CN100469871C - Low pyrogen staphylokinase and its preparation method - Google Patents

Abstract

This invention belongs to the biology project pharmacy filed, which provides a Staphylokinase (SAK) with low pyrogen and it's preparing method. The Staphylokinase has low pyrogen, high purify and strong activity, which is completely fit for the medicinal demand. This invention includes the following steps: a breaking up the material containing with the Staphylokinase and distilling the floating liquid; b. sieving the product from step a with the DEAD gelatin rob, then removing salt and condensing; c. sieving the product form step b with CM gelatin rob, collecting the sample apex, condensing the washing liquid and transforming the medium; d. sieving the product from step c with the Q gelatin rob to obtain the Staphylokinase. The Staphylokinase of this invention has good bolt dissolving effect, and it is safe and efficient for users..

Description

A kind of low pyrogen staphylokinase and preparation method thereof

Technical field

The present invention relates to the biotech medicine product field, specifically relate to a kind of staphylokinase (Staphylokinase, preparation method SAK).

Background technology

As everyone knows, the content of pyrogen directly influences the security of drug use in the medicine, and genetically engineered drug, microbe-derived medicine and other biochemical drug be because the singularity in its source, pyrogen wherein remove and control be always its production and use in key issue.Main pyrogen material intracellular toxin is all quite stable under high temperature, strong acid, highly basic, and traditional heating, distillation, filtration, anti-phase infiltration, activated carbon powder, various column chromatography method can only be removed or deactivation part intracellular toxin.Majority method all is difficult to the disposable thorough removal of intracellular toxin, even through after a plurality of steps, does not still reach clinical requirement.And because endotoxic character utmost point heterogeneity, be difficult to find a kind of method of removing pyrogen targetedly (2002 the 4th phase 100-104 of removal Chinese biological engineering magazine of pyrogen in old tin of downstream chromatography technology).The main principle that pyrogen is removed in the genetically engineered drug production at present is as far as possible intracellular toxin to be removed in the target product purge process, does not add extra depyrogenation step and chemicals as far as possible.

Staphylokinase is a kind of exoprotein of golden Portugal's bacterium lysogenic phage synthetic (de Waart.J., K.C.Winkler and C.Grootsen Nature 1962,195:407～408 Winkler K.C., deWaart.J., and C.Grootsen J.Gen.Microbiol.1965,39:321～333 Kondo.I., Kiyotaka F., Infection and Immunity 1977,18:266～272).Synthetic and secretion staphylokinase level is low, purification difficult and reason such as profuse bleeding phenomenon takes place in the test of animal model dog thrombolysis in early days owing to golden Portugal bacterium, make the clinical use of staphylokinase be restricted.Press for and meet clinical drug safety, controlled low pyrogen staphylokinase.

The method of present disclosed staphylokinase production preparation mainly contains:

One, reported method in the foreign literature

1, adopts CM-cellulose column chromatography method sodium-chlor continuous gradient wash-out single step purification (Sako TOverproduction of staphylokinase in Escherichia coli and its characterization.FEBS.1985,149 (3): 557-63), SDS-PAGE purity is about 90%, and endotoxin content is not reported.

2, adopt ion exchange chromatography and molecular sieve method purifying (Gerlach D, Kraft R, Behnke DPurification and characterization of the bacterial plasminogen activatorstaphylokinase secreted by a recombinant Bacillus subtilis.Zentralb BakteriolMikrobiol Hyg[A] .1988,269 (3): 314-22.), SDS-PAGE purity is about 90%, and endotoxin content is not reported.

3, adopt SP-Sepharose and Phenyl-Sepharose chromatography two-step purifying method (Schlott B, Hartmann M, Guhrs KH, etal.Biotechnology 1994,12 (2): 185-9.), SDS-PAGE purity is more than 95%, and intracellular toxin is less than 10Eu/mg.

4, adopt metal ion column method single step purification (10 amino acid whose staphylokinases of purifying N end disappearance) (Chattopadhyay D, Stewart JE, DeLucas LJ.Large-scale preparation of the delta10form of staphylokinase by in vitro processing of recombinant staphylokinase withpurified human plasminogen.Appl Biochem Biotechnol.1998,69 (3): 147-56.).

Two, the main purification process of domestic literature report

1, CN1096325A, the broken bacterium of thalline is used S-Sepharose ion-exchange, and binding molecule screen method purifying SDS-PAGE purity is 90～99% again.

2, CN1307129A has mainly used two step column chromatography (SP-Sepharose F.F and Phenyl-Sepharose F.F) purifying, does not report purity and endotoxin content.

3, CN1394952A removes foreign protein with the 50KD ultra-filtration membrane behind the broken bacterium of thalline, adds NaCL, directly on Phenyl-Sepharose H.P hydrophobic chromatography post through the gradient purifying.Purified staphylokinase is 10 amino acid whose derivatives of N-end disappearance, and its purity can reach (electrophoresis is pure) more than 95%.

4, CN1511952A, the broken bacterium of thalline removes foreign protein with the 100KD ultra-filtration membrane, on the filtrate pH8.0PB directly on Q-Sepharose F.F post through 0.25M NaCL wash-out purifying, purified staphylokinase is 15 amino acid whose derivatives of N-end disappearance.Its electrophoresis purity and HPLC purity all can reach more than 98%

5, CN1446912A, used anion-exchange chromatography (Q-Sepharose F.F) to wear filter, cation-exchange chromatography (SP-Sepharose F.F), gel permeation chromatography (Sephacryl S-200) three step logotypes, the purification of Recombinant staphylokinase, the result does not have assorted band for final elutriant electrophoresis detection.

Above purification process only is to improve the purity of staphylokinase, for a person skilled in the art, at genetically engineered drug, microbe-derived medicine and other biochemical drug, especially at staphylokinase, reach high purity, low heat simultaneously, still do not have suitable method report at present.

