CN1786164A - Preparation method of recombination buman tPA - Google Patents
Preparation method of recombination buman tPA Download PDFInfo
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- CN1786164A CN1786164A CN 200510019706 CN200510019706A CN1786164A CN 1786164 A CN1786164 A CN 1786164A CN 200510019706 CN200510019706 CN 200510019706 CN 200510019706 A CN200510019706 A CN 200510019706A CN 1786164 A CN1786164 A CN 1786164A
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Abstract
The invention discloses a recombination human tPA making method. It includes strain fermenting production, tPA occlusion body extracting, renaturation, purifying, condensing, and tPA production freezing dry powder making. 10g tPA crystal can be gained from 10L fermenting volume by using the making method. After denaturing and rematriatopm its total activity can reach 3-4x108IU; the total rematriatopm efficiency is more than 10%; its recovery can reach 60%; and purity can reach more than 90%. The manufacturing method has the advantages of high yield, simple operation, low cost, and high efficiency. The production technique can transform industrialization quickly.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of preparation method of recombination buman tPA, more specifically relate to the fermentative production of recombination buman tPA, and the sex change of leavened prod, renaturation, purifying process technology.
TPA is the most effective, safest at present thrombolytic drug, is widely used in the various diseases that the treatment thrombus causes, especially Acute Myocardial Infarction patient and cerebral thrombosis patient's rescue.Technology provided by the invention can be applicable to the suitability for industrialized production of tPA.
Technical background
(the tissue type plasminogen activator of tissue-type plasminogen activator, tPA) be a kind of important thrombolytics, its major function is that cutting Profibrinolysin albumen makes its activation become plasmin and make fibrinolysis, thereby reaches the purpose of thrombolysis.TPA priority activation and thrombus or insoluble fibrin bonded Profibrinolysin performance thrombus dissolving and molten fine effect, therefore the thrombus position had specificity, be difficult for causing other position hemorrhage, compared huge advantage with other thrombolytic drug such as streptokinase, urokinase, it is the most effective, safest at present thrombolytic drug, be widely used in the various diseases that the treatment thrombus causes, especially Acute Myocardial Infarction patient and cerebral thrombosis patient's rescue.1987, the tPA that genetic engineering technique is produced was in U.S.'s approval listing, and commerce is called alteplase (Activase), and being identified in the clinical observation of the mid-90 in last century is the most effective medicine of treatment myocardial infarction.At present, the tPA commodity of advanced countries such as the U.S., Britain, Germany have all been regenerated and have been tPA derivative product (promptly natural tPA molecule being carried out the tPA reorganization varient that structure of modification gets).
Be used at first production for treating with the cell system of tPA be melanoma cell (Griffiths, JB.et al.Adv.Biochem.Eng.Biotechnol.1987,34:147-166).Subsequently a series of gone on the market and be about to the listing tPA derivative products in, also be to adopt the zooblast system to produce mostly, as the TNK enzyme (Tenecteplase) of U.S.'s Genentech (Genentech) company exploitation, the nPA enzyme (Lanoteplase) of U.S.'s Bristol-mayer Si Pubo (Bristol-MyersSpuibb) company exploitation, all be to produce with Chinese hamster cell (CHO), Japan defends material (Eisai) and builds the Monteplase (Monteplase) of ripple institute exploitation and produce with body hamster kidney cell.Since the eukaryotic cell culture systems exist cell growth slow, yield poorly, characteristics such as medium component costliness, culture condition requirement height, cause the tPA cost very high, cause the tPA product price to remain high, patient is difficult to bear economically.
Intestinal bacteria (Escherichia coli) expression system becomes the first-selected host cell of most protein expression study and production owing to its easy and simple to handle, low production cost.Adopt the escherichia expression system production for treating promptly to receive in early days at the tPA drug research that the researchist pays close attention to and achieving success (Harris TJ et al.Mol.Biol.Med.1986,3:279-292 with tPA; Datar RV.et al.Biotechnology.1993,11:349-357), in the tPA product of listing, (Reteplase promptly is to be produced by coli strain K12 rPA) to the reteplase of being developed by German Pola graceful (Boehringer Mannheim) company at present.Utilize escherichia expression system to express the recombinant protein that tPA can obtain the high expression level amount usually, but the consequent is the marking protein false folding, forming does not have active albumen.The protein frequent of this non-activity occurs with the form of inclusion body together.The renaturation technology of inclusion body is normally used the denaturing agent of high density, as urea, Guanidinium hydrochloride etc., with solubilization of inclusion bodies, obtains recombinant protein at the removal denaturing agent afterwards.But contain a large amount of disulfide linkage (natural tPA contains 17 pairs of disulfide linkage) in natural tPA and the activated Recomposed tPA derivative molecular, cause the renaturation difficulty and the annealing efficiency of tPA inclusion body low, purification step increases, make that the aftertreatment technologys such as sex change, renaturation and purifying of tPA inclusion body are very complicated, therefore, adopt the cost of escherichia expression system production tPA also very high, caused tPA drug price costliness equally.
TPA does not have products production as the recombination engineering medicine with significant curative effect and huge commercial value all the time in China, and its development bottleneck is the scarcity to Research on Post-processing Techniques such as the sex change of tPA inclusion body, renaturation and purifying.At the problems referred to above, the present invention aims to provide the production technique of a cover High-efficient Production Recomposed tPA.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Recomposed tPA, tPA preparation method provided by the invention has output height, easy and simple to handle, with low cost, the efficient advantages of higher of product aftertreatment technology, for overcoming present tPA products production cost height, problems such as drug price costliness provide effective solution route.
