KR101471107B1 - A glycoprotein composition almost containing no subunit and preparation method thereof - Google Patents

Abstract

The present invention relates to a composition of a glycoprotein containing little subunits and a method of producing the same. The method comprising: raising the temperature of the tray-containing aqueous solution containing the glycoprotein that has undergone freeze-thaw treatment in vacuum to a temperature of the solution process point of 1 to 20 ° C at a temperature raising rate of 0.05 to 5 ° C / min and maintaining the tray temperature for 1 to 30 hours; And raising the tray temperature to a temperature not lower than 0 ° C at a heating rate of 0.05 to 5 ° C / min for 1 to 20 hours to obtain a lyophilized powder containing the glycoprotein.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition of a glycoprotein substantially free from subunits and a method for producing the same.

The present invention relates to the field of purification of protein drugs. And more particularly to a method for freeze-drying an aqueous solution containing a glycoprotein for treating infertility.

The glycoprotein that treats infertility is a type of structure similar to that of human gonadal gonadotropin (HCG), human postmenopausal gonadotropin (HMG), follicle stimulating hormone (FSH) and luteinizing hormone (LH) . Among them, human postmenopausal pre-stimulating hormone contains follicle-stimulating hormone and luteinizing hormone, and they are a mixture composed of a certain ratio (1: 0.1-1).

In HCG, FSH, and LH, two subunits of alpha and beta chains were bound in the form of noncovalent bonds. Their α subunits are exactly the same, with 92 amino acids and a molecular weight of about 14500 D. Asparagine at positions 52 and 78 is the amino acid of N-glucosylation. The subunit of HCG has 145 to 147 amino acids and has a molecular weight of 22200 to 39000 D, of which asparagine at positions 13 and 30 and positions 121, 127, 132 and 145 are sites where glycosylation occurs. However, the β subunit of LH is composed of 121 amino acids and the molecular weight is about 14,800 D.

In clinical practice, HCG, HMG, FSH, and LH are mainly used for infertility treatment and in vitro supplementation. They can be isolated from urine of a specific woman (after gestation or postmenopausal) and can also be produced by DNA recombinant technology.

The first generation of glycoproteins that treat infertility are Prifasi (HCG) and Pergonal (HMG), which were launched by Serono in the 60s of the 20th century. They are all relatively low in purity and contain large amounts of impurities. After the 80s of the 20th century, the Serono company introduced Metrodin-HP, a high-purity FSH formulation with very low impurity content. Gonal-F (rFSH) and Luveris (rLH), which were produced using DNA recombination technology, were also released. In addition, Ferring has also released a high purity human postmenopausal asthmatic hormone - Menopur.

Therefore, in the present market, we are working on the development and application of high purity glycoprotein hormone, thereby replacing low-purity products and trying to overcome the hypersensitivity reactions caused by mature proteins in general low-purity products. However, the high purity glycoprotein obtained through various purification means must undergo two steps. That is, the raw drug phase and the finished product phase of the preparation. In these two steps, it may be in solid form or liquid form. Liquids are generally preferred to solid form because they are detrimental to preservation and transportation and do not favor liquid form. For raw materials, the liquid must be stored below -20 ° C, otherwise it will lose its activity. The liquid form may undergo a freeze-thaw process, and even if subjected to repeated freeze-thaw cycles, Activity is lost. The liquid form also presents a risk, for example, that the packaging container ruptures easily when it approaches the brittle point at low temperatures. In the finished product of the preparation, since the liquid form is difficult to transport and the stability of the liquid form of the glycoprotein is comparatively low, the preservative must be added so that the microorganism can be prevented from breeding within the valid period. This is the same as described in Chinese patent application No. CN1309567A.

  In addition, the solid form is obtained mainly by the lyophilization method, but the high-purity glycoprotein is easily denatured by lyophilization and loses its activity. The main expression of denaturation and inactivation is that the complete molecule is broken down into two subunits of the [alpha] and [beta] chains, which belong to impurities and which are not therapeutically effective or are isomers that can cause side effects, In the preparation of the lyophilized preparation of the present invention, the amount of 10 to 30% is further increased to offset the inactivation caused during the lyophilization process. However, a certain amount of small scale is still generated in the finished product, thereby lowering the purity of the product. One of the causes of decomposition inactivation is that the combination of raw materials and auxiliary materials is unreasonable, and the other is that the technique of freeze drying is not perfect.

