Background technology
Follicle stimulating hormone (FSH) is by promoting of secreting of prepituitary gland and strengthens the most important endocrine hormone of the Follicle development and maturation in ovary, can be used for induce ovulation or super ovulation, can promote the growth of sperm man.FSH is the same with chorionic-gonadotropin hormone (hCG) with LH is all a kind of heterodimer glycoprotein, is made up of α and the β subunit of Non-covalent binding, respectively containing 92 and 111 amino acid.Glycosyl is connected to α and β two subunit regions separately in the mode of N-glycosidic link.Sugar accounts for 15% ~ 30% in glycoprotein hormones molecule, mainly comprises seminose, Fucose and semi-lactosi, N-Acetyl-D-glucosamine.Sugar-chain end, containing sialic acid, is combined in the mode of covalent linkage with albumen, is usually connected on l-asparagine, form so-called N-and connect sugar chain.The each subunit of FSH has 2 glycosyl sites (α-subunit lay respectively on the 52nd and the 78th amino acids residue, laying respectively on the 7th and the 24th amino acids residue of β-subunit), these 2 sites also have some sugar chain branches.The composition of sugar chain directly affects the biologic activity of hormone, and adopt chemistry or the method for enzymolysis to dissociate removing sugar chain, the biologic activity of FSH reduces, until disappear, and on α chain sugar chain dissociate larger than β chain on the impact of hormone.
The using value of FSH:
Due to FSH synthesis and secretion deficiency caused by the difference of FSH concentration, be the sterile major cause of masculinity and femininity.In women, the feature of this state shows as does not ovulate or abnormal ovulation.In the male sex, may cause sterile owing to there is no the generation of the great-hearted sperm of q.s.Independent injection FSH or share with LH, has been employed successfully in treatment infertility.The business-like FSH be used for the treatment of was extracted (uFSH from menopausal women) by urine source in the past.But people's Recombinant FSH (rhFSH) that application recombinant DNA technology is produced embodies better quality and validity clinically.The Gonal-F(fruit that this series products domestic has Xue Lannuo company of Switzerland to produce at present receives sweet smell) and the Puregon (Puli's health) of Dutch Ou Jianong company production.
Glycoprotein hormones is the important biomacromolecule of a class, its separation and purification, not only has important academic significance to its structure and fuction of research, and as the medicine of special applications, separation and purification goes out a large amount of high reactivity sterlings, more has huge economic benefit and practical value.But the unhomogeneity of glycoprotein hormones on sugar chain adds difficulty for its chromatographic separation, sums up the problem comprising three aspects: low levels, unstable and unhomogeneity.
Glycoprotein hormones content is in vivo all generally very low, and first this adopt certain method to carry out concentrated and enrichment with regard to requiring.Under the effect of acidity, heat, organic solvent, in glycoprotein hormones, sialic acid is easy to dissociate, and subunit is easy to dissociate, and this just requires isolation technique and the environment that must adopt neutral pH, gentleness and low temperature.Even lyophilize also can cause the inactivation of glycoprotein hormones, is generally unsuitable for adopting in separation and purification concentrating in this way, and adopts the method for ultrafiltration.This preparation for crude starting material is difficult to all to meet the requirements of.Traditional separation purification method, because oversize disengaging time is obviously to keeping biologic activity to be also disadvantageous.
In the document of the FSH of current purification of recombinant human, most dye affinity chromatography or metal-chelating filler chromatography of using is as the means of catching, and the later stage realizes this protein purification by multiple means, multi step strategy such as affinity chromatography, reversed phase chromatography, ion exchange chromatography, sieve chromatography, hydrophobic chromatographies.Such as CN201080056651 discloses anti-phase, gel-filtration, hydrophobic chromatography etc.; Mention in patent CN100554277C and utilize anion-exchange chromatography and fixing metal ions chromatography (i.e. metal chelate chromatography) and hydrophobic chromatography to carry out purifying to restructuring or natural follicle stimulating hormone.The means of catching of more uses are dye affinity chromatographies: US7939296 relates to cation-exchange chromatography, dyestuff is affine, hydrophobic, gel-filtration; EP1106623A1 relates to the means (rhFSH ultimate yield, biological activity etc. are not bright) such as dyestuff is affine, washing, gradient elution; It is affine, hydrophobic, anti-phase that CN200580037591.9 relates to dyestuff; US2009209454 chromatography method comprises that dyestuff is affine, negatively charged ion, hydrophobic, strong anion, and CN201080022681 relates to anion-exchange chromatography, hydrophobic interaction chromatography and dye affinity chromatography and repels weak anion-exchange chromatography.What it should be noted that the anion-exchange chromatography employing involved by these purification process in addition is absorption-elution mode.
