CN111690635B

CN111690635B - Preparation method of recombinant human oxk fibrinolytic enzyme

Info

Publication number: CN111690635B
Application number: CN201911398263.1A
Authority: CN
Inventors: 彭红卫; 王冲; 李晓鹏; 张磊; 万云雷; 张宝华
Original assignee: Chengdu Jingze Biopharmaceutical Co Ltd; Shanghai Jingze Biological Technology Co ltd; Jiangsu Jingze Biomedical Co ltd
Current assignee: Chengdu Jingze Biopharmaceutical Co ltd; Jingze Biomedical Hefei Co ltd; Shanghai Jingze Biological Technology Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-10-22
Anticipated expiration: 2039-12-30
Also published as: CN111690635A

Abstract

The invention provides a preparation method of recombinant human oxkerin plasmin, and particularly the preparation method comprises the steps of carrying out cation chromatography, enzyme digestion activation, hydrophobic chromatography and the like on the recombinant human oxkerin plasminogen to obtain the high-purity recombinant human oxkerin plasmin. The method has the advantages of simple steps, high protein yield and high product purity, and is suitable for industrial production.

Description

Preparation method of recombinant human oxk fibrinolytic enzyme

Technical Field

The invention relates to the field of recombinant proteins, in particular to a preparation method of recombinant human akkerplasmin.

Background

Human plasmin consists of 791 amino acids and has a molecular weight of about 88kD, forming two chains, the heavy and light chains being linked by disulfide bonds. The recombinant human akkerin is formed by cutting 249 amino acids at the C end of the human plasmin, and the molecular weight is about 27.2 kD.

Human plasminogen is one of the key components of the human fibrinolytic system. The activated plasmin has serine protease activity and can degrade various proteins such as laminin, fibronectin, collagen and the like. In addition, plasmin can activate Matrix Metalloproteinases (MMPs), and the activated MMPs can also participate in the degradation of extracellular Matrix proteins. Plasmin hydrolyzes adhesion molecules between a posterior vitreous membrane and a basement membrane of a retina to separate the vitreous body from the retina, thereby achieving the purpose of treating vitreous macular adhesion and macular traction.

Recombinant human oxkelin is a truncated plasmin that retains only the C-terminal 249 amino acids of plasminogen. The recombinant human oxkelin has the proteolytic catalytic activity of plasmin, and is more stable than the human plasmin.

The heavy chain of the recombinant human oxkelin is 19 amino acids (although the peptide chain is short, the peptide chain is derived from the heavy chain of the human oxkelin and is also called as the heavy chain), the light chain is 230 amino acids, the heavy chain and the light chain are connected through two disulfide bonds, and in addition, 4 disulfide bonds are also arranged in the light chain.

For the preparation of recombinant human oxkerplasmin, chinese patent 2003801087731 discloses the construction of plasminogen and miniplasminogen expression vectors, converting pichia pastoris with pPICZ α -MPLG1, screening high expression strain X33-MPLG1-5#, the purification steps including: plasminogen is obtained by three steps of ion exchange chromatography, hydrophobic chromatography and affinity chromatography, and then the microplasmin is obtained by quantitative activation and hydrophobic chromatography purification, but the purification steps are long, the operation is complicated and the yield is low.

Therefore, the development of a preparation method of the recombinant human oxkerin with simple steps, high protein yield and high product purity is urgently needed in the field.

Disclosure of Invention

The invention aims to provide a preparation method of recombinant human akkerin plasmin, which has simple steps, high protein yield and high product purity.

The invention provides a preparation method of recombinant human oxk plasmin, which comprises the following steps:

(a) providing a recombinant human plasminogen solution;

(b) passing the recombinant human okra plasminogen solution through a cation chromatographic column to obtain an eluent containing the recombinant human okra plasminogen;

(c) adding tranexamic acid into the eluent, and adding a plasminogen activator for enzyme digestion reaction to obtain a primary enzyme digestion solution; and

(d) and (3) passing the primary enzyme digestion solution through a hydrophobic chromatographic column to obtain a purified recombinant human oxk plasmin solution.

