An anti-egfr antibody ophthalmic injection

Abstract

The application discloses an anti-EGFR antibody eye injection, which comprises an anti-EGFR antibody with a final concentration of 1-100 mg / mL, 0.25-1 mg / mL polysorbate 20, 1-5 mg / mL buffer, 0.1-1 mg / mL isotonicity regulator, 30.0-40 mg / mL sucrose and 5-10 mg / mL glycine; and the pH value of the eye injection is 5.3-5.7. The application has good storage stability, and has few insoluble particles, which meets the requirement of USP789 eye injection, and provides a new preparation selection for the application of the anti-EGFR antibody in ophthalmology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to an anti-EGFR antibody ophthalmic injection. BACKGROUND

[0002] With the growth of human age, the aggravation of myopia or the development of related metabolic diseases, when ocular fundus diseases such as pathological myopia, age-related macular degeneration, diabetic retinopathy and the like occur, some unnecessary blood vessels are newly formed in the ocular fundus. These newly formed blood vessels invade the normal ocular fundus tissues such as choroid and retinal photoreceptor layer, causing impaired vision, and even blindness in severe cases.

[0003] Neovascularization is a common pathological change of many eye diseases, which often occurs in cornea, iris, choroid and retina, and the diseases caused thereby include corneal neovascularization, choroidal neovascularization, retinal neovascularization and the like, which can cause a series of pathological changes such as hemorrhage, exudation and proliferation in ocular tissues, thereby causing damage to the structure and function of eyeball and seriously impairing the visual function. The number of patients with ocular neovascular disease is more than 40 million, and with the aggravation of population aging, the number of patients is also rising. Patients with ocular neovascular disease almost have to re-visit or inject related drugs every month, otherwise the impairment of vision will seriously affect life.

[0004] At present, the mainstream varieties of drugs for treating ocular vascular lesions are three of ranibizumab, conbercept and aflibercept, which are all mAbs or fusion proteins targeting VEGF, and CN114652826A discloses the use of anti-EGFR antibodies, which first proposes that the monoclonal antibody targeting EGFR has a good inhibitory effect on ocular neovascularization, especially ocular fundus neovascularization, and can be used for treating diseases related to ocular fundus neovascularization. At present, the related preparations of anti-EGFR antibodies are all used for treating tumors and the like, there is no ophthalmic preparation of anti-EGFR antibodies, and the quality requirements of ophthalmic injection preparations are more stringent than those of conventional injection preparations; according to the requirements of USP789 ophthalmic injection, the insoluble particles ≥10um are required to be ≤50 particles / ml, the particles ≥25um are required to be less than ≤5 particles / ml, and the insoluble particles ≥50um are required to be ≤2 particles / ml.

[0005] CN113952450A discloses an eye preparation containing EGFR antibody, and provides specific product prescriptions with protein concentrations of 25.0 mg / mL, 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL and 5.0 mg / mL, respectively, and their specific additional agent components and pH values are as follows: 7.50 g / L glycine, 5.85 g / L sodium chloride, 2.10 g / L citric acid monohydrate, 0.97 g / L sodium hydroxide, 0.10 g / L Tween 80, pH 5.5, and it is pointed out that the preparation can be used for vitreous injection. However, the inventors found that the insoluble particle content of the preparation prepared according to the above-mentioned prescription does not meet the relevant requirements of USP 789 ophthalmic injection.

[0006] CN107773755B discloses an injection preparation of anti-epidermal growth factor receptor monoclonal antibody, which contains 10 mg / mL of anti-epidermal growth factor receptor (i.e. EGFR) monoclonal antibody, 20 mM of citric acid and sodium citrate with a total concentration of pH 6.0, 5% of trehalose by mass / volume, 0.05% of Tween 80 by mass / volume and 0.3% of sodium chloride by mass / volume. The insoluble particle data of the above-mentioned preparation is shown as follows: the insoluble particles ≥10 um after two weeks are 67 particles / ml. It can be seen that the above-mentioned preparation cannot be directly used as an ophthalmic injection, especially a preparation for vitreous injection.

[0007] Therefore, in view of the therapeutic potential of anti-EGFR antibody in ocular neovascularization diseases, it is urgent to develop an anti-EGFR antibody pharmaceutical preparation / composition that meets the quality requirements of eye injection, especially vitreous injection. SUMMARY

[0008] To solve the above-mentioned technical problems, the present application provides an anti-EGFR antibody ophthalmic injection, which comprises an anti-EGFR antibody with a final concentration of 1-100 mg / mL, 0.25-1 mg / mL of polysorbate 20, 1-5 mg / mL of a buffer, 0.1-1 mg / mL of an isotonicity adjusting agent, 30.0-40 mg / mL of sucrose and 5-10 mg / mL of glycine; and the pH value of the ophthalmic injection is 5.3-5.7.

