CN115068592A - Recombinant human interferon alpha 1b eye drops stable at normal temperature after unsealing and preparation method thereof - Google Patents

Abstract

The invention discloses a recombinant human interferon alpha 1b eye drop that is stable at room temperature after opening and a preparation method, and relates to the field of pharmaceutical preparations. One or more of the osmotic pressure regulators, the eye drops of the present invention, the present invention can be stored for a long time under low temperature conditions, and can be stored for a long time under the condition that is not protected from light at room temperature after opening, and is protected from light at 2-8 ° C It can be stored for at least 24 months under storage (regular storage requirements), after opening (resealing) at room temperature (25℃‑35℃), it can be stored for at least 9 months without being protected from light, and after opening without resealing at room temperature (25℃‑ 35°C) can be stored for at least 9 days without being protected from light, and the above can be stored means to ensure sterility during storage, no obvious changes in product pH and osmotic pressure, no visible foreign matter, and stable biological activity.

Description

Recombinant human interferon α1b eye drops stable at room temperature after opening and preparation method

technical field

The invention relates to the field of pharmaceutical preparations, in particular to a recombinant human interferon α1b eye drop that is stable at room temperature after opening and a preparation method.

Background technique

Interferon is a kind of cytokine protein drug originally produced by animal body and has broad-spectrum antiviral, antiproliferative and immunomodulatory effects. type, and each large type can be divided into several small subtypes. Among several large types, alpha type is the most widely used one. At present, this type of interferon in clinical application mainly includes interferon α2a, Interferon alpha 2b, interferon alpha 1b, etc. In 1980, foreign scientists cloned interferon alpha 2a and interferon alpha 2b genes from Europe and the United States successively, and expressed them recombinantly in Escherichia coli, making it possible to use them as drugs for clinical treatment. In 1981, Academician Hou Yunde cloned the α1-type interferon gene from healthy Chinese umbilical cord blood leukocytes using modern molecular biology techniques, which was later named α1b by the International Professional Committee on Nomenclature of Interferons. There are essential differences between recombinant human interferon α1b and recombinant human interferon α2b: although they are both proteins, recombinant human interferon α1b is a single-chain polypeptide composed of 166 amino acid residues, with a theoretical molecular weight of 19.4KD ± 1.9KD; Human interferon α2b is a single-chain polypeptide composed of 165 amino acid residues, with a theoretical molecular weight of 19.2KD±1.9KD; their primary structure differs by one amino acid, which also leads to a series of differences. Interferon α1b derived from the Chinese gene was later shown to be the most important antiviral type in the Chinese. In the research of various viral infections, it has been found that after Chinese people are infected by the virus, the antiviral substance that appears in the human body first and produces the largest amount is interferon α1b. A large number of clinical practices have shown that recombinant human interferon α1b is the most suitable for Chinese people, and the adverse reactions are significantly lower than those of recombinant human interferon α2a and recombinant human interferon α2b derived from Westerners.

Recombinant human interferon α1b has a broad-spectrum antiviral effect. It binds to cell surface receptors and activates a variety of cytoplasmic enzymes through a series of intermediate metabolisms. These enzymes can inhibit virus reproduction in cells and interfere with viral nucleic acid and protein synthesis. . Interferon can also inhibit the function of T cells and B cells, activate macrophages and NK cells, and promote the effect of killer T cells, thus achieving antiviral effects.

Ocular viral infectious diseases are inflammations caused by the infection of the eyes by viral pathogens. The most common pathogen is scab virus, followed by zona virus. Clinically, it is mainly divided into the following diseases: ① Herpes simplex ophthalmopathy: It is caused by the infection of the eye by herpes simplex virus (HSV), including eyelid herpes simplex, herpes simplex conjunctivitis, keratitis (branch) shape, map shape, disc shape, parenchymal keratitis), herpes simplex iridocyclitis, etc. ② Herpes zoster ophthalmopathy: It is caused by the infection of the eyes by the herpes zoster virus (varicella-zoster virus, VZV), including eyelid herpes zoster, herpes zoster keratitis, scleritis, etc. ③ adenovirus conjunctival keratitis. ④ epidemic hemorrhagic conjunctivitis, commonly known as pink eye.

At present, there are many ophthalmic patients with viral infections such as viral keratitis and viral conjunctivitis. There are many types of eye drops on the market, but most of them are antibiotic eye drops, which are only effective for bacterial infections. . There are fewer effective antiviral eye drops for viral infections of the eye. Interferon has broad-spectrum anti-viral, anti-tumor, and immune function-regulating effects, and has been recognized by scholars as a drug with good anti-viral efficacy.

