CN104560926A - Preparation method of trypsin and pharmaceutical composition containing trypsin - Google Patents

Abstract

The invention discloses a preparation method of trypsin and a pharmaceutical composition containing trypsin. The preparation method comprises the following processing steps: (1) mincing the raw materials to extract zymogen; (2) carrying out grading salting-in and salting-out to obtain a crude trypsin product; (3) feeding the crude product and carrying out multi-crystallization; (4) activating; (5) crystallizing; (6) dialyzing; (7) freeze drying. According to the preparation method, a set of scaled production process is established and the advantages of simplicity and low cost are provided; the titer of the prepared trypsin competitive product is above 2500 unit/mg, so that the requirement of Chinese Pharmacopoeia (2010) is met and extremely high market competitiveness is provided.

Description

A kind of tryptic preparation method and containing tryptic pharmaceutical composition

Technical field

The invention belongs to field of biological pharmacy, the tryptic bio-chemistry separation technology of designer drug.Specifically employing is saltoutd, multiple crystallization combines the technology such as dialysis to extract trypsin.

Background technology

Trypsin is a kind of protease that separation and purification obtains from ox pancreas or Pancreas Sus domestica gland.Its main function has: pus, sputum, blood clot etc. can be made to decompose, thinning, is easy to drain and gets rid of, accelerate wound surface purification, promote that granulation tissue is newborn, also have anti-inflammatory effect in addition.Clinically for local edema, hematoma and abscess etc. that empyema, hemothorax, surgery inflammation, ulcer, traumatic damage, fistula etc. produce.Spraying sucks, for respiratory tract disease.Also can be used for treating venom.Also be usually used in the front process to tissue of animal cell culture.

It is quite difficult for only extracting trypsin by traditional method.In July, 2006, A Min pharmaceutical Co. Ltd in Shanghai disclosed a kind of preparation method (200610023582.0) of Trypsin-chymotrypsin, and it adopts, and low temperature acid is carried, gradient is saltoutd, frozen water is dialysed, the Product Process of affinity chromatograph, alcohol chromatography, obtains Trypsin-chymotrypsin.The loyal core of in January, 2011 horse etc. has applied for a kind of method (200910170682.X) extracting Trypsin-chymotrypsin, which employs while CaCl2 activates that Trypsin-chymotrypsin is former and becomes Trypsin-chymotrypsin and add (NH4) 2SO4 of saturation, make it generate calcium sulfate precipitation absorption Trypsin-chymotrypsin, through eluting, saltout, freeze-dried powder that ultrafiltration and lyophilizing obtain the Trypsin-chymotrypsin of white.Product is Trypsin-chymotrypsin, does not have simple trypsin extraction separation and purification.

Summary of the invention

Object of the present invention is exactly to produce high-quality trypsin and reduce production cost, being transformed, especially the transformation of parameter to traditional handicraft, provides a kind of extraction trypsin method.

Technical scheme of the present invention is: utilize sulfuric acid solution to be extracted by the proenzyme contained in pancreatic cell, then according to the principle of isoelectric precipitation, regulate pH, ammonium sulfate fractionation salt is molten, saltout Precipitations such as trypsinogen, then by after trypsin dissolving crude product, molten through salt, saltout, activate with active chymotrypsin again, the enzymatic solution be activated again through the method for crystallization dialysis, lyophilizing, the trypsin of higher degree, comprise the steps:

1, the rough production operation of trypsin:

(1) abstraction process: fresh pancreas is rubbed into pancreas slurry, weigh.Add 0.1 ~ 0.2mol/L sulfuric acid solution of 2 ~ 3L by every kilogram of pancreas slurry, flood 20 ~ 28 hours, macerate is put sock filtration, after being filtered dry, discards filtering residue, leave and take filtrate;

(2) the molten operation of salt: add ammonium sulfate in impregnation liquid, reach 30 ~ 50% saturations, leaves standstill 10 ~ 16 hours, filters, leaves and takes filtrate;

(3) salting-out procedures: add ammonium sulfate in filtrate, reaches 50 ~ 80% saturations, leaves standstill 10 ~ 16 hours, filters, leaves and takes filter cake;

(4) every gram of filter cake adds the water dissolution of 2 ~ 5ml, repeats above-mentioned (2), (3) operation;

