WO2010145125A1

WO2010145125A1 - Highly purified human menopausal gonadotropins, method for preparing and uses thereof

Info

Publication number: WO2010145125A1
Application number: PCT/CN2009/075277
Authority: WO
Inventors: 郭照晔; 洪云海; 季斌; 严惠敏; 高霄梁; 季晓铭
Original assignee: 上海天伟生物制药有限公司
Priority date: 2009-06-18
Filing date: 2009-12-03
Publication date: 2010-12-23
Also published as: CN101928342A; KR20120042844A; CN101928342B

Abstract

Provided is a type of highly purified human menopausal gonadotropin, the HPLC purity of which is not less than 95%, and the impurity HPLC content of which is not more than 5%. Also provided are the preparing method and uses of the said highly purified human menopausal gonadotropin.

Description

High-gonadin and its methods and uses

This relates to the field of protein purification and biology. Hugh and involve high purity gonadotropins and their methods and uses. Background

The gonadotropin H ae opa sa o ado op s, H contains fo cesag ho oe FH and eze gho oe LH active ingredients, which are caused by the rest of the protein hormones FH's "and LH, chorionic gonadotropin (cho oc go ado op)" is the same amino acid with 92 amino acids at 1450 D 52 and 78. The FH is composed of 11 amino acids. 1800 D where 7 and 24 are the base amino acids, while LH is composed of 121 amino acids at 14800D and 145 amino acids at 22000 390 D

Bed H should be used for it, or it can be taken from the urine of a woman or DN wood.

The first generation containing F H is (H ) such as e o o Pe Go a which is a mixture of F H LH ratio 1. However, its ratio and purity are very low. Generally, the active ingredient in the purity of 40 300 U g protein is only 2 3. The rest are egg white. Because of the high protein content, there are potential side effects. The method can only be limited to muscle injection. High purity (PH) is particularly desirable in the art. It is also of high purity and low content, and does not contain a large amount of protein. Therefore, it does not cause a person to rest and because it can be used to improve the patient by subcutaneous injection.

However, the purification of pH is very much due to the fact that it has a certain ratio of FH and LH and needs a certain proportion of the west, but the FH and LH are different. Therefore, in the purification of the period, it is easy to make the proportion of the western in the pH. In the purification, in particular, there is no way to obtain high purity H.

Therefore, there is an urgent need in the art for a method of high purity and low purity of H to facilitate use in a patient for subcutaneous injection. content

This aim is to provide high purity, low purity methods.

Another purpose of this is to provide high purity obtained by the above method.

A further object of the present invention is to provide a high purity H compound obtained by the above method.

The purpose of this is to provide the use of high purity H obtained by the above method. In the present invention, a high purity gonadotropin ed H aMe opa sa Go ado op s is provided, the PH HP purity is not less than 95, the HPL content is not 5 HPL, the purity is not less than 96 HPL, and the content is not 4

In another, the gonadotropin or gonadin, or the source of gonadotropin or Hugh.

In another high-purity gonadotropin, the ratio of follicle stimulating hormone F H to LH-producing organisms 1 0.1—

In another high-purity gonadotropin, the proportion of follicle stimulating hormone F H to form LH is 1 0.5 1.4. In a second aspect of the present invention, there is provided a method of each of the methods of providing a high purity gonadotropin pH as described above comprising the step of operating a low purity H dye which is not purified and a high purity gonadotropin to a low purity H at 2000 FH 9 protein. the following.

In another method, including steps

1 Apply a solution containing low purity H to the undissolved dye

2 pH6-1 and

3 pH6-1 gives high purity gonadotropins.

In another solution containing low purity H, 0.1 w v of low purity H

In another solution, pH 4 8 of a solution containing low purity HM

In another solution from acetic acid, acetic acid, Na H, T s H or phosphoric acid. In the other step, the next step is included in step 3.

3 pH6 11 collection

3 Add water ethanol, Wanxin get and

3 High purity gonadotropin obtained by dehydration.

In another dye, the dye is not separated from bac o B e a ge , ee

In another, the solid phase of the dye is not precipitated from soil, glass micro, quartz micro, calcium phosphate, oxidized, polypropylene, , , or .

In another embodiment, the dye is not and precipitated from B e epha oseFF or B e epha OSe 6B. The compound of the present invention provides an effective amount of the high purity gonadotropin as provided above and the above. Acceptable rest. In the fourth aspect of the present invention, there is provided the use of the high purity gonadotropin as provided above. The high purity gonadotropin is not integrated. This product provides high-purity gonadotropins obtained by high-purity, high-purity methods for subcutaneous injection for patient use.