Summary of the invention:

First purpose of the present invention provide a kind of staphylokinase (Staphylokinase, SAK).The endotoxin content of this staphylokinase is less than 3Eu/mgSAK (the endotoxic content among the SAK of the every mg of " Eu/mgSAK " expression).

Further, the endotoxin content of this staphylokinase is equal to or less than 1Eu/mgSAK.

Further, the HPLC purity of this staphylokinase is equal to or greater than 90%.

Second purpose provides a kind of method for preparing above-mentioned staphylokinase.This method may further comprise the steps:

A, the raw material crushing that will contain staphylokinase are centrifugal, get supernatant liquor;

B, step a supernatant liquor is worn filter with the DEAE gel column, collect filtrate, desalination concentrates;

C, step b concentrated solution is crossed the CM gel column, collect the elutriant that contains staphylokinase sample peak, concentrate;

D, step c concentrated solution is worn filter with the Q gel column, collect filtrate, concentrate, dry, promptly.

Further, above-mentioned DEAE gel column is that DEAE-Sepharose F.F post, CM gel column are that CM-Sepharose F.F post, Q gel column are Q-Sepharose F.F post.

Further, the particle diameter of the chromatography media in the above-mentioned various gel columns is 50～150 μ m.

Preferably, the particle diameter of the chromatography media in the above-mentioned various gel columns is 70～120 μ m.

Further, aforesaid method may further comprise the steps:

A, the supernatant liquor ultrafiltration desalination with raw material crushing after centrifugal are phosphoric acid buffer with the filtrate transfer medium, concentrate;

B, step a concentrated solution is worn filter with DEAE-Sepharose F.F post, moving phase is phosphoric acid buffer, and filtrate is concentrated;

C, be the NaAc-HAc damping fluid, cross CM-Sepharose F.F post,, concentrate after collecting the elutriant at place, sample peak again with the NaAc-HAc buffer solution elution with step b concentrated solution transfer medium;

D, be the phosphoric acid buffer that contains NaCL, wear filter with Q-Sepharose F.F with step c filtrate transfer medium, with filtrate concentrate, dry, promptly;

The flow velocity of moving phase is 5～35ml/min in the above steps.

Further, in the aforesaid method:

Phosphoric acid buffer described in a step is 8～12mM phosphoric acid buffer of pH8.0;

Phosphoric acid buffer described in the b step is 8～12mM phosphoric acid buffer of pH8.0;

NaAc-HAc damping fluid described in the c step is the NaAc-HAc damping fluid of 18～12mM, pH5.0～6.0;

The phosphoric acid buffer that contains NaCL described in the d step is the 20mM phosphoric acid buffer that contains 0.10～0.35mol/L NaCL, pH7.0;

Flow velocity when moving phase is crossed gel column in the above-mentioned steps is 5～30ml/min.

The 3rd purpose of the present invention provides a kind of pharmaceutical composition for the treatment of acute myocardial infarction or thrombotic diseases, and it is to add the preparation that the complementary composition of acceptable pharmaceutically is prepared from by above-mentioned staphylokinase.

Further, the formulation of above-mentioned pharmaceutical preparation is injection formulations or oral preparations.

The raw material that is used to prepare staphylokinase among the present invention in the method is the various common engineering bacterias that can express staphylokinase or wild type strain that can the secreting, expressing staphylokinase, as: microbiotic calibrating staphylococcus aureus.Low pyrogen staphylokinase of the present invention is recombinant staphylokinase, natural staphylokinase or its mutant.Can to select molecular weight cut-off for use be 5000 daltonian ultra-filtration membranes to ultrafiltration step among the present invention.

Employed chromatography column is the conventional chromatography column of selling in the inventive method on market, and the various chromatography column fillers of use also are fillers commonly used in the protein purification preparation field, all adorns post according to a conventional method or buys prepacked column.Employed other solution is pyrogen-free solution after DEAE-Sepharose F.F wears the filter step in the methods of the invention, and employed plant and instrument also will be handled and check through pyrogen-free, to guarantee can not produce new pyrogen material in preparation process.The flow velocity of the inventive method damping fluid is conventional flow velocity, can do suitable adjustment.The time that each step of present method continues can be according to the chromatography column volume, the damping fluid flow velocity, and particular cases such as applied sample amount are suitably adjusted by general knowledge.

DEAE gel column among the inventive method step b wear the filter or steps d in the Q gel column wear described in the filter wearing the filter be meant that target protein mainly is present in the liquid that passes in this step, and on chromatography media can not in conjunction with or in conjunction with few, at this moment should collect the filtrate that passes target product as this step.

Staphylokinase of the present invention and pharmaceutically auxiliary added ingredients combinations such as middle corresponding pharmaceutical excipient of acceptable or carrier, and, may be made in the pharmaceutical preparation of corresponding dosage forms by corresponding corresponding pharmaceutical methods processing.For example, with the appropriate solvent that allow to use in the injectable drug and/or additives cooperate and by after the corresponding technological operation processing, promptly can be prepared into the injection agent medicine of muscle such as corresponding liquid drugs injection, powder pin or freeze-dried preparation or vein form.With pharmaceutically can received disintegrating agent, after auxiliary interpolation composition that vehicle, lubricant, tackiness agent, weighting agent etc. are commonly used mixes, handle by corresponding common process method, promptly may be made in the oral preparations of the solid preparation forms such as sustained release dosage, control-released agent of tablet, pill, capsule or appropriate form; Mix with the tensio-active agents of using always such as solubilizing agent, emulsifying agent, wetting agent, foaming or defoamer, thinner, sanitas, stablizer, correctives, thickening material etc., handle by corresponding common process method, promptly may be made in the oral pharmaceutical or the buccal lozenge of liquid preparation forms such as aqua, syrup.

