CN1587276A

CN1587276A - Purifying and producing process for high purity follicle stimulating hormone in urine

Info

Publication number: CN1587276A
Application number: CN 200410060631
Authority: CN
Inventors: 张万山; 刘兰花; 李菊根; 王启要
Original assignee: WANHUA BIOCHEMICAL PRODUCT CO Ltd NANCHANG CITY
Current assignee: WANHUA BIOCHEMICAL PRODUCT CO Ltd NANCHANG CITY
Priority date: 2004-07-23
Filing date: 2004-07-23
Publication date: 2005-03-02
Anticipated expiration: 2024-07-23
Also published as: CN100417663C

Abstract

During the purification and production of high purity uric follicle stimulating hormone, red azo dye is coupled to Sepharose 4B as the medium of affinity separating and purifying uric follicle stimulating hormone, and through dye affinity chromatography, QAE ion exchange chromatography and other technological steps, high purity uric follicle stimulating hormone product is obtained from uric gonadotropin from post-menopause. The product produced based on the said technological process has biological potency not lower than 200 IU/mg and FSH/LH higher than 100, is white and soluble, and may be used directly in producing injection.

Description

The purifying of high purity urinary fsh and production technique

Technical field

The present invention relates to a kind of purifying and production technique of high purity urinary fsh, particularly a kind of from Menotropins (HMG) crude product purifying obtain the technology of follicular stimulating hormone (FSH) product.

Background technology

(Follicle Stimulating Hormone FSH) is also referred to as follicle stimulating hormone to follicular stimulating hormone, is synthetic and excretory glycoprotein by basophilic cell.Its molecular weight is 33,000Dal, form by two non-covalent α, β subunit, the α subunit contains 92 amino acid, with corpus luteum give birth to plain (Luteinizing hormone, LH) and gonadotrophin chorionic (Human Chorionicgonadotropin, α subunit HCG) is extremely similar, the β subunit is made up of 115 amino acid, is its specific integral part of decision.Because FSH is a kind of glycoprotein analog hormone, and physico-chemical property such as its molecular weight, iso-electric point and LH, TSH (thyrotropic hormone) etc. are closely similar, therefore adopt classical ion exchange chromatography (for example DEAE resin and SP-Sephadex), gel chromatography methods such as (for example Sephadex G-100) to be difficult to its effective separation and purification.China Patent No. 88103966.7 has been described to adopt at step purifying from the gonadotropin of back, exhausted footpath such as the immunoaffinity chromatography of the monoclonal antibody of FSH and anti-phase HPLC and has been obtained human body FSH.Monoclonal antibody with antibiotic FSH in this patent is coupled on the agarose, pass through sample and steps such as washing, wash-out again, the ammonia soln that liquid is 1M is taken off in listening with respectful attention wherein, wash-out which kind of be as early as possible solution uncle pH value to be adjusted to 9.0 with Glacial acetic acid after requiring wash-out to finish in 4 times of which kind of patents of post bed, carry out ultrafiltration and concentration then, then carry out anti-phase HPLC operation and obtain the higher FSH of purity.In this patent owing to use antibody to carry out affinity chromatography, and the avidity of the antibody antigen FSH corresponding with it is very high, wash-out must elute with the FSH that (ammoniacal liquor) under the highly basic condition will be combined on the agarose, with this understanding, the easy sex change of FSH becomes does not have bioactive albumen, perhaps easily loss wherein glycosyl and become and align the material that normal physiological function has the strongly inhibited effect, so related technology is not suitable for suitability for industrialized production in this patent.China Patent No. 99101376X has described and has adopted triasine dyes to carry out the simple economic again technology of purifying FSH as the affinity chromatography of part.In this technology, the HMG crude product is removed virus with 80-90% (volume/volume) aqueous ethanolic solution earlier, on DEAE anionite-exchange resin, carry out ion exchange chromatography then, thereby wash-out obtains the FSH component, then with the solution process strongly basic anionic resin DE53 Whatman chromatography column that elutes on the triasine dyes affinity chromatography on this component solution, obtain purity than higher FSH product after elution fraction process ultrafiltration desalination and the lyophilize, the activity recovery of the FSH of report reaches 56% in this invention.

