CN113355309B - Process for preparing recombined truncated human fibrinolysin - Google Patents

Abstract

The present disclosure provides a preparation process of recombinant truncated human plasmin, which comprises the steps of 1 purifying recombinant plasminogen (Microplasminogen), 2 activating plasminogen, and 3 purifying Microplasmin. Wherein the step 1 of purifying the recombinant microplasminogen comprises MMC chromatography and PHE hydrophobic chromatography; 2, the activation of the microplasminogen in the step 2 adopts urokinase to convert the microplasminogen into microplasmin; and 3, purifying the microplasmin in the step 3, wherein the purification comprises Benzamidine affinity chromatography, SP cation exchange chromatography, ultrafiltration and concentration. The purity of the finished product microplasmin obtained by the method can reach more than 97 percent through optimizing the conditions and selecting the parameters of each step, the yield is about 20 percent, and the method is easy to be industrially amplified.

Description

Process for preparing recombined truncated human fibrinolysin

Technical Field

The invention belongs to the field of biological pharmacy, in particular relates to a preparation process of recombinant truncated human plasmin, and particularly relates to a purification and activation method of truncated human plasminogen for yeast recombinant expression and a purification method of truncated human plasmin.

Background

Vitreous Macular Adhesion (VMA) refers to adhesion between a part of the vitreous and the posterior cortex thereof and the macula when the vitreous is subjected to incomplete posterior detachment (PVD), and persistent VMA can cause a series of pathological changes of the macula such as vitreous macular traction syndrome (VMT), idiopathic macular hole, diabetic macular edema, and the like. An increasing number of studies indicate that VMA is closely related to exudative AMD, and surgical removal of VMA has a certain therapeutic effect on exudative AMD, which indicates that VMA may play a certain role in the pathogenesis of exudative AMD. In recent years, the incidence rate of VMA in exudative AMD is 27.8-36.0%, which is obviously higher than that in non-exudative AMD and normal people, and the difference has statistical significance. In addition, it has been found that exudative AMD eyes that fail photodynamic therapy and anti-Vascular Endothelial Growth Factor (VEGF) therapy often incorporate VMA.

Jetrea (ocriplasmin) is a recombinant truncated human plasmin (GW 005 is a biologically similar agent to Jetrea), has a molecular weight of 27.2 kDa, and degrades proteins involved in VMA production in the eye (the vitreal and vitreoretinal interface VRI protein components, such as laminin, fibronectin, and collagen), which facilitate the separation of the vitreous from the macula. Jetrea received FDA and european union approval for the treatment of VMA and Vitreous Macular Traction (VMT), i.e., VMA, on days 10, 17, 2012 and 3, 15, 2013, respectively. Another treatment option for the disease is vitrectomy.

Combined with the effect of anti-VEGF-A antibody drugs on treatment of eye fundus diseases such as AMD, the GW005 can be applied to patients with AMD and DR who have poor anti-VEGF-A antibody treatment effects in addition to VMA. Currently, VMA usually adopts a therapeutic means of vitrectomy, and as a specific therapeutic drug for VMA, with the progress of diagnostic techniques such as OCT and the widespread application of a vitreous injection method, it is believed that the market volume of GW005 will be further expanded.

Disclosure of Invention

The present disclosure provides a preparation process of recombinant truncated human plasmin, which comprises the steps of 1 purifying recombinant plasminogen (Microplasminogen), 2 activating plasminogen, and 3 purifying Microplasmin. Wherein the step 1 of purifying the recombinant microplasminogen comprises MMC chromatography and PHE hydrophobic chromatography; 2, the activation of the microplasminogen in the step 2 adopts urokinase to convert the microplasminogen into microplasmin; and 3, purifying the microplasmin in the step 3, wherein the purification comprises Benzamidine affinity chromatography, SP cation exchange chromatography, ultrafiltration and concentration. The purity of the finished product microplasmin obtained by the method can reach more than 97 percent through optimizing the conditions and selecting the parameters of each step, the yield is about 20 percent, and the method is easy to be industrially amplified.

Specifically, the method comprises the following steps:

in one aspect, the present invention provides a process for preparing recombinant truncated human plasmin, comprising:

step 1, purifying recombinant Microplasminogen (Microplasminogen);

step 2, activation of microplasminogen;

step 3, purifying Microplasmin;

and 2, activating the purified recombinant microplasminogen into microplasmin by adopting urokinase, reacting for 10-20min at 10-30 ℃ by using the purified recombinant microplasminogen and urokinase in a ratio of 4-8 mu g/U, and adjusting the pH value to be acidic to terminate the activation reaction.

Furthermore, the preparation process of the recombinant truncated human plasmin is characterized by comprising the following steps: the concentration of the recombinant microplasminogen purified in the step 2 is 3-6mg/mL, and the activation of the recombinant microplasminogen is carried out in a solution system with low ionic strength and near neutrality.

Furthermore, the preparation process of the recombinant truncated human plasmin is characterized by comprising the following steps: step 2 activation of the recombinant microplasminogen is to activate for 10min at 20 ℃ in a solution system of 10mmol/L Tris-HCl pH7.2-7.5, and then citric acid is added to pH3.0 after the activation is finished to terminate the reaction.

Further, the preparation process of the recombinant truncated human plasmin, disclosed by the invention, comprises the following steps of 1.1 MMC chromatography:

adopting Bestarose Diamond MMC filler, diluting zymogen fermentation liquor to the conductivity of 9.0 +/-0.5 mS/cm by using purified water, adjusting the pH value to 7.0 +/-0.1, controlling the loading capacity of 20-25 mg/ml, eluting by using an eluent containing 500mmol/L NaCl, 20mmol/L PB and pH value of 7.0, and collecting the recombinant microfibrillar plasminogen.

Further, the preparation process of the recombinant truncated human plasmin, disclosed by the invention, comprises the following step 1.2 of PHE hydrophobic chromatography:

adopting Phenyl High Performance filler, taking pH7.2 and 25mmol/L PB as a basic buffer solution, and adjusting the conductance to 125-130mS/cm by using ammonium sulfate as an equilibrium buffer solution;

adding ammonium sulfate solid into the recombinant microplasminogen collected in the step 1.1 to adjust the conductance to 125-130mS/cm, and controlling the loading capacity to 10-15 mg/ml;

the recombinant microplasminogen is eluted by gradient of 45% elution buffer solution with pH7.2 and 25mmol/L PB as elution buffer solution.

Further, the preparation process of the recombinant truncated human plasmin, provided by the invention, can further comprise the following steps of 1.3G 25 desalting: and (3) replacing the solution system of the recombinant microplasminogen obtained by the hydrophobic chromatography elution of the PHE in the step 1.2 with a solution system with low ionic strength and near neutrality.