(5) every gram of filter cake water dissolution of 1 ~ 3ml, then adds the saturated ammonium sulfate solution of 0.1 ~ 1ml in every gram of filter cake, regulates pH to 4.0 ~ 6.0;

(6) Crystallization Procedure: solution leaves standstill 24 ~ 72 hours in 20 ~ 30 DEG C of temperature, has needle-like Chymotrypsin crystallization; Centrifugal, regulate pH to 2.0 ~ 5.0, add 300 ~ 400g solid ammonium sulfate in every 1000ml solution, reach 50 ~ 80% saturations, leave standstill 12 ~ 16 hours, sucking filtration;

(7) drying process: taking precipitate, dry, obtain trypsin crude product.

2, trypsin refines production operation:

(1) feed intake: take trypsin crude product, add the water stirring and dissolving of 2 ~ 4ml by every gram of trypsin crude product, regulate pH to 3.0 ± 0.5, to help it to dissolve, stir 10 ~ 16 hours; Dosing Solution volume, adds solid ammonium sulfate in 400 ~ 500g/L ratio, after it dissolves, staticly settles 10 ~ 16 hours;

(2) salt is molten: filtered by solution, weighs filter cake weight, first adds the water of 2 ~ 4ml by every gram of filter cake, after stirring and dissolving, regulates pH to 3.0 ± 0.4, then adds the saturated ammonium sulfate solution stand precipitation 10 ~ 16 hours of 1 ~ 3ml by every gram of filter cake;

(3) saltout: solution is filtered, measures filtrate volume, add the saturated ammonium sulfate solution of equal volume amounts, staticly settle 10 ~ 16 hours;

(4) wash magnesium: discarded by supernatant, precipitate vacuum filtration, and on filter cake, add saturated Adlerika, leave standstill 1 minute, sucking filtration, treat that filtrate starts to flow out, Adlerika remaining on funnel is inclined, filter cake is taken out and weighs, to be activated;

(5) activate: claim filter cake weight to be A kilogram, filter cake is dissolved in 0.001 ~ 0.01mol/L hydrochloric acid solution that 3A ~ 5A rises, separately add 0.5 ~ 2mol/L calcium chloride solution A ~ 3A to rise and pH7.5 ~ 8.5 borate buffer 4A ~ 6A liter, add the water that 6A ~ 9A rises again, regulate pH to 7.0 ~ 8.0, the crystallized trypsin that finally every gram of filter cake adds 5 ~ 15mg activates, solution is set to 0 ~ 10 DEG C in leave standstill and activate for 60 ~ 80 hours, pH controls 7.0 ~ 8.0;

(6) deliming: the solution after activation, regulate pH to 3.0 ± 0.4, solid ammonium sulfate is added again by 200 ~ 300g/L, stirring and dissolving, deposits and makes calcium sulfate precipitation in 32 ~ 60 hours in 0 ~ 10 DEG C, filters, filtrate adds solid ammonium sulfate by 150 ~ 250g/L, in 0 ~ 10 DEG C, leave standstill 10 ~ 14 hours, filter, obtain filter cake;

(7) crystallization: add 1 ~ 2mlpH8.0 ~ 10.0 borate buffer by every gram of filter cake, regulates pH to 8.0 ± 0.4, stirs, then solution is filled bag filter, be dipped in outer transparent liquid when 0 ~ 10 DEG C, crystallization 3 ~ 7 days;

(8) dialyse: take out crystal solution and filter to obtain crystal, then add 1 ~ 3ml water dissolution by every gram of crystal, regulate pH to 3.0 ± 0.5; By solution dialysis 2 ~ 4 days, remove impurity, once, temperature controls at 0 ~ 10 DEG C every 10 ~ 14 hours dialysis Chi Huanshui;

(9) lyophilizing: take out dialysis solution, add a small amount of kieselguhr, sucking filtration, obtain filtrate, regulates pH to 6.0 ± 0.4, obtains crystallized trypsin finished product after entering freeze dryer lyophilizing.

Compared with prior art, advantage of the present invention and good effect are:

The present invention adopt saltout, multiple crystallization combines the technology such as dialysis to extract trypsin, by the improvement and bring new ideas to traditional handicraft, not only produced high-quality trypsin, had again simply simultaneously, the advantage such as low cost.

