CN1336178A - Prepn. of blood coagulation factor IX compound - Google Patents
Prepn. of blood coagulation factor IX compound Download PDFInfo
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- CN1336178A CN1336178A CN01108741A CN01108741A CN1336178A CN 1336178 A CN1336178 A CN 1336178A CN 01108741 A CN01108741 A CN 01108741A CN 01108741 A CN01108741 A CN 01108741A CN 1336178 A CN1336178 A CN 1336178A
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Abstract
The present invention relates to the preparation of plasma thromboplastin antecedent IX complex phosphate buffer solution or citrate buffer solution with 4-7 milli mole concertration and pH 5.5-6.5 is used to balance the gel used for adsorption, with fresh frozen human plasmia being added. DEAE-sepharose Fast Flow is used to proceed ion exchange adsorption separation, then same buffer solution with pH 5.5-7.0 is used to wash adsorption gel. further same buffer solution with pH 6-8 is used to elute adsorption gel, collect plasma thromboplastin antecedent IX complex containing factor II, VII, IX and X.
Description
Technical field
What the present invention relates to is a kind of improving one's methods of plasma thromboplastin component complex that be used to prepare, and particularly is fresh frozen plasma to be carried out the method for ion exchange adsorbing separation with DEAE-sepharose Fast Flow (the fast glue of diethylamide ethyl-agarose) gel.
Background technology
The dysfunction of blood coagulation that causes to hemophilia B with because of liver disease, at present mainly adopt high-purity human blood plasma thromboplastin component, or mainly contain plasma thromboplastin component complex (i.e. " the prothrombin complex ") plasma protein products of prothrombin that the K vitamin relies on, VII, IX, X.Owing to contain multiple thrombin simultaneously in latter's goods, therefore can also be used for since vitamin K deficiency and II, VII, IX, the X factor level of secondary low due to hemorrhage treatment, bigger using value is arranged.
No matter reported in literature is the single plasma thromboplastin component of preparation at present, still preparation contains the method for the prothrombin complex of blood coagulation II, VII, IX, the X factor simultaneously, all be the human plasma raw material to be carried out selective absorption according to the different needs of goods kind by ion exchange resin separate, after washing removes foreigh protein removing and/or unwanted factor composition, collect the eluent that contains the required factor composition of goods and carry out corresponding post processing again.The research report is arranged, because the isoelectric point, IP of these vitamin K dependent proteins is lower, adopting various anion exchange resin, as can being to contain DEAE (diethylamide ethyl), PEI (polymine), QAE groups such as (season aminoethanes) as Q-Sepharose Fast Flow resin, DEAE-Sepharose fast Flow resin, the DEAE-Toyopearl resin, the QAE-Toyopearl resin, the POROS-Q resin, the Fractogel-DMAE resin, Fractogel EMD-TMAE resin, and resin such as Matrex Cellufine DEAE carries out adsorbing separation and is advisable.For example, United States Patent (USP) 5,118,614 when the above-mentioned prothrombin complex (PCC) that contains II, VII, IX, the X factor of preparation, employing contains DEAE-hydroxyethyl methacrylate polymer (ethoxy isobutene. ester polymer) the chromatography exchanger resin of DEAE, with after containing 0.1 mole nacl and concentration and being 10 mM pH7.4 sodium citrate buffer solution balances, washing, can obtain the plasma thromboplastin component complex of purity 60-90% with the sodium chloride buffer that contains 0.2 mole.At United States Patent (USP) 5,409, in 990, reported DEAE-sephadex resin with the hydrophilic ethylene based polymer that contains butyl group, with containing 1 mole nacl concentration is that the sodium citrate buffer solution of 15 mM pH7 carries out balance and washing, eluting, and the activity that can make the IX factor in the prothrombin complex product is primary 76%.Emerging Shanghai blood products institute is at " China's blood transfusion magazine " 1998 (4), once proposed in the relevant research report in 196, with the citrate is anticoagulant, with DEAE-sephadex A-50 resin blood plasma is adsorbed and to separate the preparation prothrombin complex with eluting, more satisfactory effect can be arranged." China's blood transfusion magazine " 1997 (2), reported