Reversed phase chromatography needs with an organic solvent as the elution media of protein, and protein exposes for a long time in organic solvent by causing molecular structure change in various degree, serious caused irreversible denaturation, has a strong impact on the biologic activity of albumen.Metal chelate chromatography can cause high-concentration metallic ions to be introduced in protein solution, causes the reduction of protein-active in various degree, and adds the difficulty of metal ion removal.Hydrophobic chromatography is used for the separation of gonad-stimulating hormone, and the present inventor finds that activity recovery is lower, and concrete deactivation mechanism is failed to understand, for this reason, develops a kind of FSH purifying process reducing protein-active forfeiture and has important practical significance.
Summary of the invention
The technical problem that the present invention solves provides the purifying process of a kind of Gonal-F, and avoid and use reversed phase chromatography, metal chelate chromatography or the hydrophobic chromatography larger on protein-active impact, easy and simple to handle, protein-active is high and yield is high.
A kind of technical scheme of the present invention is:
A Gonal-F's purification process, comprises the following steps:
1) anion-exchange chromatography: containing the cell culture supernatant of rhFSH, loading after balance anion displacement chromatography post, the stream collected containing rhFSH wears liquid;
2) affinity chromatography: balanced by CaptureSelect affinity column (Dutch BAC Products) special for FSH, loading, then use elution buffer elution chromatography post, collects the elution peak containing rhFSH;
3) gel permeation chromatography: loading after balanced gel chromatography column, collects the component containing rhFSH.
The aglucon of the medium of anion-exchange chromatography post described in above-mentioned purification process is quaternary ammonium group or diethylaminethyl.The medium of preferred anion-exchange chromatography post is Q sepharose F.F or DEAE sepharose F.F.Containing 0.1-0.2M NaCl, pH in wherein said anion-exchange chromatography level pad used is 7.0-7.5.0.02-0.05M Tris and 0.01-0.1M methionine(Met) can also be contained further in described anion-exchange chromatography level pad used.
In affinity chromatography described in above-mentioned purification process second step: level pad used contains 0.02-0.05M Tris, pH is 7.0-7.5; The damping fluid of wash-out rhFSH contains 0.02-0.05MTris and 1.5-2.0M MgCl
2, pH is 7.0-7.5.
The medium of gel chromatography column described in above-mentioned purification process be separating ranges at 3-600kD, the gel being suitable for rhFSH purifying, preferably adopt Superdex75, Superdex200, Sephadex G-50, Sephadex G-75 or Sephadex G-100.Described gel chromatography level pad used contains 0.02-0.1M Sodium Citrate and 0.01-0.1M methionine(Met), and pH is 7.0-7.5.
Furtherly, in the step 1 of above-mentioned purification process and/or step 3: before loading, sample is carried out in ultrafiltration system concentrated and buffer exchange.Described displacement damping fluid used is containing 0.02-0.05M Tris, 0.1-0.2M NaCl and 0.01-0.1M methionine(Met), and pH is 7.0-7.5.
The present invention is raw materials used takes from the cell culture containing rhFSH obtained according to various methods, and host cell can be Chinese hamster ovary (CHO) cell.
The invention provides a kind of new purifying thinking, utilize anionresin, the chromatography combination of affine, gel-filtration can obtain pure rhFSH, cost is low, step is few, simple and easy to do,, there is not complicated refolding strategy process in steady quality, avoids and use reversed phase chromatography, metal chelate chromatography or the hydrophobic chromatography larger on protein-active impact; First utilize stream to wear the anionresin of pattern, remove a large amount of foreign protein, pigment and part residual DNA, intracellular toxin and host protein, both protected follow-up affine filler, achieve again slightly pure and enrichment method; The affinity media coupling antibody in camel source, carrying capacity is high, only specifically in conjunction with the entire molecule of FSH, and not in conjunction with single subunit, eliminates the monomelic subunit of degraded better; Gel-filtration can remove residual DNA, intracellular toxin and host protein further, and also can play the FSH albumen removing albumen aggressiveness and glycosylation deficiency, the purity of FSH is obtained after affinity chromatography 90% has brought up to 98% further.
