KR101095647B1 - Glycoprotein hormone composition - Google Patents

Abstract

The present invention relates to a solid composition containing disaccharides and glycoprotein hormones or variants thereof, wherein the content of glycoprotein hormones or variants thereof is 10 µg / mg composition or more or 100 international units / mg composition or more. The composition of the present invention has excellent stability.

Glycoprotein hormone, variant, disaccharide, follicle stimulating hormone

Description

Glycoprotein hormone composition {GLYCOPROTEIN HORMONE COMPOSITION}

The present invention relates to the field of protein purification and biopharmaceuticals. In particular, the present invention relates to compositions of glycoprotein hormones and stabilizers having excellent stability.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone produced by the pituitary gland and is composed of two subunits, α chain and β chain. The α subunit of FSH is identical to the α subunit of leuteinizing hormone (LH) and chorionic gonadotropin (CG), and has 92 amino acids and a molecular weight of about 14500D. Asparagine at positions 52 and 78 is an N- glucosylated amino acid. The β subunit of FSH consists of 111 amino acids with a molecular weight of about 18000 D, of which asparagine at positions 7 and 24 is an N- glucosylated amino acid. However, the β subunit of LH consists of 121 amino acids and has a molecular weight of about 14800D. The β subunit of CG consists of 145 amino acids and has a molecular weight of 22000-39000 D.

In clinic, FSH is mainly used for infertility treatment and extracorporeal reproduction in vitro. FSH can be isolated from the pituitary gland and postmenopausal women's urine, or can be produced through DNA recombination technology.

The first generation of products containing FSH is Menotropin (HMG), such as Sergon's Pergonal, which is a mixture with a ratio of FSH and LH of about 1. However, in patients with relatively high LH in the body and do not need external LH addition, the amount of LH is too high, which may affect the normal development of follicles. It can reduce the chance of fertilization and conception. Thus, the use of pure FSH preparations is necessary for these patients. On the other hand, excessive LH causes Polycystic Ovarian Syndrome (POOS). Studies show that excessive LH adversely affects reproductive function and causes rare menstruation, ovulation, infertility and miscarriage. Thus, treatment with pure FSH for POOS patients is safer than HMG and may reduce the risk of Ovarian Hyperstimulation Syndrome (OHSS).

Metrodin, developed by Serono, is an FSH formulation containing very low levels of LH, and Metrodin-HP, which was subsequently developed, is a high-purity FSH formulation.

Recently, high-purity menotropin (pHMG), such as Ferring's Menopur, has been developed.The ratio of purified high-purity FSH to high-purity LH or high-purity Hcg (human chorionic gonadotropin) is 1: 1. It is a product made by mixing. It replaces general HMG products and overcomes hypersensitivity reactions to human body by mixing a large amount of miscellaneous protein in general HMG products.

Therefore, although the current market is committed to the development and application of high-purity glycoprotein hormone, high-purity glycoproteins obtained by various purification means should be easily preserved and tested as a raw material, and should be formulated again. . The form of the drug substance is very important, since liquids are disadvantageous for preservation and transportation, they are generally preferred in solid form and not in liquid form. The solid form of glycoprotein hormone is mainly obtained through lyophilization, but since the high-purity glycoprotein hormone is easily denatured and loses its activity during the lyophilization process, the prior art must preserve the drug of high purity glycoprotein hormone in liquid form. It became. However, the liquid form must be stored at -20 ° C or it will lose activity. Liquid forms can undergo a freeze-dissolution process, and even after repeated freeze-thaw processes, glycoproteins are easily lost. The liquid form carries risks such as the packaging bursting easily when approaching the brittle point at low temperatures.

Therefore, there is an urgent need in the art for the development of glycoprotein hormone drug substance with excellent stability.

An object of the present invention is to provide a solid composition containing a high-purity glycoprotein hormone to be used as a raw drug.

In a first aspect of the present invention, there is provided a composition of a kind, said composition being a solid composition, including disaccharides and glycoprotein hormones or variants thereof.

In another preferred embodiment, the content of glycoprotein hormone or variant thereof is at least 10 μg / mg composition, or at least 100 international units / mg composition.

In another preferred embodiment, the content of glycoprotein hormone or variant thereof is at least 20 μg / mg composition, or at least 200 international units / mg composition.

In another preferred embodiment, the glycoprotein hormone or variant thereof is selected from follicle stimulating hormone (FSH) or variant thereof, luteinizing hormone (LH) or variant thereof, or mixtures thereof.

In another preferred embodiment, the disaccharide is two or more selected from the group consisting of lactose, sucrose, maltose, trehalose, more preferably lactose.

In another preferred embodiment, the follicle stimulating hormone or variant thereof is human follicle stimulating hormone or variant thereof.