Aqueous solutions of interferon have a problem of being unstable in long-term storage. CN1724567A discloses a stable aqueous solution of recombinant human interferon α1b, and its auxiliary agents include sodium chloride, citric acid, disodium hydrogen phosphate dodecahydrate and human serum albumin, wherein the preferred range of sodium chloride is 6.5-7.5 g/L. CN101214224A discloses a recombinant human interferon injection, which uses succinyl gelatin and Tween 80 as a stabilizer instead of human albumin. CN109010803A discloses a recombinant human interferon α2b eye drop and a preparation method thereof. The auxiliary materials include sodium hyaluronate, histidine, disodium edetate, Tween 80, bacteriostatic agent and sodium chloride. CN111617031A discloses a stable recombinant human interferon alpha 1b eye drop and a production method thereof. The eye drop consists of recombinant human interferon alpha 1b, a protective agent, an osmotic pressure regulator and water. The above-mentioned schemes all achieve the purpose of long-term stable preservation of the interferon aqueous solution by adjusting the formula of the interferon preparation, especially adding a stabilizer.

However, the above solutions, including other existing similar technologies, have a common problem: eye drops are a relatively special medicine, such as tablets, capsules, injections, etc. However, the use period of the eye drops after opening is within 24 hours for short (about 1mL in small packages) and within 1 month for long ones (about 10-30mL in large packages). Usually, it will not be put back in the storage environment required by the instructions (2-8°C away from light), and some users will not mark the opening date, which leads to the continued use of eye drops after the expiration date of opening. It can only guarantee the stability of the interferon aqueous solution in the dark environment of 2-8 ℃, and cannot effectively solve the above-mentioned problems such as room temperature and light affecting the stability, especially the problem of rapid decrease in stability after opening. Among them, the decrease in stability after opening mainly includes two factors. On the one hand, the reagents are polluted by users or the environment after opening, and the contaminants affect the reagents. On the other hand, the interferon itself receives after opening. The combined effects of various factors in the environment lead to a decrease in its own stability. In the current technology, these effects are usually reduced by adding antibacterial agents or adding a sealed bag after opening, but the effect is generally not significant, and the addition of antibacterial agents can also irritate the eyes and negatively affect the performance of eye drops.

The eye is one of the most sensitive organs in the human body. The eye drops in the eye drops applied to the eyes should add as little chemical substances as possible to reduce the irritation to the eyes under the premise of ensuring that the solution is stable at room temperature after opening.

SUMMARY OF THE INVENTION

In view of the above-mentioned problems, the present invention adds one or more of protective agents, stabilizers, additional regulators and osmotic pressure regulators to the recombinant human interferon α1b stock solution obtained by fermentation and purification, and mixes it uniformly through 0.22 μm The recombinant human interferon alpha 1b eye drops are packaged after being sterilized and filtered through a filter membrane and finally passed the inspection. The eye drops of the present invention use reagents that avoid chemicals that are irritating to the eyes, or add pure natural non-irritating ingredients, which can achieve stability for a long time after opening under normal temperature and light-proof conditions. The specific plans are as follows:

First of all, the present invention provides a recombinant human interferon α1b eye drop that is stable at room temperature after opening, including recombinant human interferon α1b, and one or two of a protective agent, a stabilizer, an additional regulator, and an osmotic pressure regulator. more than one species.

Preferably, the protective agent comprises a mixture of arginine, sitosterol, human albumin.

Preferably, the concentrations of arginine, sitosterol and human albumin in the protective agent in recombinant human interferon α1b eye drops are 1.0-1.5% (w/v), 0.3-0.5% (w/v), respectively. /v), 0.2-0.4% (w/v).

Preferably, the stabilizer comprises a mixture of water-soluble beta-1,3 glucan and lanolin.

Preferably, the concentrations of water-soluble β-1,3 glucan and lanolinine in recombinant human interferon α1b eye drops in the stabilizer are 0.5-1.2% (w/v) and 0.5-1.0%, respectively. (w/v).

Preferably, if the total concentration of the protective agent in the recombinant human interferon α1b eye drops is i, and the total concentration of the stabilizer is j, then i=2β×j, β is a constant, and takes a value of 1-1.25.

Preferably, the additional conditioning agent comprises a mixture of Tribulus terrestris extract and okra extract.

Preferably, the concentration of Tribulus terrestris extract and okra extract in recombinant human interferon α1b eye drops in the additional regulator is 0.6-0.9% (w/v), 0.1-0.5% (w/v), respectively. ).

Preferably, the preparation method of the Tribulus terrestris extract includes: crushing the Tribulus terrestris to a size of 80 or more; adding distilled water with a liquid-solid ratio of 20:1-25:1 ml/g; heating to 40-90° C. for ultrasonic extraction for 90-180min The filtrate was obtained by conventional filtration treatment, and the filtrate was centrifuged at 2000-3000 rpm for 5 min to take the supernatant, filtered through a 0.22 μm filter membrane, and freeze-dried to obtain the Tribulus terrestris extract.