Staphylokinase of the present invention can be prepared into freeze-dried preparation by following step.Extraordinarily go into 15%～30% injection human albumin by 1.5～3.0 of sak protein amount, and with pyrogen-free 0.15M NaCL pH7.0 20mM phosphoric acid buffer constant volume, be mixed with r-Sak protein liquid of 5mg/ml, with promptly getting work in-process behind the pyrogen-free 0.22 μ m membrane filtration, after the detection endotoxin content meets medicinal requirements, under 100 grades of cleanliness factors, the packing of 1ml/ bottle, lyophilize, packing gets product.

Beneficial effect of the present invention is: staphylokinase pyrogen of the present invention is low, purity is high, activity is strong, and overall quality is better than present disclosed product, and meets " 2005 editions the 3rd related requests to this series products of Chinese pharmacopoeia fully.The inventive method is clear and definite, and purifying prepares the suitable gel column kind of staphylokinase and the best use order of these gel columns, simultaneously using method is optimized, and uses the inventive method can efficiently remove pyrogen, again purified product simultaneously.This method steps is easy, and material installation is easy to get, and is with low cost, reliable for effect, is a kind of outstanding staphylokinase preparation method, can large-scale application.Staphylokinase of the present invention adds the prepared treatment acute myocardial infarction of the complementary composition of acceptable pharmaceutically or the pharmaceutical preparation of thrombotic diseases, has good thrombolysis effect through clinical research confirmation, and occur without any the untoward reaction relevant with pyrogen, be a kind of medicine for the treatment of acute myocardial infarction or thrombotic diseases safely and effectively, have good market outlook.

Description of drawings

Accompanying drawing 1 is that recombinant staphylokinase plasmid pBV220/Sak makes up synoptic diagram

Accompanying drawing 2 is worn electrophorogram that filter collects sample wherein for DEAE-Sepharose F.F: upward be the upper prop sample, merge the collection sample 2, No. 3 for penetrating sample 1-No. 6.

Accompanying drawing 3 be CM-Sepharose F.F purification of samples electrophorogram wherein: 1, purification of samples; 2, the standard protein molecular weight

Accompanying drawing 4 is worn the tomographic map of filter for Q-Sepharose F.F.

Below in conjunction with accompanying drawing, the present invention will be described by the detailed description to better embodiment of the present invention.But it is limitation of the present invention that this explanation should not be construed as, and those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from technological thought of the present invention, all belongs to the defined scope of claim of the present invention.

Embodiment

The preparation of embodiment one engineering bacteria

With the full DNA of streptococcus aureus that can secrete staphylokinase is template, with primer 1,2 for making pcr amplification: primer 1 (SEQ ID NO.1): 5 ' GGT GAA TTC ATG TCA AGT TCA TTC GAC AA3 ';

Primer 2 (SEQ ID NO.2): 5 ' GGA GGA TCC TTA TTT CTT TTC TAT AAC AA 3 '.

Primer 1 comprises the EcoRI site, and primer 2 comprises BamH I site, obtains glucokinase gene, and the dna sequence dna of this glucokinase gene (5 ' to 3 ') sees Table 1:

The dna sequence dna of table 1 glucokinase gene (SEQ ID NO.3)

Tcaagttcat tcgacaaagb aaaatataaa 30

aaaggcgatg acgcgagtta ttttgaacca 60

acaggcccgt atttgatggt aaatgtgact 90

ggagttgatg gtaaaggaaa tgaattgcta 120

tcccctcatt atgtcgagtt tcctattaaa 150

cctgggacta cacttacaaa agaaaaaatt 180

gaatactatg tcgaatggga cttagatgcg 210

acagcatata aagagtttad agtagttgaa 240

ttagatccaa gcgcaaagat cgaagtcact 270

tattttgata agaataagaa aaaagaagaa 300

acgaagtctt tccctataac agaaaaaggt 330

tttgttgtcc cagatttatc agagcatatt 360

aaaaaccctg gattcaactt aattacaaag 390

gttgttatag aaaagaaa 408

The coded aminoacid sequence (N holds the end to C) of this dna sequence dna sees Table 2:

Table 2 staphylokinase aminoacid sequence (SEQ ID NO.4)

Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser

1 5 10 15

Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val

20 25 30

Glu Gly Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro

35 40 45

Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val

50 55 60

Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu

65 70 75 80

Leu Asp Pro Set Ala Lys Ile Glu Val Thr Tyr Phe Asp Lys Asn Lys

85 90 95

Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val

100 105 110

Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile

115 120 125

Thr Lys Val Val Ile Glu Lys Lys

130 135

With the gene that obtains carry out sequencing analysis confirm errorless after, be cloned on the pBV220 carrier, inserting the site is EcoR I-BamH I, makes up plasmid pBV220/Sak (making up flow process as shown in Figure 1).Perhaps can utilize the synthetic DNA that obtains shown in SEQ ID NO.3 of existing conventional Protocols in Molecular Biology.

The recombinant vectors for preparing is imported escherichia coli DH5a with ordinary method be present in endobacillary genetic engineering bacterium with soluble form, filter out and set up successful genetic engineering bacterium to make up recombinant staphylokinase.Through stability mensuration with after N-15 amino acid analysises mensuration of end are correct, preservation r-SAK expression amount is not less than 25% engineering bacteria, as production labor journey bacterium bacterial classification, and with glycerol stock or the preservation of iced milk dry strain form.

With the engineering bacteria seed of 1ml by the glycerine preservation, be inoculated in the 400mlLB substratum (containing penbritin 100 μ g/ml), on the 250rpm shaking table, be cultured to about A6002.0 under 30 ℃, inoculate ditto to be cultured in the 3600mlLB substratum about A6003.0 and be inoculated in the B.Braun D50 fermentor tank of the high density fermentation culture medium that 36L is housed as seed liquor, control pH about 7.0, dissolved oxygen 15～75%, temperature is cultured to about A60015.0 for 30 ℃, be rapidly heated to 42 ℃ and induce the same cultivation 3～4 hours.Centrifugal (5～15 ℃, 5000rpm) collects bacterium.

