the samples are packaged, they are placed in stabilization boxes with different treatment conditions for influencing factors, taken out at different time points and tested according to the test plan. The conditions are as follows:

[0146] Oscillation stability conditions: 2°C-8°C, 300 rpm, 2 weeks, 1 month, 2 months;

[0147] Freeze-thaw stability conditions: freeze at -70℃±10℃, thaw at 25℃±2℃, and repeat freeze-thaw 5 times;

[0148] Light stability conditions: 2°C-8°C, 4500±500Lux, 2 weeks, 1 month;

[0149] Accelerated stability conditions: 25°C ± 2°C, 2 weeks, 1 month, 2 months, 3 months, 6 months;

[0150] High temperature stability conditions: 40℃±2℃, 2 weeks, 1 month;

[0151] Long-term stability conditions: 2°C-8°C, 2 weeks, 1 month, 2 months, 3 months, 6 months.

[0152] The detection method is as follows: observe the appearance of the solution with the naked eye; detect the osmotic pressure by freezing point osmometer and the protein concentration by titer method; determine the protein aggregation characteristics by SEC-HPLC and the charge heterogeneity by WCX.

[0153] 3. Stability test results

[0154] (1) Osmotic pressure, appearance, and protein concentration

[0155] At 0 h, the osmotic pressures of the samples were 310 mOsm / kg and 324 mOsm / kg, and the pHs were 5.35 and 5.36, respectively. After storage under different conditions, the osmotic pressures of all samples remained between 309 mOsm / kg and 324 mOsm / kg, and the pH was 5.29 ± 0.1, showing no significant changes compared to 0 h.

[0156] After the above samples were placed under different influencing factors for a period of time, no particles visible to the naked eye were produced and the protein concentration did not change significantly.

[0157] (2) Aggregate detection

[0158] The aggregation of each formulation was detected by SEC-HPLC, and the results are listed in Tables 17-20.

[0159] From the results in Tables 17-20, it can be seen that at 0 h, the main peak of SEC-HPLC of the above two samples accounted for 99.45%-99.69%.

[0160] After 2 months of storage under shaking conditions, 1 month of storage under light conditions, and 5 freeze-thaw cycles, the SEC-HPLC main peak proportions of the two samples were approximately 99.41%-99.71%, which was almost unchanged compared with 0 h (Table 17).

[0161] After 3 months of storage under accelerated conditions, the SEC main peak proportion of the two groups of samples was still around 99%; after 6 months of acceleration, the main peak proportion decreased slightly, only decreasing by 1.70% and 1.20% (Table 18).

[0162] After 2 weeks of high temperature treatment, the change in the SEC main peak of the two groups of samples was only about 1%. As time went on, the main peak gradually decreased after 1 month, but the decrease was still ≤2% (Table 19).

[0163] After 6 months of storage under refrigeration conditions, the changes in the main peaks of both groups of samples were less than 0.5% (Table 20).

[0164] The above results show that, in terms of aggregate formation, the fusion protein preparation of the present invention has excellent stability under various conditions such as oscillation, illumination, freeze-thaw, accelerated 6 months, and high temperature 1 month.

[0165] Table 17. SEC-HPLC test results under shaking, light, and freeze-thaw conditions

[0166] Table 18. SEC-HPLC test results under accelerated conditions

[0167] Table 19. SEC-HPLC test results under high temperature conditions

[0168] Table 20. SEC-HPLC test results under long-term refrigeration conditions

[0169] (3) Charge heterogeneity detection

[0170] The charge heterogeneity of each formulation was detected by WCX, and the results are listed in Tables 21-35.

[0171] From the results in Table 21-35, it can be seen that at 0h, the main peaks of the two groups of samples accounted for 58.1% and 57.7% respectively, the acidic peaks accounted for 14.8% and 14.7% respectively, and the basic peaks accounted for 27.1% and 27.6% respectively.

[0172] After being placed under oscillation conditions for one month, the proportions of the WCX main peak, acidic peak and alkaline peak of the two groups of samples did not change significantly; as the oscillation time increased, after two months of oscillation, the proportion of the WCX main peak only decreased by 5.3%-6.2%, the proportion of the acidic peak only increased by 0.3%-0.7%, and the proportion of the alkaline peak only increased by 5.0%-5.5% (Tables 21-23).