In another preferred embodiment, the human follicle stimulating hormone is selected from the group consisting of recombinant human follicle stimulating hormone or variants thereof, or follicle stimulating hormone derived from human urine.

In another preferred embodiment, said luteinizing hormone or variant thereof is human luteinizing hormone or variant thereof.

In another preferred embodiment, said human luteinizing hormone or variant thereof is selected from recombinant human luteinizing hormone or variant thereof, or luteinizing hormone derived from human urine or variant thereof.

In another preferred embodiment, the solid composition is a lyophilized powder.

In another preferred embodiment, the composition is a solid and consists of disaccharides and glycoprotein hormones or variants thereof. Among them, the content of glycoprotein hormone or its variants is at least 10 μg g / mg composition, or at least 100 international units / mg composition.

In a second aspect of the invention, there is provided a kind of drug composition, wherein the drug composition comprises the solid composition.

In a third aspect of the invention, there is provided the use of said solid composition for the manufacture of a medicament for the treatment of infertility syndrome.

From this, the present invention provides a form of a glycoprotein hormone raw material drug having excellent stability.

Specific Example

After extensive research, the present inventors have found that after lyophilizing a high purity glycoprotein hormone in combination with a suitable stabilizer, a stable composition can be obtained. Among them, the stabilizer is preferably disaccharide, more preferably lactose.

In addition to diluents and excipients, the inventors of the present invention, more importantly, act as a protective agent to prevent the glycoprotein molecules from being denatured by loss of activity due to the conformational changes that occur during the lyophilization process. It greatly improves the stability of the product. Based on this, the present inventors completed the present invention.

Specifically, as described in the background section, in the prior art, not only is it difficult to preserve and transport the intermediate product or drug substance of high purity glycoprotein hormone in liquid form, but also to inadvertently influence the product. do. The solid form also results in the loss of activity of the glycoprotein easily during the lyophilization process.

In the search for improvement of the prior art methods, the inventors have formulated high-purity glycoprotein and lactose in an appropriate ratio, especially when the concentration of high-purity glycoprotein and lactose is within a certain range, and lactose acts as a protective agent. It has been found that protein molecules are prevented from deactivation due to denaturation during lyophilization and loss of activity.

In the present invention, the disaccharide is formed by the glycosidic bond of two monosaccharides, for example, maltose, lactose, sucrose, trehalose and the like, preferably maltose, lactose and sucrose At least one selected from the group consisting of more preferably lactose.

In the present invention, the variant of the glycoprotein hormone refers to a glycoprotein hormone modified by deletion of amino acids or by recombinant technology, but existing medicinal effects should still exist or be enhanced.

In the composition provided by the present invention, the weights of disaccharides and glycoprotein hormones or variants thereof are 60 to 100% of the total weight of the composition, preferably 80 to 90%, and more preferably 90 to 95%.

In the composition of the present invention, the biological value of the glycoprotein hormone or variant thereof is 500 to 30000 international units / mg protein, preferably 3500 to 12000 international units / mg protein. If the hormone of the glycoprotein is only FSH or a variant thereof, the biological titer of the FSH or the variant thereof is 500 to 15000 international units / mg protein, preferably 7000 to 12000 international units / mg protein. If the glycoprotein hormone is only LH or a variant thereof, the biological titer of the LH or a variant thereof is 500 to 8000 international units / mg protein, preferably 2000-6000 international units / mg protein.

In the composition of the present invention, the purity of the glycoprotein hormone or variant thereof is 50% or more, preferably 90% or more.

The content of glycoprotein hormone or variant thereof in the composition of 1 mg sugar provided by the present invention is 100 or more international units, preferably 200 or more international units.

In the composition per 1mg provided by the present invention, the content of glycoprotein hormone or variant thereof is 10 µg or more. When the glycoprotein hormone thereof is only FSH or a variant thereof, the content of FSH or a variant thereof is 10 μg / mg composition or more, preferably 20 μg / mg composition or more. When the glycoprotein hormone is only LH or a variant thereof, the content of LH or a variant thereof is 10 µg / mg or more, preferably 35 µg / mg or more.

In the composition provided by this invention, the weight ratio of disaccharide and glycoprotein hormone or its variants is 8-99: 1, Preferably it is 12-60: 1.

In the composition provided by the present invention, the weight ratio of lactose and glycoprotein hormone or variant thereof is 8 to 99: 1, preferably 12 to 60: 1. Among them, when the glycoprotein hormone is only FSH or a variant thereof, the weight ratio of lactose to FSH or a variant thereof is 20 to 99: 1, preferably 30 to 50: 1. When the glycoprotein hormone is only LH or a variant thereof, the weight ratio of lactose and LH or a variant thereof is 8 to 99: 1, preferably 12 to 20: 1.