Further, the preparation process of the recombinant truncated human plasmin, provided by the invention, comprises the following steps of 3.1Benzamidine affinity chromatography:

adopting Benzamidine Bestarose 4FF affinity filler and taking the affinity filler containing 80mmol/L NaCl, 10mmol/L tranexamic acid, pH7.5 and 20mmol/L PB as equilibrium buffer;

adding NaCl into the activated microplasmin in the step 2 to adjust the conductance to 10 +/-1 mS/cm, adding tranexamic acid solid to 10mmol/L, adjusting the pH value to 7.5 +/-0.1, and controlling the loading capacity to 12-15 mg/ml;

using 500mmol/L tranexamic acid, pH7.5 and 25mmol/L PB as elution buffer, adopting 20% elution buffer to carry out gradient pre-washing to remove hybrid protein with weak binding, using 60% elution buffer to carry out gradient elution on target protein, and immediately using 1mol/L citric acid mother liquor to adjust the pH value of a collected elution peak to be less than 3.5.

Further, the preparation process of the recombinant truncated human plasmin, provided by the invention, comprises the following steps of 3.2 SP cation exchange chromatography:

using Sepharose SP Fastflow cation exchange chromatography packing, and using a buffer solution with pH of 3.2-3.3 and conductance of 38-40 mS/cm as an equilibrium buffer solution;

adding NaCl into the eluent obtained in the step 3.1 to adjust the conductivity to 38-40 mS/cm, adjusting the pH value to 3.2-3.3, and controlling the loading capacity to 9-15 mg/ml;

eluting the target protein by using a buffer solution containing 1mol/L NaCl and an equilibrium buffer solution according to a gradient of 35 percent of eluent.

Further, the preparation process of the recombinant truncated human plasmin, provided by the invention, can further comprise the following step 3.3 of ultrafiltration concentration:

the target protein eluted in step 3.2 was concentrated to 3.0-4.0mg/mL using tangential flow ultrafiltration and the buffer was replaced with 5mmol/L citrate buffer at pH 3.1.

In another aspect, the present invention provides the use of any one of the methods described above in the preparation of Ocripasmin or a biologically similar agent thereof.

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section. The term "plasmin" is an 88kDa non-specific protease that acts on a variety of glycoproteins including LN and FN. In addition to clearing LN and FN, plasmin can also activate endogenous metalloprotease 2. Under normal conditions, metalloprotease 2 is present in the vitreous and has binding power to collagen in the vitreous, and its activation is therefore considered to be closely related to the vitrification of the vitreous. Although plasmin can successfully induce Posterior Vitreous Detachment (PVD), the following problems also limit its application: (1) the plasmin is time-consuming and labor-consuming to obtain from the plasma of the patient, and the price is high; (2) plasmin has strong self-decomposition capability, so the plasmin must be extracted at present, otherwise the activity is rapidly lost; (3) plasmin can be reversibly combined with fibrin, thrombin, alpha 2 antiplasmin and various cells, and the specificity is not high.

The term "recombinant truncated human plasmin", also called Ocriplasmin, is originally named as microplasmin, and is characterized in that only the catalytic domain of plasmin is reserved through recombination and truncation modification on the basis of human plasmin, and the catalytic domain not only maintains the activity of human plasmin in decomposing FN, LN and collagen, but also overcomes the defects of plasmin. The price is relatively reduced because the yeast can synthesize the compound; the stability is higher, and the storage and the use are convenient; can be specifically combined with FN and LN at vitreoretinal interface, and has high specificity. In addition, it has a relatively small molecular weight of only 27.3kDa, and thus has better tissue permeability. GW005 in the present invention has the same sequence structure as ocripasmin. The GW005 sequence was derived from the patent-US 7445775, which is a counterfeit drug obtained by the present applicant during the study on octocrastin (trade name Jetrea). GW005 is a recombinant truncated human plasminogen, contains 249 amino acids in total, has a molecular weight of 27.2 kDa, is a microplasmin (namely AIa543-Asn 791) formed by deleting 12 amino acids from the N-terminal on the basis of mPLm, and has an A chain of 19 amino acids and a B chain of 230 amino acids which are complete active regions. GW005 engineering bacteria adopt X-33 as host bacteria, the inventor firstly uses plasmid pUC57-GW005 as a template, PLG/FW and PLG/RW as primers, obtains GW005 gene segments through a PCR mode, constructs recombinant plasmid pPICZ alpha A-GW005, and obtains an expression vector. Further preparing linearized pPICZ alpha A-GW005 plasmid, obtaining GW005 recombinant protein through electric shock transformation, and carrying out Zeocin (Borax toxin resistance) phenotype identification.

The term "urokinase", an enzyme protein isolated from healthy human urine, or obtained from human kidney tissue culture. Consists of two parts with the molecular weights of 33000 (LMW-tcu-PA) and 54000 (HMW-tcu-PA). Urokinase acts directly on the endogenous fibrinolysis system and can catalyze and crack plasminogen to form plasmin, and the plasmin can degrade fibrin clots and fibrinogen, blood coagulation factor V, blood coagulation factor VIII and the like in blood circulation, thereby playing a role in thrombolysis. Urokinase is plasmin activator, can directly catalyze peptide bond between arginine and valine in plasminogen molecule, and can be hydrolyzed into plasmin.

The term "MMC chromatography", Mixed-mode chromatography (MMC), is a novel method for the separation of biological macromolecules. MMC utilizes the uniquely designed micromolecule functional ligand to effectively synthesize various interactions, including hydrophobic interaction, electrostatic interaction, hydrogen bonds and the like, and realize the specific combination and dissociation of biological objects. The biomacromolecule can be combined on a medium through hydrophobic and thiophilic actions, and then the pH value and/or ionic strength of the solution are adjusted, so that the ligand and the antibody are charged with the same electric charge, and the ligand and the antibody generate electrostatic repulsion action to promote the target molecule to be eluted from the medium. The MMC uses chemical ligand, so the price is low and the performance is stable; ligand density is generally higher and adsorption capacity is greater; has the adsorption characteristic independent of salt, has low requirement on the property of the feed liquid, and is particularly suitable for the large-scale separation process. The interaction between the ligand and the biomolecule is deeply discussed, the optimal design of the MMC ligand can be realized, and the separation selectivity and the separation efficiency are improved. The MMC ligand has designability and controllability, and is a potential new platform for economic and efficient separation of biomolecules.

The term "hydrophobic chromatography" refers to adsorption chromatography from the viewpoint of the mechanism of separating and purifying a living substance. The protein surface generally has hydrophobic and hydrophilic groups, and the hydrophobic chromatography utilizes that a certain part of the protein surface has hydrophobicity and is combined with a carrier with hydrophobicity at high salt concentration. The salt concentration is gradually reduced during elution, and the salts are eluted one by one successively to purify due to different hydrophobicity, so that the method can be used for separating proteins which are difficult to purify by other methods. Binding ligands on hydrophobic chromatographic media include ether, isopropyl, phenyl, n-butyl, n-octyl, and the like. Phenyl Hp is a hydrophobic chromatography medium with Phenyl as the binding ligand.