Preferably, the preparation method of the okra extract comprises: washing, chopping, and homogenizing the okra pods; adding 3 times the mass of distilled water, and centrifuging at 3000 rpm for 5 minutes after standing for 24 hours, and taking the supernatant as the crude extract; Add 35% concentration of ethanol to the crude extract, stir while adding to produce a precipitate, centrifuge at 3200 rpm for 5 min, collect the precipitate, and vacuum dry; the obtained product is redissolved in 30-50 times the mass of distilled water, and dialyzed in 20 times the volume of distilled water for 48 hours , the medium was changed 4 times a day, lyophilized after dialysis, and the okra extract was obtained.

Preferably, the osmotic pressure regulator includes sodium chloride and borax.

Preferably, the concentrations of sodium chloride and borax in the osmotic pressure regulator in recombinant human interferon α1b eye drops are 0.1-0.3% (w/v) and 0.4-0.6% (w/v), respectively.

The percentage (w/v) in the concentration described in the present invention refers to the mass of the solute (g)÷the volume of the solution (mL)×100%.

Second, the present invention also discloses the preparation method of the above-mentioned recombinant human interferon α1b eye drops, including:

S1, take recombinant human interferon α1b stock solution, add one or more of protective agent, stabilizer, additional regulator and osmotic pressure regulator and mix well;

S2, filter sterilization;

S3, product packaging.

Preferably, said adding one or more of protective agent, stabilizer, additional regulator and osmotic pressure regulator, the order of adding is to add protective agent, stabilizer, additional regulator and osmotic pressure regulator in sequence .

Preferably, in the recombinant human interferon alfa 1b stock solution, the concentration of the recombinant human interferon alfa 1b is 10-3 million IU/ml.

In the present invention, phosphate buffer or PBS buffer can be used as a buffer system for dissolving each component to prepare the eye drops. The pH of the phosphate buffer is 6-7.5, and the concentration is 15.0-40.0 mmol/ L.

beneficial effect

The beneficial technical effects of the present invention are:

(1) Recombinant human interferon α1b eye drops of the present invention can be used for the treatment of ocular viral infectious diseases, including herpes simplex ophthalmopathy: such as eyelid herpes simplex, herpes simplex conjunctivitis, keratitis (dendritic, geographic , discoid, parenchymal keratitis), herpes simplex iridocyclitis), herpes zoster ophthalmopathy: such as eyelid herpes zoster, herpes zoster keratitis, scleritis, iridocyclitis, adenovirus conjunctival keratitis, epidemic hemorrhagic conjunctivitis;

(2) The protective agent adopted in the present invention is a mixture of arginine, sitosterol and human albumin, and the stabilizer adopted is a mixture of water-soluble β-1,3 glucan and lanolin, and such additives are effective Enhanced the stability of eye drops under normal temperature and light environment after opening;

(3) The present invention also adopts an additional regulator, which is a mixture of Tribulus terrestris extract and okra extract, both of which have had records of application and eye drops in previous work, but refused to check, and now In some work, the addition of Tribulus terrestris extract in eye drops often has the effect of improving eyesight, relieving itching, and relieving allergic conjunctivitis. The addition of okra extract in eye drops, its effect It is often to relieve dryness and fatigue of the eyes, but in the present invention, the core active ingredient of the present invention is recombinant human interferon α1b, and the only active substance that plays a therapeutic role is also recombinant human interferon α1b. The core purpose of the present invention is to improve The stability of recombinant human interferon α1b eye drops, especially the stability under normal temperature and light conditions after opening, the addition of the above two kinds of plant extracts is very small, in fact, under such additions, it is basically unable to achieve the above Conventional effect, and the purpose of adding these two plant extracts in this application is not to play a conventional therapeutic role, such an addition amount, when any one of the extracts is added alone, will affect the overall properties of the eye drops (including The performance and stability of the therapeutic properties) were not significantly affected. When the two extracts were added at the same time and added within the scope defined by the present invention, the overall stability of the eye drops was significantly improved, and the therapeutic properties did not change significantly. We Innovative use of the above two plant extracts as special stabilizers;

(4) The eye drops of the present invention can be stored for a long time under low temperature conditions, and can be stored for a long time under normal temperature and light-proof conditions after opening, and can be stored at 2-8° C. under light-proof storage (conventional storage requirements) for at least 24 months, after opening (resealing) at room temperature (25°C-35°C), it can be stored for at least 9 months without being protected from light, after opening (without resealing) at room temperature (25°C-35°C) and not protected from light It can be stored for at least 9 days, and the above can be stored refers to ensuring sterility during storage, no obvious changes in product pH and osmotic pressure, no visible foreign matter, and stable biological activity.

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

The following examples use a phosphate buffer with a pH of 7.0 and a concentration of 20.0 mmol/L.

Preparation example:

1. Preparation of recombinant human interferon α1b stock solution:

The recombinant human interferon alpha 1b stock solution is prepared by our company (Changchun Institute of Biological Products Co., Ltd.), and it can also be prepared to the required concentration using commercially available recombinant human interferon alpha 1b, or prepared by other preparation methods disclosed in the prior art, There is no significant difference in the effects and trends of other components used in combination.