Therefore, there is a desire in the art to develop a freeze-drying method of a glycoprotein suitable for the treatment of infertility, thereby obtaining a glycoprotein drug containing little or no subunits.

The present invention provides a method for freeze-drying a glycoprotein-containing aqueous solution, thereby obtaining a glycoprotein drug substantially free of subunits.

According to a first aspect of the present invention, there is provided a composition of a glycoprotein wherein the content of the subunit does not exceed 10 wt%.

In another preferred embodiment, the glycoprotein is selected from a chorionic gonadotropin, a menopausal gonadotropin, a follicle stimulating hormone, a luteinizing hormone or a mixture thereof.

In another preferred embodiment, the chorionic gonadotropin is a human chorionic gonadotropin derived from human urine and / or recombinant or a mutant thereof, wherein the menopausal gonadotropin is derived from urinary and / or recombinant human postmenopausal gonadal Stimulating hormone or a mutant thereof, wherein the follicle-stimulating hormone is human urine-derived and / or recombinant human follicle-stimulating hormone or a mutant thereof, and the luteinizing hormone is human urine-forming hormone derived from urine and / Lt; / RTI >

In another preferred embodiment, the sub-content of the composition of the glycoprotein does not exceed 5 wt%.

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

According to a second aspect of the present invention, there is provided a method for freeze-drying a glycoprotein-containing aqueous solution. The method includes the following steps:

(a) raising the tray temperature to 1 to 20 占 폚, which is lower than the solution process point temperature, at a temperature raising rate of 0.05 to 5 占 폚 / min under vacuum, and keeping it for 1 to 30 hours; And

(b) raising the tray temperature to a value greater than or equal to 0 ° C at a heating rate of 0.05 to 5 ° C / min and holding the cake for 1 to 20 hours to obtain a lyophilized powder containing the glycoprotein.

In another preferred example, the rate of temperature rise in step (a) is 0.07 to 3 占 폚 / min, the rate of temperature rise in step (b) is 0.06 to 1.5 占 폚 / Treating the aqueous solution containing the protein with a pretreatment for freezing for 0.5 to 5 hours.

In another preferred embodiment, the concentration of the glycoprotein in the glycoprotein-containing aqueous solution is from 1 / / mL to 80 mg / mL, more preferably from 500 / / mL to 60 mg / mL.

Among other preferred examples, the glycoprotein in the aqueous solution containing the glycoprotein in the step (a) is a high purity glycoprotein.

Among other preferred examples, in the step (b), a lyophilized powder containing a glycoprotein having a sub-fractional content of less than or equal to 10 wt% is obtained. More preferably, the subcomponent content is less than or equal to 5 wt%, and the subcomponent content is preferably less than or equal to 3 wt%.

Among other preferred examples, the one or more saccharide protecting agents selected from lactose, sucrose, fucose, mannitol, and dextran are contained in the aqueous solution containing the glycoprotein in the step (a). The concentration of the saccharide protecting agent in an aqueous solution containing the glycoprotein is 2 to 60 mg / ml.

In another preferred embodiment, the glycoprotein is selected from a chorionic gonadotropin, a follicle stimulating hormone, a luteinizing hormone, a menopausal gonadotropin or a mixture thereof.

The human chorionic gonadotropin is a human chorionic gonadotropin derived from human urine and / or a recombinant human gonadal gonadotropin or a mutant thereof, and the menopausal gonadotropin is human urine-derived and / or recombinant human postmenopausal gonadotropin or a mutant thereof Wherein the follicle stimulating hormone is a human follicle stimulating hormone derived from and / or recombined with human urine or a mutant thereof, and the luteinizing hormone is a human luteinizing hormone or a mutant thereof derived from urine and / or recombinant.

According to a third aspect of the present invention there is provided the use of a composition as provided in the present invention as described above for use in the manufacture of a medicament for treating a sterility syndrome.

According to a fourth aspect of the present invention, there is provided a glycoprotein produced by the method as described above.

According to a fifth aspect of the present invention, there is provided the use of a glycoprotein produced by the method as described above for producing a drug for treating a sterility syndrome.

Accordingly, the present invention provides a method for freeze-drying a glycoprotein suitable for the treatment of infertility, thereby obtaining a glycoprotein drug containing little or no subunits.