The term "sephadex G25" is a specific medium for size exclusion chromatography or gel filtration. The molecular exclusion layer is a chromatographic separation technique based on the molecular weight difference of the separated substances, the solid phase carrier is gel particles, and currently, Sephadex (Sephadex) and Sepharose (Sepharose) with various pore sizes are widely used. Sephadex chromatography is a process in which the material to be separated is passed through a Sephadex column, where the components, due to their different molecular weights, are subjected to different retardation on the column and move at different speeds in the column. Substances with molecular weight larger than the range of allowed gel meshes are completely excluded by the gel, cannot enter the interior of gel particles, have small blocking effect, and flow out of the chromatographic column firstly along with the flowing of a solvent among the gel particles, so the flow is short; the substances with small molecular weight can completely enter the meshes of the gel particles, the blocking effect is large, the flow is prolonged, and finally the substances flow out of the chromatographic column, thereby achieving the purpose of separation.

The term "Benzamidine affinity chromatography", a serine protease purification affinity medium, is prepared by coupling p-anisidine, a broad-spectrum inhibitor of serine protease, onto agarose microspheres, Focure 6B, and high-strength cross-linked agarose, Focure 4FF, referred to as Benzamidine Focure 6B and Benzamidine Focure 4 FF.

The term "ion exchange chromatography", which is a chromatography method for separating a component ion in a mobile phase from a counter ion on an ion exchanger by using the ion exchanger as a stationary phase according to the difference in the binding force between the component ion and the counter ion in the mobile phase when they are reversibly exchanged with each other. The charge of a protein is determined by the charged amino acids in the protein polypeptide. Since the charge of the amino acids in the protein is in turn dependent on the pH in the medium, the charge of the protein is also dependent on the pH of the medium. At lower pH, the negatively charged groups are neutralized and the more positively charged groups, and at higher pH, the electrical properties of the protein are reversed from those at low pH. When the protein is at a pH that makes the positive and negative charges of the protein equal, the pH at this time is called the isoelectric point. The exchanger used for ion exchange chromatography is prepared by introducing positive or negative ion groups through chemical reactions such as esterification and oxidation, and can exchange and adsorb with protein with opposite charges. Exchangers carrying cationic groups can displace negatively charged substances, known as anion exchangers, such as DEAE-cellulose resins: and vice versa as cation exchangers, such as CM-cellulose resins. Different proteins have different isoelectric points, and the types and the amounts of charges carried by the proteins after dissociation under certain conditions are different, so that the proteins can exchange and adsorb with different ion exchangers with different affinities. When the ionic groups in the buffer compete with the proteins bound to the ion exchanger, the molecules of the proteins with low affinity are desorbed first and elute, while the proteins with high affinity are desorbed later and elute. Therefore, by increasing the ionic strength of the buffer solution and/or changing the pH value, the adsorption condition of the protein can be changed, so that the proteins with different affinities can be separated.

The term "conductivity" is a parameter used to describe the ease of charge flow in a substance. The electrolyte solution can conduct electricity because the electrolyte can be ionized in the aqueous solution to generate cations and anions, and the cations and the anions move in opposite directions under the action of an electric field to form current, so that the electricity conduction phenomenon is generated. For electrolyte solutions, the concentration of the solution has a large effect on the conductivity.

The term "tangential flow" refers to a form of filtration in which the direction of liquid flow is perpendicular to the direction of filtration. Traditional liquid dead end filtration (dead end), also called perpendicular filtration, is most millipore filtration (MF, microfiltration), including the filtration form that aseptic filtration adopted, the flow direction of its liquid is unanimous with the filtration direction, and along with filterable going on, the cake layer or the gel layer thickness that the filtration membrane surface formed increase gradually, and the velocity of flow reduces gradually. When the filter medium is an ultrafiltration membrane or a microfiltration membrane with small pore diameter, the solid content in the feed liquid is very high, and the flow velocity is rapidly reduced by adopting a dead-end filtration mode, so that the dead-end filtration can only process the feed liquid with small volume. The tangential flow ultrafiltration system is mainly applied to concentration, purification, dialysis (desalination, dealcoholization and the like), buffer solution replacement, culture solution, buffer solution heat source removal and the like of biological products.

The invention achieves the following beneficial technical effects:

after yeast high expression of Microplasminogen (Microplasminogen, inactive), GW005 zymogen was purified, then, Urokinase (UK) was used as an activator to convert GW005 zymogen into active Microplasmin, GW005 enzyme, then, the activator was removed by the purification step, and finally, 5mmol/L citric acid was used to equilibrate to pH3.1, forming a stable drug solution.

Zymogen fermentation → MMC chromatography → PHE hydrophobic chromatography → desalination → urokinase activation → Benzamidine affinity chromatography → SP cation exchange chromatography → ultrafiltration concentration → stock solution. In this process: (1) the high-conductivity zymogen fermentation supernatant can be directly put on an MMC chromatographic column only by diluting 1-1.5 times and adjusting the pH, and can quickly enrich target protein and remove most of culture medium, pigment, degradation protein and host protein; (2) adding ammonium sulfate into the target protein collected by the MMC to be loaded on a PHE hydrophobic column, and further removing degraded protein, pigment and small molecular impurities by controlling elution gradient to achieve a better concentration effect; (3) desalting the PHE peak by G25, activating urokinase to form active enzyme, controlling the ratio of substrate to tool enzyme, activating temperature and time to obtain stable mixture of enzyme and zymogen, and regulating pH value to below 3.5 to inhibit urokinase enzyme activity to terminate reaction; (4) after activation, the mixture can be loaded on a Benzamidine affinity column only by adjusting to a proper pH value, zymogen and a part of nonspecific enzyme digestion bands generated in the activation process are removed, and an elution peak needs to be adjusted to be below pH3.5 for storage and detection so as to avoid continuous degradation in the detection process and influence on the judgment of a result; (5) adjusting the peak of the Benzamidine to a proper pH value and a proper conductivity value, loading the peak on an SP cation exchange chromatographic column, and removing residual urokinase and part of non-specific miscellaneous bands; (6) the SP peak obtained is concentrated by ultrafiltration and the buffer is replaced to obtain GW005 stock solution. The whole purification combination is relatively reasonable, the purity of the final product is more than 97%, the yield is about 20%, and the industrial amplification is easy.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1: MMC column chromatography different loading pH comparison SDS-PAGE detection map.

Lane 1, initial sample of fermentation broth; lane 2, run through at pH 6.0; lane 3, run through at pH 7.0; lane 4, peak eluted at ph 6.0; lane 5, peak eluted at pH 7.0.