It is that (handed over Chinese typical culture collection center preservation, its deposit number is coli strain BL21/rtPAm-1: CCTCC M205112) that the present invention utilizes the bacterial strain of producing Recomposed tPA.Recomposed tPA preparation method provided by the invention, comprise fermentative production, the tPA inclusion body of engineering strain extraction, renaturation, purifying, concentrate, the technical matters such as freezing dry powder preparation of tPA, be described below:
1. the fermentative production of engineering strain BL21/rtPAm-1 comprises following steps:
(1) inclined-plane seed culture.With LB inclined-plane solid medium, contain penbritin (Amp) 100 μ g/ml, from the original species word bank, inoculate, cultivated 18-20 hour for 37 ℃.
(2) shake-flask seed is cultivated.Use the 2YT liquid nutrient medium, contain penbritin (Amp) 100 μ g/ml.Each shakes bottle and use the 100ml substratum, and inoculation one encircles seeding, 37 ℃ shaking culture 12-14 hour.
(3) fermentation culture.Used substratum is a fermention medium: peptone 20g/L, yeast extract 8g/L, NaCl 2g/L, MgSO
40.2g/L, bubble enemy 10 μ l/L, the pure water preparation is to 10 liters of culture volumes.Regulate the pH value before the sterilization to 6.8-7.0.Steam sterilizing, cylinder was pressed 0.1MPa (120 ℃) 20 minutes.
The control condition of fermentation culture and parameter:
10 liters of culture volumes, pH6.8-7.0
Inoculum size 5%-10%
Stirrer rotating speed 40Hz
Temperature controlling range 35-37 ℃
Tank pressure 0.05-0.07MPa
Air flow 3-5 liter/minute (1: 0.3-1: 0.5)
By above-mentioned condition and parameter, being cultured to bacterium amount 6-8 grams per liter, pH value rises to 7.2-7.5, DO and drops to 20% when following, add isopropylthio-(IPTG) to final concentration 1mM, 35-37 ℃ is continued to cultivate, and induced protein was expressed 6-8 hour.Fermentation whole process is 10-12 hour, and the Recomposed tPA albumen of generation is inclusion body.
Utilize fermentation process of the present invention can obtain the Recomposed tPA inclusion body, output 1.0-1.3 gram tPA crystal (weight in wet base)/liter about.Having good stability of zymotechnique continuously fermented with 10 liters of automatic fermenters and to be produced 10 batches, and thalline output (weight in wet base) is stabilized in the 8.0-10 grams per liter, and tPA crystal (behind the purifying, weight in wet base) is stabilized in the 1.0-1.3 grams per liter, and mean yield reaches 1.1 grams per liters.
2.tPA the extraction of inclusion body and purifying.
The BL21/rtPAm-1 thalline of centrifugal collection fermentation, be suspended from the pure water, ultrasonic wave or high pressure homogenization crusher machine, the centrifugal supernatant that goes, precipitation is washed centrifugal collecting precipitation three times with the Tris-NaCl-tween 20 of pH8.0, then with pure water washing three times, centrifugal collecting precipitation, precipitation checks that through 10%SDS-polyacrylamide gel electrophoresis (10%SDS-PAGE) the tPA crystalline content is at (Fig. 2,3) more than 90%.Precipitation can be put-20 ℃ of refrigerators preservations.
3. the dissolving of inclusion body and sex change
By 5 gram tPA crystal (weight in wet base)/liter amount the tPA inclusion body is added in the 7-8M urea, regulate pH to 9-10 with NaOH, add reductive agent beta-mercaptoethanol 10-20mM, 28-32 ℃ stirring and dissolving 0.5-1 hour, dissolve and sex change.
4.tPA renaturation, method is as follows:
(1) add water with one times of sex change liquid dilution, urea concentration is reduced to below the 4M, tPA begins renaturation.
(2) remove urea and reductive agent beta-mercaptoethanol in the renaturation solution gradually with G50Sephadex molecular sieve column chromatography (available from peace agate West Asia company), make progressively renaturation of tPA, remove the following foreign protein of molecular weight 30kD simultaneously again.
(3) the tPA solution the collected 10mM-20mM Tris-Cl with pH8.0 is diluted, to tPA concentration be 1-2 restrain weight in wet base/liter, under the condition of 20-25 ℃ and pH8.0, placed 16-24 hour, carry out further renaturation.
The refolding method of tPA is a committed step among the present invention, preferably forms through test of many times.The renaturation of tPA inclusion body is under the condition of pH8.0, and (β-ME) concentration is carried out progressively to reduce urea and reductive agent beta-mercaptoethanol.The removal of urea and reductive agent adopts the G50Sephadex molecular sieve chromatography to carry out, and crosses G50 Sephadex post plays preliminary purification to tPA effect simultaneously.After G50 Sephadex column chromatography, the 10mM-20mM Tris-Cl with pH8.0 carries out Macrodilution to tPA solution again, carries out further renaturation.Through this step process, tPA purity reaches (Fig. 3) more than 95%, and the tPA activity reaches 15000IU-20000IU in every milliliter of renaturation solution, and annealing efficiency reaches about 10%.