Fig. 1 shows the liquid chromatography spectrum of FSH before lyophilization in Example 1. Fig.
Fig. 2 shows the liquid chromatography spectrum of FSH after lyophilization in Example 1. Fig.

The inventors found that during the freeze-drying process, the same lyophilization method had different effects on the double-chain structure of glycoproteins such as HCG, FSH, and LH, and single chain structure proteins such as urinary trypsin inhibitor (UTI) , The electrons were found to be almost inactivated, but the latter was more easily decomposed into subunits, resulting in inactivation. Thus, the present inventors have found through extensive studies that, surprisingly, an excessively rapid temperature rise in the lyophilization process leads to the degradation of glycoprotein, resulting in loss of activity upon denaturation. As described above, all of these glycoproteins are bound to each other in the form of noncovalent bonds, and they are maintained by weak action such as hydrophobic action and hydrogen bonding. The glycoprotein is uniformly distributed in the ice crystal of the water molecule during the freezing pretreatment process together with other compositions such as an auxiliary material. However, if the tray delivers a relatively large heat flow rate to the ice crystal structure of the product during the rapid temperature rise, The degree of vacuum in the chamber is very high, allowing the water molecules in the ice crystal to sublimate quickly into water vapor molecules in a short period of time. In this rapid sublimation process, the water molecules escape from the ice crystal structure at a very high speed, resulting in a relatively weak retention change such as hydrogen bonding and hydrophobic action between the sodas of the double-chain sugar proteins, resulting in dissociation of the sub- To lose activity.

In the course of the research, the inventors of the present invention have found that, in the lyophilization process, the decomposition of the subunit is reduced or hardly generated by proper control of the heating rate, thereby preserving the activity of the product and reducing the generation of impurities I turned my attention.

Justice

As used in the original text, "glycoprotein" includes chorionic gonadotropin (CG), follicle-stimulating hormone (FSH), luteotropic hormone (LH), human menopausal gonadotropin (Human) hormone (human menopausal gonadotropin, HMG).

As used herein, the terms "an aqueous solution containing a glycoprotein", "an aqueous solution containing a glycoprotein", "an aqueous solution containing a glycoprotein" and "an aqueous solution containing a glycoprotein" are interchangeable and refer to the same aqueous solution, Include glycoproteins or include glycoproteins and pharmaceutically / pharmacologically acceptable carriers.

As used in the text, "composition of a glycoprotein" refers to a composition containing a glycoprotein, which is a solid, including a glycoprotein or comprising a glycoprotein and a pharmaceutically / pharmacologically acceptable carrier , Preferably a lyophilized powder containing a glycoprotein.

As used in the text, "lyophilized powder containing glycoprotein" is a solid powder, including glycoproteins or including glycoproteins and pharmaceutically / pharmacologically acceptable carriers.

As used in the original text, "subunit" refers to a single α chain subunit or β chain subunit generated through the dissociation process of CG, FSH, LH, and HMG.

As used herein, a "high purity glycoprotein" refers to a glycoprotein having a purity greater than 90%, preferably greater than 95%. In a preferred embodiment of the invention, the high purity glycoprotein is lyophilized, A glycoprotein with a content less than or equal to 10 wt% is obtained.

As used in the original text, the jargon "medically acceptable" or "pharmaceutically acceptable" components apply to humans and / or animals, but are free of undue side effects (eg toxicity, irritation and adverse reactions) A substance with a reasonable benefit / risk ratio.

As used in the original text, the terminology "medically acceptable carrier" refers to a carrier for therapeutic administration and includes various excipients and diluents. This term refers to the following pharmaceutical carriers: themselves are not required active ingredients and are not excessively toxic after use. Suitable carriers are well known to those skilled in the art. There is ample discussion of medically acceptable excipients in Remington ' s Pharmaceutical Sciences, Mack Pub. Co., N. J. 1991.

The "medically acceptable carrier" above may contain liquids such as water, saline, glycerin and ethyl alcohol. In addition, auxiliary materials may be present in such carriers, such as fillers, disintegrants, lubricants, fluidizers, foams, wetting or emulsifying agents, deodorants, pH buffering substances and the like. Generally, these materials may be combined in a non-toxic, inert, and medically acceptable aqueous carrier, wherein the pH is generally about 5 to 8, more preferably about 6 to 8.