FIG. 2: comparing SDS-PAGE detection images of different sample conductances of MMC column chromatography.

Lane 1, initial sample of fermentation broth; lane 2, 1-fold diluted sample run-through; lane 3, flow through directly loaded.

FIG. 3: MMC column chromatography different eluents were compared to SDS-PAGE detection profiles.

From left to right: lane 1, eluent B1 elution peak; lane 2, eluent B2 eluted the peak.

FIG. 4: PHE column chromatography gradient elution chromatogram and SDS-PAGE detection chart.

FIG. 4A: PHE column chromatography gradient elution chromatogram.

FIG. 4B: PHE column chromatography gradient elution peak SDS-PAGE detection map.

From left to right: lanes 1, 2, 3, 4 correspond to elution peaks 1, 2, 3, 4, respectively.

FIG. 5: PHE column chromatography different flow-through SDS-PAGE detection images.

From left to right: lane 1, initial sample; lane 2, 10-15 mg/ml flow-through peak; lane 3, 15-20 mg/ml flow through peak.

FIG. 6: PHE column chromatography different elution conditions chromatogram and SDS-PAGE detection chart.

FIG. 6A: PHE column chromatography with different elution conditions.

FIG. 6B: and carrying out SDS-PAGE detection on elution peaks by PHE column chromatography under different elution conditions.

From left to right: lanes 1, 2, 3 correspond to elution peaks 1, 2, 3, respectively.

FIG. 7: the 12% reduced SDS-PAGE stained image of the digested sample.

Lane 1, enzyme digestion 10 min; lane 2, enzyme digestion 15 min; lane 3, enzyme digestion 20 min; lane 4, enzyme digestion 25 min; lane 5, enzyme digestion for 30 min.

FIG. 8: SDS-PAGE stained images were reduced 12% after digestion of the samples.

Lane 1, post-digestion sample at 20 ℃; lane 2, post-digestion sample at 25 ℃; lane 3, sample after digestion at 30 ℃; lane 4, post-digestion sample at 37 ℃.

FIG. 9: 12% reducing SDS-PAGE of digested samples at different temperatures.

Lane 1, 10min sample digested at 20 ℃; lane 2, a 2min sample at 37 ℃.

FIG. 10: benzamidine column chromatography 0.5mol/L NaCl loading chromatogram.

FIG. 11: conductivity comparison chromatography overlay chart of different loading samples of Benzamidine column chromatography.

The earliest peak was in the 5mS/cm group; the middle peak is 10mS/cm group; the latest peak was 15 mS/cm.

FIG. 12: the SDS-PAGE detection image of the Benzamidine column chromatography samples at different tranexamic acid concentrations was compared.

Lane 1, initial sample; lanes 2, 3, 4, 5, corresponding to 0mmol/L, 10mmol/L, 50mmol/L, 100mmol/L tranexamic acid concentration, respectively.

FIG. 13: the SDS-PAGE detection image of different stages of flow through of the Benzamidine column chromatography.

From left to right: lane 1, initial sample; lanes 2, 3, and 4, which are 0-30%, 30-60%, and 60% of the total loading, respectively, and the end of loading, are the flow-through peaks.

FIG. 14: schematic diagram of contour drawing device for analysis of SP column chromatography sample loading condition DOE design experiment result.

The area indicated by the box at the upper left corner in the figure is the finally selected proper sample loading interval.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1: fermentation preparation process

The production strain is inoculated in a YPD liquid culture medium for shaking culture at 30 ℃ and 220-280 rpm, and is subjected to primary culture and secondary culture until the wet weight of the strain is about 50g/L, and then the strain is subjected to tank culture, wherein the inoculation amount is 10%. If the fermentation scale is further enlarged, a seed tank culture process is added after the primary and secondary seeds, and the volume of the seed liquid is enlarged to 10% of the initial volume of the fermentation medium.

The fermentation process is divided into two stages, glycerol is used as a carbon source in the growth stage, a basic salt culture medium is cultured at the temperature of 30 ℃ and the pH value of 5.0 +/-0.2, dissolved oxygen is guaranteed to be not lower than 30% of saturation degree through aeration and stirring, after culture for about 12-18 hours, when the dissolved oxygen obviously rises, the glycerol in the culture medium is exhausted, the wet weight of the bacteria concentration is 90-120 g/L, glycerol feeding is started, the feeding speed is 12-15 ml/L/h, feeding is carried out for 2-3 hours, and the wet weight of the bacteria is 130-160 g/L. Feeding is terminated and induction of expression is initiated. The induction phase was sequentially supplemented with 10% Tween-80 solution (volume 0.1% (v/v) of the volume of the initial fermentation medium), and 1000 Xbiotin solution (volume 0.1% (v/v) of the volume of the initial fermentation medium). At this stage, closely paying attention to relevant data such as pH value, ventilation volume, stirring, dissolved oxygen, temperature, methanol, ammonia water, thallus wet weight and the like, and supplementing 25% of defoaming agent solution according to the foam condition in the fermentation process and the principle of small quantity and multiple times. The pH value of the induction stage is set to 5.5 +/-0.1, and ammonia water is used as a pH regulator. In the induction expression process, the dissolved oxygen is controlled to be in sawtooth-shaped fluctuation about 20 percent by adjusting the stirring rotating speed, the ventilation quantity and the oxygen and air ratio. The temperature is controlled to be 25 +/-1 ℃ in the initial stage of induction, and the temperature is reduced to be 22 +/-1 ℃ after 24 hours. And in the induction expression stage, yeast powder and peptone are added into the fermentation liquor once every 24 hours, the addition amounts are respectively 1% and 2% (w/v, sterilized) of the initial volume of the fermentation, and the fermentation is induced for 60-72 hours and placed in a tank.

Example 2: determination and process optimization of zymogen protein rapid capture method

The fermentation supernatant has complex components, contains expressed GW005 zymogen, culture medium, secondary metabolites, cell fragments, protease released by cell disruption and the like, and needs a purification method for efficiently capturing target protein. The method for capturing fermentation liquor, blood products and the like generally comprises the following steps: the advantages and disadvantages of ammonium sulfate fractional precipitation, BLUE affinity chromatography and MMC chromatography were compared and are shown in Table 1.

TABLE 1 comparison of methods for rapid enrichment of GW005 zymogen

Method	Advantages of the invention	Disadvantages of
			Fractional precipitation of ammonium sulfate	Simple and quick operation	The fermentation liquor has high density and viscosity, and the formed protein precipitate is difficult to separate, so that the yield is lower, generally 50 percent
BLUE affinity chromatography	Can resist higher salt sample loading, and can achieve higher yield and purity in one step	The flow-through is obvious and the loading capacity is low
			MMC chromatography	Can resist high salt and achieve higher yield and purity in one step	The elution volume is larger

Through comparison, Bestarose Diamond MMC is finally determined to be used as the chromatographic packing for rapidly capturing GW005 zymogen protein.