Summary of the invention

Purpose of the present invention just provides a kind of technology that adopts the separation and purification from the HMG crude product of dye affinity chromatography technology to obtain high purity FSH, this technology can be in the LH pollution that effectively reduces among the HMG, the biological activity that keeps FSH, the activity recovery of FSH expands the suitability for industrialized production scale to easily than higher in the product simultaneously.

The present invention includes the technology of the purifying FSH of following steps.At first with orchil (azo dyes, for example Reactive Red 120) to be made into be 0.5～3.5% (W/V) solution (initial aglucon solution) as concentration just, add 10～50% (W/V) Sepharose 4B gel then, back adding dyeing auxiliaries heats up, fixing agent and catalyzer, reaction under certain conditions.In the present invention, the dyeing auxiliaries of used the best can be NaCl in the building-up process of dyestuff is affine layer medium, and fixing agent is Na ₂CO ₃, catalyzer can be DABCO.Gel after the coupling is cleaned standby repeatedly with pure water, be loaded on the chromatography column that is generally used for affinity chromatography, and wherein affinity column is 2.5cm * 10cm in the lab scale test, is 20cm * 80cm in the pilot plant test.

After HMG crude product process sulphosalicylic acid and polyoxyethylene glycol were handled, the orchil affinity column that last borax (pH8.0) damping fluid balance is crossed carried out washing operation then.Xi Di solution glycosides propylhomoserin-NaOH damping fluid preferably wherein.Carry out the wash-out operation then, wherein best elute soln is the glycosides propylhomoserin-NaOH damping fluid that contains a kind of sylvite, obtains the FSH component.The biological activity of FSH and LH can use Serono FSH Maiclone detection kit (catalog number (Cat.No.) is 13101) to monitor with the method for radioimmunity.The detection of protein concentration adopts Lowry reagent method to carry out, and the protein standard is bovine serum albumin (Pierce company product, catalog number (Cat.No.) are 2323).

Having on the bioactive component of FSH with phosphate buffered saline buffer equilibrated QAE ion exchange column chi village cun (directly * height) of obtaining is 2cm * 18cm, in QAE ion exchange column on probation be 5cm * 30cm.Through after washing and the wash-out operation, obtain purity successively than higher FSH component.

The FSH component that elutes from the QAE ion exchange column obtains the FSH product through common ultrafiltration, membrane filtration and step such as freezing.The packing under aseptic condition of this product.Carry out the detection of pyrogen, intracellular toxin, HIV, HCV, HBV virus simultaneously.

Can access the higher FSH product of purity with method of the present invention, average biological value reaches more than the 200IU/mg, FSH/LH＞100, and Se Baiyi is molten, every index all meets or surpasses " British Pharmacopoeia " 98 editions corresponding indexs, can be directly used in the usefulness of the production of injection.And the activity recovery of FSH all is higher than at present with reported method in the technology, is more than 60%.Reduce cost when producing and lay the foundation for large-scale industrialization.

Description of drawings

Fig. 1 is the chromatography spectrogram of the affine layer of orchil purifying FSH.

Fig. 2 is the chromatography spectrogram of ion exchange chromatography purifying FSH

Embodiment

The coupling of orchil and Sepharose 4B: take by weighing a certain amount of orchil (azo dyes, for example Reactive Red 120) aglucon joins in the distilled water, make initial aglucon concentration for being between 0.5～3.5% (W/V), stirring and dissolving, add the Sepharose 4B gel that 10～50% (W/V) washing filter is done then, place the water bath with thermostatic control shaking table in 1-10min, to be raised to required temperature of reaction (40-80 ℃), add dyeing auxiliaries dyeing (for example NaCl) then, add Na again after 30 minutes ₂CO ₃(final concentration is 10-25g/L) be 15min fixedly, adds catalyzer (for example DABCO) then, reacts 2-10 hour.The pH of reaction is controlled between 9～10.Gel after the coupling is cleaned standby with pure water repeatedly.

