CN109731097A - A kind of rh-FSH preparation and preparation method thereof - Google Patents

Abstract

This patent provides a kind of rh-FSH preparation and preparation method thereof, and PLURONICS F87, auxiliary freeze-drying are added in the freeze-drying formula of rh-FSH, so that entire freeze-drying process is more easier, it is more preferable to freeze shape after preparation freeze-drying；Different from other kinds of drug, after PLURONICS F87 is added in this product, redissolves speed and be obviously improved, and insoluble granule does not also increase, and complies fully with pharmacopoeial requirements；Dissociation subunit, oxidation subunit and polymeric content detection carried out to the rh-FSH mass after redissolution, test result, which shows to be added the lyophilized preparation after PLURONICS F87, not to be had an impact the quality of destination protein rh-FSH, does not influence destination protein rh-FSH purity.

Description

A kind of rh-FSH preparation and preparation method thereof

Technical field

The present invention relates to human reproduction fields, it is more particularly related to promoting sexual gland hormone and preparation method thereof.

Background technique

According to the World Health Organization (WHO) report: there are about 1 pair, there are dysgenesia, infertilities in the every 7 couples of Mr. and Mrs in the whole world Influence the population in the whole world about 10%~15%.The disease incidence of China's infertility shows an increasing trend year by year, and one A little economically developed coastal areas cause the infertile patient to be more than due to being influenced by factors such as environmental pollution, operating pressures 20%, especially had more than in representative In Guangdong Province, 10 couples of Mr. and Mrs two suffered from because of a variety of causes for double it is infertile Sterility.Statistics display, in the female infertility cause of disease, Follicular maldevelopment syndrome caused by endocrine dysfunction is occupied entirely It is the first to occupy the female infertility cause of disease for the 48.42% of the infertile cause of disease in portion.

Gonal-F (rh-FSH) clinic is mainly used for sterility treatment, the indication of China's approval at present Have: for not ovulating (including Stein-Leventhal syndrome, PCOS) and treating nonresponder to citric acid clomiphene；For assisting giving birth to Plantation technology Ovarian hyperstimulation, in vitro fertilization-embryo implanting (IVF-ET), gamete intra-Fallopian transfer (GIFT) and ooecium slurry Sperm injection (ICSI), to obtain multiple follicular developments.

Gonal-F (rh-FSH) is developed by Merck Xue Lannuo company, trade name " Guo Nafen ", and 2000 It is listed into the country.The Merck Xue Lannuo follicle-stimulating hormone (FSH) that hospital sells at home is Gonal-F α, mainly Dosage form has injection Gonal-F (freeze-drying powder-injection) and Gonal-F's injection (aqua) two Kind dosage form.In similar domestic imitation medicine, in the preparation of injection Gonal-F (freeze-drying powder-injection), exist Freeze-drying effect it is poor and be freeze-dried after redissolve ineffective technical problem^[1]。

As above to solve the problems, such as, PLURONICS F87 is added in the reagent of this patent, and PLURONICS F87 has following effect, pool Luo Shamu 188 has more stable protective effect to the freeze-drying of carrier micelle agent^[2].Using PLURONICS F87 as carrier material Material, to the lower resveratrol of solubility (RES), the resveratrol-P188 solid dispersion body (RES-P188- that is prepared into SD), RES is compared with bulk pharmaceutical chemicals, and the solubility of RES-P188-SD improves nearly 17 times, and (RES bulk pharmaceutical chemicals solubility is 0.03mg/ ml)^[3].PLURONICS F87 is colloid when being liquid, room temperature or body temperature in low temperature, for gel of ascending the throne, there is targeting to make With, and the action time of drug can be extended, there is slow releasing function^[4-6].Prolong curative effect using P188 on drug administration by injection It is long^[7-9].Reduce toxicity and the phlebitis incidence etc. of drug^[10-11]。

PLURONICS F87, auxiliary freeze-drying, so that whole is added in this patent in the freeze-drying formula of follicle-stimulating hormone (FSH) (RH-FSH) A freeze-drying process is more easier to control, and it is more preferable to freeze shape after preparation freeze-drying；Different from other kinds of pharmaceutic adjuvant, it is husky that pool Lip river is added It is difficult to solve the problems, such as that preparation redissolves after nurse 188, this patent is added PLURONICS F87 redissolution speed and is obviously improved, and insoluble Particle does not also increase, and complies fully with pharmacopoeial requirements.