[0173] After the samples were frozen and thawed five times, the proportions of the WCX main peak, acidic peak and alkaline peak of the two groups of samples were basically the same as those at 0h (Tables 24-26).

[0174] After being placed under light conditions for one month, the proportion of the WCX main peak of the two groups of samples only decreased by 5.7%-6.1%, the proportion of the acidic peak only increased by 1.3%-1.6%, and the proportion of the alkaline peak only increased by 4.5%-4.6% (Tables 24-26).

[0175] Under accelerated conditions, as the storage time continued to increase, the main peak of WCX gradually decreased, and the content of acidic peak and basic peak increased (Table 27-29); at 6 months of acceleration, it decreased by 31.4% compared with 0h, indicating that the protein charge variable is prone to change when the sample is stored for a long time at room temperature. In the later stage, attention should be paid to the sample storage temperature to avoid sample damage caused by overheating.

[0176] When the samples were placed under high temperature conditions, as the storage time continued to increase, the WCX main peak gradually decreased, the acidic peak and alkaline peak content increased (Table 30-32), and the main peak proportion decreased by 22.8%-23.6%. This shows that the protein sample was destroyed at high temperature, resulting in a decrease in the main peak of the charge variable. In the later storage of samples, it is necessary to avoid overheating to cause sample damage.

[0177] After 6 months of storage under refrigeration conditions, the proportion of the WCX main peak of the two groups of samples only decreased by 3.6%-4.0%, the proportion of the acidic peak only increased by 0.6%-0.7%, and the proportion of the alkaline peak only increased by 3.0%-3.3% (Tables 33-35).

[0178] Table 21. Main peak detection results under oscillation conditions

[0179] Table 22. Acidic peak detection results under oscillation conditions

[0180] Table 23. Basic peak detection results under oscillation conditions

[0181] Table 24. Main peak detection results under light and freeze-thaw conditions

[0182] Table 25. Acidic peak detection results under light and freeze-thaw conditions

[0183] Table 26. Alkaline peak detection results under light and freeze-thaw conditions

[0184] Table 27. Main peak detection results under accelerated conditions

[0185] Table 28. Acidic peak detection results under accelerated conditions

[0186] Table 29. Basic peak detection results under accelerated conditions

[0187] Table 30. Main peak detection results under high temperature conditions

[0188] Table 31. Acidic peak detection results under high temperature conditions

[0189] Table 32. Alkalinity test results under high temperature conditions

[0190] Table 33. Main peak detection results under long-term refrigeration conditions

[0191] Table 34. Acidic peak detection results under long-term refrigeration conditions

[0192] Table 35. Alkaline peak detection results under long-term refrigeration conditions

[0193] The above results show that in terms of charge heterogeneity, the fusion protein preparation of the present invention has excellent stability under various conditions such as oscillation, illumination, freeze-thaw, high temperature, acceleration, and long-term refrigeration. However, attention should still be paid to the storage temperature of the preparation to avoid high temperature.

[0194] Example 4: Eye Drop Prescription Screening and Verification

[0195] To ensure that the eye drops are suitable for intraocular administration, a viscosity enhancer needs to be added to the formulation to achieve a certain viscosity. Based on the above-determined formulation, a formulation suitable for intraocular administration (eg, eye drops) is designed and screened for the NGF-Fc fusion protein.

[0196] (1) Screening of viscosity-increasing agents for eye drops

[0197] 1. Eye drops prescription design and preparation

[0198] Take NGF-Fc fusion protein 2-118-L3Fc10-M3-5, and prepare a fusion protein preparation according to the prescription in Table 36. The concentration of the fusion protein is 0.1 g / mL. The sample is filtered through a 0.22 μm filter membrane and then divided into disposable blow-fill-seal BFS bottles and sealed, 0.4 mL per bottle. Among them, prescription 12 is the NGF eye drop formula disclosed in the prior art WO2022090339A1, and prescription 13 is based on prescription 12, replacing the phosphate buffer with L-histidine buffer, and adjusting the pH to 6.2.