The present invention has set up the production technology of a set of scale, and the trypsin competitive product of preparation is tired up to more than 2500 units/milligram, meets " Chinese Pharmacopoeia " (2010 editions) requirement, has the great market competitiveness.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention will be further described.

Embodiment 1

1, the rough production operation of trypsin:

1. abstraction process:

1.1 fresh pancreases, with tweezers and shears by removings such as adipose connective tissues, put in pulverizer and rub, weigh 15Kg;

1.2 rub thing puts in Plastic Drum, floods 24 hours, the interim stirring per hour of dipping 1 time with 0.125mol/L sulfuric acid solution 30L;

Macerate is put sock filtration by 1.3, and filtering residue floods 1 hour with 0.125mol/L sulfuric acid solution 15L again, discards filtering residue after being filtered dry;

2. molten, the salting-out procedures of salt:

Twice impregnation liquid merges by 2.1, and volume is 44.9L, puts in Plastic Drum, adds solid ammonium sulfate 242g, reaches 40% saturation, add 10.866Kg solid ammonium sulfate altogether in every 1000ml solution, leaves standstill 12 hours;

2.2 filter, and leave and take filtrate, filtrate volume is 44L, and solid discards;

2.3 add solid ammonium sulfate 205g in every 1000ml clear liquid, reach 70% saturation, share ammonium sulfate 9.020kg, leave standstill 12 hours;

2.4 filter, and leave and take filter cake, weigh 269.2g, abandons filtrate;

2.5 use 807.6ml water dissolution, then by 2.1,2.2,2.3,2.4 step gradation reinforcing body ammonium sulfate nearly 40% and 70% saturation precipitations;

2.6 get 70% saturation precipitate, and weigh 178.5g, uses 267.8ml water dissolution, add 89.3ml saturated ammonium sulfate solution, and regulate pH to 5.00 with 5mol/L sodium hydroxide solution;

2.7, by solution left standstill 25 DEG C of calorstats 48 hours, have the Chymotrypsin crude crystalline of needle-like to separate out;

2.8 filter, mother solution 0.25mol/L sulfur acid for adjusting pH to 3.0, and its volume is that 328.4ml adds ammonium sulfate 100.8g, leave standstill 12 hours;

3. drying process:

Taking precipitate, puts vacuum drying oven inner drying, obtains trypsin crude product, and weigh 100.0g, and namely every kilogram of pancreas can obtain 6.67 grams of trypsin.

2, trypsin refines production operation:

1. feed intake

1.1 take trypsin crude product 100.0g, first use 300ml purified water stirring and dissolving, add 2.5mol/L sulfuric acid solution and regulate pH to 3.0, to help it to dissolve, stir 12 hours;

1.2 Dosing Solution volume 298.4ml, add solid ammonium sulfate 140.9g, after it dissolves, staticly settle 12 hours;

Solution for vacuum is filtered by 1.3, weighs filter cake weight 96.6g, adds purified water 289.9ml, adds 2.5mol/L sulfuric acid solution and regulates pH to 3.0, add saturated ammonium sulfate solution 193.2ml, quiescent setting 12 hours after stirring and dissolving;

Solution filters by 1.4, measures filtrate volume 479.3ml, adds saturated ammonium sulfate solution 479.3ml, staticly settle 12 hours;

1.5 wash magnesium: discarded by supernatant, precipitate vacuum filtration, and on filter cake, add saturated Adlerika, leave standstill 1 minute, sucking filtration, treat that filtrate starts to flow out, are inclined by Adlerika remaining on funnel, filter cake is taken out the 93.3g that weighs, to be activated;

2. activate

Filter cake is dissolved in 373.2ml0.005mol/L hydrochloric acid solution, separately add 1mol/L calcium chloride solution 186.6ml and pH8.0 borate buffer 466.5ml, add 746.4ml purified water again, regulate pH to 7.4, finally add the higher crystallized trypsin of 933mg vigor and carry out activation processing, left standstill in 5 ~ 10 DEG C by solution and activate for 72 hours, second day surveys pH value is 7.4;

