CN102533906B - Enoxaparin sodium enzymolysis method - Google Patents

Abstract

The invention discloses an enoxaparin sodium enzymolysis method, which comprises the following steps of: adding a sodium/calcium acetate solution and a mixed enzyme solution into an enoxaparin sodium solution, and performing enzymolysis at the temperature of between 29 and 31DEG C for 66 to 72 hours, wherein the pH value of the sodium/calcium acetate solution is 7; and an enzyme activity ratio of heparinase I to heparinase II to heparinase III in the mixed enzyme is (0.95-1.05):1:(0.45-0.55). An enzymolysis product obtained through the method is subjected to high performance liquid chromatography (HPLC) detection, peak areas of 17 groups of peaks are larger than 0.3 percent of the total area, and the results are obviously superior to those detected under other conditions. The invention provides the optimized enoxaparin sodium enzymolysis process, the consumption of the heparinase III is obviously reduced, the cost is saved, the process is simple, and the detection speed is improved.

Description

A kind of Enoxaparin sodium enzymolysis method

Technical field

The invention belongs to the heparin sodium production field, be specifically related to a kind of enzyme solution of Enoxaparin Sodium.

Background technology

Heparin sodium is mucopolysaccharide sulfuric acid ester anticoagulant, is the sodium salt of the CSSO3 extracted in the intestinal mucosa by pig or ox, belongs to the mucopolysaccharide material.Recent study proof heparin sodium also has reducing blood lipid.Low molecular sodium heparin refers to utilize the whole bag of tricks, by heparin sodium, is the lower heparin sodium of molecular weight that the raw material cracking obtains, and Enoxaparin Sodium is exactly a kind of low molecular sodium heparin wherein.

Heparin sodium is by being combined with ATIII and suppressing active anti-bolt, the anticoagulation brought into play of the IIa factor and Xa factor, and with ATIII in conjunction with thing by being randomly distributed in specific pentasaccharides molecular regulation in the heparin sodium molecule.Heparin sodium is combined with ATIII, Xa, makes the Xa inactivation and brings into play anti-bolt effect, and this process needs 5 glycosyl units to get final product; Same heparin sodium is combined with ATIII, IIa, make the IIa inactivation and anticoagulation occurs, but 18 glycosyl units of this process need is condition.And in the heparin sodium total amount, the amount greater than 18 glycosyl units accounts for 90%, and in the low molecular sodium heparin total amount, the amount greater than 18 glycosyl units is no more than 50%, and this has just determined that low molecular sodium heparin has higher anti-Xa factor activity and lower anti-IIa factor active.

Low molecular heparin is due to preparation method's difference, and its biochemistry has significant difference with pharmacological properties, and in body, anti-thrombus activity and hemorrhage side effect are also different.Low molecular heparin be by unfractionated heparin through cracking and fragment, NMR studies have shown that, different Low molecular heparin there are differences at the aspects such as type of molecular weight, molecular weight distribution, end structure, thioesterification, and these differences can cause corresponding bioactive difference, comprise that the ability of with ATIII, being combined, anti-IIa factor active and Xa factor are active.

Due to the structure of low molecular sodium heparin diversity relatively, therefore that the structure of low molecular sodium heparin is determined is extremely important.Enoxaparin Sodium is as a kind of low molecular sodium heparin, wherein a test item is structure detection, because the molecular weight of Enoxaparin Sodium is larger, while utilizing high performance liquid chromatography to detect, the sample ultraviolet absorption peak is overlapping serious, the peak area data that calculate are inaccurate, therefore detect after its structure need to being utilized heparinase be cracked into small segment again.Relate to Heparinase I, II and III in this step, in USP, three kinds of enzyme dosages are heparinase I (Heparinase I): heparinase II (Heparinase I I): heparinaseIII (III)=1: 1: 1, the market value of Heparinase I, II and III is approximately 6000 yuan/unit, because the market value of these three kinds of enzymes is quite high, the enzymolysis cost also according to height not under.