The present invention also all uses the damping fluid of weakly acidic pH when ultrafiltration and concentration and displacement, anionresin, affine, gel-filtration, especially neutral buffered liquid is used by FSH from affinity column wash-out, acid solution wash-out is not adopted to add the traditional method of alkali neutralization again, thus avoid albumen precipitation that pH acute variation brings and sugar to come off problem, ensure that purity and the activity of the albumen that wash-out obtains; Containing methionine(Met) in ultrafiltration displacement, anionresin damping fluid used, FSH solution replacement is also the buffer system containing methionine(Met) by gel-filtration in the lump, avoid again concentrating the rate of recovery decline of replacing and causing, and the oxidation of FSH can be reduced, be beneficial to the preservation that FSH is final.
The new technology for purifying of Gonal-F of the present invention, through verifying with active the checking of Gonal-F's purity, and shows good actual effect.The present invention prepares Gonal-F and commercially available follicle stimulating hormone
(Puli's health), through contrast, shows the present invention and prepares product and have higher biologic activity.
Embodiment
Instrument and material:
(dam Pellicon2 ultra-filtration membrane molecular weight 10K, 0.5M
2, Millipore Products), Pellicon ultrafiltration system (Millipore Products), Amicon ultra-filtration centrifuge tube (Millipore Products), AKTA Purifier chromatographic system (GE healthcare Products); TSK gelG-2000wxl post (Tosoh Products); Vydac Protein and Peptide C4 post (W. R. Grace & Co of U.S. product); High pressure liquid chromatograph (U.S. Agilent Agilent Products);
Tool FSH specific CaptureSelect filler (Dutch BAC Products), Superdex75prep grade filler, Superdex75prep grade filler and Sephadex G-75(GEhealthcare Products), Q sepharose F.F filler, DEAE sepharose FF filler (GEhealthcare Products).
One, the purifying of rhFSH
The purification route one of embodiment 1 Gonal-F albumen
1) containing the pre-treatment of the cell culture supernatant of rhFSH
Clean ultrafiltration system, and do pyrogen removal process, concentrated supernatant, replace concentrated solution with (0.02MTris+0.2MNaCl+0.1M methionine(Met)) damping fluid (pH7.0).
2) Q sepharose F.F anion-exchange chromatography
Clean Q sepharose F.F column chromatography system, and do pyrogen removal process, carry out according to the following steps successively:
Chromatography column is balanced by A phase (0.02MTris+0.2MNaCl+0.1M methionine(Met)) damping fluid (pH7.0), loading, with this understanding, impurity is combined on anion-exchange column, and Gonal-F's albumen is not combined on anion-exchange column, collects stream and wear component.
3) affinity chromatography
Clean affinity chromatography system, carries out following operation: balance chromatography column with (0.02M Tris) damping fluid (pH7.0) successively; Chromatography column is balanced with (0.02M Tris) damping fluid (pH7.0) after loading; With (0.02MTris+1.5M MgCl
26H
2o) rhFSH on damping fluid (pH7.0) elution chromatography post, collects this elution peak.
Clean ultrafiltration system, and do pyrogen removal process, uses 0.02M Tris(pH7.0) after wash cycles ultrafiltration system, the aforementioned chromatography column elution peak of ultrafiltration and concentration collects liquid, and uses 0.02M Tris(pH7.0) fully replace.
4) Superdex75prep grade chromatography
Clean Superdex75prep grade column chromatography system, and do pyrogen removal process, carry out according to the following steps successively: balance chromatography column with 0.02M Sodium Citrate+0.1M methionine(Met) damping fluid (pH7.5); The protein concentrate solution upper step collected, loading, collects the component containing rhFSH.
The purification route two of embodiment 2 Gonal-F albumen
1) containing the pre-treatment of the cell culture supernatant of rhFSH
Clean ultrafiltration system, and do pyrogen removal process, concentrated supernatant, replace concentrated solution with (0.02MTris+0.1MNaCl+0.01M methionine(Met)) damping fluid (pH7.2).
2) DEAE sepharose F.F anion-exchange chromatography
Clean DEAE sepharose F.F column chromatography system, and do pyrogen removal process, carry out according to the following steps successively:
Chromatography column is balanced by A phase (0.02MTris+0.1MNaCl+0.01M methionine(Met)) damping fluid (pH7.2), loading, with this understanding, impurity is combined on anion-exchange column, and Gonal-F's albumen is not combined on anion-exchange column, collects stream and wear component.