Bibliography

[1] the domestic Gonal-F of Xu Xuan shows up prominently [N] medication economics report, 2015-09-09 (004)

[2] Yin Na, the effect of the luxuriant auxiliary material poloxamer188 of seedling and application [J] Contemporary Chinese medicine, 2018,25 (497),10 33-36.

[3] Huanghai Sea is bright, Haitao Zhang, and progress [J] the China of Zhao Dongyong pharmaceutical polymers poloxamer continues Medical education, 2016,825183-184.

[4] Ren Hongnuan, Wang Xiaoli, Chen Xiaoru, Chen Liping, Zhou Tingting, Gao Zhen Shen, Song Xingliang resveratrol-poloxamer The preparation of 188 solid dispersions and its performance study [J] China Dispensary, 2015,26 (535), 25103-106.

[5] it is husky that Xie Yu, Ding Xiuming, An Qishun, Nie Pengfei, Jin Dejun, Sang Shenggang, Lv Haifeng, Peng Lei, Lv Chuanzhu moor Lip river Application [J] wound and emergency treatment e-magazine of the nurse in Emergency Medicine, 2013,10124-26.

[6] Zhou Qiaoyun, Zhang Chaohui, Pan Junfang, Li Yaqi poloxamer be carrier hydrophobic drug novel form study into Open up [J] Chinese Journal of Modern Applied Pharmacy, 2011,280441-45.

[7] application of Zhang Jimin, Cao Deying, Du Qing, Qi Xiaodan the poloxamer in each temperature sensitive gel delivery medicine body system of ascending the throne [J] Journal of Chinese Hospital Pharmacy, 2009,291170-72.

[8] Wu Gang, Lai Runlong, fill out progress [J] contemporary Chinese doctor of wave PLURONICS F87, and 2008, 1254-55.

[9] Wang Meng, Zhang Jun longevity, Zhou Jianping injection supplementary material PLURONICS F87 [J] pharmacy and clinical research, 2007, 0120-23.

[10] in the basis of Cheng Haixing, Wang Junping, Sun Haiyan, Qu Hualing poloxamer188 and pharmacy new development [J] State's medicine company, 2006,0970.

[11] application [J] the Chinese Journal of Pharmaceuticals of Wang Mingkun, Fang Xiaoling poloxamer in pharmacy, 2002, 12 51-54.

Summary of the invention

The purpose of this patent is that solution rh-FSH (freeze dried powder system) freeze-drying effect difference and freeze-dried preparation are multiple Molten ineffective technical problem provides a kind of rh-FSH preparation and preparation method thereof, has to reach and was entirely freeze-dried Process control is more easier, freezes the technical effect that shape is more preferable and solubility is good after preparation freeze-drying.

The technical solution that this patent provides are as follows:

A kind of rh-FSH preparation, by:

Preferably, by:

Preferably, by:

Preferably, by:

The preparation method of rh-FSH preparation, includes the following steps:

Step 1: match liquid

(1) appropriate water for injection is taken, the other compositions in addition to rh-FSH are slowly added in water for injection, and is stirred extremely It is completely dissolved.

(2) it is eventually adding the mixing of rh-FSH stoste, and is pH7.0 ± 0.5 with phosphoric acid/sodium hydroxide adjustment pH value, at this time Rh-FSH content is about 5.5 μ g/ml.

(3) prepared solution is crossed into 0.2 μm of l filter membrane aseptic filtration, in an aseptic environment with 1ml ± 0.02ml loading amount It is distributed into 2ml cillin bottle, is freeze-dried in accordance with the following steps.

Step 2: freeze-drying

(1) pre-freeze step: temperature is gradually decrease to -35 degree, pre-freeze 40min then heats to -25 degree, keeps 100min, then temperature is reduced to -45 degree, keep 180min.

(2) vacuum step: being evacuated to 0.15mbar for the vacuum degree in freeze drying box, subsequent step under the vacuum degree into Row.

(3) first step is dry: temperature is risen into -35 degree, dry 300min then heats to -33 and spends, dry 600min, It is warming up to -31 degree, dry 180min again, then is warming up to -10 degree, dry 150min, then 20 degree are warming up to, dry 30min, then 33 degree are warming up to, dry 90min.

(4) second step is dry: keeping the temperature at 33 degree, keeps 960min, entire freeze-drying will continue 48 hours or so.