The terms "follicle stimulating hormone" and "FSH" are interchangeable and refer to a hormone or a variant thereof that promotes the production of sperm or follicular cells and promotes ovarian development. The FSH in the present invention can be obtained in any manner conventionally used in the art, and can be obtained, for example, from the pituitary gland in the natural state or from the postmenopausal woman's urine or from recombinant techniques. They are naturally occurring, or obtained or synthesized by recombinant techniques, including LH or CG as impurities. In one embodiment of the present invention, the follicle stimulating hormone is a human follicle stimulating hormone or a mutant thereof, and is preferably a follicle stimulating hormone derived from urine or a recombinant human follicle stimulating hormone.

In the method of the present invention, it is preferable to separate and remove LH or other impurities other than CG by conducting rudimentary purification of FSH as a raw material by a conventional method in the art before proceeding with the purification of FSH.

As used in the original text, the terminologies "luteinizing hormone" and "LH" are interchangeable and have the same or similar structure and function as the doped natural LH in the production and acquisition of raw materials containing FSH It refers to a hormone.

Similarly, the terms "human chorionic gonadotropin" and "CG" can be interchangeably used, and hormones that have the same or similar structure and function as natural CG doped during the production or acquisition of raw materials containing FSH Point.

As used in the original text, the terminology "tray" refers to the structure of a heat transfer device of a freeze dryer, wherein a heat conduction medium such as silicone oil is included in the trays of each layer, thereby conducting heat conduction circulation. In general, a cooling / heating apparatus which is well known to those skilled in the art also has such a structure.

As used in the original text, the terminology "tray temperature" refers to the temperature reached by the heat conduction medium in the tray.

As used in the original text, the terminology "cold trap" refers to a trap that collects gas on a chilled surface in a condensing fashion. It is installed between the vacuum vessel and the pump and is a device used to adsorb gas or collect oil vapor.

As used in the original text, the terminology "sublimation drying" refers to a drying process in which ice is directly converted to water vapor by vacuum drying within a temperature range of 1 to 20 ° C below the process point.

As used in the original text, the technical term "dehumidification drying" refers to a drying process in which water molecules that have not been completely removed after sublimation drying are removed by thermal evaporation.

As used in the original text, the terminology "freeze pretreatment" refers to freezing the dried glycoprotein or its composition form prior to vacuum freeze drying. In the freeze pretreatment step, the freeze pretreatment temperature (typically 10 ° C lower than the process point of the sample) should be tightly controlled, preferably -30 to -50 ° C.

Freeze-dried

The lyophilization method of an aqueous solution containing the glycoprotein provided in the present invention includes the following steps:

(a) raising an aqueous solution containing glycoprotein pretreated under vacuum to a temperature of 1 to 20 占 폚 at a temperature raising rate of 0.05 to 5 占 폚 / min and keeping the tray temperature below the solution process point, for 1 to 30 hours; And

(b) raising the tray temperature to a value greater than or equal to 0 ° C at a temperature raising rate of 0.05 to 5 ° C / min and holding for 1 to 20 hours to obtain a lyophilized powder containing the glycoprotein.

In one preferred embodiment of the present invention, the lyophilization method of an aqueous solution containing a glycoprotein comprises the following steps:

(1) subjecting the aqueous solution containing the glycoprotein to a freeze pretreatment for 0.5 to 5 hours;

(2) raising the temperature to a sublimation drying temperature and maintaining it for 1 to 30 hours at a temperature raising rate of 0.05 to 5 deg. C / min under vacuum (most suitable when the temperature raising rate is 0.07 to 3 deg. C / min) And

(3) The temperature is raised to the desiccation drying temperature at a temperature raising rate of 0.05 to 5 캜 / minute (most suitable when the temperature raising rate is 0.1 to 1.5 캜 / minute), and the temperature is maintained for 1 to 20 hours to obtain a lyophilized powder ≪ / RTI >

More preferably, in the present invention, an aqueous solution containing a high-purity glycoprotein is lyophilized by the method provided by the present invention, and the content of the subunits contained in the glycoprotein in the obtained glycoprotein-freeze-dried powder is 10% Less than or equal to (less than or equal to 5% is most appropriate).