[0199] Table 36. NGF-FC fusion protein eye drops prescription design

[0200] 2. Stability testing method

[0201] The sample just prepared was taken as the sample at 0 h. Each sample was placed at a high temperature of 40°C ± 3°C for 7 days and then taken out to test the stability index of each sample.

[0202] The detection methods are: visual observation of the solution appearance; determination of protein aggregation characteristics by SEC-HPLC; and determination of charge heterogeneity by WCX.

[0203] 3. Test results

[0204] At 0 hours, observation of the sample appearance revealed that samples of formulations 1-5 exhibited a distinct white precipitate and the solutions became turbid. Samples of formulations 8-9 exhibited a small amount of white flocculent precipitate. No foreign matter was visible in formulations 6-7 and 10-13. To improve testing efficiency and shorten formulation test development time, samples that exhibited precipitate will no longer be tested in subsequent tests.

[0205] The proportion of the main peak detected by SEC at time 0 in the other groups was above 95%, and there was no significant difference in the main peak, acidic peak and basic peak detected by WCX (Table 37).

[0206] At room temperature 25℃, shear force 500m.s -1The viscosity of each sample was tested under the following conditions, and the results are shown in Table 37. Prescription 11 served as a control without a viscosity enhancer, so its viscosity was not tested. The viscosities of Prescriptions 6 and 7 were essentially identical, indicating no significant difference in viscosity between the samples containing both HPMC K4M and polyethylene glycol 6000 and HPMC K4M alone. Prescription 10, which used polyvinylpyrrolidone (PVP K30) as a viscosity enhancer, had a low viscosity and was not suitable for development as an eye drop.

[0207] After being placed under high temperature conditions for 7 days, the SEC-HPLC test results are shown in Table 37: After 7 days at high temperature, the SEC main peak proportion of prescriptions 12 and 13 groups decreased significantly, less than 85.0%, while the SEC main peak proportion of prescriptions 6 and 7 groups remained above 95.0%, especially prescription 6 had better stability.

[0208] Based on the above results, prescription 6 was selected as the basis for subsequent formulation development.

[0209] Table 37. Viscosity / stability test results of eye drop formulations

[0210] (II) Screening and verification of viscosity-enhancing agent concentrations in eye drop prescriptions

[0211] 1. Eye drops prescription design and preparation

[0212] Four groups of fusion protein preparations were prepared using the NGF-Fc fusion protein 2-118-L3Fc10-M3-5. The components were identical except for the addition of the thickener HPMC (K4M): 0.1 g / mL fusion protein, 20 mM histidine buffer, 8.5% trehalose, 1 mM methionine, 5 mM HPBCD, 0.03% P188, and the pH was adjusted to 5.2 with acetate.

[0213] Prescription 1 added HPMC (K4M) at a concentration of 0.2%; Prescription 2 added HPMC (K4M) at a concentration of 0.4%; Prescription 3 added HPMC (K4M) at a concentration of 0.5%; Prescription 4 did not add HPMC (K4M).

[0214] 2. Stability testing conditions and methods

[0215] At 0:00, the viscosity of each formulation was tested, and formulations 1-4 were placed under the following conditions to test protein content, pH, osmotic pressure, and relative biological activity. The biological activity assay was performed with reference to the method described in CN103376248A, using an NGF-FC fusion protein without excipients as the reference standard. The standard was stored at -80°C. The biological activity of the sample and standard was measured for each assay. The relative biological activity was calculated as the ratio of the sample's biological activity to the standard's biological activity. The relative biological activity of the sample was set at 60%-140% of that of the standard.

[0216] Oscillation stability conditions: 2°C-8°C, 300 rpm, observe for 1 week and 1 month;

[0217] Freeze-thaw stability conditions: freeze at -70℃±10℃, thaw at 25℃±2℃, and repeat freeze-thaw 5 times;

[0218] Light stability conditions: 2℃-8℃, 4500±500Lux for 1 week and 1 month;

[0219] Accelerated stability conditions: 25℃±2℃ for 1 week, 1 month, 2 months, and 3 months;

[0220] High temperature stability conditions: 40℃±2℃, 1 week, 1 month;

[0221] Long-term stability conditions: 2°C-8°C, observation for 1 week, 1 month, 2 months, and 3 months.