This patent has the beneficial effect that following aspect:

1, this patent provides a kind of rh-FSH preparation and preparation method thereof, and it is husky that pool Lip river is added in the freeze-drying formula of rh-FSH Nurse 188, auxiliary freeze-drying, so that entire freeze-drying process is more easier, it is more preferable to freeze shape after preparation freeze-drying.

2, it is different from other kinds of pharmaceutic adjuvant, after PLURONICS F87 is added, redissolves difficult problem and be resolved, and Insoluble granule does not also increase, and complies fully with pharmacopoeial requirements.

3, dissociation subunit, oxidation subunit and polymeric content detection, test have been carried out to the rh-FSH mass after redissolution The quality of destination protein rh-FSH the result shows that the lyophilized preparation being added after PLURONICS F87 does not have an impact, does not influence purpose Albumen rh-FSH purity.

Detailed description of the invention

Fig. 1 is that test example 1 is freeze-dried curve graph.

Fig. 2 is that test example 3 dissociates subunit SDS-page electrophoretogram.

Fig. 3 is that 4 polymer content of test example measures chromatogram.

Fig. 4 is that test example 5 aoxidizes subunit content measurement chromatogram.

Specific embodiment

This patent is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence To implement.

Embodiment 1

A kind of rh-FSH preparation, by:

Said components are further processed in accordance with the following steps.

Step 1: match liquid

(1) appropriate water for injection is taken, the other compositions in addition to rh-FSH are slowly added in water for injection, and is stirred extremely It is completely dissolved.

(2) it is eventually adding the mixing of rh-FSH stoste, and is pH7.0 ± 0.5 with phosphoric acid/sodium hydroxide adjustment pH value, at this time Rh-FSH content is about 5.5 μ g/ml.

(3) prepared solution is crossed into 0.2 μm of l filter membrane aseptic filtration, in an aseptic environment with 1ml ± 0.02ml loading amount It is distributed into 2ml cillin bottle, is freeze-dried in accordance with the following steps.

Step 2: freeze-drying

(1) pre-freeze step: temperature is gradually decrease to -35 degree, pre-freeze 40min then heats to -25 degree, keeps 100min, then temperature is reduced to -45 degree, keep 180min.

(2) vacuum step: being evacuated to 0.15mbar for the vacuum degree in freeze drying box, subsequent step under the vacuum degree into Row.

(3) first step is dry: temperature is risen into -35 degree, dry 300min then heats to -33 and spends, dry 600min, It is warming up to -31 degree, dry 180min again, then is warming up to -10 degree, dry 150min, then 20 degree are warming up to, dry 30min, then 33 degree are warming up to, dry 90min.

(4) second step is dry: keeping the temperature at 33 degree, keeps 960min, entire freeze-drying will continue 48 hours or so, both Obtain sample 1.

Embodiment 2

A kind of rh-FSH preparation, by:

Said components are further processed in accordance with the following steps.

Step 1: match liquid

(1) appropriate water for injection is taken, the other compositions in addition to rh-FSH are slowly added in water for injection, and is stirred extremely It is completely dissolved.

(2) it is eventually adding the mixing of rh-FSH stoste, and is pH7.0 ± 0.5 with phosphoric acid/sodium hydroxide adjustment pH value, at this time Rh-FSH content is about 5.5 μ g/ml.

(3) prepared solution is crossed into 0.2 μm of l filter membrane aseptic filtration, in an aseptic environment with 1ml ± 0.02ml loading amount It is distributed into 2ml cillin bottle, is freeze-dried in accordance with the following steps.

Step 2: freeze-drying

(1) pre-freeze step: temperature is gradually decrease to -35 degree, pre-freeze 40min then heats to -25 degree, keeps 100min, then temperature is reduced to -45 degree, keep 180min.

(2) vacuum step: being evacuated to 0.15mbar for the vacuum degree in freeze drying box, subsequent step under the vacuum degree into Row.

(3) first step is dry: temperature is risen into -35 degree, dry 300min then heats to -33 and spends, dry 600min, It is warming up to -31 degree, dry 180min again, then is warming up to -10 degree, dry 150min, then 20 degree are warming up to, dry 30min, then 33 degree are warming up to, dry 90min.

(4) second step is dry: keeping the temperature at 33 degree, keeps 960min, entire freeze-drying will continue 48 hours or so, both Obtain sample 2.

Embodiment 3

A kind of rh-FSH preparation, by:

Said components are further processed in accordance with the following steps.