The present invention also provides a method for producing a composition of a glycoprotein. The method includes the following steps:

(a) raising the temperature of the aqueous solution containing the glycoprotein that has undergone the freeze pretreatment under vacuum to a sublimation drying temperature at a temperature raising rate of 0.05 to 5 deg. C / min and holding it for 1 to 30 hours; And

(b) raising the temperature to a dehumidifying and drying temperature at a temperature raising rate of 0.05 to 5 캜 / min and holding for 1 to 20 hours to obtain a lyophilized powder containing the glycoprotein. Preferably, the composition is a lyophilized powder containing a glycoprotein. The composition is a composition of a glycoprotein having a subunit content of not more than 10 wt%.

The advantages of the present invention are as follows.

The freeze-drying method provided in the present invention is a freeze-drying method that reduces the decomposition of the subunits or hardly causes decomposition of the subunits, thereby preserving the activity of the glycoprotein and reducing the generation of impurities, To obtain a composition.

Hereinafter, the present invention will be clearly described by combining specific examples. It should be understood that these embodiments are for illustrative purposes only, and are not intended to limit the scope of the present invention. In the following examples, the experimental conditions of the specific conditions are not annotated, and the process is generally carried out according to ordinary conditions or conditions suggested by the manufacturer. Unless otherwise specified, all percentages are weighted.

Unless otherwise defined, all technical and scientific terms used in the text are intended to be familiar to those of ordinary skill in the art. Any methods and materials similar or equivalent to those described elsewhere apply to the method of the present invention. The preferred practices and materials described in the text are used for demonstration purposes only.

All of the minor content in the examples are determined by the following method:

Liquid chromatography:

Chromatographic column: Superdex 75 10 x 400 mm or similar column;

Mobile phase: 0.2 M Na ₂ HPO4 (pH 6.5): acetonitrile = 10: 2

Flow rate: 0.5 ml / min

Sample inlet concentration and volume: about 60 μg / ml x 100 μl

Detection wavelength: 215 nm

The ratio of the retention time of the subpixel pick to the main component pick is about 1.2 and the subsonic content is calculated by the area normalization method.

Example  One

FSH Freeze-drying

50 mL of 0.01 M sodium dihydrogen phosphate solution (pH adjusted to about 6.5 with NaOH) was prepared, and 2 g of lactose was added to dissolve with stirring, followed by filtration 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 Thien Biochemical Co., Ltd.) (biological concentration is 8817 international units / mg), and then the solution point of the solution is about -2 ° C, placed in a tray, placed in a freeze dryer (purchased from Virtis) and lyophilized under the following conditions:

1. Freeze pretreatment for 3 hours at -40 ℃ for tray temperature;

2. Low temperature traps at -45 ° C;

3. Vacuum pump operation;

4. The tray temperature was raised from -40 ° C to -10 ° C at a rate of 0.125 ° C / min to maintain the tray temperature at -10 ° C for 15 hours;

5. Raise the tray temperature from -10 ° C to + 20 ° C at a rate of 0.3 ° C / min to maintain the tray temperature for 5 hours after reaching + 20 ° C;

6. Remove from chamber and obtain 392 mg of lyophilized powder.

The results of the biological activity of the measured FSH are as follows:

Unit activity
(IU / mg) Total potency
(Only IU) Freeze-dried yield
(%) Substrate content
(%) Before lyophilization 8.82 0.5 After lyophilization 217 8.51 96% 1.6

Control Example

In this control example, the other steps besides the following freeze-drying method were in conformity with the method of Example 1 above.

Control Example Freeze Drying Procedure:

 1. Tray temperature reaches -40 ℃ and the pretreatment for freezing is carried out for 3 hours;

 2. Low temperature traps at -45 ° C;

 3. Vacuum pump operation;

 4. Raise the tray temperature from -40 ° C to -10 ° C to maintain the tray temperature at -10 ° C for 18 hours;

 5. Raise the tray temperature from -10 ° C to + 20 ° C and keep it for 6 hours;

 6. Remove from chamber and obtain 398 mg of lyophilized powder.

The results of the biological activity of the measured FSH are as follows:

Unit activity
(IU / mg) Total potency
(Only IU) Freeze-dried yield
(%) Substrate content
(%) Before lyophilization 8.82 0.5 After lyophilization 192 7.67 87% 11.1

As can be seen from the results of Example 1 and the control, the freeze-drying method of the present invention not only remarkably reduces the generation of subunits but also improves the freeze-drying yield.

Example  2

FSH  Preparation of freeze-dried injections

Typical examples of the production of FSH containing 100 IU of FSH lyophilized injections and containing 75 IU per bottle are as follows:

A fixed amount (calculated as biological unit) of the purified FSH product from the calculation was dissolved in 50 mL of pyrogen-free water. If necessary, the pH is adjusted to 6.5 ± 0.2 with HCl or NaOH and sterile filtration is carried out with a 0.22 μm filter.