[0222] 3. Test results

[0223] (1) Appearance, protein concentration and pH

[0224] At 0, the viscosities of the samples in groups 1 to 3 were 5.6 cp, 28.7 cp, and 34.5 cp, respectively. No viscosity test was performed on the sample 4 because no thickener was added.

[0225] After stabilizing under various influencing factors for a period of time, all samples showed no visible particles. Protein content remained essentially the same as at time 0. Regarding pH, after stabilizing under various influencing factors for a period of time, the osmotic pressure of each sample group ranged from 5.16 to 5.35, remaining within 5.2 ± 0.2, indicating a generally stable pH across all groups.

[0226] (2) Stability test results

[0227] The stability test results are shown in Tables 38-44.

[0228] Osmotic pressure: The osmotic pressure of the samples at 0 hours was between 303mOsm / kg and 317mOsm / kg. After 4 weeks of storage under oscillation (Table 38), 4 weeks under illumination (Table 39), and 12 weeks under refrigeration (Table 43), there was no significant change in the osmotic pressure of the groups. After 6 months of accelerated storage, the osmotic pressure of the samples in each group increased by 28mOsm / kg to 78mOsm / kg (Table 40). After 4 weeks under high temperature conditions, the osmotic pressure of the samples in each group increased by 20mOsm / kg to 111mOsm / kg (Table 42). The increase in osmotic pressure is due to the semipermeability of the eye drop packaging material. Water can be lost through the packaging material, resulting in an increase in the number of solute particles. The osmotic pressure of the solution depends on the number of solute particles per unit volume of solution. The more solute particles, the higher the osmotic pressure of the solution.

[0229] Relative biological activity: After being placed under oscillation conditions for 4 weeks (Table 38), accelerated conditions for 12 weeks (Table 41), high temperature conditions for 4 weeks (Table 42), and refrigerated conditions for 12 weeks (Table 44), the relative biological activities of each group of samples were within the standard range; after 4 weeks of illumination, the relative biological activities of each group of samples decreased to varying degrees (Table 39), indicating that prolonged exposure of the eye drop samples to sunlight would damage the samples and lead to reduced activity. In the later stage of sample storage, light should be avoided as much as possible, and outer packaging bags / boxes can also be added.

[0230] Table 38. Osmotic pressure and biological activity test results of eye drop preparations under oscillation conditions

[0231] Table 39. Osmotic pressure and biological activity test results of eye drops under light conditions

[0232] Table 40. Osmotic pressure test results of eye drop preparations under accelerated conditions

[0233] Table 41. Results of biological activity test of eye drop preparations under accelerated conditions

[0234] Table 42. Osmotic pressure and biological activity test results of eye drops under high temperature conditions

[0235] Table 43. Osmotic pressure test results of eye drop preparations under long-term refrigeration

[0236] Table 44. Results of biological activity test of eye drop preparations under long-term refrigeration

[0237] In summary, the NGF-Fc fusion protein eye drop formulation of the present invention can maintain good stability under conditions such as oscillation, freeze-thaw, acceleration, and long-term refrigeration, and can maintain an osmotic pressure of approximately 300 mOsm / kg. The biological activity of the fusion protein is also within the standard range (60%-140%). In particular, the viscosity of prescriptions 2 and 3 is more suitable for eye drops (25cp-50cp), ensuring that the NGF-Fc fusion protein can remain in the eye for a longer period of time and better exert its effect.

[0238] Example 5: Stability Verification of Eye Drops under Various Conditions

[0239] 1. Stability test method

[0240] The samples of recipes 2 and 3 in Example 4 (II) were placed in stabilization boxes with different influencing factor treatment conditions, taken out at different time points and tested according to the test plan. The conditions are as follows:

[0241] Oscillation stability conditions: 2°C-8°C, 300 rpm, 3 days, 1 week;

[0242] Light stability conditions (samples are packaged): 2°C-8°C, 4500±500 Lux, 1 week, 2 weeks;

[0243] Accelerated stability conditions: 25℃±2℃, 1 month;

[0244] High temperature stability conditions: 40℃±2℃, 2 weeks, 1 month;

[0245] The detection methods are: visual observation of the solution appearance; detection of protein concentration by titer assay; determination of protein aggregation characteristics by SEC-HPLC; and determination of charge heterogeneity by iCIEF assay.