among the 63-66 respectively with DEAE-sephadex A-50 and DEAE-sepharose CL-6B resin glue freezing human plasma has been adsorbed with after eluting separates, through post processings such as S/D inactivation of virus and the improvement gel absorption method that obtains prepares prothrombin complex, the activity recovery of the IX factor can be substantially near 68% in the goods again.Except that the whole bag of tricks of above-mentioned preparation plasma thromboplastin component complex, the goods that only contain single plasma thromboplastin component for preparation, United States Patent (USP) 5,457,181 have reported successively and have carried out adsorbing separation with DEAE-sephadex A-50 and DEAE-sepharose CL-6B, obtain the method for high-purity human blood IX factor goods." medicine biotechnology " 1999.6 (2), reported among the 107-109 successively respectively with DEAE-sephadex A-50, DEAE-sepharose Fast Flow and DEAE-sepharose CL-6B resin and human plasma has been adsorbed with after eluting separates the method for the high-purity human blood IX factor goods after be removed foreign protein and II, VII, the X factor.At United States Patent (USP) 5,714, in the purification process to the IX factor of 583 reports, also to adopt reinforcing YIN-essence ion exchange resin Q-Sepharose Fast Flow, and with containing variable concentrations sodium chloride and/or calcium chloride Tris (Tris) buffer to carry out balance, washing and eluting respectively be example, proposition can also be carried out purification process to the IX factor of being obtained by human plasma or recombinant product with multiple anion exchange resin chromatography.In addition, " Vox.Sang " (" sound of blood ") 1989:57:233-239 is in to the research report that is prepared the semicontinuous method that the plasma thromboplastin component complex carries out by human plasma, to batch formula absorption of carrying out with DEAE-sephadexA-50 and DEAE-sepharose Fast Flow different ions exchanger resin, propose after the comparative study of teeter column chromatography and common column chromatography, with concentration is 0.02 mole, the sodium citrate buffer solution of pH6.0 carries out balance and washing, carry out eluting with this buffer that also contains 0.4 mole nacl simultaneously, the method for teeter column chromatography can make the IX factor response rate in the goods reach 68%.
Find in the practice that the DEAE-sephadex A-50 resin that adopts in above-mentioned many preparation methoies or recommend to use is because its mechanical strength is relatively poor, service life is short, and the rubber cement separation difficulty, and the response rate is low, the production operation difficulty makes the cost of goods higher, and quality also is difficult to stablize.For this reason, West China Journal of Pharmaceutical Sciences 2000 (3), 177-179 is in the development report to the plasma thromboplastin component complex, proposition adopts DEAE-sepharose Fast Flow (the fast glue of diethylamide ethyl-agarose) gel to carry out adsorbing separation separately to people's fresh frozen plasma, activity recovery after this step separates is reached be higher than tradition to use 76.42% level of the DEAE-sephadex A-50 resin response rate.
This shows, in the various adsorption separating methods of preparation plasma thromboplastin component complex, because the difference of the Ion Exchange Medium kind that adopts, and the buffer solution system and/or the concrete index parameter that are used for balance, washing and eluting in operating process are different, can produce remarkable influence to the purity and the activity recovery thereof of resulting product.
Summary of the invention
On the basis of above-mentioned technology, through further further investigation and screening, the present invention proposes a kind ofly when preparation contains the plasma thromboplastin component complex (prothrombin complex) of II, VII, IX, the X factor, can make direct employing DEAEsepharose Fast Flow (the fast glue of diethylamide ethyl-agarose) gel carry out the ion exchange adsorbing separation and obtain the concrete grammar of ideal effect more.This method can keep the activity of II, VII, IX, the X factor on the one hand as much as possible and when reaching the higher response rate, can remove foreigh protein removing again as best one can, improves the purity of goods, to reduce the side effect of its potential hyperamization bolt.