3. deliming

Solution after activation 2.5mol/L sulfuric acid solution regulates pH to 3.1, liquor capacity is 1873ml, add solid ammonium sulfate 455.2g again, stirring and dissolving, deposits and makes calcium sulfate precipitation in 48 hours in 0 ~ 10 DEG C, filters, filtrate adds solid ammonium sulfate 369.0g, leave standstill 12 hours at 0 ~ 10 DEG C, filter, obtain filter cake weight 45.6g;

4. crystallization

Filter cake adds pH9.0 borate buffer 68.4ml, and regulate pH to 8.0 with 2.5mol/L sulfuric acid solution, stir, then solution is filled bag filter, 5 ~ 10 DEG C are dipped in outer transparent liquid, crystallization 5 days, and every 2 hours shaking ladles once;

5. dialyse

Take out crystal solution and filter to obtain crystal 28.7g, add purified water 43.1ml and dissolve, with 2.5mol/L sulfur acid for adjusting pH to 3.0; Solution is filled bag filter, is then hung in dialysis pond and dialyses 3 days, remove salt impurity, once, temperature controls at 5 ~ 10 DEG C every 12 hours dialysis Chi Huanshui;

6. lyophilizing

Take out dialysis solution, add a small amount of kieselguhr, use buchner funnel sucking filtration, obtain filtrate, regulate pH to 6.0 with 5mol/L sodium hydroxide solution, obtain crystallized trypsin finished product 6.2g after entering freeze dryer lyophilizing, its activity is tired as 2600iu/mg after measured;

Embodiment 2

1, the rough production operation of trypsin:

1. abstraction process:

1.1 fresh pancreases, with tweezers and shears by removings such as adipose connective tissues, put in pulverizer and rub, weigh 10Kg;

1.2 rub thing puts in Plastic Drum, floods 24 hours, the interim stirring per hour of dipping 1 time with 0.150mol/L sulfuric acid solution 25L;

Macerate is put sock filtration by 1.3, and filtering residue floods 1 hour with 0.150mol/L sulfuric acid solution 12.5L again, discards filtering residue after being filtered dry;

2. molten, the salting-out procedures of salt:

Twice impregnation liquid merges by 2.1, and volume is 37.4L, puts in Plastic Drum, adds solid ammonium sulfate 225g, reaches 35% saturation, add 8.415Kg solid ammonium sulfate altogether in every 1000ml solution, leaves standstill 12 hours;

2.2 filter, and leave and take filtrate, filtrate volume is 36.8L, and solid discards;

2.3 add solid ammonium sulfate 186g in every 1000ml clear liquid, reach 65% saturation, share ammonium sulfate 6.845kg, leave standstill 12 hours;

2.4 filter, and leave and take filter cake, weigh 180.2g, abandons filtrate;

2.5 use 720.8ml water dissolution, then by 2.1,2.2,2.3,2.4 step gradation reinforcing body ammonium sulfate nearly 35% and 65% saturation precipitations;

2.6 get 65% saturation precipitate, and weigh 90.5g, uses 181ml water dissolution, add 54.3ml saturated ammonium sulfate solution, and regulate pH to 5.10 with 5mol/L sodium hydroxide solution;

2.7, by solution left standstill 25 DEG C of calorstats 48 hours, have the Chymotrypsin crude crystalline of needle-like to separate out;

2.8 filter, mother solution 0.25mol/L sulfur acid for adjusting pH to 2.9, and its volume is that 234.7ml adds ammonium sulfate 82.1g, leave standstill 15 hours;

3. drying process:

Taking precipitate, puts vacuum drying oven inner drying, obtains trypsin crude product, and weigh 60.9g, and namely every kilogram of pancreas can obtain 6.09 grams of trypsin

2, trypsin refines production operation:

1. feed intake

1.1 take trypsin crude product 60.9g, first use 152.25ml purified water stirring and dissolving, add 3.0mol/L sulfuric acid solution and regulate pH to 3.1, to help it to dissolve, stir 12 hours;

1.2 Dosing Solution volume 160.3ml, add solid ammonium sulfate 64.1g, after it dissolves, staticly settle 12 hours;

Solution for vacuum is filtered by 1.3, weighs filter cake weight 50.1g, adds purified water 21.1ml, adds 2.5mol/L sulfuric acid solution and regulates pH to 3.2, add saturated ammonium sulfate solution 15.8ml, quiescent setting 12 hours after stirring and dissolving;