[0246] 3. Stability test results

[0247] (1) Appearance and protein concentration

[0248] After being placed under different influencing factors for a period of time, the samples of prescriptions 2 and 3 did not produce any particles visible to the naked eye, and the protein concentration did not change significantly.

[0249] (2) Aggregate detection

[0250] The aggregation of each eye drop formulation was detected by SEC-HPLC, and the results are listed in Table 45.

[0251] From the results in Table 45, it can be seen that at 0 h, the SEC-HPLC main peak of the above two samples accounted for 99.70%-99.80%.

[0252] After being placed under oscillation conditions for 7 days, under light conditions for 14 days, and under accelerated conditions (with outer packaging) for 1 month, the SEC-HPLC main peak of the two samples did not change compared with 0h; after being placed under high temperature conditions for 1 month, the SEC-HPLC main peak accounted for 98.80%-98.90%.

[0253] This result shows that in terms of aggregate formation, the eye drops of the present invention have excellent stability under various conditions such as oscillation, light, acceleration (with outer packaging), and high temperature.

[0254] Table 45. SEC-HPLC test results under shaking, light, acceleration, and high temperature conditions

[0255] (3) Charge heterogeneity detection

[0256] The charge heterogeneity of the eye drop formulations was detected by iCIEF method, and the results are listed in Tables 46-51.

[0257] From the results in Tables 46-51, it can be seen that at 0h, the main peaks of the two groups of samples accounted for 69.15% and 69.67% respectively, the acidic peaks accounted for 20.92% and 20.76% respectively, and the basic peaks accounted for 9.94% and 9.58% respectively.

[0258] After the samples were placed under oscillation conditions for 1 week and under light conditions (with outer packaging) for 2 weeks, there was no significant change in the proportions of the iCIEF main peak, acidic peak, and alkaline peak in the two groups of samples (Tables 46-49).

[0259] When the samples were placed under high temperature conditions, the main peak gradually decreased and the acidic peak content increased with the continuous extension of the storage time (Table 50-51). After one month of high temperature storage, the proportion of the main peak decreased by 30.18%-31.26%, indicating that the protein sample was destroyed at high temperature, resulting in a decrease in the main peak of the charge variable. During the later storage of samples, it is necessary to avoid overheating to cause sample damage.

[0260] Table 46. Main peak detection results of iCIEF method under oscillation conditions

[0261] Table 47. Detection results of acidic and basic peaks by iCIEF under oscillation conditions

[0262] Table 48. Main peak detection results of iCIEF method under illumination conditions (with outer packaging)

[0263] Table 49. Detection results of acidic and basic peaks by iCIEF under illumination conditions (with outer packaging)

[0264] Table 50. Main peak detection results of iCIEF method under high temperature conditions

[0265] Table 51. Detection results of acidic and basic peaks by iCIEF under high temperature conditions

[0266] The above results show that the eye drops of the present invention have excellent stability in terms of protein aggregation and charge heterogeneity under various conditions such as oscillation, light, acceleration, and high temperature. However, attention should still be paid to the storage temperature of the preparation to avoid high temperature.

Claims

1. An NGF-Fc fusion protein preparation, characterized in that: The preparation comprises NGF-Fc fusion protein, a stabilizer, a surfactant, an antioxidant, and a buffer, wherein the stabilizer is trehalose or sucrose, the antioxidant is methionine, the surfactant is poloxamer, and the stabilizer is a histidine buffer.

2. The NGF-Fc fusion protein preparation according to claim 1, characterized in that: The concentration of the fusion protein is 0.05 mg / ml-5.0 mg / ml, preferably 0.05 mg / ml-2.0 mg / ml, and more preferably 0.1 mg / ml-0.5 mg / ml.

