CN1189502A - Method for producing antithrombin-III, method for purifying it, and preparation containing it - Google Patents
Method for producing antithrombin-III, method for purifying it, and preparation containing it Download PDFInfo
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Abstract
A method for producing antithrombin-III from an antithrombin-III-containing aqueous solution, comprising at least one of the following steps (a) and (b): (a) heating the antithrombin-III-containing aqueous solution in the presence of a stabilizing agent so that 85% or more of the activity of antithrombin-III before the heating is maintained after the heating, and that the ratio of an antithrombin-III monomer after the heating is maintained at 95% or more; and (b) treating the antithrombin-III-containing aqueous solution with a metal chelate resin and recovering a purified antithrombin-III.
Description
Invention field
The present invention relates to the production method of antithrombin-III, purification process and contain the preparation of this enzyme.Particularly, the present invention relates to be used for the treatment of the production and the purification process of antithrombin-III of various diseases and the preparation that contains the antithrombin-III of preparation like this and purifying, reduce and cause but wherein said disease for example is a availability by antithrombin-III in the body.
Background of invention
Antithrombin-III (after this being called " AT-III ") is a kind α
2The glycoprotein of sphaeroprotein is present in the blood plasma.The AT-III molecular weight is 65,000 to 68,000, has protease inhibiting activity.AT-III is the blood coagulation activity of Trombin inhibiting consumingly.
In addition, AT-III not only has zymoplasm and suppresses active, and other thrombin such as activated factor X and activated factor IX also had suppresses active.According to another report, AT-III also has the activity of inhibition to plasminogen and trypsinase.
It is more quick under heparin coexistence situation usually that these suppress activity.
Have that these active AT-III have been used to treat unusual hypercoagulability, particularly diffusive intravascular clotting (DIC) and because but the AT-III availability reduces the various other diseases that cause.
In addition, AT-III has affinity to heparin, has reported multiple purification process, wherein based on this affinity, has used the immobilization heparin (as JP-A-63-23896; Terminology used here " JP-A " is meant " not examining open Japanese patent application ").
And, because AT-III is a kind of protein that is present in the blood plasma, when it is used as a kind of therapeutical agent, need be to potential inactivation of virus wherein.
In these viral inactivation treatment, a concrete known example is 60 ℃ of liquid thermal treatments 10 hours.But, it is reported that AT-III is stable when PH6 to 8, and can lose 80% activity (J.Biol.chem (journal of biological chemistry) in 6 hours 56 ℃ of thermal treatments, 246:3694 (1971)), on the other hand, have in many reports and point out, in the presence of Trisodium Citrate at 14.7w/v%, AT-III is carried out liquid thermal treatment 10 hours at 60 ℃, the activity of AT-III can be stablized maintenance (Thrombosis Research (thrombus research), 22:233 (1981) and J.Biol.chem .256:12140 (1981)).Yet,, observe the sex change (Vox Sang, 48:325 (1985)) of AT-III by IEA when when in the presence of 0.35 to 1.5M (10.29 to 44.12w/v%) Trisodium Citrate AT-III being heated in liquid.
Because this viral inactivation treatment also can produce metaprotein, AT-III as deactivation, polymeric protein or above-mentioned albuminoid etc., so, for example handle (JP-A-63-23896) or carry out hydrophobicity chromatography processing (JP-A-1-275600) with the existing report of the method for removing this class impurity by carrying out the immobilization heparin once more.Yet, even handling, the immobilization heparin only carries out once also can causing low activity recovery, and, the hydrophobicity chromatography has the proteic possibility of carryover contamination, resemble prealbumin, Transferrins,iron complexes, IgG or the like, so then must carry out purification step once more to remove these albumen, activity recovery can reduce thus.
Summary of the invention
An object of the present invention is to provide the production method of non-limiting examples of suitable T-III in the therapeutic treatment, when AT-III is heated in liquid state, the stable sex change (conformational change is such as dimerisation, polymerization or the like) that keeps the active of AT-III and minimizing AT-III.
In addition, another object of the present invention provides removes the single stage method of pollutent albumen such as deactivation AT-III or the like, simultaneously and keep the activity of AT-III.
In other words, the present invention relates to production method, the purification process of a kind of AT-III and contain the preparation of AT-III.
Particularly, reached these purposes of the present invention and other purposes by the method for from the aqueous solution that contains AT-III, producing AT-III, described method comprise the steps at least (a) and (b) in one go on foot:
(a) in the presence of stablizer, heat the aqueous solution that contains AT-III, still keep heating pro-antithrombin III active 85% or higher like this after the heating, and the monomeric ratio of Antithrombin III still remains on 95% or higher after the heating; With
(b) use the metal-chelating plastic resin treatment to contain the aqueous solution of AT-III, and reclaim the AT-III of purifying.
In addition, these purposes of the present invention and other purposes have been reached by a kind of pharmaceutical composition, described pharmaceutical composition contains AT-III or its pharmaceutically acceptable salt of preparation according to the method described above as active ingredient, and selectable pharmaceutically acceptable carrier.
The accompanying drawing summary
Fig. 1 is expression from the graphic representation in conjunction with the wash-out pattern of the AT-III of the resin of cupric ion.
Fig. 2 is the expression salt concn to from the graphic representation in conjunction with the influence of the AT-III wash-out pattern of the resin of cupric ion.
Fig. 3 is the spectrogram of expression in conjunction with the HPLC-GPC before and after the resin purifying AT-III of cupric ion.