Determination of pH of the sample

The GW005 zymogen has higher isoelectric point (above 8.5), the lower the pH value, the more positive charges are carried, the more favorable the protein combination is, but the too low pH value can enhance the impurity combination capability and influence the combination of the target protein and the improvement of the purity. The binding between the GW005 zymogen supernatant and the pH6.0 was compared at about pH7.0, and the results are shown in Table 2 and FIG. 1.

TABLE 2 comparison of MMC column chromatography loading pH

The results of table 2 and fig. 1 show that the pH has a large influence on the loading capacity, and under the condition of constant conductance, the pH is reduced from 7.0 to 6.0, and the loading capacity is increased by at least 47%; however, during elution, the protein is hardly eluted by the conventional buffer solution, and the MMC can be eluted only after the elution pH is increased, but the purity is obviously inferior to that of the group with the pH of 7.0, and the MMC loading pH is determined to be 7.0 by comprehensive consideration. On this basis, further attempts were made to increase the loading by optimizing the conductance.

2.2 determination of sample conductance

Under normal conditions, the conductance value of GW005 zymogen fermentation supernatant is 15-20 mS/cm, the combination condition of direct sample loading and diluted 1-time sample loading is compared, and the results are shown in table 3 and figure 2.

TABLE 3 comparison of MMC column chromatography direct loading with diluted 1-fold loading

The results in table 3 and fig. 2 show that the flow-through ratio of the fermentation liquid directly loaded (15.9 mS/cm) is larger, and the loading capacity after dilution by 1 time (9.1 mS/cm) is improved by 28%, so that the conductivity value of the MMC column chromatography loaded sample is determined to be about 9 mS/cm.

2.3 determination of elution pH

The principle of MMC chromatography is that strong and weak differential adsorption is generated between the sample surface positive and negative charge distribution difference and filler charge, and substances with different properties are separated by changing the ionic strength or pH value in a buffer system. The results of comparing the peak purity and yield of eluents of different pH values are shown in Table 4 and FIG. 3.

TABLE 4 comparison of different pH eluents of MMC column chromatography

The results in table 4 and fig. 3 show that after elution with eluent B1, eluent B2 has almost no obvious elution peak, which indicates that eluent B1 is eluted almost completely, and the content of impurities in the protein peak eluted by eluent B2 is very high, which indicates that the impurities are strongly combined, so that the MMC column chromatography eluent is determined to be 20mmol/L PB, ph7.0+500mmol/L NaCl.

Summary of MMC chromatography studies: adopting Bestarose Diamond MMC filler, diluting zymogen fermentation liquor to the conductivity of 9.0 +/-0.5 mS/cm by using purified water, adjusting the pH value to 7.0 +/-0.1 by using HCl/NaOH, and controlling the loading capacity of 20-25 mg/ml. Most of culture medium, background expression hybrid protein, pigment and the like flow through in the sample loading process; after the sample is completely loaded, 20mmol/L PB and pH7.0+500mmol/L NaCl solution are used for elution, and the elution peak is collected, namely the enriched GW005 zymogen.

Example 3: determination and process optimization of zymogen protein fine purification method

GW005 zymogen is enriched in MMC chromatography, but a small amount of degradation fragments and small molecular impurities still exist, and the most effective method for removing the impurities is to perform different column chromatography, including hydrophobic column chromatography, cation exchange column chromatography, anion exchange column chromatography and the like. MMC chromatography adopts high-salt elution, the conductivity of an elution peak is higher, and if cation or anion column chromatography is adopted, a sample needs to be diluted by a large factor or one-step desalting column chromatography is added, the production period can be obviously prolonged; hydrophobic column chromatography can be carried out by adding ammonium sulfate to proper conductivity. By experimental comparison, Phenyl High Performance was finally selected as the packing for hydrophobic column chromatography.

3.1 determination of sample conductance

Because the hydrophobicity of GW005 zymogen and hybrid protein is unknown, a higher-conductivity loading is adopted, and a method for reducing the conductivity by gradient is adopted to determine a more appropriate loading conductivity.

Name of the chromatographic column: XK 16/20;

name of the filler: phenyl HP, column volume 22 ml;

loading capacity: 10 mg/ml;

equilibration buffer and sample conductance: 150-155 mS/cm;

elution buffer: 25mmol/L PB, pH7.2, gradient elution, collection of elution peak, SDS-PAGE detection, the results are shown in figure 4 (figure 4A, figure 4B).

The result of FIG. 4B shows that the peak 1 in the chromatogram of FIG. 4A has a poor purity and contains a high proportion of impurities, while the other peaks have no obvious difference, and the elution conductance value corresponding to the peak 1 is about 122mS/cm, so that the loading conductance value needs to be selected to be about 122mS/cm, but considering that the proportion of the target protein contained in the peak 1 is also large, in order to increase the column chromatography loading as much as possible, the loading conductance value is determined to be 125-130 mS/cm.

Determination of loading capacity

After the sample loading conductance is determined to be 125-130mS/cm, the sample loading capacity is searched, and the loading capacity range is determined by an overload sample loading sectional collection flow-through method.

Filling information: phenyl HP, column volume 5 ml;

loading capacity: 20 mg/ml;

equilibration buffer and sample conductance: 125-130 mS/cm;

in the sample loading stage, the flow-through peaks of 0-10 mg/ml, 10-15 mg/ml and 15-20 mg/ml fractions were collected and subjected to SDS-PAGE, and the results are shown in FIG. 5.

The SDS-PAGE result of figure 5 shows that the flow-through protein amount of 10-15 mg/ml section is little, and the flow-through is obvious after 15mg/ml, so the PHE column chromatography loading range is determined to be 10-15 mg/ml.

Determination of elution conditions

Name of the chromatographic column: XK 16/20;

name of the filler: phenyl HP, column volume 21 ml;

loading capacity: 15 mg/ml;

equilibration buffer and sample conductance: 125-130 mS/cm;

elution buffer: gradient elution was carried out with 25mmol/L PB and pH7.2 eluent, and each elution peak was collected and subjected to SDS-PAGE, and the results are shown in FIG. 6 (FIG. 6A, FIG. 6B).

The result of fig. 6B shows that the elution peak 3 in the chromatogram of fig. 6A has a poor purity and a relatively distinct impurity band, while the elution peaks 1 and 2 do not exist, and the elution gradient corresponding to the elution peak 2 is 45%, so that the PHE column chromatography elution gradient condition is determined to be 45%.