The composition provided in the present invention, the glycoprotein hormone is not denatured during the lyophilization process, the activity is not lost, the composition has excellent stability.

The drug composition provided in the present invention includes (a) a glycoprotein hormone or a variant thereof, (b) a disaccharide and (c) a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier used for administration of a therapeutic agent and includes various excipients and diluents. The term refers to some pharmaceutical carriers that are not themselves required active ingredients and are not excessively toxic after administration. Suitable carriers are those that are well known to those skilled in the art. Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) provides information on pharmaceutically acceptable excipients. Pharmaceutically acceptable carriers in the composition include, for example, liquids such as water, saline, glycerin and ethanol. In addition, such carriers may also contain auxiliary substances such as, for example, disintegrants, wetting agents, emulsifiers and pH buffers.

The drug compositions are made in various formulations depending on different routes of administration. Such formulations may include oral, inhalation, rectal, intranasal, facial, topical, splenic, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, Schwancho, intraventricular, intrasternal and skull bones. Internal injection or infusion may be included, or administered by an external storage device. Among them, the subcutaneous administration method is preferably used.

In the drug composition of the present invention, the amount of use and the manner of use of the active ingredient are determined by various factors. The patient's age, weight, sex, health, nutrition, active ingredient active strength, dose, metabolic rate, severity of onset, and subjective judgment of the treating physician. Preferred safe effective amounts are generally above about 10 μg / kg body weight and in most situations do not exceed 10 mg / kg body weight. Preferred amounts of use are from about 10 μg / kg body weight to about 1 mg / kg body weight. The specific dosage should also consider factors such as the route of administration and the health of the patient. It is within the scope of skill of the skilled physician.

The features described in the present invention or the features described in the examples can be arbitrarily combined. All of the features described in the instructions of the present invention can be used in any composition form, and the intervening features described in the instructions can be replaced by replaceable features that can serve any same, equivalent or similar purpose. Unless otherwise stated, all features described are generic examples of equivalent or similar features.

The present invention has the following main advantages.

1. The present invention provides a glycoprotein hormone composition in solid form that is easy to store and transport.

2. The composition of the glycoprotein hormone obtained by the method of the present invention has excellent stability, so that a corresponding formulation can be prepared from the raw drug.

Hereinafter, the present invention will be described in detail with reference to specific examples.

The following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples. The conditions and test methods not listed in the following examples were performed under general conditions or conditions recommended in the manufacture. Unless otherwise specified, percentages and copies are by weight.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those recognized by those skilled in the art. In addition, any methods and materials similar or identical to those described may be used in the present invention. Preferred embodiments and materials described herein are used for demonstration purposes.

Example 1

Compositions Containing FSH

In this embodiment, high-purity FSH (purchased from Shanghai Tianyu Pharmaceutical Co., Ltd.) is used as a raw material of the FSH drug composition, and the bioactivity of the FSH is 9521 international units / mg.

After preparing 50 mL of 0.01 M sodium dihydrogen phosphate solution (adjusted to about pH6.5 with NaOH), 2 g of lactose was added thereto, stirred and dissolved, and filtered through a 0.22 μm filter. 10 mL of the filtrate was taken and completely dissolved by adding 10.0 mg of the above high-purity FSH (purchased from Shanghai C & T Biotech Co., Ltd.).

1. Preliminary freezing tray at -40 degrees Celsius for three hours,

2. Low temperature trap of -45 degrees Celsius,

3. Vacuum pump operation,

4. Hold the tray at -10 ℃ for 15 hours,

5. Hold plate at + 20 ℃ for five hours,

6. Take out 392mg of lyophilized powder from chamber.

The result of measuring the biotiter of FSH of the product is as follows.

Control

In this control, after mixing the high-purity FSH and lactose so that the FSH content is less than 50 international units / mg total amount of the composition, the lyophilization process and stability of the composition are observed.

In this comparative example, high-purity FSH (purchased from Shanghai Tianyu Biopharmaceutical Co., Ltd.) is used as a raw material of the drug composition, and the bioactivity of the FSH is 9521 international units / mg.

After preparing 500 mL of 0.01 M sodium dihydrogen phosphate solution (to adjust the pH value to about 6.5 with NaOH), 20 g of lactose was added thereto, stirred and dissolved, and filtered through a 0.22 μm filter. 100 mL of the filtrate was taken, the high-purity FSH (purchased from Shanghai Tianyu Biopharmaceutical Co., Ltd.) was completely dissolved, and then placed in a tray and placed in a lyophilizer.

Lyophilization was carried out under the same conditions as in Example 1 to obtain 3.89 g of lyophilized powder.