[0009] Further, the buffer is selected from citric acid and phosphate, preferably citric acid.

[0010] Further, the isotonicity adjusting agent is preferably sodium chloride.

[0011] Further, the concentration of the anti-EGFR antibody is preferably 1-80 mg / mL, more preferably 20-80 mg / mL, and more preferably 40-80 mg / mL.

[0012] Further, the pH value of the ophthalmic injection is 5.5.

[0013] Further, the pH regulator is sodium hydroxide.

[0014] Further, it comprises an anti-EGFR antibody with a final concentration of 80 mg / mL, 0.5 mg / ml polysorbate 20, 2.1 mg / ml citric acid, 0.3 mg / ml sodium chloride, 34 mg / ml sucrose, and 7.5 mg / ml glycine.

[0015] Further, the anti-EGFR antibody is selected from cetuximab or its biosimilars, nimotuzumab, panitumumab, and zalutumumab, preferably cetuximab or its biosimilars.

[0016] The present application finally provides a method for preparing the aforementioned anti-EGFR antibody ophthalmic injection, comprising the following steps:

[0017] 1) Weigh the raw materials according to the ratio;

[0018] 2) Take 60-80% of the formula amount of water for injection, add the buffer, glycine, sucrose, and isotonicity regulator in sequence to dissolve, then add polysorbate 20 and stir uniformly, adjust the pH value to 5.3-5.7, make up the remaining amount of water for injection, and finally add the anti-EGFR antibody stock solution under stirring to obtain the product.

[0019] Further, the stirring time in step 2) is not less than 20 min.

[0020] The anti-EGFR antibody ophthalmic injection of the present application has good storage stability, and has few insoluble particles, meeting the requirements of USP789 ophthalmic injection, and provides a new formulation selection for the application of anti-EGFR antibody in ophthalmology.

[0021] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements, or changes can be made without departing from the above basic technical ideas of the present application.

[0022] The above content of the present application will be further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following embodiments. Any technology realized based on the above content of the present application belongs to the scope of the present application. DETAILED DESCRIPTION

[0023] The raw materials and equipment in the specific embodiments of the present application can be obtained by market purchase.

[0024] Example 1 Anti-EGFR antibody ophthalmic injection

[0025] Formulation: Cetuximab 80mg / mL, polysorbate 200.5mg / ml, citric acid 2.1mg / ml, sodium chloride 0.3mg / ml, sucrose 34mg / ml, glycine 7.5mg / ml, the rest is water for injection; adjust the pH of the preparation to 5.5 (adjust with sodium hydroxide)

[0026] Preparation process:

[0027] 1) Weigh the raw materials according to the ratio;

[0028] 2) First, add 60% to 80% of the total weight of the injection to the liquid preparation container, then add the weighed citric acid, glycine, sodium chloride, sucrose to the liquid preparation container in turn, stir and dissolve, then add polysorbate 20, stir uniformly, the stirring time is not less than 20min, then adjust the pH to 5.3-5.7 with pH adjuster, finally add water for injection to constant volume to the total weight of the injection, stir uniformly, preparation is completed, after the pH is qualified, add the weighed anti-EGFR antibody stock solution, keep stirring during the addition process, the stirring time is not less than 20min to obtain the anti-EGFR antibody eye injection.

[0029] Example 2 Anti-EGFR antibody eye injection

[0030] Formulation: Cetuximab 80mg / mL, polysorbate 200.5mg / ml, citric acid 2.1mg / ml, sodium chloride 0.3mg / ml, sucrose 34mg / ml, glycine 7.5mg / ml, the rest is water for injection; adjust the pH of the preparation to 5.5 (adjust with sodium hydroxide)

[0031] Preparation process: same as example 1

[0032] Example 3 Anti-EGFR antibody eye injection

[0033] Formulation: Cetuximab 80mg / mL, polysorbate 200.5mg / ml, citric acid 2.1mg / ml, sodium chloride 0.3mg / ml, sucrose 34mg / ml, glycine 7.5mg / ml, the rest is water for injection; adjust the pH of the preparation to 5.5 (adjust with sodium hydroxide)

