CN105646671A

CN105646671A - Gonadorelin purification method

Info

Publication number: CN105646671A
Application number: CN201610102107.6A
Authority: CN
Inventors: 徐红岩; 秦敬国
Original assignee: Shanghai GL peptide Ltd; Glbetter Biochemical (shanghai) Co Ltd
Current assignee: Shanghai GL peptide Ltd; Glbetter Biochemical (shanghai) Co Ltd
Priority date: 2016-02-25
Filing date: 2016-02-25
Publication date: 2016-06-08

Abstract

The invention aims to provide a gonadorelin purification method applicable to large-scale industrial production and mainly solves the problems of low crude peptides, difficulty in product control, low yield and the like in a production process of products. The technical scheme is that gonadorelin is subjected to gradient elution purification through an SPE column and a reversion phase chromatographic column, gonadorelin fractions with higher purity are obtained, the gonadorelin fractions are converted into salt with an HPLC conversion-into-salt method, reduced-pressure rotary evaporation, concentration, freezing and drying are performed, and gonadorelin acetate is obtained. The method is used for industrially purifying gonadorelin.

Description

A kind of method of purification gonadorelin

Technical field

The present invention relates to the purification process of a kind of gonadorelin, especially relate to that a kind of cost is low, pollute less, efficiency high, the method that is suitable for industrialized purification gonadorelin.

Background technology

Gonadorelin, its chemistry 5 ' one oxo-prolyl-L-histidyl--L-tryptophanyl-L-seryl-L-tyrosyl-Gan chlorine acyl-L-arginyl-L-arginyl-L-prolyls-Aminoacetamide by name, its structural formula:

Gonadorelin can synthesize and discharge lutropin (LH) and follicule-stimulating hormone (FSH) (FSH), detectable pituitary gonadotropic hormone reserve function accordingly by Stimulation of Pituitary Gland frontal lobe. After normal person injects gonadorelin, the rising of LH is obvious, higher than FSH, preadolescence women FSH reaction higher than LH; May occur in which delayed response after gonadoliberin deficiency person injection, could react to some extent after sometimes needing intravenous drip this product a couple of days. As simulated hypothalamus secretion gonadoliberin Secretion Rhyme under normal circumstances, with low dose of pulse administration, gonadorelin can treat the Development in Puberty caused because of hypothalamus disease delay, amenorrhea and sterile; But, if adopting heavy dose of successive administration, then there is unusual effect after short-term excitement in pituitary gonadotropic hormone, suppresses hypophysis-gonad function on the contrary. Gonadorelin can be used for the diagnosis of the hypogonadism caused by Hypothalamus-pituitary pathological changes; The assessment of pituitary gonadotropic hormone function is remained after Visual function or radiotherapy; Treatment gonadotropin releasing hormone is not enough, companion's women amenorrhea caused by Secondary cases pituitary gonadotropic hormone hypofunction, infertile, male sterility; The Development in Puberty caused because of hypothalamus disease delays.

Current is use chromatography of ions and reversed phase chromatography two step purifies and separates tBOC to synthesize gonadorelin about gonadorelin purification report method. Owing to chromatography of ions pretreatment is pretty troublesome and purge process is wayward, additionally reversed phase chromatography uses trifluoroacetic acid system secondarily purified.

Summary of the invention

The method that it is an object of the invention to provide a purification gonadorelin being suitable to large-scale industrial production, mainly solves thick peptide purity in this process of producing product low, the problems such as product is wayward, yield is not high. The present invention utilizes point gonadorelin crude product at the beginning of SPE post, and applied sample amount is big and can reach to efficiently separate. The method saves a large amount of dissolving, yield height, is typically in 60��80%. Novelty of the present invention is in that first with SPE(Solid-Phase Extraction) post is easily separated, and then adopts the gradient elution separation purification of reversed-phase high-performance liquid chromatography, finally utilizes chromatography to turn salt, and it is suitable for industrialization and produces.