The method of plasma thromboplastin component complex preparation of the present invention is after with DEAE-sepharose Fast Flow gel fresh frozen plasma being carried out adsorbing separation equally, collects the prothrombin complex eluent that contains II, VII, IX, the X factor.Wherein, before adsorbing with DEAE-sepharose Fast Flow, be the 4-7 mM with concentration earlier, particularly 5-6 mM and pH5.5-6.5, the phosphate buffered solution of pH～6.0 particularly, or any in the citrate buffer solution, after in the usual way the gel that adsorbs usefulness being carried out balance, add fresh freezing human plasma again and exchange absorption, identical buffer solution with pH5.5-7.0 washs the absorption gel then, the identical buffer solution of reuse pH6-8 carries out eluting to the absorption gel, collects and contains prothrombin, VII, the plasma thromboplastin component complex of IX and X.
In the said method, in the same separating treatment process of being carried out, ion-exchange gel is carried out the used buffer solution of balance, washing and eluting should remain identical buffer solution system.Experimental result shows when adopting the phosphate-buffered liquid system even more ideal effect to be arranged.
Experimental result shows, and is above-mentioned when exchanger resin is carried out balance, washing and eluting, changes the ionic strength in the used buffer, can produce certain influence to the response rate of goods.Therefore, used various buffer solution all can be regulated its ionic strength with the alkali metal chloride of suitable kind and/or content in the said method.For example, the ionic strength of used equalizing and buffering solution can be regulated with the alkali metal chloride of 0-0.04 mole; The ionic strength of washing buffer solution can be regulated with the alkali metal chloride of 0-0.2 mole; Elution buffer solution also can be used the 0.36-0.6 mole, and particularly the alkali metal chloride of 0.4-0.5 mole is regulated.The said alkali metal chloride that is used to regulate ionic strength generally can use as sodium chloride, potassium chloride---particularly sodium chloride---these alkali metal chlorides the most commonly used.Experimental result shows, when using different salts such as sodium salt and potassium salt accent to regulate ionic strength, though the response rate to product finds no obvious influence, but the price of potassium salt is usually above sodium salt on the one hand, on the other hand, can produce more adverse effect to cardiac function if excessive potassium salt enters in the body, so the quality control requirement of product is very tight, even with the K in the goods
+Content is controlled in the regulation and claimed range that meets medicine management, also K can take place when a large amount of this product of infusion
+Accumulation also must be carried out clinical K
+Monitoring.Therefore, generally speaking to adopt sodium chloride to regulate ionic strength for well.
Proposed by the invention above-mentionedly fresh frozen plasma is carried out the operational approach of adsorbing separation, go in the method for any routines such as common column chromatography method, teeter column chromatography method or batch formula absorption method, using with the absorption gel.
Said method of the present invention adopts anionic DEAE-sepharose Fast Flow gel in the process of blood plasma being carried out adsorbing separation with ion exchange resin, not only simple to operate, and resulting product has still kept the compositing characteristic of prothrombin complex, and can keep effective ingredient II as much as possible, VII, IX, the activity of the X factor, obviously improve the response rate of this class correlation factor, lot of experiment results shows, the response rate of this step operation of the present invention can reach 93.67 ± 2.56% level, after further post processing, it is very ideal more than 80% that the overall recovery of resulting end article also can reach, and the corresponding goods of the also comparable traditional method gained of the specific activity of contained each thrombin have extremely nearly ten times raising of several times in the goods, also make the content of various foreign proteins wherein lower simultaneously, purity is higher, through external thrombus analysis and the non-stagnation model experiment confirm of animal thrombus in vivo, resulting product can reduce the danger that produces the side effect of potential hyperamization bolt significantly.