 100 g of lactose was dissolved in 2 L of injectable pyrogen-free water for injection. If necessary, adjust the pH to 6.5 ± 0.2 with HCl or NaOH and proceed with aseptic filtration through a 0.22 μm filter. Then, the solution is added to the FSH solution, the volume is adjusted to 7.5 L with pyrogen-free injection water, and the mixture is homogeneously mixed. The process point of this solution is about -2 ° C.

 The solution was dispensed in 0.75 mL of ampule bottles and lyophilized according to the following procedure.

 1. Freeze pretreatment for 3 hours at -40 ℃ for tray temperature;

 2. Low temperature traps at -45 ° C;

 3. Vacuum pump operation;

 4. The tray temperature was raised from -40 ° C to -10 ° C at a rate of 0.5 ° C / min to maintain the tray temperature for 10 hours after reaching -10 ° C;

 5. Raise the tray temperature from -10 ° C to + 35 ° C at a rate of 0.375 ° C / min to maintain the tray temperature for 10 hours after reaching + 35 ° C;

 6. Piezoelectric, removed from chamber.

Each bottle contained 75 IU of FSH and 10 mg of lactose.

The subcritical content after HPLC measurement is as follows:

Substrate content
(%) Before lyophilization 3.2 After lyophilization 3.9

Example  3

High-purity human Menopause Of Stimulation Hormone (hpHMG)  Manufacturing method

100 mL of 0.01 M sodium dihydrogen phosphate solution (pH adjusted to about 6.5 with NaOH) was prepared, and 4 g of lactose was added to dissolve with stirring, followed by filtration through a 0.22 mu m filter. 15 mL of the filtrate was taken and 35 mg of high purity human postmenopausal asthmatic stimulation hormone (purchased from Shanghai Tianyi Bio-Pharmaceutical Co., Ltd.) (FSH biological reverse transcript is 5131 IU / mg and LH biological transverse transcript is 4890 IU / mg) To complete dissolution. The process point of this solution is about -2 ° C and freeze-drying is carried out according to the following procedure:

1. Tray temperature reached -40 ℃ and the pretreatment of freezing for 3 hours;

2. Cold trap operation at -45 ° C;

3. Activate the vacuum pump;

4. The tray temperature was raised from -40 ° C to -10 ° C at a rate of 0.125 ° C / min to maintain the tray temperature for 15 hours after reaching -10 ° C;

5. Raise the tray temperature from -10 ° C to + 20 ° C at a rate of 0.3 ° C / min to maintain the tray temperature for 5 hours after reaching + 20 ° C;

6. Remove from chamber and obtain 570 mg of high purity HMG powder.

  The measured FSH biological reverse transcript is 298 IU / mg and the LH biological reverse transcript is 277 IU / mg. The sub-fraction content obtained by HPLC measurement is as follows:

Substrate content
(%) Before lyophilization 4.3 After lyophilization 4.7

Example  4

Typical examples of production processes used to make 10000 bottles of FSH lyophilized injections and also containing 75 IU of FSH and 75 IU of LH per bottle are as follows:

A hpHMG purified product of a given amount (as a unit of biological activity) was dissolved in 50 mL of pyrogen-free water for injection. If necessary, adjust the pH to 6.5 ± 0.2 with HCl or NaOH and proceed with aseptic filtration through a 0.22 μm filter.

200 g of lactose was dissolved in 2 L of pyrogen-free water for injection. If necessary, adjust the pH to 6.5 ± 0.2 with HCl or NaOH and proceed with aseptic filtration through a 0.22 μm filter. Next, the solution is added to the FSH solution, the volume is adjusted to 7.5 L with pyrogen-free injection water, and the mixture is homogeneously mixed. The process point of this solution is about -2 ° C.

Add 0.75 mL of the solution to each ampoule bottle and proceed with freeze-drying according to the following procedure:

1. Freeze pretreatment for 3 hours at -40 ℃ for tray temperature;

2. Low temperature traps at -45 ° C;

3. Vacuum pump operation;

4. The tray temperature was raised from -40 ° C to -10 ° C at a rate of 0.5 ° C / min to maintain the tray temperature for 10 hours after reaching -10 ° C;

5. The tray temperature was raised from -10 ° C to + 35 ° C at a rate of 0.375 ° C / min to maintain the tray temperature for 10 hours after reaching + 35 ° C;

6. Piezoelectric, removed from chamber.

Of the resulting ampoules, each bottle contains 75 IU of FSH, 75 IU of LH and 20 mg of lactose.