5.tPA the purifying after the renaturation and concentrated comprises step;
(1) separates the correct and incorrect tPA albumen of renaturation of renaturation with Q-Sepharose 4FF post (available from peace agate West Asia company).Pillar carries out pre-treatment through 0.5M HCl earlier, wash with water again to pH5.0-6.0,20mM Tris-Cl with the pH8.0-8.5 of 2 times of volumes carries out balance then, renaturation solution after filtering, the Tris-Cl to 20mM that adds pH8.0, upper prop, 10mM Tris-Cl with pH7.5,0.1-0.5M NaCl carries out gradient elution, collect activated tPA elution peak, or with the 10mM Tris-Cl of pH7.5,0.15-0.2M NaCl wash-out foreign protein, use the 10mM Tris-Cl of pH7.5 again, the activated tPA of 0.25-0.35M NaCl wash-out.
(2) further use hydrophobic chromatography post Phenyl-Sepharose 6FF (available from peace agate West Asia company) that tPA is carried out purifying, the pillar 5mM Tris-Cl of pH6.5-7.5,0.4-0.6M NaCl carries out balance, in the tPA liquid of Q-Sepharose 4 FF post wash-outs, add NaCl to 0.4-0.6M, regulate pH to 7.0-7.5 with HCl, upper prop carries out wash-out with the 2.5-5mMTris of pH8.0-9.0, collects activated tPA.
(3) with membrane filter (Tianjin MoTian Membrane Engineering Technology Co., Ltd, model UEIP905) the tPA elutriant is concentrated, to tPA concentration 3 * 10
5IU/ml.
Purifying process after the tPA renaturation is another committed step among the present invention, and the tPA purity of process renaturation reaches 95%, but still needs correct the separating with the incorrect albumen of renaturation of renaturation.The crucial part of this step is to use Q-Sepharose 4FF post to separate the correct and incorrect albumen of renaturation, with hydrophobic chromatography post Phenyl-Sepharose 6FF tPA is carried out further purifying then.The optimum condition of this step also is to explore via test of many times to form.In implementing this step, (not correct renaturation) albumen of non-activity directly flows out when last Q-Sepharose 4 FF posts, thus needn't be before upper prop acidifying in advance filter and remove not the correctly albumen of renaturation.Through the processing of this step, (Fig. 3, Fig. 4), specific activity is 5-6 * 10 to the Recomposed tPA purity of acquisition more than 99%
5IU/mg.
6.tPA freezing dry powder injection preparation
Preparation composition: PVP-K30 (1.5%), N.F,USP MANNITOL (0.5%), arginine (0.2%), tween-80 (0.2%), phosphoric acid salt (10mM) and the spissated tPA solution of purifying, after each composition stirring and dissolving, 0.22 the degerming of μ m membrane filtration divides to install in the freeze-drying bottle.The product freezing procedures: product precooling temperature-30 ℃, condenser temperature-45 ℃, pressure be greater than 100hpa, freeze-off time 4 hours; Sublimation drying program: product temperature-20 ℃, condenser temperature-50 ℃, vacuum tightness 0.1-0.3hpa, 40 hours time of drying; Be warming up to 25-30 ℃, continue vacuum-drying 3 hours.
In a word, the invention provides from spawn culture, to the acquisition of tPA inclusion body, to renaturation and the purifying of tPA, up to a whole set of technological process of production (Fig. 1) that obtains tPA freezing dry powder injection, wherein emphasis has solved the critical process and the technology such as renaturation, tPA purifying of tPA inclusion body.The preparation method of Recomposed tPA provided by the invention carries out on the pilot scale level, and the production technique that is obtained can be converted into industrialized producing technology very soon.Utilize preparation method provided by the present invention, can produce from the 10L fermentation volume more than tPA crystal weight in wet base 10 grams, amount to dry weight 5 grams, gross activity can be up to 3-4 * 10 after sex change, the renaturation
8IU, total annealing efficiency＞10% has the rate of recovery of active tPA to reach 60% after the renaturation, can obtain gross activity 1.5-2 * 10 after concentrating
8IU, specific activity reach 5-6 * 10
5IU/mg can make 40 of the injections of 1,500,000 IU.Through tPA the 10%SDS-polyacrylamide gel (10%SDS-PAGE) and the efficient liquid phase chromatographic analysis of renaturation and purifying, purity reaches 99%-100%.
Utilize the Recomposed tPA of the present invention's method preparation, pyrogen testing and abnormal toxicity test all reach the requirement of " Chinese biological goods rules " (version in 2000).The host cell DNA residual quantity detects to 10-100pg/ dosage (pressing every dosage 1,500,000 IU of tPA calculates), meets the requirement of " Chinese biological goods rules " (version in 2000).The content of host protein is lower than 0.01%, meets country intestinal bacteria product requirement host protein is not more than 0.1% standard.The tPA of the present invention preparation is prepared into the powdery injection via lyophilize, has satisfactory stability, 37 ℃ preserve 30 days, preserve half a year at 4 ℃, activity all descends.
Description of drawings
Fig. 1 Recomposed tPA preparation method's provided by the invention technical process comprises:
1. the fermentative production of engineering strain BL21/rtPAm-1: step 1-step 5 among Fig. 1
2.tPA the extraction of inclusion body and purifying: step 6-step 9 among Fig. 1
3. the dissolving of inclusion body and sex change: step 10 among Fig. 1
4.tPA renaturation: step 11-step 12 among Fig. 1
5.tPA the purifying after the renaturation and concentrated: step 13-step 15 among Fig. 1
6.tPA freezing dry powder injection preparation: step 16-18 among Fig. 1
The generation and the extraction of Fig. 2 Recomposed tPA inclusion body.