Fig. 4 be expression with the Ouchterlony method carry out from figure in conjunction with the impurity analysis result before and after the purifying AT-III of the resin of cupric ion.
Fig. 5 is the spectrogram in conjunction with the HPLC-GPC before and after the copper metal-chelating resin purification AT-III of the resin of expression in conjunction with cupric ion+not.
Detailed Description Of The Invention
(I) raw material
The example that contains the AT-III aqueous solution (after this often being called " the AT-III aqueous solution ") comprises the plasma protein mixture, fraction (Fr.) the II+III supernatant and the IV that obtain such as the cold Ethanol Method by Cohn, and the residue fraction that from the blood plasma that contains citrate, reclaims blood coagulation factor VIII; But raw material is not particularly limited in the blood plasma source, can be used in this AT-III purification process such as the solution that contains AT-III that obtains by methods such as genetic engineerings so yet.
US Patent No. 3,842, the concrete purification process that obtains AT-III from blood or blood plasma is disclosed among 061 (JP-A-48-35017), US Patent No. 4,340,589 (JP-A-54-95715), EP-A-339919 (JP-A-1-275600) and the EP-A-551084. For example, a used method is, will remove cold ethanol fraction IV-1, IV or the II+III supernatant that cryoprecipitate obtains and be further purified from blood plasma, as carrying out purifying by the heparin affinity chromatography method.
In addition, also can use by the cell culture method (AT-III of EP-A-53165 (JP-W-57-500768) [term of usefulness " JP-W " refers to " examine disclosed Japan internationality patent application " here] or gene engineering method [for example EP-A-90505 (JP-A-58-162529)] preparation for example.
(II) heat treatment
According to heat treating process of the present invention, the AT-III aqueous solution to be heat-treated under suitable temperature and in the suitable time, described temperature and time is enough to the wherein potential viral pollutant that contains of deactivation. Particularly, can heat-treat at 50 ℃ to 70 ℃ preferred about 60 ℃, the time is 10 minutes to 20 hours, preferred about 10 hours. The PH of solution is 5 to 10, and is preferred 6.5 to 9.0, more preferably regulates with suitable buffer solution. The ratio that the AT-III aqueous solution contains AT-III is 10 to 500 units/ml, and preferred 1 to 200 units/ml, and more preferred 25 to 100 units/ml. According to the present invention, " 1 unit " AT-III refers to be equivalent to the activity of the AT-III of contained AT-III amount among the 1ml human normal plasma. The activity of AT-III can be surveyed, and for example can use the AT-III activity measurement box (Test Team AT-III-2-kit, by Daiichi Pure chemicals Co., Ltd produces) that utilizes synthetic substrate to measure.
Heat-treat by this way, cause the gross activity of AT-III after the heat treatment to remain 85% before the heat treatment or higher, preferred 95% or higher, and the ratio of the AT-III monomer in AT-III aqueous solution total amount still remains 95% or higher, preferred 99% or higher.
The monomeric ratio of AT-III can be surveyed, and for example can pass through high performance liquid chromatography (HPLC) and use in advance with 0.3M sodium-chlor-0.05M phosphate buffered saline buffer (PH7.0) equilibrated G3,000SW
XLPost (TosohCorp. production) is measured.
For reaching the required condition of this residual activity and monomer ratio, can in the presence of following stablizer, heat-treat.
(III) stablizer
Be used for the heat treated stablizer of the present invention and be preferably selected from organic acid or its salt, carbohydrate and sodium-chlor.
Being used for organic acid of the present invention is aliphatics or aromatic saturated or undersaturated monoprotic acid (monocarboxylic acid), diprotic acid (dicarboxylic acid) or triprotic acid (tricarboxylic acid).Preferred this acid has 2 to 10 carbon atoms, more preferred 2 to 6 carbon atoms.Monacid example comprises monobasic amino acid (as glycine, L-Ala, Xie Ansuan, leucine, Isoleucine).The example of diprotic acid comprises saturated aliphatic dicarboxylic acid (as oxalic acid, propanedioic acid, succsinic acid, pentanedioic acid, hexanodioic acid), undersaturated aliphatic dicarboxylic acid (as toxilic acid, fumaric acid), aromatic dicarboxylic acid (as phthalic acid), binary amino acid (as aspartic acid, L-glutamic acid), dihydroxylic acid (as benzene tartaric acid, tartrate).The example of triprotic acid comprises trihydroxylic acid (as citric acid).Wherein, preferably apple acid, tartrate, toxilic acid, aspartic acid and citric acid, more preferably apple acid and citric acid.
In addition, can use the form of this organic acid salt.The example of organic acid salt comprises an alkali metal salt (as sodium salt, sylvite), alkaline earth salt (as calcium salt) and organic salt (as ammonium salt).Particular certain cancers and calcium salt.Organic acid salt preferred for the present invention is sodium malate and Trisodium Citrate, the most optimization citric acid sodium.
The organic acid that adds or the scope of its salt are preferably 3 to 20w/v%, and more preferably 3 to 10w/v%, and the most preferred 3 to 5w/v%.If add-on is less than 3w/v%, stabilising effect will descend.