In another preferred embodiment, before step (d), the method further comprises the steps of:

(d1) diluting the primary enzyme digestion solution with a diluent; and/or

(d2) Filtering with a filter membrane to obtain filtrate;

the resulting liquid is then subjected to step (d).

In another preferred embodiment, the recombinant human akkerin plasminogen solution comprises pichia or saccharomyces cerevisiae fermentation supernatant.

In another preferred embodiment, the recombinant human oxk plasminogen comprises a peptide fragment having the amino acid sequence shown in SEQ ID No.: 1.

In another preferred embodiment, the recombinant human oxk plasminogen solution has one or more of the following characteristics:

1) in the recombinant human oxkel plasminogen solution, the concentration of the recombinant human oxkel plasminogen is 0.01-10g/kg, preferably 0.02-8g/kg, and more preferably 0.03-6 g/kg;

2) the pH value of the recombinant human oxk plasminogen solution is 5-7, preferably 5.5-6.5;

3) the conductivity of the recombinant human oxk plasminogen solution is 10-20mS/cm, preferably 12-18 mS/cm.

In another preferred embodiment, the packing of the cationic chromatography column is selected from the group consisting of: diamond MMC Mustang, Diamond MMC, Capto MMC, or combinations thereof.

In another preferred embodiment, the ratio of the packing material of the cation chromatographic column to the sample amount of the oxk plasminogen is 1L: 0.1-100 g; preferably, 1L:1-80g, more preferably, 1L: 2-60 g.

In another preferred embodiment, in the step (b), the elution method of the cation chromatography column comprises the steps of:

i) equilibrating with 2-6 column volumes of the first equilibration solution, preferably 2-5 column volumes;

ii) rinsing with 3-10 column volumes of the mixture I of the first washing solution and the first equilibration solution, preferably 7-9 column volumes; then the

iii) eluting with 5-25 column volumes of a mixture II of the first wash solution and the first equilibration solution, preferably 7-13 column volumes;

wherein the first balance solution is a buffer solution with pH of 7 + -1, preferably 7 + -0.5, more preferably 7 + -0.2;

the first washing solution is an inorganic salt solution prepared by a buffer solution, and the concentration of the inorganic salt is 0.5-1.5mol/L, preferably 0.8-1.2 mol/L; the buffer solution is a buffer solution with pH of 7 +/-1, preferably 7 +/-0.5, more preferably 7 +/-0.2;

in the mixed solution I, the volume ratio of the first washing solution to the first balance solution is 10-30: 70-90; preferably, 15-25:75-85, more preferably, 15-20: 80-85;

in the mixed solution II, the volume ratio of the first washing solution to the first balance solution is 20-50: 50-70; preferably 25-40:60-75, more preferably 30-40: 60-70.

In another preferred embodiment, in the first wash solution, the inorganic salt is selected from NaCl, KCl, or a combination thereof.

In another preferred embodiment, the buffer is a phosphate buffer.

In another preferred embodiment, the cation chromatographic column is equilibrated with a first equilibration fluid before use.

In another preferred embodiment, in step (b), the eluate containing recombinant human akkerogen plasminogen is a fraction having a UV response at 280 + -5 nm, preferably 280 + -2 nm.

In another preferred embodiment, in step (c), the tranexamic acid is added in an amount such that the final concentration of tranexamic acid in the eluate is 0.01-0.80mol/L, preferably 0.10-0.50mol/L, more preferably 0.15-0.3 mol/L.