Summary of the invention

The objective of the invention is to provide on the basis of existing technology the Enoxaparin sodium enzymolysis method that a kind of cost is lower and effect is better.

Purpose of the present invention can reach by following measures:

A kind of Enoxaparin sodium enzymolysis method, it is specially: adding sodium acetate/calcium solution and mixed enzyme solution in the enoxaparin sodium solution, is 29～31 ℃ of lower enzymolysis 66～72 hours in temperature; The pH value of wherein said sodium acetate/calcium solution is 7, and in described mixed enzyme, the enzyme of each enzyme ratio of living is Heparinase I: Heparinase I I: Heparinase I II=0.95～1.05: 1: 0.45～0.55.

In mixed enzyme, the preferred enzyme of each enzyme ratio of living is Heparinase I: Heparinase I I: Heparinase I II=1: 1: 0.5.

The concentration of described enoxaparin sodium solution (being the aqueous solution) is 15～25mg/ml, preferred 20mg/ml.

The volume of sodium acetate/calcium solution is 3～4 times of Enoxaparin Sodium liquor capacity, preferred 3.5 times.

The mixed enzyme solution of indication of the present invention is dissolved in phosphate buffer solution and is made by Heparinase I, Heparinase I I and Heparinase I II, perhaps Heparinase I solution, Heparinase I I solution and Heparinase I II first are dissolved in respectively in phosphate buffer solution, make after remix, in described mixed enzyme solution, the concentration of each enzyme is 0.09～0.4IU/mL, preferred 0.1～0.2IU/mL.

In this law, the volume of mixed enzyme solution is 4.5～5.5 times of Enoxaparin Sodium liquor capacity, preferred 5 times.

In the present invention, the compound method of phosphate buffer solution is: after potassium primary phosphate and bovine serum albumin together are dissolved in the water, regulate pH value to 7.0, use membrane filtration after thin up; The mass ratio of described potassium primary phosphate and bovine serum albumin is 6.7～7: 1, and preferred 6.8: 1, in phosphate buffer solution, the concentration of potassium primary phosphate was 1.3mg/ml～1.4mg/ml, is preferably 1.36mg/ml; The filter membrane aperture is below 0.45 μ m.

In the present invention, the compound method of sodium acetate/calcium solution is: bovine serum albumin and lime acetate are dissolved in the water, add that to regulate the pH value with sodium hydroxide after Glacial acetic acid be 7.0, use membrane filtration after thin up; The mass ratio of bovine serum albumin and lime acetate is 1: 3.0～3.4, preferred 1: 3.2, the mass volume ratio of bovine serum albumin and Glacial acetic acid was 1: 55～60mg/ μ L, preferred 1: 58mg/ μ L, in sodium acetate/calcium solution, the concentration of lime acetate is 0.30～0.34mg/ml, preferred 0.32mg/ml; The filter membrane aperture is below 0.45 μ m.

Beneficial effect of the present invention:

The enzymolysis product that present method obtains detects by HPLC, record have 17 groups of peaks the peak area area greater than 0.3% of the total area, obviously be better than the result that other conditions record.The enzymolysis Enoxaparin Sodium technique of optimization provided by the invention, the consumption of Heparinase I II obviously reduces, and saving cost and technique are simple, the raising detection speed.

Embodiment

Instrument:

(Agilent 1200 for high performance liquid chromatograph, UV-detector), XS-205 electronic balance (plum Teller), PURELAB Classic ultrapure water machine, ultrasonic cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd.), glass core suction funnel (Tian Jinjin rises glassware company limited), PHS-3C type pH meter.

Reagent:

Enoxaparin Sodium standard substance (USP), Heparinase I, Heparinase I I, Heparinase I II, ultrapure water, SODIUM PHOSPHATE, MONOBASIC (analytical pure), sodium perchlorate (analytical pure), phosphoric acid (analytical pure), lime acetate (analytical pure), bovine serum albumin (biochemical reagents), Glacial acetic acid (analytical pure), sodium hydroxide (analytical pure), potassium hydroxide (analytical pure).