Below in conjunction with result of the test shown in the drawings,, again foregoing of the present invention is described in further detail by the specific embodiment of some examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only is confined to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 is the HPLC contrast collection of illustrative plates that the inventive method and traditional method resulting product are carried out
Fig. 2 is the SDS-PAGE contrast collection of illustrative plates that the inventive method and traditional method resulting product are carried out
Fig. 3 is the IE contrast collection of illustrative plates that the inventive method and traditional method resulting product are carried out
Fig. 4 is the CIE contrast collection of illustrative plates that the inventive method and traditional method resulting product are carried out
The specific embodiment
Example 1～example 10 adopts is common column chromatography mode, and its method of operating is: with in advance molten In the DEAE-sepharose Fast Flow gel chromatography column of swollen good and dress post, with the long-pending phase of 4-5 times of colloid bodies Answer phosphate buffer or citrate buffer, in the usual way balance chromatography exchange column. Then, with fresh Freezing human plasma under 37 ℃ of conditions so that the unlikely immediate mode separated out of fibrinogen melt and use same volume Be after buffer solution is adjusted to pH7.0, inject the chromatography exchange column and keep the listed corresponding time of repose of each example, complete Become the absorption exchange process. Suitable buffer solution with same system washs chromatography with 2.5 ml/min flow velocitys then Post. After peak to be washed finishes, again in order to sodium chloride be adjusted to desired ion intensity same system buffer solution with 2.5 the speed of ml/min is carried out wash-out operation to the chromatography exchange column, and collect contain required prothrombin, The eluent of the PCC of VII, IX and X. Employing phosphate buffer and citrate buffer Each routine concrete operations condition and the rate of recovery are respectively as shown in Table 1 and Table 2.
Table 1 adopts each routine concrete operations condition and rate of recovery of phosphate buffered liquid system column chromatography
| Operating condition | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| Buffer concentration (mM) | 7 | 6 | 4 | 5 | 6 |
| Equilibrium liquid ionic strength (M) | 0 | 0.02 | 0.04 | 0 | 0 |
| Equilibrium liquid pH | 6 | 6.5 | 5.5 | 5.5 | 6.0 |
| Cleaning solution ionic strength (M) | 0.2 | 0 | 0.1 | 0.1 | 0 |
| Cleaning solution pH | 7 | 5 | 6 | 5.5 | 6.0 |
| Eluent ionic strength (M) | 0.45 | 0.5 | 0.4 | 0.36 | 0.45 |
| Eluent pH | 6 | 6.5 | 7 | 8 | 6.0 |
| The standing adsorption time (min) | 30 | 35 | 25 | 30 | 30 |
| The rate of recovery (%) | 71.0 | 83.2 | 80.4 | 88.7 | 93.7 |
Table 2 adopts each routine concrete operations condition and rate of recovery of citrate buffer system column chromatography
| Operating condition | Example 6 | Example 7 | Example 8 | Example 9 | Example 10 |
| Buffer concentration (mM) | 7 | 6 | 4 | 5 | 6 |
| Equilibrium liquid ionic strength (M) | 0 | 0.02 | 0.04 | 0 | 0 |
| Equilibrium liquid pH | 6 | 6.5 | 5.5 | 5.5 | 6.0 |
| Cleaning solution ionic strength (M) | 0.2 | 0 | 0.1 | 0.1 | 0 |
| Cleaning solution pH | 7 | 5 | 6 | 5.5 | 6.0 |
| Eluent ionic strength (M) | 0.45 | 0.5 | 0.4 | 0.36 | 0.45 |
| Eluent pH | 6 | 6.5 | 7 | 8 | 6.0 |
| The standing adsorption time (min) | 30 | 35 | 25 | 30 | 30 |
| The rate of recovery (%) | 58.3 | 70.6 | 69.0 | 74.5 | 78.4 |
The formula adsorption method is criticized in being that example 11～example 20 adopts, and it is operating as: long-pending with 3-4 times of colloid bodies Corresponding phosphate buffer or citrate buffer, in the usual way to the good DEAE-of swelling After sepharose Fast Flow gel carries out balance, with the Fresh Frozen human plasma under 37 ℃ of conditions so that fiber The unlikely immediate mode of separating out of proteinogen is melted and is adjusted to pH7.0 with the same system buffer solution, and adds absorption Gel, stirring and adsorbing was finished absorption exchange process, then suction filtration in 60 minutes under 4 ℃ of conditions. Use again 3 times to glue Behind the same system buffer solution washing absorption gel of sample volume, suction filtration. Amass and usefulness to colloid bodies with 8～10 times at last The same system buffer solution that sodium chloride is adjusted to desired ion intensity carries out the suction filtration operation of wash-out, and collection contains required The eluent of the PCC of prothrombin, VII, IX and X. Adopt phosphate buffer and citron The routine concrete operations condition of each of phthalate buffer and the rate of recovery are respectively shown in table 3 and table 4.