The processing of the purification step 1-crude product of FSH: the FSH activity in the HMG crude product is about 1IU/mg, and the activity of LU is about 1IU/mg.Owing to contain a lot of foreign proteins in the HMG crude product, and impurity such as kaolin, determine the technology of following crude product processing; Be the solution of 2% (W/V) with deionized water with dissolving crude product earlier, slowly using the pH value furnishing 3.5 of 0.2mol/L iodo salicylic acid solution with crude product solution under the stirring condition then, 4 ℃ of operational conditions are spent the night and are left standstill.Centrifugal collection supernatant liquor, supernatant liquor are prepared affinity column after dialysing 24 hours night with 1mmol/L, pH8.0 borax buffering earlier.

The purification step 2-dye affinity chromatography of FSH: dried dye affinity chromatography medium is crossed the liquid swelling with pure water earlier in about 20 ℃, adorn post then.The dyestuff affinity column that contains the affine layer of dyestuff medium is gone up sample then earlier with borax (pH8.0) the damping fluid balance of 20mM, and wherein, sample is the pretreated HMG solution of process, and the protein concentration of last sample solution is a 10mg/mL glue.Borax (pH8.0) damping fluid circulation 2-4 column volume with 20mM.Earlier with 50mM glycosides propylhomoserin-NaOH damping fluid (pH 10) washing 5 column volumes, 50mM glycosides propylhomoserin-NaOH then, 0.2M KCl (pH9) damping fluid washs 4 column volumes.

Adopt 50mM glycosides propylhomoserin-NaOH, 0.2～3M KCl (pH9) carries out gradient elution.Monitor proteinic elution peak with Ultraviolet Detector, the FSH component is carried out the ultrafiltration desalination, and it is assorted to adjust its pH with the damping fluid of pH 7.3, sampling simultaneously detects the activity of FSH.

The chromatography spectrogram of orchil affinitive layer purification FSH as shown in Figure 1.

The purification step 3-QAE displacement chromatography of FSH:

10-50mM PB damping fluid balance QAE ion exchange column with pH7.3.Go up sample then, the protein concentration of last sample solution is about 10-100mg/mL glue.10-50mM PB damping fluid with pH7.3 washs 5 column volumes.

With the 10-50mM PB of pH7.3,20-100mM NaCl damping fluid carries out wash-out.Monitor proteinic elution peak with Ultraviolet Detector, sampling simultaneously detects the activity of FSH.

The chromatography spectrogram of QAE ion exchange chromatography purifying FSH as shown in Figure 2.

The FSH component obtains to meet the FSH product of " British Pharmacopoeia " 98 editions specifications through common steps such as ultrafiltration, membrane filtration and lyophilize.

Claims

1, a kind of purifying of high purity urinary fsh and production technique is characterized in that processing step is as follows:

A, at first red azo class dyestuff is made into certain density solution, add 10～50% gels then, the back of heating up successively adds dyeing auxiliaries, fixing agent and catalyzer, carries out linked reaction, gel after the coupling is cleaned with pure water repeatedly, is loaded on the chromatography column of affinity chromatography;

B, will urinate the augmentor crude product and handle through sulphosalicylic acid and polyoxyethylene glycol after, the orchil affinity column that last borax cushioning balance is crossed carries out washing operation and wash-out then and operates;

Phosphate buffered saline buffer equilibrated ion exchange column is used in having of C, acquisition on the bioactive component of FSH, ion exchange column through after washing and the wash-out operation, obtains purity than higher FSH component successively;

D, the FSH component that elutes from ion exchange column with the packing under aseptic condition of this product, carry out the detection of pyrogen, intracellular toxin, HIV, HV, HBV virus through common ultrafiltration, membrane filtration and freezing, dry FSH product simultaneously.

2, the purifying of high purity urinary fsh according to claim 1 and production technique is characterized in that dyeing auxiliaries used in the building-up process of dye affinity chromatography medium is NaCl, and fixing agent is Na ₂CO ₃, catalyzer is DABCO.

3, the purifying of high purity urinary fsh according to claim 1 and production technique is characterized in that in the washing operation, and the solution of washing is glycosides propylhomoserin-NaOH damping fluid, and in the wash-out operation, elute soln is the glycosides propylhomoserin-NaOH damping fluid that contains sylvite.