What must use is organic acid or its salt and carbohydrate, can not use sodium-chlor.The adding of carbohydrate or sodium-chlor can reduce the organic acid content that adds and can suppress to cause the Denaturation (dimerization of AT-III and polymerization) of AT-III deactivation.The example of carbohydrate comprises monose, disaccharides, sugar alcohol and aminosugar.The example of monose comprises glucose, fructose, semi-lactosi, seminose, pectinose and inositol.The example of disaccharides comprises sucrose, lactose and maltose.The example of sugar alcohol comprises mannitol, sorbyl alcohol and Xylitol.The example of aminosugar comprises glycosamine and amino sugar derivative N-acetyl-D-glycosamine.Wherein, preferably sucrose, lactose, sorbyl alcohol, inositol, seminose, N-acetyl-D-glycosamine and mannitol.
The amount preferable range that adds sugar is 10 to 60w/v%, and more preferably 20 to 50w/v%, and most preferably 20 to 30w/v%.If this amount is less than 10w/v%, stabilising effect will descend so, if should measure more than 60w/v%, so also is disadvantageous, though because stability can obtain to improve, viral pollutant also can be stabilized.
The scope that adds the sodium-chlor amount is preferably 0.5 to 3.0M, and more preferred 1.0 to 2.5M, and most preferably 1.5 to 2.0M, and most preferably 1.5 to 2.0M.If this amount is less than 0.5M, then stabilising effect descends, and this is measured more than 3.0M, also is disadvantageous, though because stability can obtain to improve, the solvability of AT-III can reduce.
But, according to the present invention, added consistent dose is not limited in the above-mentioned scope especially, as long as they can satisfy following conditions, be that the gross activity of AT-III after the thermal treatment keeps 85% before the thermal treatment or higher, the monomeric ratio of AT-III keeps 95% or higher after the thermal treatment.
(IV) purifying
The purification process of AT-III of the present invention is included in and uses metal-chelating plastic resin treatment AT-III in the AT-III purification step.More specifically, when this method is included in from the aqueous solution that contains AT-III purifying AT-III, the AT-III aqueous solution is contacted with the metal-chelating resin, and reclaim unabsorbed part.
The protein concentration that the AT-III aqueous solution can have about 0.1 is to 5w/v%.And preferred use has been purified to the AT-III aqueous solution of a certain degree, and it is to use the AT-III aqueous solution of said fixing heparin carrier prepurification In-particular.In addition, advantageously before solution is carried out metal-chelating plastic resin treatment of the present invention, this AT-III aqueous solution is carried out viral inactivation treatment, preferably adopt above-mentioned heat treating process to carry out by thermal treatment (50-70 ℃, 5 to 30 hours).
And, when the AT-III aqueous solution contains a kind ofly during as this sequestrants such as citric acids, preferably use by dialysis treatment or similar method and remove the AT-III aqueous solution behind the sequestrant, to prevent wash-out metal ion from the metal resin.
By metal ion is incorporated on the resin with chelate bond, comprise and can purchase resin (as the iminodiacetic acid (salt) acid type), can obtain being used for metal-chelating resin of the present invention.Object lesson comprises chelating-agarose F.F. (chelating-sepharoseF.F.) (being produced by Pharmacia) and AF-chelat Toyopearl 650M (being produced by Tosoh Corp.).
The metal ion that is bonded on the resin is not particularly limited, as long as metal ion can effectively separate AT-III.These examples comprise cupric ion, nickel ion and cobalt ion, and from the angle of purification efficiency, cupric ion is particularly preferred.
The inventive method is that the AT-III aqueous solution is contacted with the metal-chelating resin.All can implement this contact by post method or batch method of treatment, but from degree of purification and the preferred use post of purification efficiency method.
About contact conditions, reaction can carry out PH6 to less than 8 preferred 6.5 to 7.7 and salt concn be 0 to 1M preferred 0.05 to 0.5M.Preferred solvent comprises the 0.1M phosphate buffered saline buffer (PH7.0 to 7.5) that contains 0.05 to 0.5M sodium-chlor, but sodium-chlor is optional.In order to improve the separation efficiency between metal-chelating resin and AT-III, can in this damping fluid, add antagonist, as glycine, imidazole, and the preferred antagonism dosage that adds is 20mM or still less.
When using the post method to carry out purification process of the present invention, the adjusted AT-III aqueous solution to above-mentioned condition is applied on the metal-chelating resin column, this post is balance under similarity condition (same buffer remains in 2 ℃ to room temperature as developping solution, reclaims not absorbed portion then).Can regulate used metal-chelating amount of resin according to the foreign matter content in containing the aqueous solution of AT-III, AT-III content etc.; But generally speaking, every 4mlAT-III aqueous solution uses about 1ml metal-chelating resin.Elution rate according to the controlled amount system post of the loading level of metal-chelating resin and post.
When using batch method to carry out purification process of the present invention, 2 ℃ to room temperature, make the AT-III aqueous solution that is adjusted to above-mentioned condition contact centrifugal then recovery supernatant liquor with equilibrated metal-chelating resin under similarity condition.Can regulate duration of contact according to the foreign matter content in containing the aqueous solution of AT-III, AT-III content etc., but can contact usually 10 minutes to 1 hour just enough.
In addition, in some cases, metal ion can be from seepage on the metal-chelating resin; But the metal ion of seepage can easily be removed, for example, and by using not resin or the method for ion-exchanger processing or the method that adding is dialysed then as this sequestrant of EDTA of bind metal ion.
AT-III by purification process of the present invention purifying like this is substantially free of impurity albumen, sex change AT-III, AT-III polymkeric substance etc., therefore can obtain highly purified AT-III by a step.