From the above examples and the control example, the FSH composition obtained in the present invention almost lost the activity in the freeze-drying process, but it can be seen that the control example definitely lost the activity.

Stability comparison

The stability of the samples of Example 1 and the control was observed at 30 ° C. The results are as follows.

As can be seen from this, the FSH composition obtained in the present invention has excellent stability compared to the control example.

Example 2

Compositions Containing LH

In this embodiment, LH (purchased from Shanghai Tianyu Biopharmaceutical Co., Ltd.) of high purity was used as a raw material of the LH drug composition, and the bioactivity of the LH is 4307 international units / mg.

500 mL of 0.01 M sodium dihydrogen phosphate solution (adjusted to about pH6.5 with NaOH) was prepared, followed by stirring and dissolving 2 g of lactose, followed by filtration through a 0.22 μm filter. 10 mL of the filtrate was taken and completely dissolved by adding the high-purity LH (purchased from Shanghai Tianyu Biopharmaceutical Co., Ltd.), and then placed in a tray and placed in a lyophilizer. Lyophilization was carried out under the same conditions as in Example 1 to obtain 402 mg of lyophilized powder.

The biotiter and stability of LH of the product were observed at 30 ° C. The results are as follows.

From this, it can be seen that the activity of the LH composition obtained in the present invention is almost unchanged and has excellent stability.

Example 3

Compositions Containing FSH, LH Mixtures

After preparing 100 mL of 0.9% sodium chloride (to adjust the pH value to about 7.0 with NaOH or HCl), 4 g of maltose was added thereto, stirred and dissolved, and filtered through a 0.22 μm filter. Take 10 mL of the filtrate, add 10.0 mg of high purity FSH (purchased from Shanghai Tianyu Biopharmaceutical Co., Ltd., biotiter is 9521 international units / mg), and then purchase 25.0 mg of high purity LH (Shanghai Tianyu Biopharmaceutical Co., Ltd.) , Biotiter was 4307 international units / mg), completely dissolved, and placed in a tray and placed in a lyophilizer. After lyophilization under the same conditions as in Example 1, 405 mg of lyophilized powder was obtained, and the measured bioactivity of FSH was 223 IU / mg, and LH was 242 IU / mg.

The stability results of the product at 30 ℃ is as follows.

Example 4

Preparation of FSH Lyophilized Injections

An example of preparing 100 bottles of FSH lyophilized injections containing 75 IU of FSH in each bottle is as follows.

35 mg of the FSH lyophilized composition prepared in Example 1 was dissolved in 10 mL of pyrogen-free water for injection, then aseptically filtered with a 0.22 μm filter, and the filter was washed with pyrozin-free water for injection. It was.

1 g lactose was dissolved in 30 mL of pyrosine free injection water. If necessary, adjust the pH value to 6.5 ± 0.2 with HCl or NaOH. After sterile filtration with a 0.22 μm filter, it was added to the FSH solution, adjusted to 75 ml using pyrosine-free injection water, and mixed uniformly.

0.75 mL of the solution was dispensed into ampoules, and then lyophilized.

Among the ampoules obtained, each bottle contained 75 IU FSH and 10 mg lactose.

The above is a preferred embodiment of the present invention and does not limit the practical technical scope of the present invention. The substantial technical details of the invention are broadly defined in the appended claims, and in the event that any person uses a technical entity or method, any change in form that is exactly the same as or equivalent to the definition of the claims of the invention is also contemplated. The claims belong to the claims.

Claims (12)

Lactose and glycoprotein hormones, wherein the glycoprotein hormone is selected from follicle stimulating hormone, luteinizing hormone, or a mixture thereof, the biological titer of the follicle stimulating hormone is 7000 to 12000 IU / mg protein, luteinizing hormone The biological titer of is 2000-6000 IU / mg protein, the weight ratio of lactose and follicle stimulating hormone in the composition is (30 to 50): 1, and the weight ratio of lactose to luteinizing hormone is (12 to 20): 1. Solid composition. The composition of claim 1, wherein the follicle stimulating hormone is human follicle stimulating hormone. 3. The composition of claim 2, wherein the human follicle stimulating hormone is selected from recombinant human follicle stimulating hormone or follicle stimulating hormone derived from human urine. The composition of claim 1, wherein the luteinizing hormone is human luteinizing hormone. The composition according to claim 4, wherein the human luteinizing hormone is selected from recombinant human luteinizing hormone or human urinary luteinizing hormone. The composition of claim 1 wherein the solid composition is a lyophilized powder. A pharmaceutical composition for treating infertility syndrome, comprising the solid composition according to any one of claims 1 to 6. delete delete delete delete delete