The renaturation of Fig. 3 Recomposed tPA and purifying
Swimming lane M. protein molecule quantitative character (Marker).Swimming lane 1 is presented at isopropylthio-(IPTG) induce after, the albumen in the ultrasonic disruption cell precipitation, the tPA albumen size that obtains is about 38kD.Swimming lane 2 shows that purity reaches more than 90% through the tPA inclusion body of Tris-NaCl-tween 20 and pure water washing.The tPA albumen that swimming lane 3 shows behind the G50Sephadex column chromatography purification, purity is about 95%.Swimming lane 4 shows that purity is more than 99% through the tPA albumen behind Q-Sepharose 4 FF and the Phenyl-Sepharose 6 FF column chromatography purifications.Protein electrophorese is to carry out on 10%SDS-polyacrylamide gel (10%SDS-PAGE).Protein content is that electrophoresis result is scanned, and obtains with KODAKID 3.5 software analysis.
Fig. 4 high pressure liquid chromatographic analysis is through the Recomposed tPA of renaturation and purifying
Undertaken by test center of Wuhan University.Reach 100% through the Recomposed tPA purity behind renaturation, the purifying.
The molten fine circle method of Fig. 5 detects the activity through the tPA of renaturation and purifying
The result shows that the Recomposed tPA that the present invention prepares has the activity that plasminogen activation becomes plasmin.Point sample is in proper order: 1. negative control, 2 μ l physiological sodium chloride solutions; 2. through renaturation, purifying and spissated tPA solution 2 μ l; 3.tPA standard substance 20IU (2 μ l); 4.tPA standard substance 5IU (0.5 μ l).
Fig. 6 tPA freezing dry powder stability of formulation
A 1. negative controls, physiological sodium chloride solution 1 μ l
2.tPA standard substance 10IU (1 μ l)
3. the tPA of prepared fresh (1 μ l)
1.37 ℃ of tPA (1 μ l) that place 10 days of B
2.4 ℃ 10 days tPA (1 μ l) of placement
3.tPA standard substance 10IU (1 μ l)
1.37 ℃ of tPA (1 μ l) that place 20 days of C
2.4 ℃ 20 days tPA (1 μ l) of placement
3.tPA standard substance 10IU (1 μ l)
1.37 ℃ of tPA (1 μ l) that place 30 days of D
2.4 ℃ 30 days tPA (1 μ l) of placement
3.tPA standard substance 10IU (1 μ l)
The result shows that tPA freezing dry powder preparation stability is good, places 30 days at 37 ℃, and the active nothing of tPA obviously descends.
Fig. 7 solid phase spot hybridization detects the foreign DNA residual quantity
1-4. positive control.E. coli chromosomal dna, point sample amount are 10ng, 1000pg, 100pg, 10pg.
5,6. negative control does not contain the 10mM Tris-Cl of DNA, point sample 1 μ l.
7,8.tPA sample, the point sample amount is by the tPA amount of 1,500,000 IU.
The result shows that the tPA sample of every dosage contains foreign DNA between 10-100pg.
Embodiment
The fermentation manufacturing technique of embodiment 1, Recomposed tPA
The bacterial strain that the present invention produces Recomposed tPA is that coli strain BL21/rtPAm-1 (transfers to Chinese typical culture collection center preservation, its deposit number is CCTCC M205112), the fermentation manufacturing technique step is shown in step 1-step 5 among Fig. 1, its effect is the primordial seed that will preserve through slant culture, shake-flask culture, the fermentation culture fermentative production level that progressively increases, and induces then to produce Recomposed tPA albumen.Be specifically described as follows:
1. inclined-plane seed preparation:
(1) substratum:
The LB substratum: peptone 10g/L, yeast extracts 5g/L, NaCl 10g/L,
Penbritin (Amp) 100mg/L
The LB solid medium: peptone 10/L, yeast powder 5g/L, NaCl 10g/L, agar powder 2g/L,
Penbritin (Amp) 100mg/L
(2) a little dry powder of picking from coli strain BL21/rtPAm-1 freezing dry powder seed joins in the 200 μ lLB substratum, suspends evenly, and inoculation one ring was cultivated 18-20 hour for 37 ℃ on every inclined-plane LB solid medium, obtained the inclined-plane seed.The inclined-plane seed can use for 2 weeks in 2-4 ℃ of preservation.
2. shake-flask seed preparation:
(1) substratum:
The 2YT substratum: peptone 16g/L, yeast extract 8g/L, NaCl 5g/L, the tap water preparation is regulated pH to 7.0 with 5M NaOH.1.034 * 10
5Pa high pressure steam sterilization 20 minutes.
(2) connect one from the inclined-plane seed and encircle 100ml and shake in the bottle, add penbritin (Amp) simultaneously to final concentration 100 μ g/ml, 37 ℃, 200rmp cultivated 12-14 hour, obtained shake-flask seed.
3. fermentation culture
(1) fermention medium: peptone 20g/L, yeast extract 8g/L, NaCl 2g/L, MgSO
40.2g/L, bubble enemy 20 μ l/L, the pure water preparation is to 10 liters of culture volumes.Regulate the pH value before the sterilization to 6.8-7.0.Steam sterilizing, cylinder is pressed 0.1Mpa, 120 ℃, 20 minutes.