In another preferred embodiment, in step (c), the enzyme reaction has one or more of the following characteristics:

1) the weight ratio of the recombinant human Orkel plasminogen activator to the recombinant human Orkel plasminogen activator is 1:120-500, preferably 1:150-400, more preferably 1: 200-350;

2) the temperature of the enzyme digestion reaction is 15-30 ℃, preferably 20-25 ℃;

3) the temperature of the enzyme digestion reaction is 2-12h, preferably 4-8 h; and/or

4) The recombinant human oxk plasminogen activator is streptokinase, staphylokinase, urokinase, or a combination thereof, preferably staphylokinase or urokinase.

In another preferred example, in the step (d1), the diluent is tranexamic acid and (NH) in the buffer solution₄)₂SO₄And (3) solution.

In another preferred embodiment, the buffer in the dilution is a buffer with a pH of 7 + -1, preferably 7 + -0.5, more preferably 7 + -0.2.

In another preferred embodiment, the concentration of tranexamic acid in the diluent is 0.1 to 0.3mol/L, preferably 0.15 to 0.25 mol/L.

In another preferred embodiment, (NH) in the diluent₄)₂SO₄The concentration of (B) is 0.1 to 5mmol/L, preferably 0.5 to 4mmol/L, more preferably 0.5 to 3 mmol/L.

In another preferred embodiment, the volume ratio of the diluent to the primary enzyme is 1-5:1, preferably 1-3: 1.

In another preferred embodiment, in the step (d1), the concentration of the protein in the diluted liquid is 0.1-1g/L, preferably 0.1-0.8 g/L.

In another preferred example, in the step (d2), the filter membrane is 0.22-0.45 um.

In another preferred embodiment, the packing of the hydrophobic chromatography column is selected from the group consisting of: capto ButyL imprcs, Capto phenyl imprcs, or combinations thereof.

In another preferred example, the ratio of the filling material of the hydrophobic chromatography column to the sampling amount of the oxk plasminogen is 1L: 0.1-100 g; preferably, 1L:1-80g, more preferably, 1L: 2-60 g.

In another preferred embodiment, in the step (d), the elution method of the hydrophobic chromatography column comprises the steps of:

I) balancing with 1-4 times of column volume of the second balancing solution, preferably 2-3 times of column volume;

II) leaching with a mixture III of 2-6 times of the column volume of the second washing solution and the second equilibrium solution, preferably 3-5 times of the column volume; then the

III) gradient elution with a second eluent;

wherein the second equilibrium solution is tranexamic acid and (NH) prepared by buffer solution₄)₂SO₄The concentration of the tranexamic acid in the solution is 0.1-0.3mol/L, preferably 0.15-0.25 mol/L; and (NH)₄)₂SO₄The concentration of (B) is 0.1 to 5mmol/L, preferably 0.5 to 4mmol/L, more preferably 0.5 to 3 mmol/L;

the second washing solution is a tranexamic acid solution prepared by a buffer solution, preferably, the concentration of the tranexamic acid in the second washing solution is 0.1-0.3mol/L, preferably, 0.15-0.25 mol/L;

in the mixed solution III, the volume ratio of the second washing solution to the second equilibrium solution is 10-30: 70-90; preferably, 15-25:75-85, more preferably, 15-20: 80-85; and

the second eluent is formed by a two-phase gradient mixing, wherein the two phases comprise: phase A: a second balancing liquid; and phase B: a second washing solution;

wherein the gradient elution is that the B phase elutes with a (10-20%) to (80-90%) (V/V) linear gradient, based on the total volume of the second eluent;

each buffer is independently a buffer having a pH of 7. + -.1, preferably 7. + -. 0.5, more preferably 7. + -. 0.2.

In another preferred embodiment, the hydrophobic chromatography column is equilibrated with a second equilibration solution before use.

In another preferred example, the method further comprises the steps of:

(e) concentrating the purified recombinant human oxkerin plasmin solution obtained in the step (d) to obtain high-concentration recombinant human oxkerin plasmin.

In another preferred embodiment, the concentration is ultrafiltration concentration.