The preparation of reagent:

Mobile phase A: precision takes the 0.280g SODIUM PHOSPHATE, MONOBASIC, adds after the 950ml ultrapure water dissolves and uses phosphorus acid for adjusting pH to 3.0, is diluted to 1000ml, obtains.

Mobile phase B: precision takes 0.280g SODIUM PHOSPHATE, MONOBASIC and 140g sodium perchlorate, adds after the 950ml ultrapure water dissolves and uses phosphorus acid for adjusting pH to 3.0, is diluted to 1000ml, obtains.

Mobile phase A and Mobile phase B with 0.45 μ m membrane filtration after, degassed rear use.

Phosphate buffer solution (pH7.0): precision takes potassium primary phosphate 68mg, add bovine serum albumin 10mg, put in the 50ml volumetric flask, adding the 30ml ultrapure water dissolves, measure pH, regulate pH7.0 with potassium hydroxide if needed, with ultrapure water, be diluted to scale, shake up, with using after 0.45 μ m membrane filtration.

Sodium acetate/calcium solution (pH7.0): precision takes bovine serum albumin 10mg, adds the 32mg lime acetate, after adding the ultrapure water dissolving of 60ml, add 580 μ L Glacial acetic acid, regulating pH with the sodium hydroxide of 2M is 7.0, and solution is transferred in the 100ml volumetric flask, be diluted to scale with ultrapure water, shake up.With using after 0.45 μ m membrane filtration.

Heparinase I solution: taking heparin enzyme I1 bottle, add phosphoric acid buffered soln (pH7.0) appropriate, dissolve the solution that obtains 0.4IU/ml (validity period :-20 ℃ 3 months, take out before using).

Heparinase I I solution: taking heparin enzyme II1 bottle, add phosphoric acid buffered soln (pH7.0) appropriate, dissolve the solution that obtains 0.4IU/ml (validity period :-20 ℃ 3 months, take out before using).

Heparinase I II solution: taking heparin enzyme III1 bottle, add phosphoric acid buffered soln (pH7.0) appropriate, dissolve the solution that obtains 0.4IU/ml (validity period :-20 ℃ 3 months, take out before using).

Chromatographic condition:

Chromatographic column	Waters spherisorb SAX(4.6mm×250mm)
		Instrument	Agilent 1200
Detector	UV-detector
		Column temperature	50℃

Flow velocity	1ml/min
		Sampling volume	10μl
Wavelength	234nm

Program setting:

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	97	3
20	65	35
			50	0	100
60	0	100
			61	97	3
79	97	3

The heparinase hybrid mode:

The preparation of Enoxaparin Sodium sample solution:

Precision takes Enoxaparin Sodium standard 200mg, puts in the volumetric flask of 10ml, adds ultrapure water and dissolves and be diluted to scale, shakes up, and obtains (20mg/ml).Accurate draw solution 20 μ L, add 70 μ L sodium acetate/calcium solutions (pH7.0), adds the heparinase mixed solution of 100 μ L different methods, mixes gently, presses the different methods experiment, finally enters liquid chromatography and record chromatographic peak.

Experimental technique:

Experimental result:

Enoxaparin Sodium with method 1 method 30 ℃ of lower enzymolysis results:

Enoxaparin Sodium with method 2 methods 25 ℃ of lower enzymolysis results:

Enoxaparin Sodium with method 2 methods 30 ℃ of lower enzymolysis results:

Enoxaparin Sodium with method 3 methods 25 ℃ of lower enzymolysis results:

Enoxaparin Sodium with method 3 methods 30 ℃ of lower enzymolysis results:

Interpretation of result:

Utilize similar factors to analyze result, the similar factors calculation formula: $f2=50\×\lg \left\{\right[1+(1/n){\mathrm{\Σ}}_{n-1}^n{\left(100\right)}^2]-1/2\×100\}$ Calculate:

Method	method 1(30℃)	method 2(25℃)	method 2(30℃)	method 3(25℃)	method 3(30℃)
						f2	95	98	99	80	85

Can find out from similar factors: the similar factors when method 2 reacts under 30 ℃ is the highest, peak area is the most approaching completely with final enzymolysis for the peak area at explanation obtains under the method each peak, and the peak number amount that only the sample enzymolysis splits minute under the method is maximum, and the most complete of under the method sample enzymolysis is described.