. 1 PH purity. Hugh way

Explicit and in-depth study of dyes and high purity H. HPL purity HPL content 5 is currently known to have the highest purity of H and the biological ratio of FH and LH can be within the required solids such as 1 0.1 1 3 . Hugh's low purity H dye is not purified and is high Purified gonadotropin. As used herein, "low purity" means that the organism H below 200 FHU 9 is at 40 50 FHU 9. Low purity H can be obtained by methods well known in the art, such as, but not limited to, high soil adsorption in urine, ah, ethanol solution, including 10,000 It can also be obtained from the water. It can also be obtained from this channel. The biological ratio of FH and H in low purity H is 1 0.1 3 and the solid content is 1 0.5 1.4. The gonads ", " and H" used in this paper can be used. It refers to a protein hormone that is released by life or contains active ingredients of FH and LH. Can be a gonadin or a resting person, or a human source

Gonadin or Hugh.

As used herein, follicle stimulating hormone "and FH" can be used to refer to a hormone that promotes or follicles, promotes ovarian ovarian hormones, or can be extracted from the urine of a woman who can be secreted before the break in natural conditions. Got it.

As used herein, serotonin and "LH" can be used to refer to a hormone or sputum that is secreted by premenstrual cells and acts on mature ovulation.

As used herein, "except for gonadotropins, refers to proteins other than FH LH such as merging, polymerization, etc. The method of determining the high purity gonadotropin purity of the high-liquid phase HPL method provided by the HPL under the HPL One method gives the content.

The methods of providing high purity gonadotropins of this invention are the purification of low purity H dyes.

The method of each high purity gonadotropin in this one includes steps

1 A solution containing low purity H is applied to the dye and the supernatant and 2 pH 6 11 to obtain a high purity gonadotropin. The dye is not precipitated and can be well known in the art such as, but not limited to, ee, ag. Residence B e residence B e epha ose 6B live in a solution of 0.01 5 from acetic acid solution, Na H solution, or T s H solution.

In this one, high purity gonadotropin

The solution containing low purity H is applied to the undissociated dye

The T and pH of pH 6 1 yield high purity gonadotropins. In each of the other high purity gonados method steps

A solution containing low purity H onto a low-purity H solution on the undegraded B e ephaose is to dissolve low purity H at pH 4 8

In the step

0.03 0.07 Na H of pH6 11

Low pH6 11 0.03 0.07 T s H v high pH6 11 0.03 0.07 T s H

Collect

Add water ethanol, get the heart and get

High purity gonadotropin that will be dehydrated.

Of or sulphuric acid, which is from Na, K, or generally used in the same step in step v and step v

Low 0 0.5 Relative to residence 0.1 0.5 Residence 0.3 0.5 Height 0.5 5 Comparison of residence 2 5 Residence 3 5

The low purity H solution in the high purity gonadotropin method provided herein is a solution of low purity H dissolved in 0.01 0.03 acetic acid to obtain a solution containing low purity H. 4 8 high gonads pHMG

The method can obtain high purity gonadotropin HP content 5 living land HPL content 4 The high-purity gonadotropin obtained by the follicle stimulating hormone FH sputum LH-producing organism ratio 1 0.1 3 The living follicle stimulating hormone FH sap to produce LH biological proportion 1 0.5 1.4. Composition

As used herein, wood acceptable rest includes various forms and dilutions. The woods themselves are not essential active ingredients and are not applied. He is known as Re go 'ha ace, which is well known in the art. Ca ce ce ack b. Co. .J. 1991) can be found in the above-mentioned acceptable shape. The acceptable rest in the compound includes water, water, and ethanol. Sexual things such as, conditions, emulsification, p, etc.

The compounds can vary from one to the other. Oral, topical, non-subcutaneous, intravenous, and intramuscular injection or input or by means of an external device may be administered in one of the following ways. Among them, the subcutaneous injection method.

The high-purity gonadotropin obtained by the present method can be used for various uncombined administrable applications including (but not limited to), subcutaneous injection, and the like. Apply by subcutaneous injection. The form of application is matched. The amount of application of the active substance is usually 0.01 5 ug kg per day compared with the place of residence 0.05 0. g kg rest 0.2 0. g kg rest.

This can be used in other ways. The special features mentioned above or mentioned may be arbitrary. All of the features disclosed in any of the specific forms disclosed herein may be substituted for any equivalent, equivalent or similar purpose. Therefore, there is a special average or similar generality that is specifically disclosed.

This taxi is

6 1. Provide a high-purity, low-purity injection method that provides a high-purity purification method. Let's take a break from the rest. Understand this and not use it to limit the solid. The methods specified below are usually built as usual or in accordance with manufacturing. Unless otherwise otherwise all percentages, ratios, ratios, or.

The percentage of the vertical break in the present invention is well known in the art, for example, in the 100 ml solution.

Unless otherwise used in this article, all of this field

Familiar and the same. In addition, any methods and materials that are similar or equal in content can be used in the method. The preferred method in the text is used for materials. The H H L organism in the present invention

Methods .

The ratio of H or pH in the present invention refers to how much F H or LH in the milligram protein. The method for determining the protein content is based on y or derived methods. The HPL purity or HPL content in this specification refers to the percentage of the surface method.

The method for determining the purity of gonads in the present invention is 2.5

Pe dex75 10 300 L

Phase 0.2 phosphoric acid solution pH7.0 200 1000

Dissolve the phase to 500 U L

00 U

.