The preparation of embodiment two staphylokinases

1, material and facility

Broken bacterium instrument: homogenizer;

Ultrafiltration system: Millipore Pellicon 0.5m ²* 3, retaining molecular weight is 5000;

SDS-PAGE electrophoresis scanner: DS-700 Bio-Rad;

Chromatography column: 50 * 400mm, 100 * 400mm Shanghai Yarong Biochemical Instrument Plant;

Chromatography media: DEAE-Sepharose F.F, CM-Sepharose F.F, Q-Sepharose F.F are Pharmacia Biotech product, and particle diameter is 90 μ m.

The chromatography detection system: Ultraviolet Detector (HD-93-1 type) Shanghai gold reaches biochemical instrument factory.

2, preparation process

The weight in wet base of getting the fermentation gained is the 1610g engineering bacteria, with 10000ml pH8.0 10mM phosphoric acid buffer suspension thalline, get supernatant liquor 9000ml behind the broken bacterium, supernatant liquor is 36.9% through SDS-PAGE electrophoresis scanning r-SAK content, protein concentration 16.60mg/ml, total protein concentration is 149400mg, and r-SAK content is 55129mg.

With above-mentioned supernatant liquor with 15% (NH ₄) ₂SO ₄After the precipitation process, be the pH8.010mM phosphoric acid buffer through ultrafiltration (5KD) desalination, transfer medium.Be concentrated into 1500ml, protein concentration is 86mg/ml (total protein concentration 129000mg).

DEAE-Sepharose F.F wears filter

The chromatography column specification is that 100 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 300mm of DEAE-Sepharose F.F post, moving phase are the 10mM phosphoric acid buffer (flow velocity 10ml/min) of pH8.0.Collection penetrates sample, merges the staphylokinase sample according to the gel electrophoresis result, gets 3000ml solution, concentration 35mg/ml (total protein concentration 105000mg).The ultrafiltration desalination concentrates and transfer medium is the NaAc-HAc damping fluid of 20mM, and pH is 5.0.Gel electrophoresis the results are shown in Figure 1 (wherein: " and on " be the upper prop sample, merge 2, No. 3 sample for penetrating sample 1-No. 6).

CM-Sepharose F.F purifying chromatography column: 50 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 200mm of CM-Sepharose F.F post, moving phase pH5.0～6.0 20mM NaAc-HAc damping fluids (flow velocity 20ml/min).Earlier wash foreign protein with containing 0.15mol/L NaCl moving phase, with containing 0.3mol/L NaCl moving phase wash-out, collect elution peak again, get the 2400ml protein solution, concentration is 10.1mg/ml, and total protein concentration is 24240mg.CM sample ultrafiltration and concentration and transfer medium are for containing 0.15mol/L NaCL pH7.0 20mM phosphoric acid buffer, volume 1500ml.The results are shown in Figure 2

It is that 100 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 250mm of Q-Sepharose F.F post, moving phase contain 0.15mol/L NaCL pH7.0 20mM phosphoric acid buffer (flow velocity 5ml/min) that Q-Sepharose F.F wears the filter chromatography column.Collection penetrates sample peak (see figure 3).

Getting sample 2510ml through above-mentioned steps, is that protein concentration is the stoste of 8.8mg/ml, and its staphylokinase purifying yield is 40.1% (respectively goes on foot the purification effect contrast and see Table 3)

Table 3 respectively goes on foot the purification effect contrast

Step	Volume (ml)	Protein concentration (mg/ml)	Protein content (mg)	Staphylokinase amount (mg)	Yield
						Broken bacterium supernatant	9000	16.60	149400	55129
DEAE-Sepharose F.F wears filter	3000	35.00	105000
						CM-Sepharose F.F purifying	2400	10.10		24240
Q-Sepharose F.F wears filter	2510	8.8		22088	40.1％

Purifying gained protein product quality detection project and the results are shown in Table 4

Table 4 product quality inspection result

Project	Index	Verification result
			Specific activity (Au/mg)	≥4.0×10 4	5.1×10 4
Electrophoresis purity	≥95.0％	100
			HPLC purity	≥95.0％	99.15
Molecular weight (K D)	15.0±10％	14.8
			Exogenous DNA residual quantity	＜100pg/ dosage	Up to specification
Host's mycoprotein residual quantity	<0.1％	Up to specification
			The residues of antibiotics activity	Negative	Negative
Bacteria endotoxin content (Eu/mg)	<3	0.5Eu/mg
			Iso-electric point (primary area band)	5.5～7.5	6.2
Uv scan (nm)	λ Max:277±3	278
			N terminal amino acid sequence (15)	SSSFDKGKYKKGDDA	SSSFDKGKYKKGDDA
Peptide figure	Meet the r-SAK figure	Meet the requirements

Detection or measuring method all adopt " the method for the same race that 2005 editions three appendix of Chinese pharmacopoeia are included.Detected result shows that the staphylokinase that present embodiment makes all meets related request.

The preparation of embodiment three staphylokinases

1, material and facility (with embodiment two)

2, preparation process

Get the engineering bacteria that weight in wet base is 800g,, get supernatant liquor 5000ml behind the broken bacterium with 5500ml pH8.0 10mM phosphoric acid buffer suspension thalline, supernatant liquor is 38.9% through SDS-PAGE electrophoresis scanning r-SAK content, protein concentration 10.40mg/ml, total protein concentration are 52000mg, and r-SAK content is 20228mg.

With above-mentioned supernatant liquor with 15% (NH ₄) ₂SO ₄After the precipitation process, be pH8.0 10mM phosphoric acid buffer through ultrafiltration (5KD) desalination, transfer medium.Be concentrated into 1000ml, protein concentration is 43.4mg/ml (total protein concentration 43400mg).