For achieving the above object, technical scheme is as follows:

A kind of method of purification gonadorelin, comprises the steps:

1) gonadorelin crude product is first carried out gradient elution purification through SPE post, first with percent by volume 10% acetonitrile+percent by volume 0.1%TFA(trifluoroacetic acid) aqueous solution eluting, then with percent by volume 40% acetonitrile+percent by volume 0.1%TFA aqueous solution eluting, the peptide solution of purpose peak value is collected;

2) the inverted performance liquid chromatographic column of target peptide solution collected in step 1) is carried out gradient elution purification, mobile phase A is percent by volume 0.01-0.5% phosphate aqueous solution, Mobile phase B is chromatographic grade acetonitrile, gradient elution is B%(percent by volume): 15%-55%, collects the peptide solution of target peak;

3) target peak peptide solution utilizes HPLC turn salt method and turn salt, mobile phase A is percent by volume 0.05-0.3% aqueous acetic acid, Mobile phase B is chromatographic grade acetonitrile, gradient is permanent gradient washes 10-25 minute of 5%, then with aqueous acetic acid and acetonitrile system eluting, gradient is 10%-70%, collects acetic acid gonadorelin peptide solution; 4) the above-mentioned peptide solution concentrated frozen of vacuum rotary steam dries and obtains acetic acid gonadorelin.

The scheme being more highly preferred to is: above-mentioned steps 1) in SPE post be HyperSepPEP or MCXSPE post.

The scheme being more highly preferred to is: above-mentioned steps 3) in chromatographic column fixed phase be octadecyl silane.

The scheme being more highly preferred to is: above-mentioned steps 3) mobile phase A is percent by volume 0.1% aqueous acetic acid.

The invention has the beneficial effects as follows: the present invention utilizes SPE column separating purification gonadorelin crude product peptide, low price is easy to operate. Not only efficiently separate purification gonadorelin crude product, reach certain separating effect, thus protecting Reversed Phase High Performance. Utilizing HPLC secondarily purified simultaneously and adopt chromatography to turn salt, can obtain the HPLC purity fine peptide more than 98.0%, and improve yield 5%, applied sample amount improves 10%, reduces cost, have found a purification process being suitable to industrialization gonadorelin.

Accompanying drawing explanation

Fig. 1 is product chromatogram of the present invention.

Detailed description of the invention

Embodiment 1

1. sample treatment

Weigh 5g gonadorelin crude product to add percent by volume 5% acetonitrile ultrasonic dissolution and clarify to liquid, be that to collect filtrate after 0.45um membrane filtration standby with aperture.

2. a purification

Chromatographic column: SPE post. Purge process: by above-mentioned filtrate loading SPE post, SPE post is HyperSepPEP, first with percent by volume 10% acetonitrile+percent by volume 0.1%TFA aqueous solution eluting, then with percent by volume 40% acetonitrile+percent by volume 0.1%TFA aqueous solution eluting, the peptide solution of purpose peak value is collected. the fraction vacuum rotary steam collected is concentrated into 40-50mg/mL standby.

3. secondarily purified

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 50cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.01-0.5% phosphate aqueous solution, Mobile phase B is trifluoroacetic acid aqueous solution, and gradient elution is B%:15%-55%. Flow velocity: 80-100ml/min. Detection wavelength: 220nm. Purge process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, 15%B balances 10min loading, and applied sample amount is 40-60mL sample solution. Linear gradient elution 60min, collects target product. Vacuum rotary steam after the target product right rail merging collected is concentrated into about 60-80mg/mL standby.

4. turn salt

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 50cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.1% aqueous acetic acid, and Mobile phase B is trifluoroacetic acid aqueous solution. Flow velocity: 80-100ml/min. Detection wavelength: 220nm. Turn salt process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, the permanent gradient washes of gradient 5%B 10-25 minute, with aqueous acetic acid and acetonitrile system eluting after loading, applied sample amount is 60-80mL, gradient is 10%-70%, collects gonadorelin solution.By turning all peptide solutions of gained after salt, carrying out being evaporated to 80-100mg/mL, thickening temperature is less than 35 DEG C, and then lyophilization obtains the purity gonadorelin more than 98.0%, purification yield 77.2%. Product chromatogram is shown in Fig. 1.

Embodiment 2

1. sample treatment

Weigh 15g gonadorelin crude product to add percent by volume 5% acetonitrile ultrasonic dissolution and clarify to liquid, be that to collect filtrate after 0.45um membrane filtration standby with aperture.

2. a purification

Chromatographic column: SPE post. Purge process: by above-mentioned filtrate loading SPE post, for MCXSPE post, first with percent by volume 10% acetonitrile+percent by volume 0.1%TFA aqueous solution eluting, then with percent by volume 40% acetonitrile+percent by volume 0.1%TFA aqueous solution eluting, the peptide solution of purpose peak value is collected. the fraction vacuum rotary steam collected is concentrated into 40-50mg/mL standby.