In addition, can use ordinary method such as anion exchanger facture or be further purified AT-III according to the purpose that will reach through the inventive method purifying like this with containing the insoluble vehicle treated method of hydrophobic group as its part.
(V) aftertreatment
Compound method according to commonly used can be prepared into pharmaceutical preparation with the AT-III through viral inactivation treatment and/or the inventive method purifying.The example of pharmaceutical preparation comprises the aqueous solution, and suspension and freeze-dried preparation by selectively mixing with pharmaceutically acceptable carrier or additive (as releasing rare dose, tonic, tensio-active agent), can prepare these medicaments in a usual manner.Can carry out the administration of AT-III preparation by injection or the conventional prescription of intravenous drip AT-III, for example carry out administration by slow intravenous injection or drip liquid body preparation.
The AT-III of production of the present invention and/or purifying can be used for treating that AT-III reduces the diffusive intravascular clotting syndrome (DIC) that forms in the thrombotic trend that caused by congenital AT-III disappearance and the comitative aspect.
Usually, with the speed administration AT-III of unit every day 1,000 to 3,000 (or 20 to 60 units/kg body weight), but can change this dosage arbitrarily according to different conditions such as age, body weight, symptom etc.And when preparation of the present invention was used to obstetrics or surgery DIC emergency treatment, preferred dosage was that every day 40 is to 60 units/kg body weight.The osmotic pressure of preferred liquid preparation is identical or close with humans and animals patient's physiological status.
(VI) invention effect
When by the inventive method with the AT-III of liquid state heating during with inactivation of viruses, the Denaturation (conformational change such as dimerization, polymerization etc.) that the activity of AT-III can be stablized maintenance and AT-III can be reduced, thus, say from medical angle, can prepare AT-III with tight security and availability.
In addition, according to purification process of the present invention, the AT-III of contained impurity albumen and sex change and all can effectively be removed by the polymkeric substance that thermal treatment forms in the AT-III aqueous solution can obtain the AT-III of high security thus.And, because purification step is simple and be suitable for scale operation, so obviously can be used as industrial process.
Because pharmaceutical preparation of the present invention contains the AT-III of aforesaid method purifying, so, the AT-III with excellent safety can be provided pharmaceutical preparation.
With reference to the following examples the present invention has been carried out more detailed description, still, these embodiment do not limit the present invention in any way, and raw material and method used in testing below are as follows:
Raw material and method
(1) AT-III raw material
Raw material used herein is to prepare by the solution that concentrates from Cohn ' s fraction II+III supernatant liquor or fraction IV, this fraction has been used immobilization heparin column purifying, with solution in 60 ℃ and 5% Trisodium Citrate, 2MNaCl and 30% sorbyl alcohol exists and the condition of PH7.8 under heating 10 hours, carry out viral inactivation treatment.
(2) be used for the chromosorb of purifying
Chelating-agarose F.F. (being produced by Pharmacia) or AF-chelate Toyopearl 650M (being produced by TosohCorp.) are used as the chelating chromosorb.
(3) AT-III determination of activity and remaining rate are calculated
Use S-2238 to measure by synthetic substrate method.S2238 and used reacting terminating solution are to attach that (Daiichi Pure Chemicals Co. is on Ltd) at Test Team AT-III-2-kit.One pipe (20mg) S-2238 is dissolved in the 21ml water.Be distributed into several parts and under-40 ℃, deposit with thrombin solution (1.2U/ml) with by the standard A T-III solution (1U/ml) that Green Cross Corpoation produces, when using, melt again.Testing sample is adjusted to 1 to 10mU/ml with the solution that contains 40mM Tris, 60mM EDTA, 0.14M NaCl, 0.2%HSA (human serum albumin) and 2.4U/ml heparin and PH8.4, one share, 50 μ l samples and 100 μ l thrombin solutions are mixed, and following warm blue or green 5 minutes in 37 ℃.Then, mixed and incubation 5 minutes accurately then, adds 1000 these reactions of μ l reaction terminating with 100 μ l substrate solutions.Measure solution absorbance down in 405nm, calculate sample AT-III activity according to the standard straight-line of using standard A T-III solution to make.
The percentage of the ratio of AT-IIL gross activity is AT-III reactive residual rate before the AT-III gross activity of each AT-III sample of thermal treatment [U/ml * liquid volume (ml)] and the thermal treatment, the results are shown in table 1.
(4) AT-III specific activity
By measuring every duplicate samples at A
280nm(after this be called " A
280") under absorbancy calculate protein content in the testing sample.The AT-III activity of top gained is able to the specific activity (U/A280) of every duplicate samples divided by protein content, the results are shown in table 1.
(5) gel-filtration HPLC analyzes
By G3000 SW
XLThe SystemGold (Beckman production) of (φ 7.8mm * 30mm, Tosoh produces) post equipment uses the solution that contains 50mM sodium phosphate and 0.3M NaCl and PH7.0 to carry out this analysis as developping solution.In all cases, flow velocity is 0.7ml/min, detects under the 280nm absorbancy.
Analyze the AT-III monomer ratio that draws by HPLC and be shown in table 1.From table 1 result, also can obtain the denaturation degrees (conformational change such as dimerization, poly etc.) of AT-III.