Step 1: match liquid

(1) appropriate water for injection is taken, the other compositions in addition to rh-FSH are slowly added in water for injection, and is stirred extremely It is completely dissolved.

(2) it is eventually adding the mixing of rh-FSH stoste, and is pH7.0 ± 0.5 with phosphoric acid/sodium hydroxide adjustment pH value, at this time Rh-FSH content is about 5.5 μ g/ml.

(3) prepared solution is crossed into 0.2 μm of l filter membrane aseptic filtration, in an aseptic environment with 1ml ± 0.02ml loading amount It is distributed into 2ml cillin bottle, is freeze-dried in accordance with the following steps.

Step 2: freeze-drying

(1) pre-freeze step: temperature is gradually decrease to -35 degree, pre-freeze 40min then heats to -25 degree, keeps 100min, then temperature is reduced to -45 degree, keep 180min.

(2) vacuum step: being evacuated to 0.15mbar for the vacuum degree in freeze drying box, subsequent step under the vacuum degree into Row.

(3) first step is dry: temperature is risen into -35 degree, dry 300min then heats to -33 and spends, dry 600min, It is warming up to -31 degree, dry 180min again, then is warming up to -10 degree, dry 150min, then 20 degree are warming up to, dry 30min, then 33 degree are warming up to, dry 90min.

(4) second step is dry: keeping the temperature at 33 degree, keeps 960min, entire freeze-drying will continue 48 hours or so, both Obtain sample 3.

Test example 1

It is freeze-dried state according to the condition measurement embodiment 1 in table 1, test result is shown in Fig. 1.

Table 1 is freeze-dried condition

Serial number	Time (h:m)	Temperature (DEG C)	Vacuum degree (mbar)
				1	0:05	-35	NA
2	0:20	-25	NA
				3	0:10	-45	NA
4	0:30	-35	0.1500
				5	0:20	-33	0.1500
6	0:20	-31	0.1500
				7	0:40	-10	0.1500
8	1:00	25	0.1500
				9	0:30	33	0.1500
10	6:00	33	0.0200

The freeze-drying curve of embodiment 1 as shown in Figure 1 shows that entire freeze-drying process is smooth, and embodiment 1 freezes shape after being lyophilized Good, the freeze-drying of embodiment 1 is more successful.

Test example 2

Redissolve: sterilized water for injection 1.0ml dissolution is added in sample (1 lyophilized preparation) in Example 1, redissolves the time No more than 3 seconds, transparent supernatant liquid is obtained

Particulate determination: it checks (0,903 first method of " Chinese Pharmacopoeia " (version in 2015) general rule) in accordance with the law, meets rule It is fixed.

Test example 3

Dissociate subunit measurement

Embodiment 1 is diluted to debita spissitudo with water for injection, mixes, appropriate 1:1 is taken to add 2 × loading buffer, It mixes, is centrifuged 5 minutes under the conditions of 10000rpm.

1, preparative separation glue

According to different molecular weight needs, separation sol solution (using 12.5% separation gel) is made according to the form below 2, is poured into mold To certain altitude, add water seal top, at room temperature polymerization (room temperature is different, and polymerization time is different).

2 separation gel solution concentration table of table

2, preparation concentration glue

After sol solution polymerization to be separated, water layer above is sucked with filter paper, then pour into concentration sol solution (formula is seen the above table), It is inserted into sample comb, pays attention to that bubble is avoided to occur.

3, it is loaded: carefully extracting sample comb after sol solution polymerization to be concentrated, electrode buffer is filled into electrophoresis tank front and back slot, Test solution/reference substance solution 10ug or more (dying method with coomassie brilliant blue) is added in well.

4, electrophoresis: this law is vertical slab electrophoresis, constant pressure, initial voltage 80V, is adjusted to 150-200V when into separation gel, Stop electrophoresis when bromophenol blue is migrated to glue bottom.

5, dyeing and decoloration: electrophoresis finishes, and takes out film, is placed in Coomassie brilliant blue dye liquor 2 hours, then de- with destainer It is stored in after color to gel background transparent in dry glue liquid.

Test result is as shown in Fig. 2, show the rh-FSH preparation after redissolution by dissociating subunit SDS-page electrophoretogram Foreign protein is not generated, is had no adverse effects to stoste rh-FSH purity.