PHE chromatography study summary: adopting Phenyl High Performance filler, adding ammonium sulfate solid to adjust the conductivity of an MMC elution peak to 125-130mS/cm, controlling the loading capacity of the sample to be 10-15 mg/ml, controlling the equilibrium buffer solution to be 25mmol/L PB and pH7.2, adding ammonium sulfate to the conductivity to be 125-130mS/cm, controlling the elution buffer solution to be 25mmol/L PB and pH7.2, adopting the gradient of 45% elution buffer solution to elute the target protein, and using the gradient of 100% elution buffer solution to elute the protein with strong binding and degraded impurities.

Desalination

Name of the chromatographic column: XK 26/20;

filling information: g25, column volume 50ml

Sample loading amount: 10 to 20 percent

The recombinant microplasminogen obtained by PHE hydrophobic chromatography elution is desalted by G25, and the solution system is replaced by a low-ionic strength near-neutral solution system (10 mmol/L Tris-HCl pH7.5).

Example 4: determination of zymogen activation method and process optimization

GW005 zymogen becomes enzyme with bioactivity after being activated, wherein the peptide bond between R19 and V20 is hydrolyzed and broken by urokinase, staphylokinase or streptokinase, so that the molecular conformation is changed and the enzyme has fibrinolytic bioactivity. Urokinase (UK) was used as an activator in the search. The process of enzyme digestion activation of UK is essentially an enzymatic process, and the enzymatic process is generally related to a reaction system (mainly pH value, ionic strength and the like), temperature, substrate concentration, enzyme concentration and reaction time. According to experience and simple experiments, UK has higher activity under the pH near-neutral condition, and almost completely deactivates at the pH value of 3.0; obviously influencing the UK enzyme digestion under the condition that 0.5mol/L ammonium sulfate exists; therefore, 10mmol/L Tris-HCl pH7.5 with low ionic strength was selected as the activation reaction system. Generally the enzymatic reaction rate is positively correlated with the reaction temperature, substrate concentration and enzyme concentration; the reaction time required for the reaction in which the enzymatic rate is fast is relatively short. Therefore, the reaction temperature, the substrate (i.e., GW005 proenzyme) concentration, the enzyme (i.e., UK) concentration and the reaction time were intensively studied.

Enzyme digestion time pretest

Performing enzyme digestion reaction according to the parameters in the table 5, sampling electrophoresis every 5min of enzyme digestion, adding citric acid into the sampled product until the pH value is close to 3.0, stopping enzymatic reaction, and inspecting for 30 min; the samples were examined by 12% reduction SDS-PAGE and the results are shown in FIG. 7.

TABLE 5 parameter set-up for enzyme digestion time Pre-run

The results of fig. 7 show that: after 20min of digestion, the amount of GW005 zymogen (indicated by the dotted arrow) was reduced, but the amount of GW005 zymogen (indicated by the solid arrow) was not increased or reduced, but the non-specific cleavage band (indicated by the parenthesis) was increased. The analysis reason is as follows: in the initial reaction stage, the GW005 zymogen concentration is high, UK mainly activates zymogen, and a small amount of non-specific enzyme digestion exists; with the lapse of time, the concentration of GW005 enzyme is higher than that of zymogen, and at the moment, UK can further carry out enzymolysis on GW005 enzyme, so that the concentration of GW005 enzyme is almost unchanged or reduced, and the non-specific enzyme digestion band is gradually increased. Therefore, the enzyme digestion takes a suitable time, and the longer the time is, the more GW005 enzyme is obtained.

Temperature pre-run of enzyme digestion

The digestion reactions were performed according to the parameters of Table 6, with digestion experiments at 20 deg.C, 25 deg.C, 30 deg.C and 37 deg.C, respectively, with one sample at every 2min for digestion at 37 deg.C, and the others at 10 min. The results of 12% reduction SDS-PAGE examination of the digested samples are shown in FIG. 8, and the calculated GW005 enzyme yield is shown in Table 7.

TABLE 6 parameter set-up for enzyme digestion temperature Pre-test

TABLE 7 GW005 enzyme yield table

The results in fig. 8 and table 7 show that: as the temperature increases, the nonspecific cleavage increases significantly, while the amount of the GW005 enzyme obtained (yield) does not increase correspondingly.

The results of comparing the samples digested at 20 ℃ for 10min with the samples digested at 37 ℃ for 2min by 12% reduction SDS-PAGE staining are shown in FIG. 9. FIG. 9 shows that after digestion, GW005 enzyme yield is comparable, and nonspecific digestion at a lower temperature is much less, so digestion conditions were chosen: the temperature is 20 ℃ and the enzyme digestion time is 10 min.

Example 5: GW005 enzyme crude purification method determination and process optimization

After the proenzyme is activated by urokinase, the components are complex, besides the needed GW005 enzyme, the proenzyme also has residual non-activated proenzyme and non-specific hybrid bands are generated in the activation process, and a relatively efficient purification method is needed for separating the GW005 enzyme and other impurities. The literature reports that serine protease can have specific affinity action with the Benzamidine filler, and GW005 enzyme belongs to the serine protease family, so the Benzamidine Bestarose 4FF is selected as the crude purification filler of the activated enzyme.

Determination of sample conductance

In the affinity purification process, a certain amount of non-specific adsorption exists, and in order to reduce the non-specific adsorption, a certain concentration of salt is generally added into the sample, so that the condition that the concentration of the loading salt is 0.5mol/L NaCl is firstly tested.

A chromatographic column: 5ml of a pre-packed column;

filling: benzamidine 4 FF;

loading capacity: 2 mg/ml;

sample information: NaCl is added into the sample to 0.5mol/L, and the pH value is adjusted to 7.4 by 1mol/L NaOH solution;

and (3) an equilibrium buffer: 50mmol/L Tris-HCl +0.5mol/L NaCl, pH7.4;

elution buffer: 50mmol/L Glycine, pH3.0.

The elution profile is shown in FIG. 10, and the results show that the sample was run through during loading, so the salt concentration had to be reduced.

The combination of the three salt concentrations of 5mS/cm, 10mS/cm and 15mS/cm is continuously searched and compared, a chromatographic overlay is shown in FIG. 11, the result shows that the flow-through of three groups of samples is slightly increased along with the increase of the conductance value, but the overall difference is not obvious, the compromise condition is realized, and finally 10mS/cm is selected as the sample loading conductance of the Benzamidine column chromatography.

5.2 determination of the concentration of tranexamic acid in the sample

Tranexamic acid blocks the lysine binding site of Plasminogen (Plasminogen), thereby reducing its occurrence of self-cleavage; since GW005 zymogen is a truncated product of Plasminogen, it is necessary to add some tranexamic acid to reduce self-shearing in the purification process after GW005 zymogen activation, but tranexamic acid may also inhibit the binding between GW005 and Benzamidine filler, so the effect of different tranexamic acid concentrations on binding capacity is compared, and the results are shown in table 8 and fig. 12.