(6) Ouchterlony analyzes
With common method, by each sample concentration to be measured absorbancy about 30 to the 280nm is carried out this analysis.Used antibody following (numbering with Fig. 4 in consistent): anti-human plasma copper-protein (1) (Hoechst N-antiserum(antisera) ceruloplasmin); Anti-human transferrin (2) (Hoechst N-antiserum(antisera) Transferrins,iron complexes); Anti-human albumin (3) (HoechstN-antiserum(antisera) albumin); Albumen (4) (Hoechst N-antiserum(antisera) prealbumin) before the anti-people; Anti-people's haptoglobin (5) (DAKO A030); Anti-people's alpha2 Macroglobulin (6) (DAKO A033); Anti-people's alpha1 Anti-trypsin (7) (DAKO A012); Anti-people IgA (8) (DAKO A02621); Anti-human IgG (9) (DAKO A04231); Anti-people IgM (10) (Hoechst resists-IggM (μ chain) rabbit anteserum); Anti-human prothrombin former (11) (DAKO A325); With anti-Cryodesiccant Human Fibrinogen (12) (DAKO A080).
The a fraction IV-110kg mashed prod that is obtained by the Cohn cohn fractionation is suspended in 100 liters of physiological saline, adding barium sulfate to concentration in this suspension is 5w/v%, this miscellany was stirred under room temperature 30 minutes, and the thrombogen that trace exists is removed in the adsorption of the thrombogen by barium sulfate.The supernatant liquor that so obtains is adjusted to PH6.5, and with 13w/v% polyoxyethylene glycol #4,000 (molecular-weight average measured value: 2,600 to 3,800, by Nippon Soda Co., Ltd produces) is mixed, the centrifugal gained throw out of removing; With the supernatant liquor that obtains once more with the polyoxyethylene glycol #4 of 30w/v%, 000 is mixed, and the throw out that forms thus of centrifugal recovery.
The throw out that is reclaimed is dissolved in about 20 liters cold saline, is added on to use in advance on physiological saline equilibrated heparin-agarose (pharmacia production) post, to adsorb AT-III on post.To remove impurity albumen, then the 2.0M sodium chloride solution is passed through this post with the 0.4M sodium chloride solution washing post for preparing like this to reclaim the AT-III part of wash-out.
So the AT-III aqueous solution that obtains is mixed with the stablizer that is shown in table 1, is adjusted to PH7.8, then in 60 ℃ of following thermal treatments 10 hours.(ButylToyopearl 650 with the AT-III aqueous solution so handled and butyl-type polyethylene support, Tosoh Corp. produces) contact, this carrier uses 20mM sodium citrate buffer solution (PH7.5) balance that contains 3M sodium-chlor in advance, and uses identical damping fluid not to be adsorbed part as developping solution to reclaim.It is to the AT-III of 0.5% sodium citrate buffer solution (PH7.5) dialysed overnight with the acquisition purifying then.
The activity of the AT-III sample of every part of purifying after the mensuration thermal treatment is to calculate the active remaining rate for the AT-III before the thermal treatment.Also can obtain the monomer ratio of every part of AT-III sample.
Table 1
| Stablizer | Specific activity (U/A 280) | Remaining rate (%) | HPLC (％) | ||
| Before the thermal treatment | 8.8 | 100 | 99.7 | ||
| 19w/v％Na-Cit，2M?NaCl?pH7.8 | 6.9 | 78.4 | 78.9 | ||
| ?5w/v％ Na-Cit | 60w/v％?Sorb.pH7.8 | 7.5 | 84.7 | 86.4 | |
| 2M NaCl | 10w/v％Sorb.pH7.8 | 8.0 | 85.0 | 90.0 | |
| ?20w/v％Sorb.pH7.8 | 8.4 | 88.4 | 95.0 | ||
| ?30w/v％Sorb.pH7.8 | 8.6 | 90.3 | 95.2 | ||
| 40w/v％Sorb.pH7.8 | 7.8 | 88.0 | 96.7 | ||
| ?50w/v％Sorb.pH7.8 | 8.1 | 92.0 | 99.3 | ||
| ?60w/v％Sorb.pH7.8 | 8.2 | 93.6 | 99.7 | ||
| 10w/v％ Na-Cit | 40w/v％Sorb.pH7.8 | 7.6 | 85.7 | 97.7 | |
| ?50w/v％Sorb.pH7.8 | 7.7 | 87.7 | 91.9 | ||
| ?60w/v％Sorb.pH7.8 | 8.1 | 91.4 | 98.9 | ||
| ?2M?NaCl，50w/v％Sorb.pH7.8 | 7.9 | 89.8 | 96.7 | ||
Remaining rate (%): the percentage of the ratio of AT-III gross activity before AT-III gross activity and the thermal treatment after the thermal treatment.
HPLC analyzes (%): the AT-III monomer ratio
When adding Trisodium Citrate and sodium-chlor, can observe synergy, so dimerization and polymerization can be inhibited, and under lower concentration Trisodium Citrate condition, can be observed stabilising effect.In the last table, Na-Cit refers to Trisodium Citrate, and Sorb refers to sorbyl alcohol.
Embodiment 2
Use chelating-agarose F.F. purifying AT-III:
(φ 0.9 * 2.3cm) goes up that to add the water-soluble concentration of same bed volume (1.5ml) be the CuSO of 10mg/ml to chelating-agarose F.F. post of 1.5ml
45H
2O is with bond.Use this post of water washing of 2 times of bed volumes (3ml) then.Then, the solution that contains 0.1M sodium phosphate and 0.5MNaCl and PH8.0 by 3 times of bed volumes comes this post of balance.Utilize PD-10 post (pharmacia production), with by same buffer-exchanged through heat treated 3ml AT-III concentrated solution (A
280=4.20) be added on the post of preparation thus, collect elutriated fraction with 1.5ml/Fr (Fraction fraction) speed.Then, on post, add every part be 3 times of bed volumes (4.5ml) be adjusted to PH7.5,7.0 and 6.0 the solution that contains 0.1M sodium phosphate and 0.5M NaCl, collect elutriated fraction with the same manner with 1.5ml/Fr speed.Remove bond by 50mMEDTA2Na with 2 times of bed volumes, then, with 3 times of bed volume water washings used chelate column of regenerating.