The recombinant plasmid containing recombinant human interferon α1b liquid gene was constructed by using E. coli expression technology, and transformed into E. coli to obtain stable engineering bacteria. After fermentation of the engineering bacteria, the polymerization in the interferon was removed through preliminary extraction and several purification processes. Recombinant human interferon α1b stock solution was obtained, and the concentration was 100,000 IU/ml, and the purity complied with the provisions of "Pharmacopoeia of the People's Republic of China" (2015 edition). Operations such as plasmid preparation, transformation, fermentation, and purification are all routine operations in the prior art.

2. Preparation of Tribulus terrestris extract: pulverize Tribulus terrestris to 120 mesh; add distilled water at a liquid-to-material ratio of 20:1 ml/g; heat to 70 ° C for ultrasonic extraction for 120 min; conventional filtration treatment to obtain filtrate, and the filtrate is centrifuged at 3000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter, and freeze-dried to obtain the Tribulus terrestris extract.

3. Preparation of okra extract: wash, chop, and homogenize okra pods; add 3 times the mass of distilled water, stand for 24 hours and then centrifuge at 3000 rpm for 5 min, take the supernatant as a crude extract; add to the crude extract 35% concentration of ethanol was added and stirred to produce a precipitate, centrifuged at 3200 rpm for 5 min, collected the precipitate, and vacuum-dried; the obtained product was redissolved in 50 times the mass of distilled water, dialyzed in 20 times the volume of distilled water for 48 hours, and the liquid was changed 4 times a day, Lyophilized after dialysis to obtain okra extract.

In addition to the above components, other components including buffers are commercially available.

Embodiment 1 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, also including protective agent, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

Comparative Example 1 Recombinant Human Interferon α1b Eye Drops

Including recombinant human interferon alpha 1b, and also including one or more of protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes arginine.

The concentration of arginine in the protective agent in recombinant human interferon alpha 1b eye drops was 1.5% (w/v).

Comparative Example 2 Recombinant Human Interferon α1b Eye Drops

Including recombinant human interferon alpha 1b, and also including one or more of protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes sitosterol.

The concentration of sitosterol in the protective agent in recombinant human interferon α1b eye drops is 0.5% (w/v).

Comparative Example 3 Recombinant Human Interferon α1b Eye Drops

Including recombinant human interferon alpha 1b, and also including one or more of protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine and sitosterol.

The concentrations of arginine and sitosterol in the protective agent in the recombinant human interferon α1b eye drops are 1.5% (w/v) and 0.5% (w/v), respectively.

Comparative Example 4 Recombinant Human Interferon α1b Eye Drops

Including recombinant human interferon alpha 1b, and also including one or more of protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine and human albumin.

The concentrations of arginine and human albumin in the protective agent in the recombinant human interferon α1b eye drops were 1.5% (w/v) and 0.4% (w/v), respectively.

Comparative Example 5 Recombinant Human Interferon α1b Eye Drops

Including recombinant human interferon alpha 1b, and also including one or more of protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of sitosterol and human albumin.

The concentrations of sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 0.5% (w/v) and 0.4% (w/v), respectively.

Embodiment 2 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, also including protective agent, stabilizer, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolinine in the stabilizer in recombinant human interferon α1b eye drops were 1.2% (w/v) and 1.0% (w/v), respectively.

Embodiment 3 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, also including protective agent, stabilizer, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.0% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 1.0% (w/v) and 0.8% (w/v), respectively.

Comparative Example 6 A recombinant human interferon α1b eye drop

Including recombinant human interferon alpha 1b, also including protective agent, stabilizer, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes water-soluble beta-1,3 glucan.

The concentration of water-soluble β-1,3 glucan in the recombinant human interferon α1b eye drops in the stabilizer is 1.2% (w/v).

Comparative Example 7 A recombinant human interferon α1b eye drop

Including recombinant human interferon alpha 1b, also including protective agent, stabilizer, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes lanolinine.

The concentration of lanolinine in the recombinant human interferon α1b eye drops in the stabilizer is 1.0% (w/v).

Embodiment 4-7 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, also including protective agent, stabilizer, osmotic pressure regulator.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

Assuming that the total concentration of protective agent in recombinant human interferon α1b eye drops is i, and the total concentration of stabilizer is j, then i=2β×j, β is a constant, and the value is 1-1.25.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are respectively 1.0-1.5% (w/v) and 0.3-0.5% (w/v) , 0.2-0.4% (w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5-1.2% (w/v) and 0.5-1.0% (w/v) respectively. v).

The difference of embodiment 4-7 lies in the concentration of each component of protective agent and stabilizer:

Example 4: The concentrations of arginine, sitosterol and human albumin in the protective agent in recombinant human interferon α1b eye drops were 1.5% (w/v), 0.5% (w/v), 0.4 % (w/v); the concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5% (w/v) and 0.5% (w respectively). /v).

Example 5: The concentrations of arginine, sitosterol, and human albumin in the protective agent in recombinant human interferon α1b eye drops were 1.4% (w/v), 0.4% (w/v), 0.4%, respectively %(w/v); the concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5(w/v) and 0.5(w/v, respectively) ).