Table 3 adopts the phosphate buffered liquid system to criticize formula to adsorb each example concrete operations condition and rate of recovery
| Operating condition | Example 11 | Example 12 | Example 13 | Example 14 | Example 15 |
| Buffer concentration (mM) | 7 | 6 | 4 | 5 | 6 |
| Equilibrium liquid ionic strength (M) | 0 | 0.02 | 0.04 | 0 | 0 |
| Equilibrium liquid pH | 6 | 6.5 | 5.5 | 5.5 | 6.0 |
| Cleaning solution ionic strength (M) | 0.2 | 0 | 0.1 | 0.1 | 0 |
| Cleaning solution pH | 7 | 5 | 6 | 5.5 | 6.0 |
| Eluent ionic strength (M) | 0.45 | 0.5 | 0.4 | 0.36 | 0.6 |
| Eluent pH | 6 | 6.5 | 7 | 8 | 6.0 |
| The rate of recovery (%) | 65.4 | 76.1 | 74.5 | 80.2 | 89.9 |
Table 4 adopts the citrate buffer system to criticize formula to adsorb each example concrete operations condition and rate of recovery
| Operating condition | Example 16 | Example 17 | Example 18 | Example 19 | Example 20 |
| Buffer concentration (mM) | 7 | 6 | 4 | 5 | 6 |
| Equilibrium liquid ionic strength (M) | 0 | 0.02 | 0.04 | 0 | 0 |
| Equilibrium liquid pH | 6 | 6.5 | 5.5 | 5.5 | 6.0 |
| Cleaning solution ionic strength (M) | 0.2 | 0 | 0.1 | 0.1 | 0 |
| Cleaning solution pH | 7 | 5 | 6 | 5.5 | 6.0 |
| Eluent ionic strength (M) | 0.45 | 0.5 | 0.4 | 0.36 | 0.6 |
| Eluent pH | 6 | 6.5 | 7 | 8 | 6.0 |
| The rate of recovery (%) | 60.9 | 72.3 | 75.0 | 76.2 | 82.3 |
In each above-mentioned example, with after regulating sodium chloride that ionic strength uses and changing into potassium chloride, to the rate of recovery Find no obvious impact.
The PCC goods that the product that said method of the present invention is collected obtains after post processing are with front State the testing result contrast of clotting factor specific activity and the foreign protein content of the corresponding goods of reported in literature method gained, As shown in table 5.