Solution filters by 1.4, measures filtrate volume 36.5ml, adds saturated ammonium sulfate solution 36.5ml, staticly settle 15 hours;

1.5 wash magnesium: discarded by supernatant, precipitate vacuum filtration, and on filter cake, add saturated Adlerika, leave standstill 1 minute, sucking filtration, treat that filtrate starts to flow out, are inclined by Adlerika remaining on funnel, filter cake is taken out the 48.2g that weighs, to be activated;

2. activate

Filter cake is dissolved in 241.0ml0.01mol/L hydrochloric acid solution, separately add 1.5mol/L calcium chloride solution 96.4ml and pH8.5 borate buffer 192.8ml, add 337.4ml purified water again, regulate pH to 7.5, finally add the higher crystallized trypsin of 480mg vigor and carry out activation processing, left standstill in 5 ~ 10 DEG C by solution and activate for 72 hours, second day surveys pH value is 7.4;

3. deliming

Solution after activation 3.0mol/L sulfuric acid solution regulates pH to 3.2, liquor capacity is 820ml, add solid ammonium sulfate 205.0g again, stirring and dissolving, deposits and makes calcium sulfate precipitation in 54 hours in 0 ~ 10 DEG C, filters, filtrate adds solid ammonium sulfate 160.0g, leave standstill 12 hours at 0 ~ 10 DEG C, filter, obtain filter cake weight 24.0g;

4. crystallization

Filter cake adds pH9.0 borate buffer 48.0ml, and regulate pH to 8.1 with 3.0mol/L sulfuric acid solution, stir, then solution is filled bag filter, 5 ~ 10 DEG C are dipped in outer transparent liquid, crystallization 5 days, and every 2 hours shaking ladles once;

5. dialyse

Take out crystal solution and filter to obtain crystal 15.1g, add purified water 30.2ml and dissolve, regulate pH to 2.9 with 3.0mol/L sulfuric acid solution; Solution is filled bag filter, is then hung in dialysis pond and dialyses 3 days, remove salt impurity, once, temperature controls at 5 ~ 10 DEG C every 12 hours dialysis Chi Huanshui;

6. lyophilizing

Take out dialysis solution, add a small amount of kieselguhr, use buchner funnel sucking filtration, obtain filtrate, regulate pH to 6.2 with 5mol/L sodium hydroxide solution, obtain crystallized trypsin finished product 3.8g after entering freeze dryer lyophilizing, its activity is tired as 2590iu/mg after measured;

Embodiment 3

Trypsin 1,250 ten thousand unit of preparation in Example 2, take 15g mannitol, add 500ml water for injection to dissolve, adjust ph to 6.0 after mixing, and then inject water to 1000ml, poly (ether-sulfone) ultrafiltration membrane aseptic filtration, is sub-packed in 1000 processed good cillin bottles, lyophilizing, rolls and covers.

The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims (11)

1. tryptic preparation method and containing a tryptic pharmaceutical composition, is characterized in that, comprise the following steps: (1) raw material rubs, extracting proenzyme; (2) classification salt molten, saltout; (3) crystallization, obtains trypsin crude product; (4) crude product feeds intake, multiple crystallization; (5) activate; (6) crystallization; (7) dialyse; (8) lyophilizing.

2. preparation method according to claim 1, it is characterized in that, each step is specially:

(1) extract: the pancreas of animal is rubbed into pancreas slurry, with sulfuric acid solution dipping, filter, leave and take filtrate;

(2) salt molten, saltout: add ammonium sulfate in filtrate, make the saturation of ammonium sulfate reach 30-50%, leave standstill, filter, leave and take filtrate; Add ammonium sulfate in filtrate, make the saturation of ammonium sulfate reach 50-80%, leave standstill, filter, leave and take filter cake; Gained filter cake is dissolved in water, and add saturated ammonium sulfate solution, adjust ph is to 4.0-5.5;

(3) crystallization: solution left standstill is to there being needle-like Chymotrypsin crystallization; Centrifugal, adjust ph, to 2.0-5.0, adds solid ammonium sulfate in the solution, sucking filtration; Taking precipitate, dry, obtain trypsin crude product;