DEAE-Sepharose F.F wears filter chromatography column: 100 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 300mm of DEAE-Sepharose F.F post, moving phase pH8.0 10mM phosphoric acid buffer (flow velocity 15ml/min).Collection penetrates sample, merges the staphylokinase sample according to the gel electrophoresis result, gets 2100ml solution, concentration 16.8mg/ml (total protein concentration 35280mg).The ultrafiltration desalination concentrates and transfer medium is a 20mM NaAc-HAc damping fluid, pH5.0.

CM-Sepharose F.F purification column chromatography: 50 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 200mm of CM-Sepharose F.F post, moving phase are pH5.0～6.0 20mM NaAc-HAc damping fluids.Earlier wash foreign protein with containing 0.15mol/L NaCl moving phase, with containing 0.3mol/L NaCl moving phase wash-out (flow velocity 15ml/min), collect elution peak again, get the 1100ml protein solution, concentration is 8.2mg/ml, and total protein concentration is 9020mg.CM sample ultrafiltration and concentration and transfer medium are for containing 0.15mol/L NaCL pH7.0 20mM phosphoric acid buffer, and volume is 700ml.

Through Q-Sepharose F.F wear filter chromatography column: 100 * 400mm (Shanghai Yarong Biochemical Instrument Plant), the high 250mm of Q-Sepharose F.F medium post, moving phase contains 0.15mol/L NaCL pH7.0 20mM phosphoric acid buffer (flow velocity 25ml/min).Get sample 790ml, the albumen stoste of concentration 10.57mg/ml.

The time that above-mentioned steps continues meetings such as variation according to applied sample amount, damping fluid flow velocity under different situations change to some extent.Present embodiment is 41.2% (respectively go on foot the purification effect contrast and see Table 5) through the staphylokinase purifying yield after the above-mentioned steps

Each step purification effect contrast of table 5

Step	Volume (ml)	Protein concentration (mg/ml)	Protein content (mg)	Staphylokinase amount (mg)	Yield
						Broken bacterium supernatant	5000	10.40	52000	20228
DEAE-Sepharose F.F wears filter	2100	16.80	35280
						CM-Sepharose F.F purifying	1100	8.20		9020
Q-Sepharose F.F wears filter	790	10.57		8350	41.2％

Purifying gained protein product quality detection project and the results are shown in Table 6.Detection or measuring method all adopt " the method for the same race that 2005 editions three appendix of Chinese pharmacopoeia are included.Detected result shows that the staphylokinase that present embodiment makes all meets related request.

Table 6 product quality inspection result

Project	Index	Verification result
			Specific activity (Au/mg)	≥4.0×10 4	5.8×10 4
Electrophoresis purity (%)	≥95.0	98.1
			HPLC purity (%)	≥95.0	99.4

Molecular weight (KD)	15.0±10％	14.7
			Exogenous DNA residual quantity	＜100pg/ dosage	Up to specification
Host's mycoprotein residual quantity (%)	<0.1	Up to specification
			The residues of antibiotics activity	Negative	Negative
Bacteria endotoxin content (Eu/mg)	<3	0.5Eu/mg
			Iso-electric point (primary area band)	5.5～7.5	6.1
Uv scan (nm)	λ Max：277±3	277
			N terminal amino acid sequence (15)	SSSFDKGKYKKGDDA	SSSFDKGKYKKGDDA
Peptide figure	Meet the r-SAK figure	Up to specification

The pyrogen content confirmatory experiment of the staphylokinase of embodiment four the present invention preparation

Get the foregoing description 2 second step products therefrom and final step products therefrom with reference to " three relevant endotoxin measurement method limulus reagent tests of Chinese pharmacopoeia (0.5 Eu/ml) carry out heat-original determinating, the results are shown in Table 7.

Table 7 pyrogen confirmatory experiment result

In the preparation process of engineered protein medicine, each step all will be noted pyrogen.By table 5 as seen, after the processing through the inventive method first two steps, the pyrogen substances content of staphylokinase stoste is very low, and most intracellular toxins are removed, but can not reach officinal intracellular toxin control requirement, and the extremely difficult removal of remaining a small amount of intracellular toxin.On the basis of step, the Q-Sepharose F.F of final step wears filter can make the product pyrogen reach requirement remaining endotoxin removal to very low level, is to one of influential committed step of staphylokinase pyrogen amount in the inventive method in front.Simultaneously, final step also makes the purity of staphylokinase reach more than 99% (the HPLC purity) simultaneously, has satisfied the needs that large scale purification is produced.Certainly, the obtaining of such effect needs and preceding several steps cooperates just and can reach.

Implement the preparation of the staphylokinase preparation of five the present invention preparation

Get the sak protein stoste 790ml (the r-SAK amount is 8350mg) that obtains among the embodiment three, add 20% injection human albumin by 2.0 times protein contents, and be settled to 1670ml with pyrogen-free 0.15M NaCL pH7.0 20mM phosphoric acid buffer, be mixed with r-Sak protein liquid of 5mg/ml, rapidly with promptly getting work in-process behind the pyrogen-free 0.22 μ m membrane filtration, after the detection endotoxin content meets medicinal requirements, under 100 grades of cleanliness factors, the packing of 1ml/ bottle, (-40 ℃ of pre-freezes vacuumized after 2～6 hours, slowly were warming up to-20 ℃ in lyophilize, kept 4～8 hours, be rapidly heated in 1 hour to 0 ℃, kept 2 hours, after the end freeze-drying at 20～35 ℃, but vacuum or fill nitrogen and seal), get product.

Adopt that " standard method of the same race of including of 2005 editions three appendix of Chinese pharmacopoeia is examined and determine.Verification result (seeing Table 8) shows that the staphylokinase that present embodiment makes all meets related request.