The results are shown in Fig. 1.As shown in Figure 1, most of albumen is adsorbed in PH8.0, and on the copper chelate column in PH7.5 or when being lower than PH7.5 by wash-out.
Table 2 illustrates the result from the GPC-HPLC analysis of the elutriant of copper chelate column.Just as shown in table 2, before being added to the copper chelate column, unmodified (complete) AT-III purity is 88.0%; And this purity becomes 100% under all tested pH values elutriated fraction from post.That is to say, solution is lower than 8 preferred PH7.5 or can removes sex change AT-III and foreign protein significantly effectively by pillar when lower in PH, and these impurity even when PH6.5, also can remain in the pillar
Table 2
| The level branch | 1-5 | ?6 | ?7 | ?8 | ?9 | ?10 | ?11 | ?12 | ?13 | ?14 |
| The AT-III purity (%) that pH is complete | 8.0 - | ????7.5 ????100 | ????7.0 ????100 | ????6.5 ????100 | ||||||
Be added on the complete AT-III purity in the sample on the pillar: 88%.
Embodiment 3
The influence of salt concn
Be to measure the influence of the salt concn in the chromatogram damping fluid, use 0.1M sodium phosphate buffer (PH7.5) or measure copper chelating chromatographic behavior through thermal treatment and spissated AT-III solution by the damping fluid that adding 0.1M NaCl or 0.5M NaCl in 0.1M sodium phosphate (PH7.5) prepare.That is to say, will carry out exchange of solvent, its A through thermal treatment and spissated AT-III solution and every kind of buffered soln by the PD-10 post
280Value is transferred to is about 5, then, with every part of 5ml solution, is added in and uses same buffer equilibrated 1ml copper chelate column (on the φ 0.8 * 2cm).Merge the post solution excessively of initial 3ml part, remainder is gathered into several parts of 1ml.Then, use damping fluid washing pillar separately respectively, the elutriant during with washing is gathered into several parts of 1ml.The wash-out pattern is shown in Fig. 2.The GPC-HPLC measurement result of complete AT-III purity is shown in table 3 in every part of elutriant fraction.
Table 3
| The level branch | Eluate cumulative volume (ml) | Complete AT-III purity (%) | ||
| NaCl concentration (M) | ||||
| ????0 | ????0.1 | ????0.5 | ||
| ????1 ????2 ????3 ????4 ????5 | ????3 ????4 ????5 ????6 ????7 | ????100.0 ????100.0 ????100.0 ????100.0 ????100.0 | ????100.0 ????100.0 ????100.0 ????100.0 ????100.0 | ????100.0 ????100.0 ????100.0 ????100.0 ????100.0 |
The purity that is added in the complete AT-III of the sample on the pillar is: 81.15%.
As shown in Figure 2, do not contain the NaCl situation and contain 0.1M or 0.5M NaCl situation between elution samples in do not find notable difference.And just as shown in table 3, in all cases, the purity of complete AT-III is 100.0% in the elutriant.Therefore, demonstrate that the variation of salt concn does not almost exert an influence to chromatographic behavior in the damping fluid.
In addition, use and to be adjusted to PH7.3,7.5 or 7.7 0.1M sodium phosphate buffer (containing 0.1MNaCl) and to have measured PH and change influence chromatographic behavior; But its variation that demonstrates PH in this scope does not almost exert an influence to chromatographic behavior.Use 0.1M sodium phosphate buffer (containing 0.1MNaClPH7.5) also to measure chromatographic behavior under 4 ℃ and the room temperature; But its temperature variation that demonstrates in this scope does not almost exert an influence to chromatographic behavior.
Embodiment 4
The assessment of the definite and purification yield of degree of purification in scale-up
By PD-10 post (A
280=4.05) will carry out buffer-exchanged with the 0.1M sodium radio-phosphate,P-32 solution that contains 0.1MNaCl and PH7.5 through thermal treatment and spissated AT-III solution, (on the φ 1.5 * 2.8cm), this post has been used the same buffer balance then the 25ml of this solution partly to be added on the copper chelating Toyopearl 650M post of 5ml.The 2.5ml that will be equivalent to the attached column bed volume partly discards, and then, the elutriant that application of samples is obtained simultaneously and the washing water that contain level pad of 2 times of column volumes combine (32.5ml), as the AT-III of purifying.A
280Value and AT-III activity recovery are shown in table 4, and the GPC-HPLC collection of illustrative plates is shown in Fig. 3 before and after the chromatogram.
Just as shown in table 4, obtained the good rate of recovery, promptly for A
280Being 80.3%, is 89.6% for the AT-III activity.And as shown in Figure 3, the post elutriant of GPC-HPLC demonstrates the simple spike of complete AT-III, and does not contain polymkeric substance, sex change AT-III and other impurity albumen.