In another preferred embodiment, the ultrafiltration concentration comprises the steps of:

adjusting the pH of the purified recombinant human oxkerin solution obtained in step (d) to 2.5-4, preferably 2.5-3.5, more preferably 2.8-3.0; and

the ultrafiltration is performed using a 4-6kDa ultrafiltration membrane, preferably 5 kDa.

In another preferred example, in the ultrafiltration liquid, the pH regulator is citric acid, phosphoric acid and acetic acid buffer solution.

In another preferred example, in the ultrafiltration liquid, the pH regulator is a mixed solution of citric acid and tranexamic acid.

In another preferred example, in the ultrafiltration exchange solution, the pH regulator is a mixed solution of 1.5-3mol/L citric acid and 0.1-0.3mol/L tranexamic acid.

In another preferred embodiment, the pH of the solution within the membrane of the ultrafiltration membrane is the same as the solution to be concentrated.

In another preferred embodiment, the solution in the membrane of the ultrafiltration membrane comprises an inorganic salt having a concentration of 60 to 200mmol/L, preferably 80 to 150mmol/L, more preferably 80 to 120 mmol/L.

In another preferred embodiment, the inorganic salt is selected from the group consisting of: NaCl, KCl, or a combination thereof.

In another preferred example, in the step (e), the "high concentration" refers to that the concentration of the recombinant human oxkelin in the obtained concentrated solution is more than or equal to 2g/L, preferably, 2-20g/L, and more preferably, 3-18 g/L.

In another preferred embodiment, the purified recombinant human akkerin plasmin has a purity of 95% or more, preferably 96% or more, 97% or more, and more preferably 99% or more, and most preferably 99.5-99.99%.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the amino acid sequence and disulfide bond linkage site of recombinant human oxk plasmin;

FIG. 2 is a chromatogram of oxkerin (a) obtained in example 1 and a commercial product (JETREA, lot No. 15088) (b);

FIG. 3 is an Optical Coherence Tomography (OCT) image of 28 days post-dose intravitreal detachment of example 4, with 75 μ g/eye (a), 125 μ g/eye (b);

FIG. 4 is an Optical Coherence Tomography (OCT) image of intravitreal detachment 2 days after dosing for example 5 with vehicle control (a), 62.5 μ g/eye (b);

FIG. 5 is a posterior vitreal detachment Optical Coherence Tomography (OCT) image 2 days post dose, with 75 μ g/eye (a), 125 μ g/eye (b);

FIG. 6 is a graph of collagen fiber retention on the surface of the posterior limiting membrane under a scanning electron microscope (1500 times) 2 days after administration, wherein the vehicle controls (a), 62.5. mu.g/eye (b), and 125. mu.g/eye (c).

Detailed Description

The inventor provides a preparation method of recombinant human oxk plasmin through extensive and intensive research and a large number of screening and tests. The method comprises the steps of carrying out cation chromatography, enzyme digestion and hydrophobic chromatography on the recombinant human plasminogen solution to obtain the recombinant human oxk plasmin with high protein yield and high purity (> 99.7%), wherein the obtained recombinant human oxk plasmin can be directly used for preparing intraocular injection preparations. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The weight volume percentage units in the present invention are well known to those skilled in the art and refer to, for example, the weight of solute in a 100 ml solution.

In the present invention, the term "buffer formulated … solution" refers to a solution comprising a buffer system that maintains the pH of the solution within a certain range, and is not intended to limit the manner in which the solution is formulated.

Recombinant human oxk plasminogen solution

Refers to the cell culture supernatant of a recombinant cell expressing a protein comprising the amino acid sequence of SEQ ID No. 1 before any purification steps are taken. The expression includes the crude form of the supernatant (separated from the cells) as well as the concentrated and/or filtered and/or ultrafiltered supernatant. The "solution" may contain small amounts of insoluble impurities. The solution may be suitably adjusted (e.g., concentration, pH, conductivity, etc.) prior to carrying out the method of the invention.