[0034] Preparation process: same as example 1

[0035] Example 4 Anti-EGFR antibody eye injection

[0036] Formulation: Cetuximab 80mg / mL, polysorbate 200.5mg / ml, citric acid 2.1mg / ml, sodium chloride 0.3mg / ml, sucrose 34mg / ml, glycine 7.5mg / ml, the rest is water for injection; adjust the pH of the preparation to 5.5 (adjust with sodium hydroxide)

[0037] Preparation process: same as Example 1

[0038] Example 5 Anti-EGFR antibody ophthalmic injection

[0039] Formulation: Nab-paclitaxel 1 mg / mL, polysorbate 200.5 mg / mL, citric acid 2.1 mg / mL, sodium chloride 0.3 mg / mL, sucrose 34 mg / mL, glycine 7.5 mg / mL, and the rest is water for injection, and the pH of the preparation is adjusted to 5.5 (adjusted with sodium hydroxide)

[0040] Preparation process: same as Example 1

[0041] The beneficial effects of the present application are specifically illustrated by the following test examples.

[0042] Test Example 1: Anti-EGFR antibody ophthalmic injection study

[0043] Amino acid sequence of cetuximab:

[0044] Heavy chain (SEQ ID No: 1):

[0045] QVQLKQSGPG LVQPSQSLSITCTVSGFSLT NYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLight chain (SEQ ID No: 2):

[0046] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY ASESISGIPSRFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGAGTK LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

[0047] In the following tests, the JZB09 protein is cetuximab.

[0048] Insoluble particle detection method: insoluble particle detection method in Chinese Pharmacopoeia 2020 edition third part general rule 0903, light resistance method

[0049] Purity SEC-HPLC: refer to Chinese Pharmacopoeia 2020 edition third part general rule 0514 molecular exclusion chromatography for detection;

[0050] Purity CE-SDS: refer to Chinese Pharmacopoeia 2020 edition fourth part general rule 0542 capillary electrophoresis for detection;

[0051] Purity rCE-SDS: refer to Chinese Pharmacopoeia 2020 edition fourth part general rule 0542 capillary electrophoresis for detection; protein content detection: refer to Chinese Pharmacopoeia 2020 edition fourth part general rule 0401, A280 method for detection.

[0052] Based on the conventional screening method of existing antibody preparation, the present application preliminarily selects polysorbate 20, citric acid, sodium chloride, glycine and pH regulator as excipients to design the prescription, so as to screen out the ophthalmic preparation meeting the USP requirement for JZB09 protein.

[0053] Surfactant dosage preliminary screening: take polysorbate 20 as surfactant, and design the following table 1 prescription.

[0054] Table 1 surfactant screening prescription

[0055]

[0056] Table 2 surfactant screening prescription insoluble particle and stability detection results

[0057]

[0058] Result analysis: From the results of Table 2 above, it can be seen that when the concentration of polysorbate 20 is 0.5 and 0.75 mg / ml, compared with the concentration of polysorbate 20 being 0.25 mg / ml, the preparation has fewer insoluble particles, but the amount of insoluble particles still does not meet the requirements of the USP pharmacopoeia for insoluble particles of ophthalmic injection, therefore, the concentration of polysorbate 20 is preliminarily selected as 0.5 and 0.75 mg / ml for screening of other excipients to obtain a preparation formula with insoluble particles meeting the USP pharmacopoeia.

[0059] Based on this, the following Table 3 prescription is further designed for excipient screening to control the osmotic pressure to be 250-360 mOsmol / kg.

[0060] Table 3 Excipient screening prescription design

[0061]

[0062] Table 4 Excipient screening prescription insoluble particle and stability test results

[0063]

[0064]

[0065]

[0066] Result analysis: From the above 8 groups of prescription data under the condition of high temperature 40℃, it can be seen that the 09P20220605 prescription has no significant change in appearance, protein content, purity (SEC, CE-SDS, rCE-SDS) at 1W / 2W / 4W (1 week / 2 weeks / 4 weeks) compared with 0 days, and has no obvious difference compared with the data of the other 7 groups of prescriptions, while the 09P20220605 prescription is superior to the other 7 groups of prescriptions in terms of insoluble particle detection items, therefore, the final injection prescription of JZB09 is determined as: 0.5 mg / ml polysorbate 20, 2.1 mg / ml citric acid, 0.3 mg / ml sodium chloride, 34.0 mg / ml sucrose, 7.5 mg / ml glycine, 0.88 mg / ml sodium hydroxide aqueous solution.

[0067] The prescription is used in the clean room environment under the background of B+A for the production of engineering batches, the engineering batch prescription is shown in the following Table 5, and the insoluble particle and stability conditions under different protein concentration conditions are focused on, and the engineering batch insoluble particle and stability test results are shown in the following Table 6.