In addition, when above-mentioned sample being carried out the Ouchterlony analysis, all not detecting all potential impurity albumen (is ceruloplasmin, Transferrins,iron complexes, albumin, prealbumin, haptoglobin, α
2Macroglobulin, α
1The Parenogen (see figure 4) of antitrypsin, IgA, IgG, IgM, thrombogen.
Therefore, it demonstrates at PH7.5 and can remove all impurity albumen, polymkeric substance and sex change AT-III by copper chelating Toyopearl 650M chromatogram.
Table 4
| Step | Vol (ml) | ????A 280 | The AT-III activity | |||||
| A 280 | A 280×vol | Yield (%) | ??U/ml | Gross activity | ??U/A 280 | Yield (%) | ||
| When being added on post | ????25 | ?4.05 | ??101.3 | ??80.3 | ??29.9 | ??748 | ??7.38 | ??89.6 |
| When flowing through post | ????32.5 | ?2.50 | ??81.3 | ??20.6 | ??670 | ??8.26 | ||
Remove the copper of seepage from copper resin post
Known in copper chelate column chromatogram, in adhesion protein, have micro-seepage usually with pillar bonded copper.Existing people proposes to remove the method for the copper of seepage, wherein, the resin of a little not bond (but be the sequestrant bond, but metal does not exist) post is connected with a metal-chelating resin column.So, detect the effect of the method for this removing seepage copper as follows.To be dissolved in the Fr.II+III supernatant liquor and the spissated AT-III solution (A that obtain by thermal treatment of 20ml part in the 0.1M sodium phosphate buffer that contains 0.1MNaCl and PH7.5
280=4.96) be added to the copper chelating the Toyopearl post (φ 1.5 * 2.8cm, 5ml) on, this post has been used the same buffer balance, and the fraction of will not adsorb merges (27.5ml, A
280=3.18).23.5 parts wherein are added to have been crossed with the same procedure balance) do not add metalchelated Toyopearl 650M post (φ 0.8 * 2.0cm, 1ml) on, merge the fraction (25ml, the A that are not adsorbed
280=2.63).The copper Determination on content the results are shown in table 5 in per step.
Table 5
| Step | Copper content (ppb) |
| Negative control (chromatogram buffering) when flowing through not the bond chelate column when flowing through the copper chelate column when being added on the copper chelate column | ????320 ????10,300 ????15 ????13 |
Just as shown in table 5, though in the copper chelate column that is passed through, found 10, the seepage copper of 300ppb, but find this solution can be removed fully by the Toyopearl 650M post of bond chelating not the copper of seepage, because leakage has been reduced to 15ppb, this is very near control level (13ppb)
Raw material and the GPC-HPLC spectrogram of elutriant that does not add the chelate column of metal are shown in Fig. 5.As shown in Figure 5, in addition obtain raw material by the Fr.II+III supernatant liquor also can be by copper chelate column purifying, because except that the peak of complete AT-III, do not find other clearly peak.The purity that goes out from calculated by peak area is 99.9%.
The purification yield that obtains by the copper chelate column and the joint pin of bond Toyopearl post not
Consider to the efficient that makes chromatographic step improves and automatization, at the copper chelate column with do not add on the joint pin of metal ground chelate column and detect.To dialyse 1600 times to the 0.1M sodium phosphate buffer that contains 0.1M NaCl and PH7.5 from the solution of Fr.II+III supernatant liquor or Fr.IV through thermal treatment and spissated AT-III, and (multiple of always dialysing is 160 once more same damping fluid to be dialysed 100 times, 000), uses Centyiprep-30 (Amicon production) to concentrate and dilute (Fr.IV:A
280=5.23, FR.II+III supernatant liquor: A
280-5.50).By using Centriprep-30, improved the dialysis multiple, because remaining citric acid makes the big 1600 times of dialysis of seepage quantitative change of copper.With every part of 20ml of these solution partly put on placed in-line 5ml (φ 1.5 * 2.8cm) copper chelatings ((on the Toyopearl post of bond chelating, these posts have not been used same damping fluid balance to φ 0.8 * 2.0cm) for Toyopearl and 1ml.For the solution of wash-out, when applying sample, discard the attached column volume of total column capacity, and the fraction of wash-out merges subsequently, and they are mixed with the washing fraction that 10m is equivalent to 2 times of copper chelate column bed volumes.
From the purification yield shown in the table 6 obviously as seen, all demonstrate good activity recovery from two kinds of raw materials of Fr.II+III supernatant and Fr.IV, it is 90% or higher.And specific activity is about 10U/A
280, it equates with the activity of pharmaceutical preparation in using.
Table 6
| Raw material | Step | Amount of liquid (ml) | A 280 | ||
| A 280 | TotalA 280 | Yield (%) | |||
| The II+III supernatant liquor | When being added on post | 20.0 | 5.50 | 110 | 100 |
| When flowing through post | 27.0 | 3.22 | 86.9 | 79.0 | |
| IV | When being added on post | 20.0 | 5.23 | 105 | 100 |
| When flowing through post | 26.0 | 2.92 | 75.9 | 72.3 | |
| Raw material | Step | The AT-III activity | |||
| U/ml | Gross activity | Yield (%) | Specific activity (U/A 280) | ||
| The II+III supernatant liquor | When being added on post | 49.0 | 981 | 100 | 8.9 |
| When flowing through post | 33.9 | 914 | 93.2 | 10.5 | |
| IV | When being added on post | 40.9 | 818 | 100 | 7.8 |
| When flowing through post | 29.1 | 757 | 92.5 | 10.0 | |
The preparation of AT-III pharmaceutical preparation
To make purifying AT-III desalination among the embodiment 5, concentrate and filter by BMM (bemberg MicroporousMembrane) film (producing) by Asahi chemical Industry Co.Ltd..Zhi Bei AT-III sample 50 units/ml mixes with 2w/v% N.F,USP MANNITOL, 0.52w/v% Trisodium Citrate and 0.5w/v% sodium-chlor thus, and this mixture is regulated PH7.2 to 7.5 with 1N sodium hydroxide, by sterilization Millipore strainer filter-sterilized, be divided into n part 500 units, lyophilize is to obtain freeze-dried pharmaceutical formulation then.