(2) control condition of fermentation culture and parameter
10 liters of culture volumes (sterilization back), pH6.8-7.0
Inoculum size 5%-10%
Stirrer rotating speed 40Hz
Temperature controlling range 35-37 ℃
Tank pressure 0.05-0.07MPa
Air flow 3-5 liter/min (0.3-1: 0.5)
By above-mentioned condition and parameter, be cultured to that bacterium amount 6-8 grams per liter, pH value rise to 7.2, DO drops to 20% when following, adds isopropylthio-(IPTG) to final concentration 1mM, 35-37 ℃ is continued cultivation, induced protein expression 6-8 hour.Fermentation whole process is 10-12 hour, and the tPA albumen of generation is inclusion body, all appears in the precipitation of broken cell (Fig. 2).
The extraction and the purifying of embodiment 2.tPA inclusion body
See step 6-step 9 among Fig. 1, its effect is a Recomposed tPA albumen of collecting fermentative production, and carries out preliminary purifying.Embodiment is as follows:
1. the thalline of centrifugal collection fermentation culture, be suspended from the pure water (200 gram weight in wet bases/liter).
2. ultrasonic disruption thalline, centrifugal 8 minutes of 12000rmp removes supernatant.
3. precipitation is washed three times with Tris-NaCl (pH8.0)-tween 20, wash by the amount of gram precipitation/15ml lavation buffer solution at every turn, and each centrifugal 6 minutes, centrifugal collecting precipitation.
4. the pure water washing is three times, and each centrifugal 6 minutes, collecting precipitation.
The 5.10%SDS-polyacrylamide gel electrophoresis is checked rtPAm-1 albumen, as seen its purity more than 90% (Fig. 2, Fig. 3).Precipitation is put-20 ℃ of refrigerators preservations.
The sex change and the dissolving of embodiment 3 inclusion bodys
See the step 10 among Fig. 1.In 5 gram tPA crystal (weight in wet base)/liter ratio the inclusion body of centrifugal collection is dissolved in 7-8M urea, with NaOH with pH regulator to 9-10, add the reductive agent beta-mercaptoethanol (10-20mM of β-ME), 30 ℃, stirred 0.5-1 hour, carry out sex change and dissolve.
The renaturation of embodiment 4tPA
See step 11 and step 12 among Fig. 1, its effect is that sex change dissolved Recomposed tPA is carried out renaturation, obtains activated, soluble Recomposed tPA albumen, can carry out purifying to Recomposed tPA albumen simultaneously.Method is as follows:
1. add water with one times of sex change liquid dilution, urea concentration is reduced to below the 4M, tPA begins renaturation.
2. remove urea and reductive agent beta-mercaptoethanol (β-ME) with the G50Sephadex molecular sieve chromatography.Pillar Tris-Cl (pH8.0) balance of 10mM, upper column quantity is a column volume 20%.
3. with 10mM Tris-Cl (pH8.0) washing pillar, detect the solution that collection contains tPA with nucleic acid protein detector (wavelength 280nm).Recomposed tPA directly flows out (band black tea look) at first from post, be the following albumen (colourless) of molecular weight 30KD subsequently, and at last effusive is urea and β-ME (colourless).
With the tPA solution collected by 2 gram tPA (weight in wet base)/liter amount be adjusted to designated volume with 10mM Tris-Cl (pH8.0), renaturation is further carried out in 20-25 ℃ of placement 16-24 hour.
Through this step process, the annealing efficiency of Recomposed tPA reaches about 10%, the tPA purity of protein can reach about 95% (the 10%SDS-polyacrylamide gel electrophoresis, Fig. 3).
Purifying after the embodiment 5.tPA renaturation and concentrated
See step 13-step 15 among Fig. 1, Recomposed tPA albumen needs the correct and incorrect albumen sepn of renaturation also need be further purified simultaneously through after the renaturation.Embodiment is as follows:
1. separate the correct and incorrect tPA albumen of renaturation of renaturation with Q-Sepharose 4FF chromatography column.Pillar washes with water to pH5.0-6.0 more earlier through 0.5M HCl pre-treatment, uses 20mM Tris-Cl (pH8.0) balance of 2 times of volumes then.
2. renaturation solution adds Tris-Cl (pH8.0) after filtering to 20mM, and upper prop is to approaching saturated.
3. with 10mM Tris-Cl (pH7.5), 0.1-0.5M NaCl gradient elution, detect the activated tPA elution peak of collection with nucleic acid protein detector (wavelength 280nm), or with 10mM Tris-Cl (pH7.5), 0.15-0.2M NaCl, 3 times of column volume wash-out foreign proteins are used 10mM Tris-Cl (pH7.5) again, the activated tPA of 0.25-0.35M NaCl wash-out.
4. further tPA is carried out purifying with hydrophobic chromatography post Phenyl-Sepharose 6FF.Pillar 5mMTris, 0.5M NaCl (pH6.5-7.5) balance.
5. in the tPA liquid of Q-Sepharose 4FF post wash-out, add NaCl to 0.5M, regulate pH to 7.0-7.5, upper prop with HCl.
6. carry out wash-out with 5mMTris (pH8.5), detect with nucleic acid protein detector (wavelength 280nm) and collect activated tPA elution peak.
7. with membrane filter the tPA elutriant is concentrated, to tPA concentration 3 * 10
5IU/ml.