(4) feed intake: get trypsin crude product, be dissolved in water, regulate pH, stir; Add ammonium sulfate, dissolve, leave standstill; Filter, filter cake adds water, stirring and dissolving, regulates pH, adds saturated ammonium sulfate solution, leaves standstill; Filter, in filtrate, add saturated ammonium sulfate solution, leave standstill; Wash magnesium, to be activated;

(5) activate: filter cake is dissolved in hydrochloric acid solution, adds calcium chloride solution and borate buffer, then add water, regulate pH, finally add crystallized trypsin and activate, by solution left standstill;

(6) crystallization: the solution after activation is regulated pH, adds solid ammonium sulfate, stirring and dissolving, leaves standstill, and filter, filtrate adds solid ammonium sulfate, leaves standstill, and filters, obtains filter cake; Add borate buffer in filter cake, regulate pH, stir, then solution is filled bag filter, be dipped in outer transparent liquid;

(7) dialyse: filter to obtain crystal, be dissolved in water, regulate pH; Solution is dialysed, removes impurity;

(8) lyophilizing: take out dialysis solution, sucking filtration, obtains filtrate, regulates pH, obtains crystallized trypsin finished product after lyophilizing.

3. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, it is characterized in that, in step (1), described animal pancreas is fresh Pancreas Bovis seu Bubali, fresh Pancreas Sus domestica; The concentration of sulfuric acid solution is 0.1 ~ 0.2mol/L; Pancreas slurry is 1:2 ~ 1:3 with the weight ratio of sulfuric acid solution; Dip time is 20 ~ 28 hours.

4. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, it is characterized in that, in described step (2), the weight ratio of water and filter cake is 1:1 ~ 3:1; Adjust ph to 4.0 ~ 6.0; The saturated ammonium sulfate solution added and the weight ratio of filter cake are 1:10 ~ 1:1.

5. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, is characterized in that, dissolves that to leave standstill ambient temperature is 20 ~ 30 DEG C in described step (3); Adjust ph to 2.0 ~ 5.0; The weight adding solid ammonium sulfate in every 1000ml solution is 300 ~ 400g.

6. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, it is characterized in that, in step (4), the weight ratio of trypsin crude product and water is 1:2-1:4, regulate pH to 3.0 ± 0.5, stir 10 ~ 16 hours; Often liter of solution adds 400 ~ 500g solid ammonium sulfate, after it dissolves, staticly settles 10 ~ 16 hours.

7. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, it is characterized in that, in step (5), claim filter cake weight, add 0.001 ~ 0.01mol/L hydrochloric acid solution and filter cake is dissolved, add 0.5 ~ 2mol/L calcium chloride solution, and the borate buffer of pH7.5 ~ 8.5, then add water, regulate pH7.0 ~ 8.0, the crystallized trypsin that finally every gram of filter cake adds 5 ~ 15mg carries out activation processing, and pH value controls 7.0 ~ 8.0; The addition of described hydrochloric acid solution is 3 ~ 5 times of filter cake weight, and the addition of described calcium chloride solution is 1 ~ 3 times of filter cake weight, and the addition of described borate buffer is 4 ~ 6 times of filter cake weight.

8. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, it is characterized in that, in step (6), the solution after activation regulates pH to 3.0 ± 0.4, solid ammonium sulfate is added again by 200 ~ 300g/L, stirring and dissolving, deposits and makes calcium sulfate precipitation in 32 ~ 60 hours in 0 ~ 10 DEG C, filters, solid ammonium sulfate is added by 150 ~ 250g/L in filtrate, place 10 ~ 14 hours when 0 ~ 10 DEG C, filter, obtain filter cake; Add 1 ~ 2mlpH8.0 ~ 10.0 borate buffer by every gram of filter cake, regulate pH to 8.0 ± 0.4, stir, then solution is filled bag filter, be dipped in outer transparent liquid, temperature controls at 0 ~ 10 DEG C.

9. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, is characterized in that, in step (7), 1 ~ 3ml water dissolution is added by every gram of crystal, regulate pH to 3.0 ± 0.5, temperature controls at 0 ~ 10 DEG C, dialyses 2 ~ 4 days.

10. the tryptic preparation method of one according to claim 2 and containing tryptic pharmaceutical composition, is characterized in that, in step (8), regulate pH to 6.0 ± 0.4.

11. 1 kinds of pharmaceutical compositions, its trypsin prepared containing with good grounds claim 1-10 as active component, also containing mannitol.