The clotting factor specific activity of table 5 goods and foreign protein content control experiment result
| Project | The goods of the inventive method gained | The goods of conventional method gained |
| The IX factor is solidified specific activity (U/mg protein) | 7.36±0.96 (n＝6) | 0.747±0.348 (n＝7) 5.07±0.53 (n＝7) |
| II solidifies specific activity (U/mg protein) | 6.16±0.44 (n＝6) | 0.799±0.349 (n＝7) |
| The VIII factor is solidified specific activity (U/mg protein) | 2.08±0.10 (n＝6) | 0.343±0.252 (n＝7) |
| The X factor is solidified specific activity (U/mg protein) | 4.32±0.23 (n＝6) | 0.581±0.199 (n＝7) |
| Fibronectin (Fn) content (mg/L) | 65.61±9.83 (n＝6) | 70.89±51.31 (n＝7) |
| Plasminogen (Pg) content (mg/L) | 5.43±0.71 (n＝6) | 10.71±0.57 (n＝7) |
| Fibrinogen (Fg) content (mg/L) | 0.11±0.02 (n＝6) | / |
The PCC goods of the inventive method gained and the height that is undertaken by aforementioned documents method resulting product Hydraulic fluid phase chromatography (HPLC) results of comparison, shown in two collection of illustrative plates among Fig. 1, wherein A is conventional method system The collection of illustrative plates of product, B are the collection of illustrative plates of the inventive method goods. Peak shape contrast by A, B two collection of illustrative plates is clearly aobvious Illustrate, the purity of the inventive method resulting product is higher than the goods of conventional method. Maximum absorption band in each collection of illustrative plates Molecular weight corresponding to 2 (retention times 28 minutes) is 4-7 ten thousand, just prothrombin, VII, IX, X branch Son amount in-scope; Be positioned at maximum absorption band 2 peak 1 and peak afterwards 3 before and correspond to respectively HMW Albumen and LMWP. The analysis that two collection of illustrative plates are carried out shows, prothrombin in the inventive method goods, The increase of VII, IX, X active ingredient peak shape Area Ratio conventional method goods 16.366%, show that its content has Improve, and the peak shape area of HMW foreign protein has reduced 15.233%, shows that its content reduces to some extent, It is big that the changes of contents of little molecule foreign protein becomes.
SDS-PAGE (the dodecyl sodium sulfate polypropylene that goods by the distinct methods gained are further carried out The acid amides electrophoresis), the contrast collection of illustrative plates of IE (immunoelectrophoresis) and CIE (crossed immunoelectrophoresis) respectively as Fig. 2, Fig. 3 and shown in Figure 4, shown in the A among each figure, the B is conventional method goods and the inventive method system respectively The sample of product, 1 is the LMWP standard. These collection of illustrative plates have equally also clearly shown the inventive method Goods in foreign protein content obviously be less than the goods of conventional method, purity significantly improves.
To through said method of the present invention goods with by in the corresponding goods of aforementioned documents report method gained The results of comparison that detects of activated clotting factor content, as shown in table 6.
The results of comparison of activated clotting factor content in table 6 goods
| Activation factor | The inventive method resulting product (n=3) | Conventional method resulting product (n=3) |
| The specific activity of the IIa factor (mU/ml) | 34.20±1.56 | 79.00±8.45 |
| The specific activity of the IXa factor (mU/ml) | 8.28±0.45 | 69.20±3.26 |
| The specific activity of Xa factor (mU/ml) | 7.50±0.89 | 30.92±3.87 |
The result of table 6 shows that the content of the activated clotting factor in the inventive method goods is well below tradition side The goods of method show that the PCC goods that obtain by preparation method of the present invention can significantly be reduced in to make With in potential thrombus danger.
By external thrombus analysis and body indoors modeling test, can show the thrombogenicity size that goods are potential. (NAPTT) experiment and fibrin ferment when wherein, the external thrombus analysis comprises inactive partial thromboplastin (TGt50) experiment during generation. To through the resulting said products of the inventive method with the conventional method gained The contrast and experiment of these two experiments that corresponding goods carry out is as shown in table 7, and wherein the NAPTR in the table is Ratio during inactive part blood coagulation, that is: the ratio of sample NAPTT value/blank NAPTT value.