3) affinity chromatography
Clean affinity chromatography system, carries out following operation: balance chromatography column with (0.02M Tris) damping fluid (pH7.2) successively; Chromatography column is balanced with (0.02M Tris) damping fluid (pH7.2) after loading; With (0.02MTris+2.0M MgCl
26H
2o) rhFSH on damping fluid (pH7.2) elution chromatography post, collects this elution peak.
Clean ultrafiltration system, and do pyrogen removal process, uses 0.02M Tris(pH7.2) after wash cycles ultrafiltration system, the aforementioned chromatography column elution peak of ultrafiltration and concentration collects liquid, and uses 0.02M Tris(pH7.2) fully replace.
4) Superdex200prep grade chromatography
Clean Superdex200prep grade column chromatography system, and do pyrogen removal process, carry out according to the following steps successively: balance chromatography column with 0.1M Sodium Citrate+0.01M methionine(Met) damping fluid (pH7.0); The protein concentrate solution upper step collected, loading, collects the component containing rhFSH.
The purification route three of embodiment 3 Gonal-F albumen
1) containing the pre-treatment of the cell culture supernatant of rhFSH
Clean ultrafiltration system, and do pyrogen removal process, concentrated supernatant, replace concentrated solution with (0.05MTris+0.2MNaCl+0.1M methionine(Met)) damping fluid (pH7.5).
2) Q sepharose F.F anion-exchange chromatography
Clean Q sepharose F.F column chromatography system, and do pyrogen removal process, carry out according to the following steps successively:
Chromatography column is balanced by A phase (0.05MTris+0.2MNaCl+0.1M methionine(Met)) damping fluid (pH7.5), loading, with this understanding, impurity is combined on anion-exchange column, and Gonal-F's albumen is not combined on anion-exchange column, collects stream and wear component.
3) affinity chromatography
Clean affinity chromatography system, carries out following operation: balance chromatography column with (0.05M Tris) damping fluid (pH7.5) successively; Chromatography column is balanced with (0.05M Tris) damping fluid (pH7.5) after loading; With (0.05MTris+2.0M MgCl
26H
2o) rhFSH on damping fluid (pH7.5) elution chromatography post, collects this elution peak.
Clean ultrafiltration system, and do pyrogen removal process, uses 0.05M Tris(pH7.5) after wash cycles ultrafiltration system, the aforementioned chromatography column elution peak of ultrafiltration and concentration collects liquid, and uses 0.02M Tris(pH7.5) fully replace.
4) Superdex75prep grade chromatography
Clean Superdex75prep grade column chromatography system, and do pyrogen removal process, carry out according to the following steps successively: balance chromatography column with 0.05M Sodium Citrate+0.1M methionine(Met) damping fluid (pH7.0); The protein concentrate solution upper step collected, loading, collects the component containing rhFSH.
The detection of embodiment 4 target protein
1) SDS-PAGE detects
The protein sample of separation and purification is carried out SDS-PAGE analysis, by embodiment 1,2,3 final obtain the detected result of target protein and protein Marker as shown in Figure 5, target protein apparent molecular weight is consistent with theoretical value, about 40kD.
2) WesternB loting detects
The protein sample of separation and purification is carried out reduced form WesternBloting analysis, by embodiment 1,2,3 final obtain the detected result of target protein and protein Marker as shown in Figure 6, target protein apparent molecular weight is consistent with theoretical value, about 40kD.
3) purity and yield
A, ELISA detect
This method is used for FSH analysis of immunogenicity and quantitative analysis, utilizes antigen-antibody to combine the content detecting FSH.Use the western Tang in follicle stimulating hormone ELISA kit(Shanghai biotechnology Products), testing process by specification carries out.
B, SEC-HPLC method
Chromatographic column: TSK gel G-2000wxl post (30cm × 7.8mm)
Moving phase (phosphate buffered saline buffer): the Na of 0.1mol/L
2sO
414.2g, adds the water dissolution of 950ml, adds 6.75ml phosphoric acid, is adjusted to pH6.7, is settled to 1000ml with water, 0.45um membrane filtration with saturated NaOH (5-6ml).
Flow velocity: 0.5mL/min,
Determined wavelength: 214nm,
STOP:30min。
By embodiment 1,2,3 final obtain target protein detected result as shown in Figure 2,3, 4, target protein goes out peak in 15.9min.Contrast medicine Puli health detected result as shown in Figure 1.
