CN109897095A

CN109897095A - A method of improving menotropins potency

Info

Publication number: CN109897095A
Application number: CN201711293332.3A
Authority: CN
Inventors: 刘乃山; 夏衬来; 于晓娜
Original assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Current assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2019-06-18

Abstract

The invention discloses a kind of methods using chromatographic technique purifying menotropins.It specifically, is exactly the menotropins potency that potency is about 30iu/mg to be increased to about 600iu/mg, and indices meet Chinese Pharmacopoeia regulation with affinity chromatography and cation exchange chromatography low liter menotropins.The method of the present invention is easy to operate, and production cost is low, and product purity is high, is more able to satisfy clinical and scientific research demand.

Description

A method of improving menotropins potency

Technical field

The present invention relates to field of biotechnology, and it is steady to prepare potency height, yield to relate in particular to a kind of application chromatography The method of fixed menotropins.

Background technique

Menotropins (HMG) are by a kind of merocrine glycoprotein promoting sexual gland hormone of people's anterior lobe of hypophysis, usually It is made from purifying extracted in the urine of menopause or postmenopausal women.HMG contains follicular stimulating hormone (FSH) and corpus luteum Generate plain (LH), contained ratio approximation 1: 1.Clinic is mainly used for treating the sterility caused by women is not ovulated because of functionality； Male because hormonal readiness it is low caused by spermacrasia or vigor it is insufficient.In addition, being also used to treat such as women's amenorrhoea, menstruation The diseases such as uncomfortable, men and women sexual dysfunction.

Ascbheim in 1928, which first reported, has found active material from climacteric women urine, though it reports later It much, but really to the understanding of HMG and preparation, and is gradually formed and compares since the Donimi in the 60's of 20th century It is prepared by the technique of standardization.China begins one's study from the 70's of 20th century, and the 80's formally entered clinical test rank Section, starts mass production the nineties.

Currently, more classical preparation process is kaolin-Ethanol Method preparation HMG semifinished product, then with various purifying sides Method is purified.Domestic currently used purification process yield is relatively low (generally less than 50%), and finished product potency is on the left side 100iu/mg It is right.Chinese patent CN101792482A is described " a kind of purification process of promoting sexual gland hormone ", and this method uses alumina silicate powder As adsorbent extraction purification HMG, but its potency is only capable of reaching 150iu/mg.

In actual production, purification by chromatography menotropins are selected, a variety of foreign proteins therein can be more effectively removed, And its physicochemical property and bioactivity is set to keep stablizing, so that the indices of product be made to meet Chinese Pharmacopoeia standard.More Importantly, by technique continuously improve and it is perfect so that menotropins potency be not less than 600iu/mg, bring huge warp Benefit of helping and social benefit.

Summary of the invention

The present invention provides a kind of methods of chromatographic purifying menotropins, make menotropins potency that can stably reach 600iu/ Mg or more.

The technical scheme is that low liter menotropins obtain after product dissolution, affinity chromatography, ion-exchange chromatography To high-titer menotropins, include the following steps:

(1) menotropins crude product, which is used, contains 0.1 ~ 0.3mol/L NaCl, 0.01 ~ 0.03 mol/L EDTA containing pH 6.0 ~ 8.0 Tris-HCl buffer solution, centrifugation go to precipitate, obtain supernatant；

(2) affinity chromatography: Argmatin- is balanced containing 0.1 ~ 0.3mol/L NaCl Tris-HCl buffer with pH 6.0 ~ 8.0 Sepharose column after supernatant upper prop, contains 0.1 ~ 0.3mol/L NaCl, 0.1 ~ 0.3mol/L guanidine hydrochloride with pH 6.0 ~ 8.0 The elution of Tris-HCl buffer, eluent adds NaCl and 1mol/L Tris-HCl to be diluted to through ultrafiltration desalination to no chloride ion The Tris-HCl buffer of 7.0 ~ 9.0 NaCl Han 0.05 ~ 0.30mol/L of pH；

(3) ion-exchange chromatography: above-mentioned buffer is splined on DEAE-Sepharose Fast Flow column, with pH 6.0 ~ 8.0 The Tris-HCl equilibrium liquid of the NaCl containing 0.01 ~ 0.20mol/L rinses, and uses the NaCl containing 0.1 ~ 0.3mol/L of pH 6.0 ~ 8.0 instead Tris-HCl buffer elution.

(4) ultraviolet detection collects enzymatic activity part, and freeze-drying is after ultrafiltration concentration to get finished product；

Specific embodiment

The present invention is further explained in the light of specific embodiments, but the scope of protection of present invention is not limited to In the range of embodiment statement.

Embodiment 1

(1) pre-process: take menotropins crude product 1:15(w/v by weight) ratio be added pH 7.0 NaCl containing 0.2mol/L, The Tris-HCl buffer solution of 0.02mol/L EDTA, centrifugation go to precipitate, and obtain supernatant；

(2) affinity chromatography: Argmatin- Sepharose is balanced with the 7.0 Tris-HCl buffer of NaCl containing 0.2mol/L of pH Column after supernatant upper prop, is eluted with the Tris-HCl buffer of 7.0 NaCl containing 0.2mol/L of pH, 0.2mol/L guanidine hydrochloride, Eluent adds NaCl and 1mol/L Tris-HCl to be diluted to pH 7.0 containing 0.20mol/L through ultrafiltration desalination to no chloride ion The Tris-HCl buffer of NaCl；

(3) ion-exchange chromatography: above-mentioned buffer is splined on DEAE-Sepharose Fast Flow column, is contained with pH 7.0 The Tris-HCl equilibrium liquid of 0.10mol/L NaCl rinses, and uses the Tris-HCl buffer of 7.0 NaCl containing 0.2mol/L of pH instead Elution.