The result of table 7NAPTT experiment and TGt50 experiment
| Detect index | The goods of the inventive method gained | The goods of conventional method gained |
| NAPTT (second) | 235.65±6.07 (n＝6) | 204.47±8.82 (n＝6) |
| NAPTR | 0.93±0.05 | 0.82±0.04 (n＝6) |
| TGt50 (branch) | 15.78±2.50 (n＝6) | 7.10±2.21 (n＝6) |
Experimental study in the body that the goods of above-mentioned distinct methods is carried out with the non-stagnation model of mouse has confirmed too The potential thrombus danger that they are different. With after in the goods of the inventive method and the conventional method goods input mouse body SFMC (SFMC), FDP (fibrin degradation product (FDP)), D-Dimer (D-Dimer) and the experiment of t-PA (tissue plasminogen activator) indices content dynamic change situation knot Really, respectively shown in table 8～table 11, wherein with the negative contrast of 25% albumin.
The dynamic change result of SFMC content in the mouse body behind the different goods of table 8 input
| Group | 10 minutes | 25 minutes | 35 minutes | 50 minutes | 80 minutes |
| The growth rate of the inventive method goods group | 0.50 | 1.00 | 1.75 | 2.00 | 1.75 |
| The growth rate of the goods group of conventional method | 0.50 | 1.75 | 2.25 | 3.00 | 2.75 |
| The growth rate of infusion albumin control group | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
The dynamic change result of FDP content in the mouse body behind the different goods of table 9 input
| Group | 10 minutes | 25 minutes | 35 minutes | 50 minutes | 80 minutes |
| The inventive method goods group (μ g/ml) | 1.88±0.27 | 4.38±0.43 | 4.38±0.56 | 8.75±1.05 | 4.38±0.60 |
| The goods group of conventional method (μ g/ml) | 2.19±0.54 | 7.5±0.29 | 13.75±2.07 | 60.00±7.95 | 45.00±8.33 |
| Infusion albumin control group (μ g/ml) | 2.19±0.54 | 4.38±0.37 | 5.31±0.48 | 5.31±0.79 | 3.44±0.56 |
The dynamic change result of D-Dimer content in the mouse body behind the different goods of table 10 input
| Group | 10 minutes | 30 minutes | 50 minutes | 60 minutes | 80 minutes |
| The inventive method goods group (mg/L) | 0.035±0.016 | 0.044±0.029 | 0.113±0.058 | 0.152±0.069 | 0.184±0.107 |
| The goods group (ms/L) of conventional method | 0.030±0.015 | 0.041±0.018 | 0.649±0.121 | 0.660±0.187 | 0.788±0.115 |
| Infusion albumin control group (ms/L) | 0.034±0.012 | 0.040±0.014 | 0.043±0.021 | 0.037±0.011 | 0.036±0.014 |
The dynamic change result of t-PA content in the mouse body behind the different goods of table 11 input
| Group | 10 minutes | 30 minutes | 50 minutes | 60 minutes | 80 minutes |
| The inventive method goods group (μ g/L) | 15.79±11.38 | 27.85±8.14 | 27.27±7.68 | 25.70±13.61 | 43 33±11.31 |
| The goods group of conventional method (μ g/L) | 31.82±8.16 | 36.71±9.47 | 40.31±7.37 | 29.20±4.43 | 84.09±17.54 |
| Infusion albumin control group (μ g/L) | 41.61±8.41 | 42.31±9.37 | 46.50±9.08 | 41.87±7.95 | 34.79±8.93 |
Find after the comparative analysis, import indices behind 25% albumin change little, and input high dose The inventive method goods and the conventional method goods after, increment has in various degree appearred respectively in indices, and The dynamic change of indices in 0-80 minute of conventional method goods group all is higher than the inventive method goods Group has shown that the conventional method goods in vivo than the easier hypercoagulative state that is in of the goods of the inventive method, have indicated The potential danger that thrombus takes place is bigger, and this experimental result then demonstrates the goods of the inventive method can be significantly Reduce the potential danger that thrombus takes place after entering in the body.