[0068] Table 5 Engineering batch prescription

[0069]

[0070] Table 6 Engineering batch insoluble particle and stability test results

[0071]

[0072]

[0073]

[0074] Result analysis: using diluent prescription: 0.5mg / ml polysorbate 20, 2.1mg / ml citric acid, 0.3mg / ml sodium chloride, 34.0mg / ml sucrose, 7.5mg / ml glycine, 0.88mg / ml sodium hydroxide aqueous solution, the clean area in B+A background can produce products meeting the requirements of USP789 ophthalmic injection (≥10um not more than 50 particles / ml, ≥25um not more than 5 particles / ml, ≥50um not more than 2 particles / ml).

[0075] Comparative experiment 1

[0076] On the basis of the prescription of the application, sucrose is replaced by trehalose, and the insoluble particles and stability of the preparation are investigated, and the prescription is shown in Table 7.

[0077] Table 7 prescription of comparative experiment 1

[0078]

[0079] Table 8 results of insoluble particle and stability detection (0d) of comparative experiment 1 prescription

[0080]

[0081]

[0082] Result analysis: from the above table, on the basis of the prescription of the application, sucrose is replaced by trehalose, there is no significant difference in protein content, the purity is slightly lower, but the insoluble particles are significantly different from the prescription of the application. The particles greater than ≥10um are still more than 50 particles when the protein concentration is 1mg / ml, which does not meet the requirements of USP ophthalmic injection.

[0083] Comparative experiment 2:

[0084] Aflibercept is an anti-VEGF fusion protein preparation for intravitreal injection, therefore, in the prescription screening, the excipients of the drug aflibercept prescription are used, and the prescriptions of the application JZB09 protein concentration of 10, 40, 50, 80mg / ml are designed respectively, see the following table, and the insoluble particles and stability are investigated.

[0085] Table 9 prescription of comparative experiment 2

[0086]

[0087] Table 10: Results of insoluble particles and stability test of prescription of comparative experiment 2

[0088]

[0089] Result analysis: From the above table, it can be seen that when the protein concentration is 10 mg / ml, the number of insoluble particles ≥10 um is more than 50, and with the increase of concentration, the number of insoluble particles ≥10 um also increases, which does not meet the requirements of USP ophthalmic preparation.

[0090] Comparative experiment 3:

[0091] Based on the prescription disclosed in CN113952450A, the formulations of JZB09 protein with concentrations of 5, 10, 40, 50 and 80 mg / ml were prepared, and the insoluble particles and stability were investigated.

[0092] Table 11: Prescription of comparative experiment 3

[0093]

[0094] Table 12: Results of insoluble particles and stability test of prescription of comparative experiment 3

[0095]

[0096] Result analysis: From the above table, it can be seen that when the protein concentration is 10 mg / ml, the number of insoluble particles ≥10 um is more than 50, and with the increase of concentration, the number of insoluble particles ≥10 um also increases, which does not meet the requirements of USP ophthalmic preparation.

[0097] In summary, the specific excipients and their amounts in the present application make the anti-EGFR antibody ophthalmic injection have good storage stability, and the number of insoluble particles is small, which meets the requirements of USP 789 ophthalmic injection.

Claims

1. An anti-EGFR antibody ophthalmic injection, characterized in that: It includes the final concentration. The ophthalmic injection contains 80 mg / mL anti-EGFR antibody, 0.5 mg / mL polysorbate 20, 2.1 mg / mL citric acid, 0.3 mg / mL sodium chloride, 34.0 mg / mL sucrose, 7.5 mg / mL glycine, and the remainder is water for injection; the pH value of the ophthalmic injection is 5.

5. The anti-EGFR antibody is cetuximab.

2. The anti-EGFR antibody ophthalmic injection according to claim 1, characterized in that: The pH adjuster is sodium hydroxide.

3. A method for preparing the anti-EGFR antibody ophthalmic injection according to claim 1 or 2, characterized in that: 1) Weigh the raw materials according to the proportions; 2) Take 60-80% of the formula amount of water for injection, add citric acid, glycine, sucrose and sodium chloride in sequence to dissolve, then add polysorbate 20 and stir evenly, adjust the pH value to 5.5, add the remaining water for injection, and finally add the anti-EGFR antibody stock solution under stirring conditions to obtain the final product.

4. The method according to claim 3, characterized in that: Step 2) Stirring time should be no less than 20 minutes.

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