3. The NGF-Fc fusion protein preparation according to claim 1 or 2, characterized in that: The concentration of the histidine buffer is 8 mM-50 mM, preferably 10 mM-30 mM.

4. The NGF-Fc fusion protein preparation according to any one of claims 1 to 3, characterized in that: The concentration of the sucrose or trehalose is 6%-12%, preferably 7%-10%.

5. The NGF-Fc fusion protein preparation according to any one of claims 1 to 4, characterized in that: The concentration of methionine is 0.05 mM-7 mM, preferably 0.1 mM-5 mM, more preferably 1 mM-3 mM.

6. The NGF-Fc fusion protein preparation according to any one of claims 1 to 5, characterized in that: The concentration of the poloxamer is 0.005%-0.3%, preferably 0.01%-0.2%, and more preferably 0.01%-0.03%.

7. The NGF-Fc fusion protein preparation according to any one of claims 1 to 6, characterized in that: The pH of the preparation is 5.0-6.5, preferably 5.0-6.2, more preferably 5.0-5.

4.

8. The NGF-Fc fusion protein preparation according to any one of claims 1 to 7, characterized in that: The formulation further comprises cyclodextrin, preferably hydroxypropyl-beta-cyclodextrin.

9. The NGF-Fc fusion protein preparation according to claim 8, characterized in that: The cyclodextrin concentration is 0.05mM-8mM, preferably 1mM-6mM, and more preferably 5mM.

10. The NGF-Fc fusion protein preparation according to claim 1, characterized in that: The preparation is any one of the following preparations: (1) The concentration of the fusion protein is 0.05 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.10 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.14 mg / ml, 0.16 mg / ml, 0.18 mg / ml or 2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0-6.2; (2) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 Mm, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0-6.2; (3) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%, the concentration of methionine is 0.1 mM-5 Mm, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0-6.2; (4) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, the concentration of poloxamer is 0.01%-0.2%, and the preparation The pH is 5.0-6.2; (5) the concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%, 0.02%, 0.03%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, and the pH of the preparation is 5.0-6.2; (6) The concentration of the fusion protein is 0.05 mg / ml-2.0 mg / ml, the concentration of the histidine buffer is 10 mM-30 mM, the concentration of sucrose or trehalose is 7%-10%, the concentration of methionine is 0.1 mM-5 mM, the concentration of poloxamer is 0.01%-0.2%, and the pH of the preparation is 5.0, 5.1, 5.2, 5.3, 5.4, 5.6, 5.8, 6.0 or 6.

2.

11. The NGF-Fc fusion protein preparation according to claim 1, characterized in that: The preparation is any one of the following preparations: (1) The concentration of the fusion protein is 0.1 mg / ml, the concentration of the histidine buffer is 10 mM, the concentration of sucrose or trehalose is 7%, the concentration of methionine is 1.5 mM, the concentration of poloxamer is 0.01%, and the pH of the preparation is 5.4; (2) the concentration of the fusion protein is 0.1 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 9%, the concentration of methionine is 1.0 mM, the concentration of poloxamer is 0.03%, and the pH of the preparation is 5.2; (3) The concentration of the fusion protein is 0.5 mg / ml, the concentration of the histidine buffer is 30 mM, the concentration of sucrose or trehalose is 10%, the concentration of methionine is 3 mM, the concentration of poloxamer is 0.02%, and the pH of the preparation is 5.0; (4) The concentration of the fusion protein is 2.0 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 8%, the concentration of methionine is 2.0 mM, the concentration of poloxamer is 0.1%, and the pH of the preparation is 5.8; (5) The concentration of the fusion protein is 0.5 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 10%, the concentration of methionine is 0.1 mM, the concentration of poloxamer is 0.2%, and the pH of the preparation is 6.2; (6) The concentration of the fusion protein is 0.05 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of sucrose or trehalose is 8.5%, the concentration of methionine is 5.0 mM, the concentration of poloxamer is 0.04%, and the pH of the preparation is 5.

2.

12. The NGF-Fc fusion protein preparation according to any one of claims 1 to 11, characterized in that: The cyclodextrin concentration is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM or 6 mM.