Table 8 quality of the pharmaceutical preparations verification result

Project	Index	Verification result
			Outward appearance	White loose body, solution should be colourless or little opalescent clarified liq of being with, and should not contain macroscopic insolubles	Up to specification
Tire by (Au/ bottle)	80～150% of labelled amount	26.2 ten thousand
			Moisture	≤3.0％	1.96％
The pH value	6.5～7.5	7.0
			Sterility test	Meet " biological products sterility test rules " requirement	Up to specification
Abnormal toxicity test	Meet " biological products abnormal toxicity test rules " requirement	Up to specification
			Pyrogen test	Meet " biological products pyrogen test rules " requirement	＜0.5EU/mg is up to specification

The effect experiment of the staphylokinase preparation of experimental example one the present invention preparation

Staphylokinase of the present invention (recombinant staphylokinase,) be a kind of novel thrombolytic agent, it forms mixture by combining with Profibrinolysin in the human plasma, this mixture combines with the scleroproein on thrombus surface specifically, makes fibrin degradation, thrombolysis, it is strong to have the thrombolysis ability, characteristics such as the scleroproein binding specificity is good, and the fibrinogen degradation activity is low, and immunogenicity is low.

Get the foregoing description five prepared staphylokinase preparations and carry out clinical trial, the result is as follows.

One, I clinical trial phase and result

Through bureau of drug administration of the Ministry of Health [No. 36 file of (96) system Shen body] approval, staphylokinase preparation I clinical trial phase of the present invention has been carried out in China Medical Sciences Academy Fu Wai Hospital clinical pharmacology base year September in June, 1999 to 1999.

Test-results shows all healthy volunteers to staphylokinase preparation 20mg well-tolerated of the present invention, and no serious adverse reaction does not have because of adverse events drug withdrawal person.Staphylokinase preparation human pharmacokinetics of the present invention meets intravenous injection two Room open models.Drug main will be distributed in the blood, plays a role in blood vessel, and it is apparent poor that the elimination phase transformation period does not have 2.5 to 20mg, average out to 47.21 ± 10.28min.

Two, II phase clinical preliminary test and result

Through National Drug Administration's [No. the 08th, (2000) system Shen body word] approval, January in August, 2000 to calendar year 2001,10 routine 20mg staphylokinase preparation for treating of the present invention acute myocardial infarction (Acute Myocardial Infarction, AMI) patient's II clinical trial phase trial test have been carried out at China Medical Sciences Academy Fu Wai Hospital and The Third Affiliated Hospital of Peking University.

Test-results shows: coronarography reached thrombolysis in myocardial infarction test (thrombolysisin myocardial ischemia, TIMI) TIMI3 level blood flow person 7 examples, TIMI2 level blood flow person 2 examples, TIMI1 level blood flow person 1 example in 90 minutes.Respond well.

Three, II clinical trial phase and result

Ratify through National Drug Administration, China Medical Sciences Academy Fu Wai Hospital is as group leader unit, Rui Jin hospital of Shanghai Second Emdical University, The Third Affiliated Hospital of Peking University, Capital University of Medical Sciences's ZhaoYang Hospital, General Hospital, Shenyang Military Command, Liaoning People's Hospital, Xijing hospital of The Fourth Military Medical University, Attached Hospital No.1, dalian Medical Univ., loyal hospital is pacified by the Capital University of Medical Sciences, the Shanghai City Sixth People's Hospital, Friendship Hospital of the Capital University of Medical Sciences, No. 1 Hospital Affiliated to Chinese Medical Univ is as the ginseng unit of grinding, carried out the II clinical trial phase of recombinant staphylokinase of the present invention year October in January, 2002 to 2003, this test is a multicenter, parallel control research at random.

1, subjects and grouping

Be divided at random

1. staphylokinase preparation group of the present invention was at 30 minutes internal jugular vein infusion 20mg; When on-test, because hematencephalon takes place 1 example, staphylokinase preparation group using dosage of the present invention is adjusted into 15mg by 20mg, 3mg intravenous injection wherein, and 12mg was at 30 minutes internal jugular vein infusions;

2. recombinant human tissue plasminogen activator (Recombinant human tissue plasminogenactivator, rt-PA) group, infusion 50mg in 90 minutes, first quiet notes 8mg buckles, remaining 42mg in 90 minutes quiet drip off complete;

3. direct percutaneous tranluminal coronary angioplasty (Percutaneous TransluminalCoronary Angioplasty, PTCA) group.

12 clinic test center include staphylokinase preparation group 108 examples of the present invention altogether in, rt-PA organizes 107 examples, direct ptca group 111 examples.(wherein two thrombolytic group are received the standard of including in that the age violates scheme greater than 70 years old case and are rejected 3 examples owing to mistake).3 routine staphylokinase formulation dosage 20mg of the present invention (wherein 1 example age rejected greater than 70 years old person) are arranged.Only to meeting treatment casebook invention staphylokinase preparation group 104 examples of inclusion criteria, rt-PA organizes 106 examples and adds up.

Wherein, age mean and constituent ratio when staphylokinase preparation of the present invention and rt-PA group case are selected, sex, the body weight mean, weight index, height, blood pressure (systolic pressure and diastolic pressure), pulse, heart function when being admitted to hospital (Killip) classification, hypertension history, the past stenocardia history, the past myocardial infarction history, smoking history reaches now still smoker's ratio, diabetic history, the insulinize history, familial history of coronary artery disease, the hypercholesterolemia history, CK, the MB-CK peak value, symptom begins to arriving hospital's time, arrive hospital to the pitch time for the treatment of, symptom begins all do not have marked difference to the pitch time of treatment, has good comparability.Staphylokinase preparation group 99 examples of the present invention, rt-PA organizes 101 examples and has finished 90 minutes coronarographies.