Example 6: The concentrations of arginine, sitosterol and human albumin in the protective agent in recombinant human interferon α1b eye drops were 1.5% (w/v), 0.5% (w/v), 0.4 % (w/v); the concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5% (w/v) and 0.7% (w, respectively). /v).

Example 7: The concentrations of arginine, sitosterol, and human albumin in the protective agent in recombinant human interferon α1b eye drops were 1.3% (w/v), 0.3% (w/v), 0.4%, respectively % (w/v); the concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5% (w/v) and 0.5% (w respectively). /v).

Embodiment 8 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, and also including protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolinine in the stabilizer in recombinant human interferon α1b eye drops were 1.2% (w/v) and 1.0% (w/v), respectively.

The additional conditioning agent includes a mixture of Tribulus terrestris extract and okra extract.

The concentrations of Tribulus terrestris extract and okra extract in the additional regulator in recombinant human interferon α1b eye drops are 0.9% (w/v) and 0.5% (w/v), respectively.

Comparative Example 8 A recombinant human interferon α1b eye drop

Including recombinant human interferon alpha 1b, and also including protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolinine in the stabilizer in recombinant human interferon α1b eye drops were 1.2% (w/v) and 1.0% (w/v), respectively.

The additional conditioning agent includes Tribulus terrestris extract.

The concentration of Tribulus terrestris extract in recombinant human interferon alpha 1b eye drops in the additional regulator is 0.9% (w/v).

Comparative Example 9 A recombinant human interferon α1b eye drop

Including recombinant human interferon alpha 1b, and also including protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolinine in the stabilizer in recombinant human interferon α1b eye drops were 1.2% (w/v) and 1.0% (w/v), respectively.

The additional conditioning agent includes a mixture of okra extracts.

The concentration of okra extract in recombinant human interferon alpha 1b eye drops in the additional regulator is 0.5% (w/v).

Embodiment 9 A kind of recombinant human interferon α1b eye drops stable at room temperature after opening

Including recombinant human interferon alpha 1b, and also including protective agents, stabilizers, additional regulators, and osmotic pressure regulators.

The osmotic pressure regulator includes sodium chloride and borax.

The concentrations of sodium chloride and borax in the osmotic pressure regulator in the recombinant human interferon α1b eye drops are 0.2% (w/v) and 0.5% (w/v), respectively.

Assuming that the total concentration of protective agent in recombinant human interferon α1b eye drops is i, and the total concentration of stabilizer is j, then i=2β×j, β is a constant, and the value is 1-1.25.

The protective agent includes a mixture of arginine, sitosterol, and human albumin.

The concentrations of arginine, sitosterol and human serum albumin in the protective agent in recombinant human interferon α1b eye drops are 1.5% (w/v), 0.5% (w/v), 0.4% ( w/v).

The stabilizer includes a mixture of water-soluble beta-1,3 glucan and lanolinine.

The concentrations of water-soluble β-1,3 glucan and lanolin in the stabilizer in recombinant human interferon α1b eye drops are 0.5% (w/v) and 0.5% (w/v), respectively.

The additional conditioning agent includes a mixture of Tribulus terrestris extract and okra extract.

The concentrations of Tribulus terrestris extract and okra extract in the additional regulator in recombinant human interferon α1b eye drops are 0.9% (w/v) and 0.5% (w/v), respectively.

Comparative Example 10 A recombinant human interferon α1b eye drop

Includes recombinant human interferon alpha 1b, human albumin, sodium chloride (and buffer).

The concentration of human serum albumin in recombinant human interferon α1b eye drops is 2% (w/v); the concentration of sodium chloride in recombinant human interferon α1b eye drops is 0.85% (w/v).

The method for preparing recombinant human interferon alpha 1b eye drops with the formula described in the above embodiment/comparative example is:

S1, take recombinant human interferon α1b stock solution, add one or more of protective agent, stabilizer, additional regulator and osmotic pressure regulator and mix well;

S2, filter sterilization;

S3, product packaging.

Described adding one or more of protective agent, stabilizer, additional regulator and osmotic pressure regulator, the order of adding is to add protective agent, stabilizer, additional regulator and osmotic pressure regulator in sequence, when the formula When one of the components is not included, the component can be removed, and it and the components are added in the original order.

Each component can be prepared into a semi-finished product with a relatively high concentration with a buffer solution, and then a certain amount can be added to the system for preparing eye drops after concentration and volume calculation to obtain the required concentration.

The sterile filtration includes:

1) Connect two 293 filters in series, tighten the screw caps on the filters, and connect the filter inlet pipeline with the sterilization tank supply pipeline. The filtrate pipeline is sent into the buffer chamber of the aseptic operation isolator through the pipeline hole of the aseptic operation isolator, and the pipeline hole is sealed. Send the liquid inlet pipeline connected to the 50L-100L semi-finished product tank through the pipeline hole of the aseptic operation isolator into the buffer chamber of the aseptic operation isolator, and seal the pipeline hole. Put the sample vials and bottle caps into the buffer compartment of the aseptic operation isolator through the buffer compartment door. Open the aseptic operation isolator VHP and sterilize for 30-40 minutes.