TABLE 8 comparison of the results of different tranexamic acid concentrations in Benzamidine column chromatography

The results in Table 8, FIG. 12 show that as the concentration of tranexamic acid increases, the flux is significant, with less than 10% flux at a concentration of 10mmol/L, after the trade-off, it was decided to use 10mmol/L for the final added concentration of the loading tranexamic acid.

Determination of loading capacity

A chromatographic column: 5ml of a pre-packed column;

filling: benzamidine 4 FF;

loading capacity: 20 mg/ml;

sample information: sample conductance was adjusted to 10mS/cm, pH7.5 and tranexamic acid was added to 10 mmo/L;

SDS-PAGE was performed by stepwise collecting flow-through peaks at different ratios of the total amount of sample in the collection process, and the results are shown in FIG. 13.

The result of figure 13 shows that the section flow-through peaks of 0-30% and 30-60% are mainly zymogen, and a small amount of target protein flow-through appears after 60%, and the target protein is calculated to be about 5mg, so that the loading capacity of the finally used Benzamidine 4FF filler is determined to be 12-15 mg/ml.

5.4 determination of elution conditions

A chromatographic column: XK16/20

Filling information: benzamidine 4FF, 17 ml;

loading capacity: 15 mg/ml;

sample information: sample conductance was adjusted to 10mS/cm, pH7.5 and tranexamic acid was added to 10 mmo/L;

and (3) an equilibrium buffer: 20mmol/L PB, pH7.5+80mmol/L NaCl +10mmol/L tranexamic acid;

elution buffer: 20mmol/L PB, pH7.5+500mmol/L tranexamic acid.

After sampling, the samples were sequentially eluted by gradients of 20% B, 60% B, 80% B, 100% B, etc., and the elution peaks were collected, and the content, recovery rate, and purity of the elution peaks were measured, and the results are shown in table 9.

TABLE 9 Benzamidine column chromatography different elution gradient results

The results of recovery and purity counted according to table 9 show that the 60% gradient elution peak has higher purity and yield, and substantially completely elutes the target protein, while the 20% and 100% gradient elution peak have poorer purity, which indicates that some impurities contained in the sample are stronger than the target protein, and some impurities are weaker than the target protein, so the finally determined elution conditions are: 20% prewashing, 60% eluting target protein, 100% regeneration.

Summary of the chromatographic development of Benzamidine: adopting Benzamidine Bestarose 4FF affinity filler, adding NaCl solid to adjust the conductivity of a sample after urokinase activation to 10 +/-1 mS/cm, adding tranexamic acid solid to 10mmol/L, adjusting the pH value to 7.5 +/-0.1 by using 1mol/LNaOH solution, and controlling the loading capacity to be 12-15 mg/ml. The equilibrium buffer solution is 20mmol/L PB, the pH value is 7.5+80mmol/L NaCl +10mmol/L tranexamic acid, the elution buffer solution is 25mmol/L PB, the pH value is 7.5+500mmol/L tranexamic acid, the weak-binding hybrid protein is pre-washed by adopting the gradient of 20% elution buffer solution, the target protein is eluted by the gradient of 60% elution buffer solution, and finally, the strong-binding impurity is regenerated and removed by the gradient of 100% elution buffer solution. The collected elution peak needs to be immediately adjusted to a pH value below 3.5 by using 1mol/L citric acid mother liquor for storage and detection.

Example 6: GW005 enzyme purification method determination and process optimization

GW005 enzyme is enriched in Benzamidine affinity chromatography, but a small amount of degradation fragments and residual urokinase still exist, the most effective method for removing the impurities is to adopt ion exchange column chromatography, the principle of the method is that strong and weak differential adsorption is generated between the GW005 enzyme and filler charges by utilizing the characteristic of positive and negative charge distribution difference on the surface of a sample, and substances with different properties are separated by changing the ionic strength or pH value in a buffer system. Considering that GW005 enzyme is stable at low pH value and is seriously degraded after the pH value is increased, cation exchange chromatography is more suitable, and Sepharose SP Fastflow is finally selected as filler for GW005 enzyme fine purification chromatography through experimental comparison.

Determination of the Loading conditions

According to the pH value stability result of GW005 enzyme, the GW005 enzyme has better stability when the pH value needs to be adjusted to be below pH4.0 at room temperature, so the pH value of the sample is lower than 4.0; meanwhile, as the isoelectric point of GW005 enzyme is higher (more than 8.5), and too low pH value can cause protein to have very much positive charge, so that the protein is very firmly combined with the filler, and the elution yield is influenced, the binding capacity of the sample and the filler is reduced by increasing the conductivity value of the sample; and (3) synthesizing the two conditions, and performing experimental design and result analysis by adopting a DOE method in two ranges of pH value of 3.2-3.8 and conductance of 20-40 mS/cm to optimize the sample loading condition.

According to the sample loading condition designed by DOE, a 5ml small column is adopted, each group of experiments are loaded according to the flow-through protein amount of 5%, and the loading capacity is calculated according to the actual protein amount of the loaded samples. After the equilibration, the elution is carried out by using 20mmol/L citric acid, 28mmol/L sodium citrate and 1mol/L NaCl and a buffer solution with the pH value of 3.8, elution peaks are collected and the concentration is measured, and the content of each elution peak and the protein recovery rate are calculated, and the results are shown in Table 10.

TABLE 10 SP column chromatography results for different loading conditions

According to the statistical data result in the table 10, the loading capacity and the yield form an obvious negative correlation relationship, the loading capacity and the yield are weighed, firstly, two points with the loading capacity of more than 9mg/ml and the yield of more than 65% are selected to draw a contour line drawing instrument graph (as shown in fig. 14), a region (pH 3.2-3.8 and conductance 33-40 mS/cm) surrounded by two lines in fig. 14 is a region meeting the conditions, but the stability of GW005 enzyme is further considered, so that the pH value interval is reduced to pH 3.2-3.3, the conductance value is changed to 38-40 mS/cm, namely a filling region of a square frame at the upper left corner in the upper graph, the corresponding loading capacity is 9-15 mg/ml, the yield is 66.4-72.6%, the pilot test production requirements are met, and therefore the sampling conditions of the SP are finally determined to be pH 3.2-3.3 and the conductance is 38-40 mS/cm.

Determination of elution conditions

A chromatographic column: SP FF, 5ml self-contained column;

loading capacity: 9-15 mg/ml;

equilibration buffer and sample: controlling the pH value to be 3.2-3.3 and the conductance to be 38-40 mS/cm;

elution buffer: 20mmol/L citric acid pH3.2-3.3, containing 1mol/L NaCl solution gradient elution, collecting each elution peak, detection elution peak content, recovery rate, purity, the results are shown in Table 11, 12.