The subcritical content after HPLC measurement is as follows:

Substrate content
(%) Before lyophilization 4.5 After lyophilization 4.9

Example  5

HCG  Preparation of freeze-dried injections

Typical examples of the production process of HCG used to make 10000 bottles of HCG freeze-dried vaccine and also containing 5000 IU per bottle are as follows:

An aliquot of the HCG from the calculation (in units of biological titer) was dissolved in 50 mL of pyrogen-free water for injection. If necessary, adjust the pH to 6.5 ± 0.2 with HCl or NaOH, and proceed with aseptic filtration through a 0.22 μm filter.

After dissolving 300 g of mannitol and 10.9 g of NaH ₂ PO ₄ · H ₂ O in free water for injection rosin of 3 L pie, adjust pH with NaOH to 7.0 ± 0.2 and then subjected to sterile filtration with a 0.22 ㎛ filter . Then, the solution is added to the FSH solution, and the volume is adjusted to 7.5 L with pyrogen-free injection water.

  Add 0.75 mL of this solution to each ampoule bottle, and proceed with lyophilization according to the following procedure.

1. Freeze pretreatment for 3 hours at -40 ℃ for tray temperature;

2. Cold trap operation at -45 ° C;

3. Vacuum pump operation;

4. The tray temperature was raised from -40 ° C to -10 ° C at a rate of 0.5 ° C / min to maintain the tray temperature for 10 hours after reaching -10 ° C;

5. The tray temperature was raised from -10 ° C to + 35 ° C at a rate of 0.375 ° C / min to maintain the tray temperature for 10 hours after reaching + 35 ° C;

6. Piezoelectric, removed from chamber.

Among the obtained ampule bottles, each bottle contains 0,000 IU of HCG and 20 mg of mannitol.

The subcritical content after HPLC measurement is as follows:

Substrate content
(%) Before lyophilization 3.3 After lyophilization 3.5

All references cited herein are incorporated by reference in this application and are incorporated herein by reference in their entirety. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (13)

(a) raising an aqueous solution containing glycoprotein pretreated under vacuum to a temperature of 1 to 20 占 폚 at a temperature raising rate of 0.05 to 5 占 폚 / minute to maintain the temperature of the tray below the solution process point and holding it for 1 to 30 hours; And
(b) raising the temperature of the tray to a value greater than or equal to 0 ° C at a heating rate of 0.05 to 5 ° C / min and maintaining the temperature for 1 to 20 hours to obtain a lyophilized powder containing the glycoprotein A method for freeze-drying an aqueous solution containing
Wherein said glycoprotein is selected from a human gonadotropin, a menopausal gonadotropin, a follicle stimulating hormone, a luteinizing hormone, or a mixture thereof. The method according to claim 1, wherein the rate of temperature rise in step (a) is 0.07 to 3 占 폚 / min, and the rate of temperature rise in step (b) is 0.06 to 1.5 占 폚 / min. 3. The method according to claim 2, comprising the step of pre-treating the aqueous solution containing the glycoprotein for 0.5 to 5 hours before the step (a). The method according to claim 1, wherein the concentration of the glycoprotein in the aqueous solution containing the glycoprotein is 1 쨉 g / mL to 80 mg / mL. 5. The method according to claim 4, wherein the concentration of the glycoprotein in the aqueous solution containing the glycoprotein is 500 μg / mL to 60 mg / mL. The method according to claim 1, wherein an aqueous solution containing the glycoprotein in step (a) contains one or more saccharide protecting agents selected from lactose, sucrose, fucose, mannitol, and dextran, , Wherein the concentration of the saccharide protecting agent is 2 to 60 mg / ml. The method according to claim 1,
The human chorionic gonadotropin is a human chorionic gonadotropin derived from human urine. The menopausal gonadotropin is a human postmenopausal gonadotropin derived from human urine. The follicle stimulating hormone is a human follicle stimulating hormone derived from human urine , Wherein the luteinizing hormone is a human luteinizing hormone derived from urine. delete delete delete delete delete delete

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