(4) it is detected and is recorded with Ultraviolet Detector, elute enzymatic activity peak, sediment is packed into disk into freeze dryer after ultrafiltration, It is lyophilized up to menotropins.

According to " Chinese Pharmacopoeia " two, page 2015,530, menotropins quality standard, product meets the requirements.According to general rule 1216 and 1217 measure follicular stimulating hormone (FSH) and lutropin (LH), and potency is respectively 657iu/mg and 642iu/ Mg, HMG potency reach 600iu/mg or more.

Embodiment 2

(1) menotropins crude product is slow with the Tris-HCl of NaCl containing 0.15mol/L, 0.015 mol/L EDTA containing pH 6.6 Fliud flushing dissolution, centrifugation go to precipitate, and obtain supernatant；

(2) affinity chromatography: Argmatin- is balanced with the 6.6 Tris-HCl buffer of NaCl containing 0.15mol/L of pH Sepharose column, after supernatant upper prop, with 6.6 NaCl containing 0.15mol/L of pH, the Tris-HCl of 0.15mol/L guanidine hydrochloride Buffer elution, eluent add NaCl and 1mol/L Tris-HCl to be diluted to pH 7.5 and contain through ultrafiltration desalination to no chloride ion The Tris-HCl buffer of 0.15mol/L NaCl；

(3) ion-exchange chromatography: above-mentioned buffer is splined on DEAE-Sepharose Fast Flow column, is contained with pH 6.6 The Tris-HCl equilibrium liquid of 0.05mol/L NaCl rinses, and uses the Tris-HCl buffering of 6.6 NaCl containing 0.15mol/L of pH instead Liquid elution.

According to " Chinese Pharmacopoeia " two, page 2015,530, menotropins quality standard, product meets the requirements.According to general rule 1216 and 1217 measure follicular stimulating hormone (FSH) and lutropin (LH), and potency is respectively 662iu/mg and 637iu/ Mg, HMG potency reach 600iu/mg or more.

The above described is only a preferred embodiment of the present invention, being not that the invention has other forms of limitations, appoint What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc. Imitate embodiment.But without departing from the technical solutions of the present invention, according to the technical essence of the invention to above embodiments institute Any simple modification, equivalent variations and the remodeling made, still fall within the protection scope of technical solution of the present invention.

Claims

1. a kind of method for improving menotropins potency, which is characterized in that method includes: by low liter menotropins by affine Chromatography, ion-exchange chromatography are purified, and the menotropins of high-purity are finally obtained, which is characterized in that this method includes as follows Step:

Tris-HCl buffer solution of the menotropins crude product containing NaCl, EDTA, centrifugation go to precipitate, and obtain supernatant；

Affinity chromatography: balancing Argmatin- Sepharose column with the Tris-HCl equilibrium liquid containing NaCl, after supernatant upper prop, With the Tris-HCl buffer elution containing NaCl, guanidine hydrochloride, eluent is through ultrafiltration desalination to no chloride ion；

(3) ion-exchange chromatography: NaCl and 1mol/L Tris-HCl is added to be diluted to the solution after above-mentioned ultrafiltration desalination certain dense Degree, is splined on DEAE-Sepharose Fast Flow column, is rinsed with the Tris-HCl equilibrium liquid containing NaCl, uses instead containing different dense Spend the Tris-HCl elution of NaCl；Ultraviolet detection collects enzymatic activity part, after ultrafiltration concentration freeze-drying to get at Product.

2. the method as described in claim 1, which is characterized in that buffer as described in step (1) is containing for pH 6.0 ~ 8.0 The Tris-HCl buffer of 0.1 ~ 0.3mol/L NaCl, 0.01 ~ 0.03 mol/L EDTA.

3. the method as described in claim 1, which is characterized in that equilibrium liquid described in step (2) is pH 6.0 ~ 8.0 containing 0.1 ~ 0.3mol/L NaCl Tris-HCl buffer.

4. the method as described in claim 1, which is characterized in that eluent described in step (2) is pH 6.0 ~ 8.0 containing 0.1 The Tris-HCl buffer of ~ 0.3mol/L NaCl, 0.1 ~ 0.3mol/L guanidine hydrochloride.

5. the method as described in claim 1, which is characterized in that described in step (3) dilute after final concentration of pH 7.0 ~ The Tris-HCl buffer of 9.0 NaCl Han 0.05 ~ 0.30mol/L.

6. the method as described in claim 1, which is characterized in that equilibrium liquid described in step (3) is that pH 6.0 ~ 8.0 contains The Tris-HCl buffer of 0.01 ~ 0.20mol/L NaCl.

7. the method as described in claim 1, which is characterized in that eluent described in step (3) with pH 6.0 ~ 8.0 containing 0.1 ~ The Tris-HCl buffer of 0.3mol/L NaCl.