TABLE 11 SP column chromatography different elution gradient results (60 mg loading)

TABLE 12 SP column chromatography different elution gradient results (55 mg loading)

Combining the results of recovery and purity in tables 11 and 12, the 30% and 35% gradient elution peak purity were comparable, with a slightly higher recovery of 35%, so it was decided to finally use 35% as the final SP column chromatography elution gradient.

Summary of SP chromatographic process studies: adding a NaCl solid to adjust the elution peak of the Benzamidine to the conductance of 38-40 mS/cm by using a Sepharose SP Fastflow cation exchange chromatography filler, adjusting the pH value to 3.2-3.3 by using 1mol/L citric acid or sodium citrate mother liquor, and controlling the loading capacity of 9-15 mg/ml. After the sample is loaded, the buffer solution containing 1mol/L NaCl and the equilibrium solution with the same pH value are used for eluting the target protein according to the gradient of 35% eluent, and the 100% eluent is used for eluting and combining strong degradation impurities and residual urokinase.

Example 7: concentrating by ultrafiltration

GW005 dosage form adopts water injection dosage form, concentration is 2.5mg/ml, and buffer solution in the prescription is 5mmol/L citrate (citric acid and sodium citrate buffer pair, pH3.1). Since the SP cation exchange chromatography peak in the last step had a low protein concentration (about 1.5 mg/ml) and the buffer was different, it was necessary to replace the buffer with the SP chromatography peak and concentrate the protein. The replacement buffer solution generally comprises methods such as gel filtration chromatography, dialysis, ultrafiltration and the like, wherein the protein concentration is reduced by the former two methods, the ultrafiltration can concentrate samples while effectively replacing the buffer solution, and the treatment time can be shortened by increasing the membrane area of the ultrafiltration membrane. Therefore, tangential flow ultrafiltration was used to displace the buffer and concentrate GW005 to 3.0-4.0 mg/ml.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Miwei (Shanghai) Biotech Co., Ltd

Jiangsu Maiweikang New drug research and development Limited company

<120> preparation process of recombinant truncated human plasmin

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Thr Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

Claims (6)

1. A preparation process of recombinant truncated human plasmin, comprising the following steps:

step 1, purifying recombinant Microplasminogen (Microplasminogen) from the supernatant of yeast fermentation liquor with high expression of Microplasminogen, comprising step 1.1 MMC chromatography and step 1.2 PHE hydrophobic chromatography;

step 2, activation of microplasminogen;

step 3, purifying Microplasmin, comprising step 3.1 of affinity chromatography of Benzamidine and step 3.2 of cation exchange chromatography of SP;

wherein the content of the first and second substances,

step 1.1 MMC chromatography: adopting Bestarose Diamond MMC filler, diluting zymogen fermentation liquor to the conductivity of 9.0 +/-0.5 mS/cm by using purified water, adjusting the pH value to 7.0 +/-0.1, controlling the loading capacity of 20-25 mg/ml, eluting by using an eluent containing 500mmol/L NaCl, 20mmol/L PB and pH value of 7.0, and collecting recombinant microfibrillar plasminogen;

step 1.2 PHE hydrophobic chromatography: adopting Phenyl High Performance filler, taking pH7.2 and 25mmol/L PB as a basic buffer solution, and adjusting the conductance to 125-130mS/cm by using ammonium sulfate as an equilibrium buffer solution; adding ammonium sulfate solid into the recombinant microplasminogen collected in the step 1.1 to adjust the conductance to 125-130mS/cm, and controlling the loading capacity to 10-15 mg/ml; eluting the recombinant microplasminogen by gradient of 45% elution buffer solution with pH7.2 and 25mmol/L PB as elution buffer solution;

step 2, activating the purified recombinant microplasminogen into microplasmin by adopting urokinase, wherein the proportion of the purified recombinant microplasminogen to the urokinase is 4-8 mug/U, reacting for 10-20min at 10-30 ℃, and adjusting the pH value to acidity to terminate the activation reaction;

step 3.1 affinity chromatography of Benzamidine: adopting Benzamidine Bestarose 4FF affinity filler and taking the affinity filler containing 80mmol/L NaCl, 10mmol/L tranexamic acid, pH7.5 and 20mmol/L PB as equilibrium buffer; adding NaCl into the activated microplasmin in the step 2 to adjust the conductance to 10 +/-1 mS/cm, adding tranexamic acid solid to 10mmol/L, adjusting the pH value to 7.5 +/-0.1, and controlling the loading capacity to 12-15 mg/ml; using 500mmol/L tranexamic acid, pH7.5 and 25mmol/L PB as an elution buffer solution, adopting a gradient pre-washing of 20% of the elution buffer solution to remove hybrid protein with weak binding, using 60% of the elution buffer solution to carry out gradient elution on target protein, and immediately adjusting the pH value of a collected elution peak to be below 3.5 by using 1mol/L citric acid mother liquor;

step 3.2 SP cation exchange chromatography: using Sepharose SP Fastflow cation exchange chromatography packing, and using a buffer solution with pH of 3.2-3.3 and conductance of 38-40 mS/cm as an equilibrium buffer solution; adding NaCl into the eluent obtained in the step 3.1 to adjust the conductivity to 38-40 mS/cm, adjusting the pH value to 3.2-3.3, and controlling the loading capacity to 9-15 mg/ml; eluting the target protein by using a buffer solution containing 1mol/L NaCl and an equilibrium buffer solution according to a gradient of 35 percent of eluent;

the amino acid sequence of the recombinant truncated human plasmin is shown in sequence 1.

2. The process for preparing recombinant truncated human plasmin according to claim 1, wherein said recombinant truncated human plasmin further comprises: the concentration of the recombinant microplasminogen purified in the step 2 is 3-6mg/mL, and the activation of the recombinant microplasminogen is carried out in a solution system with low ionic strength and near neutrality.

3. The process for preparing recombinant truncated human plasmin according to claim 1, wherein said recombinant truncated human plasmin further comprises: step 2 activation of the recombinant plasminogen is to activate the recombinant plasminogen in a solution system of 10mmol/L Tris-HCl with pH7.2-7.5 at 20 ℃ for 10min, and after the activation is finished, citric acid is added to pH3.0 to terminate the reaction.

4. The process for preparing recombinant truncated human plasmin according to claim 1, wherein step 1 further comprises the steps of 1.3G 25 desalting: and (3) replacing the solution system of the recombinant microplasminogen obtained by the hydrophobic chromatography elution of the PHE in the step 1.2 with a solution system with low ionic strength and near neutrality.

5. The process for preparing recombinant truncated human plasmin according to claim 1, wherein step 3 further comprises step 3.3 of ultrafiltration concentration:

the target protein eluted in step 3.2 was concentrated to 3.0-4.0mg/mL using tangential flow ultrafiltration and the buffer was replaced with 5mmol/L citrate buffer at pH 3.1.

6. Use of the process according to any one of claims 1-5 for the preparation of Ocripasmin or a biologically similar agent thereof.

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