SEQ ID No.:1：

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

The term "recombinant" refers to a substance produced by using recombinant DNA techniques.

The term "proenzyme" refers to an inactive precursor of an enzyme that is synthesized or initially secreted in the cell. The active center of the enzyme may be formed or exposed by a cleavage modification of the proenzyme.

In the present invention, the recombinant human oxk plasminogen is biologically inactive.

In another preferred embodiment, the recombinant human oxk plasminogen solution is obtained by expressing pichia or saccharomyces cerevisiae.

1) in the recombinant human oxkel plasminogen solution, the concentration of the recombinant human oxkel plasminogen is 0.01-10g/kg, preferably 0.02-0.8g/kg, and more preferably 0.03-6 g/kg;

Recombinant human oxk fibrinolytic enzyme

The recombinant human oxk plasmin can be obtained by digesting the zymogen with a recombinant human oxk plasminogen activator. Wherein the activator cuts the peptide bond between the arginine at the 19-position and the valine at the 20-position of the zymogen, and the two peptide bonds are connected, thereby obtaining the recombinant human oxkerin with biological activity.

In the present invention, the recombinant human oxk plasminogen activator is selected from the group consisting of: streptokinase, staphylokinase, urokinase, or a combination thereof.

Preparation method

(a) providing a recombinant human plasminogen solution;

(d1) diluting the primary enzyme digestion solution with a diluent; and/or

(d2) Filtering with a filter membrane to obtain filtrate;

the resulting liquid is then subjected to step (d).

In another preferred embodiment, the buffer is a phosphate buffer.

In another preferred embodimentIn the diluent, (NH)₄)₂SO₄The concentration of (B) is 0.1 to 5mmol/L, preferably 0.5 to 4mmol/L, more preferably 0.5 to 3 mmol/L.

In another preferred embodiment, the ligand of the packing of the hydrophobic chromatography column is selected from phenyl, octyl, butyl, isopropyl and ether groups.

In another preferred embodiment, the filler for hydrophobic chromatography is selected from the group consisting of: capto Butyl, Capto Butyl Impress, Phenyl Sepharose 6FF (low sub), Butyl Sepharose High Performance, Butyl Sepharose 6FF, Phenyl Sepharose High Performance, Capto Phenyl (High sub), or combinations thereof.

III) gradient elution with a second eluent;

wherein the second equilibrium solution is tranexamic acid and (NH) prepared by buffer solution₄)₂SO₄The concentration of tranexamic acid in the solution is 0.1-0.3mol/L, preferably 0.15-0.25 mol/L; and (NH)₄)₂SO₄The concentration of (B) is 0.1 to 5mmol/L, preferably 0.5 to 4mmol/L, more preferably 0.5 to 3 mmol/L;

the second washing solution is a tranexamic acid solution prepared in a buffer solution, preferably, the concentration of the tranexamic acid in the second washing solution is 0.1-0.3mol/L, preferably, 0.15-0.25 mol/L;

In another preferred example, the method further comprises the steps of:

Preferably, the recombinant human oxkelin bioactivity is 70-130%, 80-120%, or 90-110%, or 95-100%, more preferably, 95-110%, most preferably, 95-105% of a reference substance, and the activity percentage is compared with the activity of the reference substance.

Preparation

The recombinant human oxkerin prepared by the invention can be used in any suitable preparation. Ophthalmic preparations such as injections, eye drops and the like are preferred. The injection may be in the form of a lyophilizate or a solution.

Typically, the formulation further comprises one or more pharmaceutically acceptable carriers.

Particularly preferably, the recombinant human oxkerin prepared by the invention can be configured as follows: dissolving in pyrogen-free water, and adjusting to isotonic with osmotic pressure regulator (such as NaCl) to pH 5-8 with pH regulator. Can be directly injected or made into lyophilized preparation. The formulation may be administered by intravitreal injection.