2) Send the filtrate pipeline connected to the filter and the liquid inlet pipeline connected to the 50L-100L semi-finished product tank to the main cabin of the aseptic operation isolator through the pipe hole between the main cabin and the buffer cabin after unpacking, Close the pipe hole. Items such as sample vials and bottle caps are put into the main compartment of the aseptic operation isolator through the door between the two compartments. Remove the innermost layer of packaging and connect the two pipes with clips.

3) Place the 50L-100L semi-finished product tank on the scale to clear it, open the tank bottom valve and liquid supply valve of the sterilization tank, and start sterilization and filtration. Observe the upper and lower filter plates of the filter and the exhaust valve, and the next step can be performed only when there is no liquid overflow. Adjust the peristaltic pump to gear 3, filter the liquid in the sterilization tank into the sterilized 50L-100L semi-finished product tank to the required weight (convert the required preparation volume into weight according to the specific gravity). Turn off the peristaltic pump, clamp the filter filtrate silicone tube with hemostatic forceps, the filtrate in this bucket is the semi-finished product of recombinant human interferon α1b eye drops. Then carry out the semi-finished product inspection, and then proceed to the next step of sub-packaging after passing the test.

Product packaging:

The finished product of recombinant human interferon α1b eye drops is 200,000 IU/2.0ml/bottle. Product filling volume requirements: Fill 2.15ml/bottle to ensure that the filling volume of each bottle is not less than the quantity indicated on the label.

At the beginning of filling, confirm the environment of the filling room, confirm the temperature, humidity and pressure difference between the filling room, the temperature should be controlled at 15-26 ℃, the relative humidity should be controlled at 30%-70%, and the filling room and other areas should be kept at a certain level. Differential pressure (not less than 10Pa); confirm equipment: confirm that the eye drop filling machine and electronic balance are operating normally; confirm that the inner packaging material is sterilized by ethylene oxide; confirm that the filling utensils, shaking bucket and runway are autoclaved; confirm Semi-finished products to be filled: Before filling, the name, batch number, and quantity of semi-finished products to be filled must be carefully checked, and whether the opening and body of the cans are well sealed.

After the above preparations are confirmed, connect the connected filling pipeline to the peristaltic pump and the semi-finished tank to be filled in turn. Attach the filling needle to the needle clamp. Fix the sterilized bottle shaking hopper, inner plug shaking hopper and outer cap shaking hopper on the shaker, and then pour the bottle, bottle stopper and bottle cap into the shaking hopper. Open the compressed air valve. Turn on the key of the filling host, and power on the filling host. Open the inner plug and outer lid shakers and adjust them to the appropriate speed. Turn on the bottle unscrambler power, turn on the bottle unscrambler shaker and adjust it to a proper speed, and turn on the bottle unscramble switch. After the eye drop bottles, stoppers and caps on the filling and sealing machine have sufficient spare quantities at the entrance, you can prepare to start the machine and start filling.

At the beginning of filling, the electronic balance is used to detect the filling amount. During the filling process, the filling amount should be observed frequently, and the filling amount must be checked every 1 hour of filling. To achieve accurate loading, to prevent uneven loading. Filling speed ≤ 75 bottles/min. Keep 55-60 bottles/min with the equipment running stably. Filling must be completed within 24 hours. After filling, the semi-finished product tank to be filled should be opened to check whether there is sediment or other foreign matter. Strict attention should be paid to aseptic operation during the filling process.

Product quality inspection: (0 months and 0 days in the time involved indicate that the inspection will be carried out on the same day after production or after opening)

1. According to the method described in Chinese patent CN202010591909.4 (our earlier work), the samples prepared in the above examples and comparative examples were subjected to the stability investigation test of recombinant human interferon α1b eye drops, which was characterized by aseptic Do not open the package after operation, and proceed under specific conditions.

In order to further determine the stability of the formulation and preparation method of the present invention, whether the drug is stable at a storage temperature of 2-8° C. is judged by summarizing and analyzing the detection data, which provides a theory for the determination of the packaging, transportation, storage conditions and validity period of the product. basis, and reset the expiration date of the product during the stabilization period.

1) Accelerated stability test of recombinant human interferon α1b eye drops at 23-27°C:

According to the physicochemical properties of recombinant human interferon α1b, combined with the particularity of the eye drop dosage form, an accelerated stability test at 23-27°C was carried out on the present invention. Three batches of recombinant human interferon α1b eye drops (production batch numbers: S20220201, S20220202, S20220203) prepared according to the optimized formula and process of Example 2 were used as the investigation objects, and the accelerated stability investigation test at 23-27°C was carried out.