3. secondarily purified

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 100cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.01-0.5% phosphate aqueous solution, Mobile phase B is trifluoroacetic acid aqueous solution, and gradient elution is B%:15%-55%. Flow velocity: 180-200ml/min. Detection wavelength: 220nm. Purge process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, 15%B balances 10min loading, and applied sample amount is 200-250mL sample solution. Linear gradient elution 80min, collects target product. Vacuum rotary steam after the target product right rail merging collected is concentrated into about 60-80mg/mL standby.

4. turn salt

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 50cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.1% aqueous acetic acid, and Mobile phase B is trifluoroacetic acid aqueous solution. Flow velocity: 80-100ml/min. Detection wavelength: 220nm. Turn salt process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, the permanent gradient washes of gradient 5%B 10-25 minute, with aqueous acetic acid and acetonitrile system eluting after loading, applied sample amount is 250-300mL, gradient is 10%-70%, collects gonadorelin solution. By turning all peptide solutions of gained after salt, carrying out being evaporated to 80-100mg/mL, thickening temperature is less than 35 DEG C, and then lyophilization obtains the purity gonadorelin more than 98.0%, purification yield 75.2%. Product chromatogram is shown in Fig. 1.

Embodiment 3

1. sample treatment

Weigh 25g gonadorelin crude product to add percent by volume 5% acetonitrile ultrasonic dissolution and clarify to liquid, be that to collect filtrate after 0.45um membrane filtration standby with aperture.

2. a purification

3. secondarily purified

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 150cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.01-0.5% phosphate aqueous solution, Mobile phase B is trifluoroacetic acid aqueous solution, and gradient elution is B%:15%-55%. Flow velocity: 450-500ml/min. Detection wavelength: 220nm. Purge process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, 15%B balances 10min loading, and applied sample amount is 500-600mL sample solution. Linear gradient elution 100min, collects target product.Vacuum rotary steam after the target product right rail merging collected is concentrated into about 60-80mg/mL standby.

4. turn salt

Chromatographic column: C18 chromatographic column, pillar diameter and length is: 50cm �� 25cm. Mobile phase: mobile phase A is percent by volume 0.1% aqueous acetic acid, and Mobile phase B is trifluoroacetic acid aqueous solution. Flow velocity: 80-100ml/min. Detection wavelength: 220nm. Turn salt process: after using percent by volume 90% acetonitrile flushing chromatographic column clean, the permanent gradient washes of gradient 5%B 10-25 minute, with aqueous acetic acid and acetonitrile system eluting after loading, applied sample amount is 400-450mL, gradient is 10%-70%, collects gonadorelin solution. By turning all peptide solutions of gained after salt, carrying out being evaporated to 80-100mg/mL, thickening temperature is less than 35 DEG C, and then lyophilization obtains the purity gonadorelin more than 98.0%, purification yield 74.6%. Product chromatogram is shown in Fig. 1.

Claims

1. the method for a purification gonadorelin, it is characterised in that comprise the following steps:

1) gonadorelin crude product is first carried out gradient elution purification through SPE post: first with percent by volume 10% acetonitrile+percent by volume 0.1% trifluoroacetic acid aqueous solution eluting, then with percent by volume 40% acetonitrile+percent by volume 0.1% trifluoroacetic acid aqueous solution eluting, the peptide solution of purpose peak value is collected;

2) the inverted performance liquid chromatographic column of target peptide solution collected in step 1) is carried out gradient elution purification: mobile phase A is percent by volume 0.01-0.5% phosphate aqueous solution, Mobile phase B is chromatographic grade acetonitrile, gradient elution is B%:15%-55%, collects the peptide solution of target peak;

3) target peak peptide solution utilizes HPLC turn salt method and turn salt: mobile phase A is percent by volume 0.05-0.3% aqueous acetic acid, Mobile phase B is chromatographic grade acetonitrile, gradient is permanent gradient washes 10-25 minute of 5%, then with aqueous acetic acid and acetonitrile system eluting, gradient is 10%-70%, collects acetic acid gonadorelin peptide solution;

4) the above-mentioned peptide solution concentrated frozen of vacuum rotary steam dries and obtains acetic acid gonadorelin.

2. the method for a kind of purification gonadorelin according to claim 1, it is characterised in that: in described step 1), SPE post is HyperSepPEP or MCXSPE post.

3. the method for a kind of purification gonadorelin according to claim 1, it is characterised in that: in described step 3), chromatographic column fixed phase is octadecyl silane.

4. the method for a kind of purification gonadorelin according to claim 1, it is characterised in that: described step 3) mobile phase A is percent by volume 0.1% aqueous acetic acid.