2, test-results:

A, main terminal point

(1) coronarography evaluation of result:

90 minutes TIMI3 levels of staphylokinase preparation group of the present invention blood flow is 57.58%, and the rt-PA group is 48.51%, P=0.1929, no significance difference between two groups;

Staphylokinase preparation group of the present invention 90 minutes patency rate (TIMI2 level and TIMI3 level blood flow) is 77.78%, and the rt-PA group is 63.37%, and P=0.0277 has the significance difference between two groups, and staphylokinase preparation group of the present invention is better than the rt-PA group.

90 minutes radiographies of staphylokinase preparation group of the present invention are proofreaied and correct TIMI blood flow frame number 38.89 ± 21.49, and the rt-PA group is 45.52 ± 37.63, P=0.2665, no significance difference between two groups.

The relevant coronary artery of 90 minutes infarct of staphylokinase preparation group of the present invention is remaining narrow 80.22 ± 19.82%, and the rt-PA group is 82.94 ± 20.49%, P=0.3412, no significance difference between two groups.

(2) clinical compound terminal point:

Dead 9 examples of staphylokinase preparation group of the present invention (8.65%) in January, dead 6 examples of control group rt-PA (5.66%), P=0.3997, no significance difference between two groups.

Non-lethality re-infarction staphylokinase preparation of the present invention group 3 examples (2.88%) in January, control group rt-PA4 example (3.77%), P=1.000, no significance difference between two groups.

Stenocardia recurrence staphylokinase preparation group 9 examples of the present invention (8.65%) in January, control group rt-PA17 example (16.04%), P=0.1043, no significance difference between two groups.

Carry out target vessel Reconstruction (TVR) staphylokinase preparation of the present invention group 32 examples (30.77%) in January, control group rt-PA26 example (24.53%), P=0.3119, no significance difference between two groups.

Above-mentioned four sum total (compound clinical endpoint) staphylokinase preparation group of the present invention 46 examples (44.23%), control group rt-PA42 example (39.62%), P=0.5185, no significance difference between two groups.

B, secondary endpoints

(1) heart failure is called on or the pulmonary edema incidence: staphylokinase preparation group 14 examples of the present invention (13.46%), control group rt-PA12 example (11.32%), the difference not statistically significant that P=0.6377 is two groups.

(2) January time left side chamber EF: staphylokinase preparation group 0.56 ± 0.12 of the present invention, control group rt-PA 0.70 ± 0.80, the difference not statistically significant that P=0.1969 is two groups.

(3) hemorrhage: hemorrhage 30 examples (28.85%) take place in staphylokinase preparation group of the present invention, and control group rt-PA 29 (27.36%), P=0.8105, two groups difference not statistically significant.

Serious or life-threatening hemorrhage: staphylokinase preparation group 2 examples of the present invention, control group rt-PA 4 examples comprise the severe haemorrhage at encephalic, gi tract, genitourinary tract, pars oralis pharyngis, skin and conduit position.Staphylokinase preparation group 1 example of the present invention (0.96%) of wherein intracranialing hemorrhage, recovery from illness is left hospital; Control group rt-PA 4 examples (3.77%) wherein have 2 examples because of the death of intracranialing hemorrhage, and 2 example recoveries from illness are left hospital (1 example is made a definite diagnosis without CT for suspicious intracranialing hemorrhage), P=0.3691, two groups difference not statistically significant;

Hemorrhage staphylokinase preparation group 4 examples of the present invention of moderate, control group rt-PA 4 examples; Hyporrhea staphylokinase preparation of the present invention group 24 examples, control group rt-PA 21 examples, P=0.4597, difference not statistically significant between two groups.

(4) other complication: cardiac rupture, asystole, atrial fibrillation, auricular flutter, sustained ventricular tachycardia, ventricular fibrillation, 2-30 atrioventricular block, continue ypotension, cardiogenic shock, heart failure is called on and two groups of the incidences of pulmonary edema, the medium complication of ischemic cerebral apoplexy between the difference not statistically significant.

C, adverse events

(1) death incident: dead 9 examples of staphylokinase preparation group of the present invention (8.65%), dead 4 examples of cardiac rupture wherein, dead 5 examples of cardiogenic shock; Dead 6 examples of control group rt-PA (5.66%), dead 3 examples of cardiogenic shock wherein, dead 1 example of interventional therapy complication, dead 2 examples of intracranialing hemorrhage, P=0.3997, two groups difference not statistically significant.

(2) hemorrhage: hemorrhage 30 examples (28.85%) take place in staphylokinase preparation group of the present invention, control group rt-PA29 example (27.36%), P=0.8105, two groups difference not statistically significant.Serious or life-threatening hemorrhage staphylokinase preparation group 2 examples of the present invention, control group rt-PA 4 examples comprise the severe haemorrhage at encephalic, gi tract, genitourinary tract, pars oralis pharyngis, skin and conduit position; Staphylokinase preparation group 1 example of the present invention (0.96%) of wherein intracranialing hemorrhage, recovery from illness is left hospital; Control group rt-PA 4 examples (3.77%) wherein have 2 examples because of the death of intracranialing hemorrhage, and 2 example recoveries from illness are left hospital (1 example is made a definite diagnosis without CT for suspicious intracranialing hemorrhage), P=0.3691, two groups difference not statistically significant; Hemorrhage staphylokinase preparation group 4 examples of the present invention of moderate, control group rt-PA 4 examples; Hyporrhea staphylokinase preparation of the present invention group 24 examples, control group rt-PA 21 examples, P=0.4597, two groups difference not statistically significant.

(3) staphylokinase preparation group of the present invention and rt-PA group is not all observed untoward reactions such as tangible blood system and liver, kidney function damage and allergy, heating, shiver with cold.Any untoward reaction that is caused by pyrogen materials such as intracellular toxins does not appear.

(4) give in staphylokinase preparation 20mg person 3 examples of the present invention, (wherein 1 example age rejected greater than 70 years old person), hematencephalon 1 example, survival does not have dead.