The inspection items refer to the Pharmacopoeia of the People's Republic of China (2015 edition), and select the appearance, visible foreign matter, filling volume, pH value, osmolarity, biological activity and sterility inspection as the inspection indicators, and carry out a 6-month stability inspection. The specific inspection and inspection items and inspection time are shown in Table 1.

Table 1 Investigation on the accelerated stability of recombinant human interferon α1b eye drops

Note: "√" in the table means to test; "/" means not to test.

23-27°C accelerated stability test results: Through the 6-month accelerated stability test at 23-27°C, the physical and chemical properties such as appearance, visible foreign matter, filling volume, pH value have not changed; sterility inspection is also qualified; penetration The molar concentration fluctuated slightly but was relatively stable, and the biological activity basically did not fluctuate. The specific inspection results are shown in Table 2.

Table 2 The results of accelerated stability investigation of recombinant human interferon α1b eye drops at 23-27°C

2) Long-term stability test of recombinant human interferon α1b eye drops at 2～8℃:

According to the physicochemical properties of recombinant human interferon α1b, combined with the particularity of the dosage form of the eye drop, the long-term stability test at 2-8°C of the present invention was carried out. Three batches of recombinant human interferon α1b eye drops (production batch numbers: 20200101, 20200102, 20200103) produced in 2020 prepared according to the optimized formula and process in Example 2 were tested for long-term stability at 2-8°C. The inspection items refer to the "Pharmacopoeia of the People's Republic of China" (2015 edition), and select identification, appearance, visible foreign matter, filling volume, pH value, osmolarity, biological activity and sterility inspection as the inspection indicators, and carried out a period of 24 months. The stability of the inspection, the specific inspection and inspection items and inspection time are shown in Table 3.

Table 3 Investigation on the long-term stability of recombinant human interferon α1b eye drops at 2～8℃

Note: "√" in the table means to test; "/" means not to test.

Results of long-term stability study at 2-8°C: Through the 24-month long-term stability study at 2-8°C, the physical and chemical properties such as identification, appearance, visible foreign matter, and filling volume have not changed, and the sterility inspection has not changed, and the pH value has not changed. Very stable. Although there are slight fluctuations in osmolarity, it is relatively stable. Although there are fluctuations in biological activity, it is within the quality standard range within 24 months. The specific inspection results are shown in Table 4.

Table 4 Results of long-term stability investigation of recombinant human interferon α1b eye drops at 2～8℃

Analysis of the stability test results of recombinant human interferon α1b eye drops: The stability of three batches of finished recombinant human interferon α1b eye drops (20200101, 20200102, 20200103) was investigated. It can be observed that the finished product of recombinant human interferon α1b eye drops has better stability. During the monitoring period, the test results of the finished eye drops were all within the qualified range stipulated in Part III of the Pharmacopoeia of the People's Republic of China (2015 edition). The test data shows that the finished eye drops meet the quality standards in terms of identification, appearance, visible foreign matter, filling volume, and sterility inspection. The pH value is very stable, and although the osmolarity has a slight fluctuation phenomenon, it is relatively stable during the detection period, and the biological activity is stable. Although the biological activity of these three batches of eye drops fluctuated during the monitoring period, they all met the quality standards within 24 months.

Conclusion: Through the accelerated stability study of recombinant human interferon α1b eye drops at 23～27℃ and long-term stability at 2～8℃, there is no significant change in the indexes of recombinant human interferon α1b eye drops during the detection period. Therefore, it can be proved that the recombinant human interferon α1b eye drops are very stable when stored in the dark at 2-8°C, and the effective use period can be set as 0-24 months.

Quality Control Evaluation

1) Identification test

According to the "People's Republic of China Pharmacopoeia" three (2015 edition) immunospot method (general rule 3402), it should be positive.

2) Visual inspection

Should be a colorless liquid.

3) Visible foreign body

According to "People's Republic of China Pharmacopoeia" three (2015 edition) (general rule 0904), it should be qualified.

4) Loading capacity

According to the "People's Republic of China Pharmacopoeia" three (2015 edition) (general rule 0105), not less than the labeled amount.

5) pH value

According to the "People's Republic of China Pharmacopoeia" three (2015 edition) (general rule 0631), it should be

6) Osmolality

According to the "People's Republic of China Pharmacopoeia" three (2015 edition) (general rule 0632), it should be

7) Biological activity

According to "Pharmacopoeia of the People's Republic of China" Part III (2015 Edition) (General Rule 3523), the first method of cytopathic inhibition method should be the labeled amount

8) Sterility check

According to "People's Republic of China Pharmacopoeia" three (2015 edition) (general rule 1101), it should be grown aseptically.

Based on the special purpose of the present invention, we took one batch of samples from the above examples and comparative examples, and carried out long-term stability tests under sealed/unsealed normal temperature (25°C-35°C) storage without light after opening, The specific inspection and inspection items and inspection time are shown in Table 5 and Table 6. The results are shown in Table 7 and Table 8.