As can be seen from the above table, when method 2 reacts under 30 ℃ 72 hours with the similar factors of 96 hours near 100, illustrate 72 hours basically identical with the result of 96 hours enzymolysis, namely enzymolysis was complete at 72 hours for sample, the time continue to extend hydrolysis result again without affecting.

Can draw by above-mentioned analysis, optimum enzymatic hydrolysis condition is Heparinase I: Heparinase I I: Heparinase I II=1: 1: 0.5, temperature was 30 ℃, and enzymolysis time is 66 to 72 hours.

Claims (7)

1. an Enoxaparin sodium enzymolysis method, is characterized in that adding sodium acetate/calcium solution and mixed enzyme solution in the enoxaparin sodium solution, in temperature, is 29～31 ℃ of lower enzymolysis 66～72 hours; The pH value of wherein said sodium acetate/calcium solution is 7, and in described mixed enzyme, the enzyme of each enzyme ratio of living is heparinase I: heparinaseⅡ: heparinase III=1:1:0.5; The concentration of wherein said enoxaparin sodium solution is 15～25mg/ml; The volume of described sodium acetate/calcium solution is 3～4 times of Enoxaparin Sodium liquor capacity.

2. Enoxaparin sodium enzymolysis method according to claim 1, it is characterized in that described mixed enzyme solution is dissolved in phosphate buffer solution and is made by Heparinase I, Heparinase I I and Heparinase I II, perhaps Heparinase I solution, Heparinase I I solution and Heparinase I II first are dissolved in respectively in phosphate buffer solution, make after remix; In described mixed enzyme solution, the concentration of each enzyme is 0.09～0.40 IU/ml.

3. Enoxaparin sodium enzymolysis method according to claim 1 and 2, the volume that it is characterized in that described mixed enzyme solution is 4.5～5.5 times of Enoxaparin Sodium liquor capacity.

4. Enoxaparin sodium enzymolysis method according to claim 2 is characterized in that the compound method of described phosphate buffer solution is: after potassium primary phosphate and bovine serum albumin together are dissolved in the water, regulate pH value to 7.0, use membrane filtration after thin up; The mass ratio of described potassium primary phosphate and bovine serum albumin is 6.7～7:1; In phosphate buffer solution, the concentration of potassium primary phosphate is 1.3mg/ml～1.4mg/ml; The aperture of described filter membrane is below 0.45 μ m.

5. Enoxaparin sodium enzymolysis method according to claim 4, the mass ratio that it is characterized in that described potassium primary phosphate and bovine serum albumin is 6.8:1; In phosphate buffer solution, the concentration of potassium primary phosphate is 1.36mg/ml; The filter membrane aperture is 0.45 μ m.

6. Enoxaparin sodium enzymolysis method according to claim 1, the compound method that it is characterized in that described sodium acetate/calcium solution is: bovine serum albumin and lime acetate are dissolved in the water, add that to regulate the pH value with sodium hydroxide after Glacial acetic acid be 7.0, use membrane filtration after thin up; The mass ratio of bovine serum albumin and lime acetate is 1:3.0～3.4, and the mass volume ratio of bovine serum albumin and Glacial acetic acid is 1:55～60mg/ μ L; In sodium acetate/calcium solution, the concentration of lime acetate is 0. 30～0.34mg/ml; The filter membrane aperture is below 0.45 μ m.

7. Enoxaparin sodium enzymolysis method according to claim 6, the mass ratio that it is characterized in that bovine serum albumin and lime acetate is 1:3.2, the mass volume ratio of bovine serum albumin and Glacial acetic acid is 1:58mg/ μ L, and in sodium acetate/calcium solution, the concentration of lime acetate is 0.32mg/ml; The filter membrane aperture is 0.45 μ m.