With the purity of 10%SDS-PAGE and high performance liquid chromatography detection process purifying and spissated Recomposed tPA, measure tPA activity (seeing embodiment 6) with molten fine circle method.The result shows that the Recomposed tPA purity of acquisition reaches 99-100% (10%SDS-polyacrylamide gel electrophoresis, Fig. 3; High performance liquid chromatography, Fig. 4), specific activity is about 5-6 * 10
5IU/mg, annealing efficiency be more than 10%, activity recovery about 60%.
Embodiment 6 Recomposed tPAs are active to be detected
Adopt the solusphere method to carry out, step is as follows:
1.tPA standard substance: available from Beijing pharmaceutical biological product calibrating institute of the Ministry of Health, every bottle contains tPA30000IU, and with the dissolving of 3ml physiological sodium chloride solution, making tPA concentration is 10IU/ μ l.
2. human thrombin: be made into 100IU/ml with physiological sodium chloride solution, in-20 ℃ of preservations.
3. Profibrinolysin:, be made into 0.5mg/ml with physiological sodium chloride solution available from Beijing pharmaceutical biological product calibrating institute.
4. human fibrinogen: 30mg/ props up, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's preparation standard reagent.Configuration before the experiment, earlier at 37 ℃ of water-bath preheating 15min, physiological sodium chloride solution is preheating simultaneously also before the configuration, and every adds the 5ml physiological sodium chloride solution then, and insulation 30min is left standstill in 37 ℃ of water-baths dissolves it fully, is configured to 6mg/ml solution.
5. fibrin plate preparation: take by weighing the 125mg agarose, add the 23ml physiological sodium chloride solution, boil dissolving, 60 ℃ of water-bath balances, add 280ul scleroproein (0.5mg/ml), add 14 μ l zymoplasms (100IU/ml), shake up, muddy back is fallen dull and stereotyped, horizontal positioned, and room temperature is solidified.
6. active the detection: punch on fibrin plate with punch tool, diameter 2mm, in tPA sample and standard substance point hand-hole, 37 ℃ 8 hours, measurement transparent circle diameter.Transparent circle diameter according to standard tPA and rtPAm-1 sample calculates the sample activity.
The results are shown in Figure 5, can produce molten fine circle by plasminogen activation through the rtPAm-1 behind sex change, renaturation and the purifying, through with the conversion of standard tPA, activity reaches 5-6 * 10
5IU/mg reaches the level (5 * 10 of international like product
5IU/mg).
The preparation and the Detection of Stability thereof of embodiment 7tPA freezing dry powder injection
See step 16-step 18 among Fig. 1, its objective is Recomposed tPA albumen is made the dry powder injection, be beneficial to preservation.Specific implementation method is as follows:
1. preparation composition and prescription: PVP-K30 (1.5%), N.F,USP MANNITOL (0.5%), arginine (0.2%), tween-80 (0.2%), phosphoric acid salt (10mM) and the spissated tPA solution of purifying, after the stirring and dissolving, 0.22 the degerming of μ m membrane filtration divides to install in the 20ml freeze-drying bottle every bottled 5ml.
2. preparation freeze drying process.The product freezing procedures: product precooling temperature-30 ℃, condenser temperature-45 ℃, pressure be greater than 100hpa, freeze-off time 4 hours.Sublimation drying program: product temperature-20 ℃, condenser temperature-50 ℃, vacuum tightness 0.1-0.3hpa, 40 hours sublimation drying time; Be warming up to 30 ℃, continue vacuum-drying 3 hours.The freeze-drying prods that obtains is a white, and inside is evenly fluffy, surfacing.
3.tPA freezing dry powder stability of formulation
The tPA freezing dry powder places 37 ℃, gets one bottle every 10 days with physiological sodium chloride solution dissolving, surveys actively with the solusphere method, compares with the same batch of product and the tPA standard substance of 4 ℃ of preservations, calculates every bottle of gross activity decline.
The results are shown in Figure 6, placed 30 days at 37 ℃, the active nothing of tPA obviously descends.Lyophilized powder is preserved half a year at 4 ℃, does not also find the active decline of tPA.The tPA freezing dry powder that shows preparation has satisfactory stability.
Embodiment 8 Recomposed tPA animal experiments
1. pyrogen testing:
Each tPA sample is injected 3 of rabbit by 30,000 units/kg body weight ear vein, other establishes three ear vein injections of control group stroke-physiological saline solution, injection back take temperature changes, every 30 minutes take temperatures 1 time, survey more than 6 times continuously, 3 rabbit body temperature rise of test group are all in 0.6 ℃, and the intensification summation of 3 rabbits is no more than 1.40 ℃, reach the requirement of " Chinese biological goods rules " (version in 2000).
2. abnormal toxicity test:
Undertaken by tail vein injection and two test group of abdominal injection, by the injection of per kilogram of body weight 50,000 tPA of unit dosage, 20 of each sample injections are that contrast is rented with 20 injection stroke-physiological saline solution in addition, observe continuously 7 days.The mouse of two test group of result is all strong depositing in 7 days, no abnormal reaction, and body weight gain, movable normal with feed, reach the requirement of " Chinese biological goods rules " (version in 2000).
Embodiment 9 foreign DNA residual quantities detect
Use the solid phase spot hybridization.
1. extract the chromosomal DNA of e. coli bl21 (DE3).Press Sa nurse Brooker etc., the method for molecular cloning experiment guide (second edition,, Science Press in 1992) is carried out.