30 C

Flow rate 0.5 L

215

Same as 60

Gonadotropin retention with 18 1g r Each of the high-gonadin 1 uses low-purity H (from Shanghai Bio-Bio Co., Ltd.) as the starting material for p. The organism of F is 273 U g LH of the organism 319 U g

5 L B e epha ose FF (from E e sha ) was equilibrated in a balance (0.02 acetic acid pH 7.0).

Equilibrate 09 low-purity L equilibrium to dissolve H or Na solution p 7.0 and then top up B e epha ose FF up to 5 times equilibrium 0. 5 yc Na H PH 8 times off with 0.05 T s H , 0.4 a PHg 8 times off, most 0. 5 T s H , 4 a pHg, fraction collection collected, for example 300, one will contain active ingredients The part of the well is the effective component of 10000, adding 5 times of rest of the water, the ethanol is collected, and the water is dehydrated to obtain 236 9 which is high purity gonadotropin 1.

The biological method is determined according to the method of mercury and mercury N in 2005. F H LH organism.

Its 1

1 derived creature and snake

F Biological Biology F H LH Biology

P snake ( u mg) ( u mg ) ratio

High snake gonadotropin

5012 5036 0.99 97.19 shows that purification by this method can greatly improve the purity.

2

Each high purity gonadotropin 11 loads 5 LB e epha ose 6B (from E e sha ) (0.02 acetic acid pH 7.0) equilibrated.

Equilibrate the same source of low-purity 600 in the same source in the same source. Dissolve the H or Na solution p 7.0 and then top up the B e epha ose 6B and balance it up to 5 times. 5 yc Na H PH 5 times rest and reuse 0. 5 T s H , 0.2 Na PHg 5 times rest, and then use 0.5 T s H , 0.5 Na PHg 7 times rest most 0.05 T s H , 4 Na PHg , and collect the effective components of the wells 10000 to 0, That is, high purity gonadotropin 11 is obtained. This H. H H biological and protein content ratio.

Protein content determination method Lo y .

F H specific activity method F H biological U L ml Hugh protein content g L

2

2 creatures and snakes

F biological organism F ratio

Protein content F H LH

( U mg egg P snake (mg mL)

( mL) ( mL) White)

High snake

Sex 6076 5843 0.502 12104 1.04 97.85 Adenine 11

It shows that the purification by this method can greatly improve the purity. 3

Each of P G

Made at 10 pH and 75 F and 75 L per bottle as follows

Dissolve 1 high purity gonadotropin 1.64 9 in 5 injections of raw water at 0.22 U of raw water.

9 Dissolve 19 lactose in the injected raw water if required H or Na pH 6.5 + 0.2 0.22U. Then add the raw water to the upper F solution to 75.

The upper solution was dispensed into 0.75 frozen bottles per bottle.

The obtained ones were 75 F H 75 LH and 9 lactose per bottle.

The preferred content of the above-mentioned power is not to limit the wood content of the solid content of the wood content, but also the requirement that the solid wood or the method of any other person to complete the same or the same The history of the exchange will be fixed.

Claims

Claim

1. High purity gonadotropin p edH a e opa sa o ado op s, pH is characterized by its HPL purity not lower than its HPL content is not 5

HPL purity is not less than 96 HPL content is not 4

2. The high-purity gonadotropin required for 1 is characterized by the absence of gonadotropin or gonadotropin, or the source of gonadotropin or Hugh.

3. Requires 1 high-purity gonadotropin, which is characterized by a high degree of gonadotropin, follicle stimulating hormone F H, and LH-producing organisms.

4. The high-purity gonadotropin of claim 1 is characterized by high-purity gonadotropin. The proportion of follicle stimulating hormone F H to LH-producing organisms is 1 0.5 1.4.

5. Each of the methods for requesting the high-purity gonadotropin pH of any of the four methods includes a step of operating the low-purity H of the low-purity H dye and the high-purity gonadotropin purified at 200 F H .

9 protein or less.

6. The method of claim 5, the method of which includes the steps

1 Apply a solution containing low purity H to the undissolved dye

2 pH6 11 and

3 pH6-1 gives high purity gonadotropins.

7. The method of claim 6 is characterized in that 0.1-w v of low purity H in a solution containing low purity H

8. The method of claim 6 is characterized in that the pH of the solution containing low purity H is 4 4

9. The methods of claim 6 are specific to acetic acid solution, acetic acid solution, Na H solution, T s HC solution or phosphoric acid solution.

10. The method of any of the methods of claim 5 is characterized in that the dye is not separated from the dye and is formulated from bac o B ea ge RG ee

11. The method of any of the 5-9 is characterized in that the solid phase of the dye is not precipitated from soil, glass micro, quartz micro, calcium phosphate, oxidized, polypropylene, , , or .

12. The method of any of the 5-9 is characterized in that the dye is not separated from the B e epha ose FF or the B e epha ose 6B

13. The composition of the composition comprises an effective amount of a high purity gonadotropin which is required to be any one of the above and an acceptable one.

14. The use of high-purity gonadotropins in any of the requirements of 1 4 is particularly uncommon.