Average purity and yield:
Detected by ELISA, SEC-HPLC and calculate, target protein total yield 44%, wherein: target protein concentration 12mg/L in culture supernatants; Through ultrafiltration and concentration and buffer exchange, the target protein rate of recovery 85%; The Q-Sepharose FF chromatography target protein rate of recovery 90%; The affinity chromatography target protein rate of recovery is greater than 80%, lipidated protein about 90%, the target protein rate of recovery 80% in ultrafiltration immediately and buffer exchange step; The Sephadex G-75 gel permeation chromatography target protein rate of recovery more than 90%, target protein purity is greater than 98%.
4) protein active detects
Biological activity assay is according to luciferase (Luciferase) chemoluminescence method.Its principle is: the clone of stably express FSHR is when jointly hatching with FSH, to start the expression with the luciferase gene (reporter gene) of FSHR coupling under the induction of FSH, the close relation of this acceptor-reporter gene forms the theoretical basis of this detection method.Measured the expression level of reporter gene by luciferase quantification kit, carry out the Relative biological activity of indirect reaction upstream inductor FSH.At synchronous detection many concentration FSH international standard substance, and after drawing out the relevant typical curve of Exact concentrations, namely by Determination reading and typical curve, obtain the accurate activity of unknown FSH sample.Puli's Kombi activity value is 90833IU/mg, by embodiment 1,2,3 final obtain target protein Activity determination the results are shown in Table 1.
The Gonal-F of table 1 purifying of the present invention and the former expression activitiy grinding medicine Puli health
|
Embodiment 1 |
Embodiment 2 |
Embodiment 3 |
Active quantities |
11916IU/mg |
98333IU/mg |
11583IU/mg |
With the ratio of Puli's health |
131.1% |
108.2% |
127.5% |
5) content of RP-HPLC oxygen determination subunit
Containing methionine residues in the primary structure of rhFSH, so be easily subject to the oxidation of oxygen in air.5% is not more than with the content ratio of α subunit according to quality criteria requirements oxidation subunit content.
1.RP-HPLC chromatographic condition
Chromatographic column: Vydac Protein and Peptide C4 post (25cm × 4.6mm, 5um)
Mobile phase A (0.1mol/l TEAP damping fluid): 85% phosphoric acid 6.75ml, add water 9500ml, adjusts pH to 6.00 ± 0.05, be settled to 1000ml with water with triethylamine, after 0.45um membrane filtration, places after 24 hours and use.
Mobile phase B: acetonitrile
Moving phase C: water: acetonitrile: trifluoroacetic acid=20:82:0.1%
Flow velocity: 1.0mL/min;
Determined wavelength: 214nm;
Column temperature: 40 DEG C;
Table 2:RP-HPLC gradient table
2. the preparation of sample solution: get rhFSH appropriate is that 0.1mg/ml is as sample solution with injection water dilution
3. assay method: pipette samples solution 100 μ l, injection liquid chromatography, record color atlas.FSH α subunit goes out peak after β subunit; , also will there is the small peak of oxidation products between α, β subunit, about 30-35min in the sample containing oxidation products.
4. result: the reference examples containing oxidation products, detected result is as Fig. 9, and its α subunit goes out peak in about 39 points, and its oxidation products goes out peak in about 34 points; By final the obtained target protein of embodiment 2 and 3, oxidation subunit content detected result is shown in Fig. 7,8, α subunits all go out peak after 35 points, does not occur oxidation products peak between α, β subunit, visible, oxidized methionine residues do not detected.
6) other quality examination
By final the obtained target protein solution of embodiment 1,2,3, other quality measurements is as follows: intracellular toxin <2EU/5ug, host DNA remains <1pg/5ug, host protein remains <0.1%, above detection method is all carried out according to 2010 editions pharmacopeia, and result all meets the quality standard of pharmacopeia.
Experiment shows, uses method of the present invention, and operation steps is simple, and the activated protein rate of recovery is relatively high, and other quality control indexs all reach relevant laws and regulations requirement.
The affinity chromatography that this purifying process adopts and ion exchange chromatography and gel permeation chromatography, relative to reversed phase chromatography, technological process is simple, low to the requirement of equipment, and decreasing organic solvent affects the sex change that target protein produces; Relative to metal chelate chromatography, avoid metal ion hanging column process, simplify purifying process, and avoid metal ion remaining in protein soln.By our 300L cell culture system, carry out the large scale purification of Gonal-F by method of the present invention, respond well.
In sum, this purification process of Gonal-F is convenient, and filler cost is low, and target protein activity is high, is applicable to being applied to commercial production scale.