(5) the 3 routine patients of Ti Chuing are patient more than 70 years old, staphylokinase preparation group 2 examples wherein of the present invention, and rt-PA organizes 1 example, and 90 minutes coronarographies are TIMI3 level blood flow behind the thrombolysis, do not have dead.

D, experiment conclusion:

Staphylokinase preparation of the present invention is at 30 minutes internal jugular vein infusion 15 mg treatment AMI, 90 minutes vascular patencies 77.8% after its medication, be better than contrasting medicine rt-PA (63.4%, P=0.0277); TIMI3 level blood flow is 57.6%, and is similar to rt-PA (48.5%), and clinical compound terminal point does not have marked difference for two groups.Hemorrhage (comprising hematencephalon), incidence and rt-PA did not also have marked difference.The result shows that staphylokinase preparation of the present invention is the thrombolytic drug of a safe and effective treatment acute myocardial infarction.

Show that by above example staphylokinase pyrogen of the present invention is low, each batch endotoxin content all＜0.5EU/mg, pyrogen content meets " three requirements to like product of Chinese pharmacopoeia fully; The purity height, its HPLC purity is more than 99%; Active strong, the height of tiring.Add the prepared treatment acute myocardial infarction of the complementary composition of acceptable pharmaceutically or the preparation of thrombotic diseases by staphylokinase of the present invention, in the human body practical clinical, showing in the good curative effect, any untoward reaction that is caused by pyrogen materials such as intracellular toxins does not appear yet, be a kind of medicine for the treatment of acute myocardial infarction or thrombotic diseases safely and effectively, have good market outlook.

Above detailed description of the present invention does not limit the present invention, those skilled in the art can thought according to the present invention make various changes and distortion, such as selecting product different with the trade(brand)name of filler in the gel column used in the present invention but that kind is identical and index is identical or approaching for use, regulate the flow velocity of chromatographic step etc., only otherwise break away from spirit of the present invention, all belong to the defined scope of claims of the present invention.

A kind of low pyrogen staphylokinase and preparation method thereof .ST25

SEQUENCE LISTING

＜110〉Chengdu Diao 9 Wang pharmaceutical factory

＜120〉a kind of low pyrogen staphylokinase and preparation method thereof

<130>050234

<160>4

<170>PatentIn version 3.2

<210>1

<211>29

<212>DNA

<213>Artificial

<220>

＜223〉primer sequence 1

<400>1

<210>2

<211>29

<212>DNA

<213>Artificial

<220>

＜223〉primer sequence 2

<400>2

<210>3

<211>408

<212>DNA

＜213〉streptococcus aureus

<400>3

<210>4

<211>136

<212>PRT

＜213〉streptococcus aureus

<400>4

Claims (10)

1, a kind of preparation method of staphylokinase is characterized in that it may further comprise the steps:

A, the raw material recombinant bacterial strain intestinal bacteria fragmentation that will contain staphylokinase are centrifugal, get supernatant liquor;

B, step a supernatant liquor is worn filter with the DEAE gel column, collect filtrate, desalination concentrates;

C, step b concentrated solution is crossed the CM gel column, collect the elutriant that contains staphylokinase sample peak, concentrate;

D, step c concentrated solution is worn filter with the Q gel column, collect filtrate, concentrate, dry, promptly.

2, method according to claim 1 is characterized in that described DEAE gel column is a DEAE-Sepharose F.F post; The CM gel column is a CM-Sepharose F.F post; The Q gel column is a Q-Sepharose F.F post.

3, method according to claim 2, the particle diameter that it is characterized in that the gel filler in the described various gel column are 50～150 μ m.

4, method according to claim 3, the particle diameter that it is characterized in that the gel filler in the described various gel column are 70～120 μ m.

5, according to each described method of claim 1-4, it is characterized in that may further comprise the steps:

A, the supernatant liquor ultrafiltration desalination with raw material crushing after centrifugal are phosphoric acid buffer with the filtrate transfer medium, concentrate;

B, step a concentrated solution is worn filter with DEAE-Sepharose F.F post, moving phase is phosphoric acid buffer, and filtrate is concentrated;

C, be the NaAc-HAc damping fluid, cross CM-Sepharose F.F post,, concentrate after collecting the elutriant at place, sample peak again with the NaAc-HAc buffer solution elution with step b concentrated solution transfer medium;

D, be the phosphoric acid buffer that contains NaCL, wear filter with Q-Sepharose F.F with step c concentrated solution transfer medium, with filtrate concentrate, dry, promptly;

Flow velocity when moving phase is crossed gel column in above-mentioned b, c, each step of d is 5～40ml/min.

6, method according to claim 5 is characterized in that:

Phosphoric acid buffer described in a step is 8～12mM phosphoric acid buffer of pH8.0;

Phosphoric acid buffer described in the b step is 8～12mM phosphoric acid buffer of pH8.0;

NaAc-HAc damping fluid described in the c step is the NaAc-HAc damping fluid of 18～12mM, pH5.0～6.0;

The phosphoric acid buffer that contains NaCL described in the d step is the 20mM phosphoric acid buffer that contains 0.10～0.35mol/L NaCL, pH7.0;

Flow velocity when moving phase is crossed gel column in the above-mentioned steps is 5～30ml/min.

7, the staphylokinase of each described method preparation of claim 1-6, it is characterized in that: its endotoxin content is less than 3Eu/mg SAK, and its HPLC purity is equal to or greater than 99%.

8, staphylokinase according to claim 7 is characterized in that: its endotoxin content is equal to or less than 1Eu/mg SAK.

9, a kind of pharmaceutical composition for the treatment of acute myocardial infarction or thrombotic diseases, it is to add the preparation that the complementary composition of acceptable pharmaceutically is prepared from by claim 7 or 8 each described staphylokinases.

10, pharmaceutical composition according to claim 9 is characterized in that described preparation is injection formulations or oral preparations.

Images