Table 5 Recombinant human interferon α1b eye drops were re-sealed after opening, and the long-term stability was investigated under normal temperature illumination

Note: "√" in the table means to test; "/" means not to test.

Table 6 Recombinant human interferon α1b eye drops are not resealed after opening, long-term stability under normal temperature illumination

Note: "√" in the table means to test; "/" means not to test.

Table 7 Recombinant Human Interferon α1b Eye Drops are re-sealed after opening, and the results of long-term stability investigation under normal temperature illumination

Table 8 Recombinant human interferon α1b eye drops are not resealed after opening, and the results of long-term stability investigation under normal temperature illumination

Analysis of the test results for the stability of the recombinant human interferon α1b eye drops at room temperature after opening: the change in properties is mainly reflected by the biological activity and sterility inspection results. The stabilizing effects of sitosterol and human albumin have no promoting effect, and the mixing of sitosterol and human albumin has no effect of superposition or promotion. Only when arginine is added, and sitosterol and human albumin exist at the same time, it can achieve the effect of the present invention. The two components in the stabilizer did not achieve significant effect when used alone, and the synergistic effect of the two was significant. The defined ratio between the stabilizer and the protective agent can effectively increase the stability of the system. The two plant extracts are used separately as additional regulators to be added to the system, which do not contribute to the stability. Adding the two at the same time can significantly increase the stability. We have not yet made a breakthrough discovery in terms of the principle, but we guess that it may be the two Some of the substances in the extract interact with each other to produce targeted substances with antibacterial activity, thereby increasing the stability of the system, and both extracts are pure natural ingredients that are non-irritating to the eyes, this finding It may play a role in promoting many of our future work. Re-sealing after unpacking in the present invention refers to unpacking the packaged reagents in the air in a non-sterile environment, and then re-sealing after leaving the open for 30s (simulating the standard use scene of reagents in the hands of users); Unsealed means that the packaged reagents are unsealed in the air in a non-sterile environment and left open.

The preferred specific embodiments and examples of the present invention have been described in detail above, but the present invention is not limited to the above-mentioned embodiments and examples, and within the scope of knowledge possessed by those skilled in the art, it can also be used without departing from the concept of the present invention. Various changes are made under the premise.

Claims (10)

1. An unsealed recombinant human interferon alpha 1b eye drop stable at normal temperature is characterized in that: comprises recombinant human interferon alpha 1b, and also comprises one or more than two of a protective agent, a stabilizing agent, an additional regulator and an osmotic pressure regulator.

2. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 1, which is characterized in that: the protective agent comprises a mixture of arginine, sitosterol and human serum albumin.

3. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 2, which is characterized in that: the concentrations of arginine, sitosterol and human serum albumin in the protective agent in the eye drops of the recombinant human interferon alpha 1b are respectively 1.0-1.5% (w/v), 0.3-0.5% (w/v) and 0.2-0.4% (w/v).

4. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 1, which is characterized in that: the stabilizer comprises a mixture of water-soluble beta-1, 3-glucan and lysine.

5. The kaifeng ambient temperature stable recombinant human interferon alpha 1b eye drops according to claim 4, which is characterized in that: the concentrations of the water-soluble beta-1, 3 glucan and the lazine in the stabilizing agent in the eye drops of the recombinant human interferon alpha 1b are 0.5-1.2% (w/v) and 0.5-1.0% (w/v), respectively.

6. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 3 or 5, which is characterized in that: if the total concentration of the protective agent and the total concentration of the stabilizing agent in the eye drops of the recombinant human interferon alpha 1b are i and j, i is 2 beta multiplied by j, and beta is a constant and takes a value of 1-1.25.

7. The kaifeng recombinant human interferon alpha 1b eye drops which are stable at normal temperature according to claim 1, which is characterized in that: the additional regulator comprises a mixture of tribulus terrestris extract and okra extract.

8. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 1, which is characterized in that: the concentrations of the tribulus terrestris extract and the okra extract in the eye drops of the recombinant human interferon alpha 1b in the additional regulator are 0.6-0.9% (w/v) and 0.1-0.5% (w/v), respectively.

9. The kaifeng recombinant human interferon alpha 1b eye drops stable at normal temperature according to claim 1, which is characterized in that: the osmotic pressure regulator comprises sodium chloride and borax; the concentrations of sodium chloride and borax in the osmotic pressure regulator in the eye drops of the recombinant human interferon alpha 1b are 0.1-0.3% (w/v) and 0.4-0.6% (w/v), respectively.

10. A preparation method of recombinant human interferon alpha 1b eye drops stable at normal temperature after being unsealed as claimed in any one of claims 1 to 9, which is characterized in that: the method comprises the following steps:

s1, taking a stock solution of the recombinant human interferon alpha 1b, adding one or more than two of a protective agent, a stabilizing agent, an additional regulator and an osmotic pressure regulator, and uniformly mixing;

s2, filtering and sterilizing;

s3, subpackaging the product;

and the protective agent, the stabilizing agent, the additional regulator and the osmotic pressure regulator are added in sequence.