Claims (10)
1. the preparation method of plasma thromboplastin component complex, the fast glue of diethylamide ethyl-agarose with anionic carries out the ion exchange adsorbing separation to fresh frozen plasma, it is characterized in that with before the fast glue absorption of diethylamide ethyl-agarose, be after a kind of in the phosphate buffered solution of 4-7 mM and pH5.5-6.5 or the citrate buffer solution carries out balance to the gel that adsorbs usefulness in the usual way earlier with concentration, add fresh freezing human plasma again and exchange absorption, identical buffer solution with pH5.5-7.0 washs the absorption gel then, the identical buffer solution of reuse pH6-8 carries out eluting to the absorption gel, collects and contains prothrombin, VII, the plasma thromboplastin component complex of IX and X.
2. the preparation method of plasma thromboplastin component complex as claimed in claim 1 is characterized in that used buffer solution is that concentration is the phosphate buffer of 5-6 mM.
3. the preparation method of plasma thromboplastin component complex as claimed in claim 1 is characterized in that used buffer solution is that concentration is the citrate buffer of 5-6 mM.
4. as the preparation method of the described plasma thromboplastin component complex of one of claim 1 to 3, it is characterized in that the pH of used equalizing and buffering solution and washing buffer solution is 6.0.
5. as the preparation method of the described plasma thromboplastin component complex of one of claim 1 to 3, it is characterized in that to regulate its ionic strength with alkali-metal chloride in the used various buffer solution.
6. the preparation method of plasma thromboplastin component complex as claimed in claim 5 is characterized in that the ionic strength of used equalizing and buffering solution is regulated with the sodium chloride of 0-0.04 mole.
7. the preparation method of plasma thromboplastin component complex as claimed in claim 5 is characterized in that the ionic strength of used washing buffer solution is regulated with the sodium chloride of 0-0.2 mole.
8. the preparation method of plasma thromboplastin component complex as claimed in claim 5 is characterized in that used elution buffer solution regulates with the sodium chloride of 0.36-0.6 mole.
9. the preparation method of plasma thromboplastin component complex as claimed in claim 8, the ionic strength that it is characterized in that used elution buffer solution is the 0.4-0.5 mole.
10. as the preparation method of the described plasma thromboplastin component complex of one of claim 1 to 3, it is characterized in that the adsorbing separation operation fresh frozen plasma carried out with the absorption gel, can adopt any in common column chromatography method, teeter column chromatography method or batch formula absorption method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396494A (en) * | 2013-04-27 | 2013-11-20 | 江苏省疾病预防控制中心 | Monoclonal antibody of blood coagulation factor IX |
| CN103688177A (en) * | 2011-06-17 | 2014-03-26 | 学校法人东日本学园 | Method of measuring blood coagulation time to detect lupus anticoagulants |
| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
-
2001
- 2001-08-16 CN CN01108741A patent/CN1126758C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103688177A (en) * | 2011-06-17 | 2014-03-26 | 学校法人东日本学园 | Method of measuring blood coagulation time to detect lupus anticoagulants |
| CN103688177B (en) * | 2011-06-17 | 2017-07-04 | 学校法人东日本学园 | The assay method of lupus anticoagulant analyte detection blood coagulation time |
| US9970046B2 (en) | 2011-06-17 | 2018-05-15 | School Juridical Person Higashi-Nippon-Gakuen | Method of measuring blood coagulation time to detect lupus anticoagulants |
| CN103396494A (en) * | 2013-04-27 | 2013-11-20 | 江苏省疾病预防控制中心 | Monoclonal antibody of blood coagulation factor IX |
| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
| CN104560925B (en) * | 2014-12-30 | 2017-11-03 | 山东泰邦生物制品有限公司 | A kind of method that activation prepares human thrombin in chromatography waste liquid from humanclottingfactorⅨ |
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| Publication number | Publication date |
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| CN1126758C (en) | 2003-11-05 |
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