2. bacterial chromosomal dna conduct after enzyme is cut digestion prepares the template of dna probe, the probe of digoxigenin labeled is with DIG-High Prime DNA Labeling and Detection Starter Kit I test kit (available from Roche company) preparation, method reference reagent box specification sheets.
3. e. coli chromosomal dna is done a series of dilutions, as the positive control and the dna content standard of dot hybridization, positive control dna point sample amount is 10ng, 1000pg, 100pg, 10pg.
4. Zhi Bei tPA sample calculates (dosage that the tPA medicine is common) by the every injection amount of 1,500,000 IU, handles degraded tPA albumen through Proteinase K earlier, and 100 ℃ of water-baths are 10 minutes then, the ice bath cooling, and centrifugal 5 minutes, with suction filtration sample injector point film.
5.120 ℃ crosslinked half an hour.
6. step such as probe hybridization, colour developing is undertaken by the test kit specification sheets.
The result shows that the present invention prepares the foreign DNA content of tPA sample at 10-100pg/ dosage (Fig. 7), be lower than the 100pg/ dosage (the existing rules of China are adjusted, and host cell DNA residual quantity standard is less than 10ng/ human dosage) of " Chinese biological goods rules " (version in 2000) regulation.
Embodiment 10 host cell proteins assays
Adopt the double-antibody sandwich euzymelinked immunosorbent assay (ELISA).
1. (carbonate buffer solution, pH9.6) dilution rabbit enterobacteria thalline to the 10 μ g/ml of the Chinese People's Anti-Japanese Military and Political College adds in the 96 hole enzyme plates 4 ℃ of placements, 16-18 hour with 100 μ l/ holes with coating buffer.
2. wash plate 3 times.With washings (phosphoric acid buffer-0.05% soil temperature 20 pH7.4) is joined 1% bovine serum albumin, adds in the hole with 200 μ l/ holes, put 37 ℃ 2 hours.
3. (0.5% bovine serum albumin-phosphoric acid buffer-0.05% soil temperature 20 pH7.4) begins to do double dilution with the standard tropina from 500ng/ml, until minimum concentration 7.8125ng/ml obtains series of standards tropina concentration gradient with diluent.
4. with diluent tPA stoste is diluted to about 250 μ g/ml.
5. will seal good enzyme plate washing 3 times, standard substance and sample will all be added in the hand-hole with 100 μ l, put 37 ℃ 2 hours.
6. wash plate 3 times,, add in the plate with 100 μ l/ holes with diluent 1: 1000 dilution horseradish peroxidase (HRP) rabbit Chinese People's Anti-Japanese Military and Political College enterobacteria protein antibodies, put 37 ℃ 2 hours.
7. wash plate 10 times.Add the substrate solution (pyrocatechol 8mg dissolves in citric acid-phosphate buffered saline buffer, and pH5.0 adds 30% hydrogen peroxide, 30 μ l) of provisional configuration with 100 μ l/ holes, 37 ℃ 40 minutes.
8. add 1M sulfuric acid termination reaction with 50 μ l/ holes.
9. in microplate reader, select 492nm wavelength photometry absorption value (A), do typical curve, according to the host protein content in the A value calculating tPA sample of testing sample with the A value and the concentration gradient of standard protein.
Measured host protein content in 5 batches of tPA samples by above-mentioned steps.The result is respectively 0.0087%, 0.0092%, 0.0039%, 0.0055%, 0.0074%, 0.0032%, all is lower than 0.01%, meets country intestinal bacteria product requirement host protein is not more than 0.1% standard.
Claims (3)
1. the preparation method of a recombination buman tPA, it comprises the following steps:
A, utilize engineering strain BL21/rtPAm-1, carry out the fermentative production Recomposed tPA, engineering strain BL21 (DE3)/rtPAm-1, CCTCC M205112, utilize engineering strain BL21/rtPAm-1 fermentative production recombination buman tPA to comprise step: at first to be the inclined-plane seed culture: to use the LB substratum, cultivated 18-20 hour for 37 ℃; Next is that bottle shakes the seed preparation: use the 2YT substratum, shake-flask culture was cultivated 12-14 hour for 37 ℃; The 3rd is fermentation culture: use fermention medium, 37 ℃ are carried out fermentation culture and induced protein expression;
The extraction of B, tPA inclusion body and purifying:
Centrifugal collection thalline, the ultrasonic disruption thalline, the centrifugal supernatant that goes, precipitation is respectively washed three times with Tris-NaCl-tween 20 and the pure water of pH8.0;
The dissolving of C, inclusion body and sex change:
By 5-10 gram tPA weight in wet base/liter amount the tPA inclusion body is added in the 7-8M urea, regulate pH to 9-10 with NaOH, add the reductive agent beta-mercaptoethanol to 10-20mM, 28-32 ℃ stirring and dissolving 0.5-1 hour, dissolve and sex change;
D, tPA renaturation, its method is as follows:
(a) add water with one times of sex change liquid dilution, urea concentration is reduced to below the 4M, tPA begins renaturation;
(b) remove urea and reductive agent beta-mercaptoethanol in the renaturation solution gradually with G50 Sephadex molecular sieve column chromatography, make progressively renaturation of tPA, remove the following foreign protein of molecular weight 30kD simultaneously again;
(c) the tPA solution the collected 10mM-20mM Tris-Cl with pH8.0 is diluted, to tPA concentration be 1-2 restrain weight in wet base/liter, under the condition of 20-25 ℃ and pH8.0, placed 16-24 hour, carry out further renaturation;
E, column chromatography purification and concentrated:
(a) separate the correct and incorrect tPA albumen of renaturation of renaturation with Q-Sepharose 4 FF posts, pillar carries out pre-treatment with 0.5M HCl earlier, wash with water again to pH5.0-6.0,20mM Tris-Cl with the pH8.0-8.5 of 2 times of volumes carries out balance then, renaturation solution after filtering, the Tris-Cl to 20mM that adds pH8.0, upper prop, with the 10mM Tris-Cl of pH7.5,0.1-0.5M NaCl carries out gradient elution, collects activated tPA elution peak, or with the 10mM Tris-Cl of pH7.5,0.15-0.2M NaCl wash-out foreign protein is used the 10mM Tris-Cl of pH7.5, the activated tPA of 0.25-0.35MNaCl wash-out again;
(b) further tPA is carried out purifying with hydrophobic chromatography post Phenyl-Sepharose 6 FF, the pillar 5mM Tris-Cl of pH6.5-7.5,0.4-0.6M NaCl carries out balance, in the tPA liquid of Q-Sepharose 4 FF post wash-outs, add NaCl to 0.4-0.6M, regulate pH to 7.0-7.5 with HCl, upper prop carries out wash-out with the 2.5-5mMTris of pH8.0-9.0, collects activated tPA;
(c) with membrane filter the tPA elutriant is concentrated, to tPA concentration 3 * 10
5IU/ml;
F, the preparation of tPA freezing dry powder injection:
Adopt freeze-drying that spissated tPA solution is made the freezing dry powder preparation.