Though described the present invention in detail, and with reference to specific embodiment, the various changes and modifications of being done are conspicuous to those skilled in the art under the situation that does not deviate from spirit and scope of the invention.
The application is based on the Japanese publication number flat 8-309281 that submitted on November 20th, 1996 and the flat 9-163426 that submitted on June 4th, 1997, and the full text of these applications is hereby incorporated by reference.
Claims (10)
1. method that from the aqueous solution that contains antithrombin-III, prepares antithrombin-III, its comprise the following steps (a) and (b) at least one step:
(a) in the presence of stablizer, heating contains the aqueous solution of antithrombin-III, cause antithrombin-III active 85% of still keeping after the heating before the heating or higher, and the monomeric ratio of antithrombin-III after the heating remains 95% or higher; With
(b) contain the aqueous solution of antithrombin-III with the metal-chelating plastic resin treatment, and reclaim the antithrombin-III of purifying.
2. according to the process of claim 1 wherein that stablizer is organic acid or its salt and carbohydrate, and selectively be sodium-chlor.
3. according to the method for claim 2, wherein organic acid or its salt are selected from glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, oxalic acid, propanedioic acid, succsinic acid, pentanedioic acid, hexanodioic acid, toxilic acid, fumaric acid, phthalic acid, aspartic acid, L-glutamic acid, oxysuccinic acid, tartrate, citric acid and salt thereof.
4. according to the method for claim 2, wherein carbohydrate is selected from glucose, fructose, semi-lactosi, seminose, pectinose, inositol, sucrose, lactose, maltose, mannitol, sorbyl alcohol, Xylitol, glycosamine and N-ethanoyl-D-glucosamine.
5. according to the method for claim 2 or 3, wherein the content of organic acid or its salt is 3 to 20W/V% in solution.
6. according to the method for claim 2 or 4, wherein contents of saccharide is 10 to 60W/V% in solution.
7. according to the method for claim 2 or 5, wherein sodium chloride content is 0.5 to 3.0mol/l in solution.
8. according to the process of claim 1 wherein that the metal ion of metal-chelating resin is selected from cupric ion, nickel ion and cobalt ion.
9. will contain the aqueous solution of antithrombin-III according to the process of claim 1 wherein preceding the heat-treating of step (b).
10. pharmaceutical composition, it contains antithrombin-III or its pharmaceutically acceptable salt as active ingredient of any described method preparation of with good grounds claim 1 to 9, and selectable pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97126456 CN1189502A (en) | 1996-11-20 | 1997-11-20 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP309281/96 | 1996-11-20 | ||
| JP163426/97 | 1997-06-04 | ||
| CN 97126456 CN1189502A (en) | 1996-11-20 | 1997-11-20 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1189502A true CN1189502A (en) | 1998-08-05 |
Family
ID=5177667
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 97126456 Pending CN1189502A (en) | 1996-11-20 | 1997-11-20 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
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| Country | Link |
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| CN (1) | CN1189502A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229922A (en) * | 2011-06-07 | 2011-11-02 | 山东思诺拜特生物科技有限公司 | Process for preparing high-stability liquid alpha-galactosidase |
| CN103059129A (en) * | 2013-01-28 | 2013-04-24 | 贵州泰邦生物制品有限公司 | Method for preparing human antithrombin-III product |
| CN105315365A (en) * | 2015-11-17 | 2016-02-10 | 上海洲跃生物科技有限公司 | Preparation method for human antithrombin III |
| CN106568765A (en) * | 2015-10-12 | 2017-04-19 | 上海长岛生物技术有限公司 | Thrombin chromogenic substrate solution, thrombin aqueous solution, method and kit for determination of antithrombin III activity |
-
1997
- 1997-11-20 CN CN 97126456 patent/CN1189502A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229922A (en) * | 2011-06-07 | 2011-11-02 | 山东思诺拜特生物科技有限公司 | Process for preparing high-stability liquid alpha-galactosidase |
| CN102229922B (en) * | 2011-06-07 | 2013-08-07 | 山东思诺拜特生物科技有限公司 | Process for preparing high-stability liquid alpha-galactosidase |
| CN103059129A (en) * | 2013-01-28 | 2013-04-24 | 贵州泰邦生物制品有限公司 | Method for preparing human antithrombin-III product |
| CN103059129B (en) * | 2013-01-28 | 2015-02-11 | 贵州泰邦生物制品有限公司 | Method for preparing human antithrombin-III product |
| CN106568765A (en) * | 2015-10-12 | 2017-04-19 | 上海长岛生物技术有限公司 | Thrombin chromogenic substrate solution, thrombin aqueous solution, method and kit for determination of antithrombin III activity |
| CN105315365A (en) * | 2015-11-17 | 2016-02-10 | 上海洲跃生物科技有限公司 | Preparation method for human antithrombin III |
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