Test example 4

Polymer content measurement

1, sample preparation

Test sample is diluted to 0.25mg/mL with mobile phase, is transferred in sample injection bottle after mixing.(" middle traditional Chinese medicines are measured in accordance with the law Allusion quotation " (version in 2015) general rule 0512), condensate peak area is not higher than the 2.0% of the gross area

2, chromatographic condition is shown in Table 3.

3 polymer content of table measures chromatographic condition

Test result is shown in Fig. 3, in the experiment of polymer content measurement, can't detect FSH condensate in experimental result completely Content, the only independent main peak of destination protein RH-FSH also meet the requirement for being not higher than the gross area 2% in this way.

Test example 5

Aoxidize subunit content analysis

1, sample preparation

Test sample: sample is diluted to 0.25mg/mL with water.

Reference material: reference material is diluted to 0.25mg/mL with water.

Quality-control product: taking 400 μ 1 of 0.25mg/mL reference material solution, is added 1,37 DEG C of 1% hydrogenperoxide steam generator, 10 μ and incubates 30 Minute,

10ul 0.33mg/ml 2, the ethanol solution of 4- dichlorobenzoic acid, sample introduction immediately after mixing is added.

2, chromatographic condition is shown in Table 4.

Table 4 aoxidizes subunit content chromatographic condition

Test result is shown in Fig. 4, aoxidizes in subunit peak the either equal very little of oxidation subunit peak area of a subunit or b subunit, Also it is significantly lower than the 5% of the gross area.

Interpretation of result: embodiment 1 is melted again with 1mL water for injection, then carries out ultrafiltration with super filter tube, removes destination protein Then all formulations auxiliary material part other than rh-FSH takes appropriate progress test example 3-5.Dissociate subunit, oxidation subunit and polymerization The content of body is to detect three experiments of rh-FSH purity of protein, and according to experimental result, the content for dissociating subunit is obviously no more than 5%, and the equal very little of oxidation subunit peak area of either a subunit or b subunit in subunit peak is aoxidized, also it is significantly lower than the gross area 5%, and polymer content measurement experiment in, can't detect rh-FSH polymer content in experimental result completely, only An independent main peak of destination protein rh-FSH also meets the requirement for being not higher than the gross area 2% in this way.

The above experiment shows that it is feasible that PLURONICS F87 is added in my pharmaceutical formulation, and absolutely not shadow Ring destination protein rh-FSH purity.

Although the embodiment of the invention patent has been disclosed as above, not only in the description and the implementation Listed utilization.It can be applied to the field of various suitable the invention patent completely.For those skilled in the art, Other modifications may be easily implemented.Therefore without departing from the general concept defined in the claims and the equivalent scope, this hair Bright patent is not limited to specific details and legend shown and described herein.

Claims (5)

1. a kind of rh-FSH preparation, which is characterized in that by:

2. rh-FSH preparation according to claim 1, which is characterized in that by:

3. rh-FSH preparation according to claim 1, which is characterized in that by:

4. rh-FSH preparation according to claim 1, which is characterized in that by:

5. the preparation method of rh-FSH preparation described in -4 any claims according to claim 1, which is characterized in that including such as Lower step:

Step 1: match liquid

(1) appropriate water for injection is taken, the other compositions in addition to rh-FSH are slowly added in water for injection, and is stirred to complete Dissolution.

(2) it is eventually adding the mixing of rh-FSH stoste, and is pH7.0 ± 0.5 with phosphoric acid/sodium hydroxide adjustment pH value, at this time rh- FSH content is about 5.5 μ g/ml.

(3) prepared solution is crossed into 0.2 μm of l filter membrane aseptic filtration, is distributed into an aseptic environment with 1ml ± 0.02ml loading amount In 2ml cillin bottle, it is freeze-dried in accordance with the following steps.

Step 2: freeze-drying

(1) pre-freeze step: being gradually decrease to -35 degree for temperature, and pre-freeze 40min then heats to -25 degree, keeps 100min, then Temperature is reduced to -45 degree, keeps 180min.

(2) vacuum step: the vacuum degree in freeze drying box is evacuated to 0.15mbar, subsequent step carries out under the vacuum degree.

(3) first step is dry: temperature being risen to -35 degree, dry 300min then heats to -33 degree, dry 600min, then rises Temperature extremely -31 degree, dry 180min, then -10 degree are warming up to, dry 150min, then 20 degree are warming up to, drying 30min, then be warming up to 33 degree, dry 90min.

(4) second step is dry: keeping the temperature at 33 degree, keeps 960min, entire freeze-drying will continue 48 hours or so.