2, the preparation method of a kind of recombination buman tPA according to claim 1 is characterized in that the used substratum of fermentation culture, and its composition is as follows:
Peptone 20g/L, yeast extract 8g/L, NaCl 2g/L, MgSO4 0.2g/L, bubble enemy 10 μ l/L, the pure water preparation to 10 liters of culture volumes, is regulated pH to 6.8-7.0 before the sterilization, fermention medium adopts steam sterilizing, and cylinder is pressed 0.1mpa, 120 ℃, 20 minutes.
3, the preparation method of a kind of recombination buman tPA according to claim 1 is characterized in that the control condition of fermentation culture:
10 liters of culture volumes, pH6.8-7.0;
Inoculum size 5%-10%;
Stirrer rotating speed 40Hz;
Temperature controlling range 35-37 ℃;
Tank pressure 0.05-0.07Mpa;
Air flow 3-5 liter/minute;
By above-mentioned condition, be cultured to bacterium amount 6-8 grams per liter, pH value and rise to 7.2-7.5, DO and drop to 20% when following, add isopropylthio-to final concentration 1mM, 35-37 ℃ is continued cultivation, induced protein expression 6-8 hour.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101220081B (en) * | 2008-01-28 | 2011-01-05 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN102366628A (en) * | 2010-12-31 | 2012-03-07 | 华东理工大学 | Quality stabilization technique of antitumor protein medicines |
| CN102757474A (en) * | 2011-04-25 | 2012-10-31 | 珠海健康元生物医药有限公司 | Method for removing chaotropic agent used in protein preparation process |
| CN103694339A (en) * | 2013-12-04 | 2014-04-02 | 珠海联邦制药股份有限公司 | Renaturation method of insulin glargine precursor |
| CN111909920A (en) * | 2020-08-24 | 2020-11-10 | 武汉人福药业有限责任公司 | Expression and purification method of recombinant human tissue-type plasminogen activator |
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| JPH0699324B2 (en) * | 1985-10-02 | 1994-12-07 | 持田製薬株式会社 | Tissue plasminogen activator lysis method |
| US5260419A (en) * | 1991-03-20 | 1993-11-09 | The Dupont Merck Pharmaceutical Company | Purification of active and inactive/latent forms of plasminogen activator inhibitor-1 |
| US6372473B1 (en) * | 1997-05-28 | 2002-04-16 | Human Genome Sciences, Inc. | Tissue plasminogen activator-like protease |
| CN1281048A (en) * | 2000-08-24 | 2001-01-24 | 武汉大学 | Reconstituted human t-PA molecule and its engineering bacterium strain to express it |
| CN1260247C (en) * | 2003-11-05 | 2006-06-21 | 上海中科伍佰豪生物工程有限公司 | Large-scale cytorrhyctes compound purifying technology for recombining tPA derivative |
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| CN101220081B (en) * | 2008-01-28 | 2011-01-05 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN102366628A (en) * | 2010-12-31 | 2012-03-07 | 华东理工大学 | Quality stabilization technique of antitumor protein medicines |
| CN102757474A (en) * | 2011-04-25 | 2012-10-31 | 珠海健康元生物医药有限公司 | Method for removing chaotropic agent used in protein preparation process |
| CN102757474B (en) * | 2011-04-25 | 2014-10-15 | 珠海健康元生物医药有限公司 | Method for removing chaotropic agent used in protein preparation process |
| CN103694339A (en) * | 2013-12-04 | 2014-04-02 | 珠海联邦制药股份有限公司 | Renaturation method of insulin glargine precursor |
| CN111909920A (en) * | 2020-08-24 | 2020-11-10 | 武汉人福药业有限责任公司 | Expression and purification method of recombinant human tissue-type plasminogen activator |
| CN111909920B (en) * | 2020-08-24 | 2022-04-15 | 武汉人福药业有限责任公司 | Expression and purification method of recombinant human tissue-type plasminogen activator |
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