13. The NGF-Fc fusion protein preparation according to any one of claims 1 to 12, characterized in that: The preparation is a liquid preparation, preferably, the preparation is an injection preparation or an eye drop preparation.

14. The NGF-Fc fusion protein preparation according to claim 13, characterized in that: The preparation is an eye drop preparation, and further comprises a viscosity increasing agent; 15. The NGF-Fc fusion protein preparation according to claim 14, characterized in that: The viscosity increasing agent is hypromellose; more preferably, the viscosity increasing agent is hypromellose K4M.

16. The NGF-Fc fusion protein preparation according to claim 15, characterized in that: The concentration of the viscosity enhancer is 0.4%-0.5%.

17. The NGF-Fc fusion protein preparation according to claim 15, characterized in that: The eye drop preparation contains the following components: the concentration of the fusion protein is 0.1 mg / ml, the concentration of the histidine buffer is 20 mM, the concentration of trehalose is 8.5%, the concentration of methionine is 1.0 mM, the concentration of poloxamer is 0.03%, the concentration of cyclodextrin is 5 mM, the concentration of hydroxypropyl methylcellulose is 0.4%, 0.45% or 0.5%, and the pH of the preparation is 5.

2.

18. The NGF-Fc fusion protein preparation according to any one of claims 1 to 17, characterized in that: The NGF-Fc fusion protein comprises an NGF portion and an Fc portion from N-terminus to C-terminus, wherein the Fc portion is derived from the Fc portion of IgG or a mutant of the Fc portion of IgG, and the mutant of the Fc portion of IgG includes a mutation in a site associated with ADCC / CDC activity.

19. The NGF-Fc fusion protein preparation according to claim 18, characterized in that: The NGF part is human nerve growth factor, preferably a human nerve growth factor mutant.

20. The NGF-Fc fusion protein preparation according to claim 19, characterized in that The human nerve growth factor mutant, relative to the amino acid position of the wild-type human nerve growth factor, has a mutation site including Phe12Glu; preferably, the human nerve growth factor comprises the amino acid sequence of SEQ ID NO:

2.

21. The NGF-Fc fusion protein preparation according to any one of claims 18 to 20, characterized in that: The NGF portion is fused to the Fc portion via a polypeptide linker, and the polypeptide linker comprises the amino acid sequence of SEQ ID NO:

3.

22. The NGF-Fc fusion protein preparation according to any one of claims 18 to 21, characterized in that: The Fc portion is derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO:

4.

23. The NGF-Fc fusion protein preparation according to any one of claims 18 to 22, characterized in that: The NGF-Fc fusion protein comprises the amino acid sequence shown in SEQ ID No:

1.

24. The NGF-Fc fusion protein preparation according to any one of claims 1 to 23, characterized in that: The NGF-Fc fusion protein preparation is used in preparing drugs for treating NGF-related diseases.

25. According to the use described in claim 24, the NGF-related disease is a nervous system disease; preferably, the nervous system disease is selected from the group consisting of neonatal hypoxic-ischemic encephalopathy, cerebral palsy, critical illness myopathy, neurological deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down syndrome, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, diffuse brain injury, thymic dysplasia, optic nerve contusion, follicular dysplasia, spinal cord injury, glaucoma, neurotrophic keratitis, optic nerve injury, neuromyelitis optica, retinal related diseases, urinary incontinence, Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, hypertensive cerebral hemorrhage neurological dysfunction, cerebral small vessel disease, acute ischemic stroke, corneal endothelial dystrophy, diabetic neuropathy, diabetic foot ulcer, neurogenic skin ulcer, pressure sore, neurotrophic corneal ulcer, diabetic corneal ulcer and macular hole.

26. According to the use according to claim 24, the NGF-related disease is a non-neurological disease; preferably, the non-neurological disease is selected from the group consisting of splenic atrophy, splenic contusion, decreased ovarian reserve function, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicular hypoplasia, spermatogenesis disorders (such as oligospermia, asthenozoospermia, oligoasthenozoospermia), ischemic ulcers, stress ulcers, rheumatoid ulcers, liver fibrosis, corneal ulcers, burns, oral ulcers and lower extremity venous ulcers.