Indications of

The recombinant human oxkerplasmin can be used for preparing pharmaceutical compositions or preparations for treating and/or preventing eye diseases.

In another preferred embodiment, the ocular disease is selected from the group consisting of: retinal detachment, retinal rupture, vitreous hemorrhage, diabetic vitreous hemorrhage, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, age-related macular degeneration, macular hole, vitreous macular traction, macular pucker, macular exudate, cystic macular edema, fibrin deposition, retinal vein occlusion, retinal artery occlusion, subretinal hemorrhage, amblyopia, endophthalmitis, retinopathy of prematurity, glaucoma, retinitis pigmentosa, or a combination thereof.

The main advantages of the invention include:

1. the preparation method of the invention has simple steps, and the obtained recombinant human oxkerin has high purity and high yield, and is suitable for industrial production.

2. Compared with the plasmin purification method in the prior art, the method creatively simplifies the purification steps before enzyme digestion, and removes part of impurities in the hydrophobic chromatography after enzyme digestion, thereby reducing the operation steps, greatly improving the protein yield (more than 95%), and successfully obtaining the recombinant human oxk plasmin with high purity (more than or equal to 99.7%) by optimizing the elution method in the hydrophobic chromatography.

3. The high-purity recombinant human oxkerin plasmin prepared by the preparation method can be directly used for preparing intraocular injection preparations, reduces the incidence rate of symptomatic vitreous macular adhesion of eyes, is convenient for patients to use, and reduces the pain caused by operations.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Reagent

The recombinant human oxk plasminogen solution can be obtained by gene recombination, cloning and pichia pastoris expression, and has an amino acid sequence shown in SEQ ID No. 1. The recombinant human oxk plasminogen solution is pichia pastoris fermentation supernatant.

The recombinant human akkerogen plasminogen activator is glucokinase: purchased from biosciences, inc.

Reference product: from Shanghai Jingze Biotechnology, Inc., product batch number 32S171102-RM 02.

Column chromatography

Column chromatography equipment: APPS Process, manufacturer: lisui science and technology (Suzhou) Inc.

Cation chromatographic column: purchased from scion technologies (suzhou) ltd: column diameter/height: 14cm by 12 cm; chromatography packing: diamond MMC Mustang; the manufacturer: boglon (shanghai) biotechnology limited; the goods number is: AI 0163.

Hydrophobic chromatographic column: purchased from scion technologies (suzhou) ltd: column diameter/height: 10cm by 23 cm; chromatography packing: capto ButyL imprs; the manufacturer: general Electric Company; the goods number is: 17-3719-03.

Detection method

The invention relates to a method for measuring biological potency, purity and enzyme kinetics, which comprises the following steps:

and (3) biological activity:

the determination method of the biological activity of the recombinant human akkerplasmin refers to the determination method of the related biological activity in Chinese pharmacopoeia 2015 edition.

And (3) purity detection:

SEC-HPLC, Agilent 1260 liquid chromatography using an Advance Bio SEC column (

2.7 μm, 7.8 × 300mm), flow rate: 0.5mL/min, detection wavelength: 280nm, isocratic elution, sample volume 20 u l.

Enzyme kinetics

The enzyme kinetics is measured using a chromogenic substrate S-2403 plasmin/plasminogen-SK (Shanghai Kangfu Biotechnology Co., Ltd.), and the measurement method belongs to the methods well known to those skilled in the art.

Example 1

Preparation of high-purity recombinant human oxk plasmin I

Low-purity recombinant human oxk plasminogen (see sequence FIG. 1, purity about 0.15mg/ml) was used as starting material, wherein the proenzyme was inactive and the bio-titer was 0 IU/mg.

A50 kg of the above described low purity recombinant human Ock plasminogen solution containing about 8.2g recombinant human Ock plasminogen, pH6, conductivity 15mS/cm was loaded onto a 2L cationic chromatography column packed with Diamond MMC Mustang packing, equilibrated well with an equilibration solution of 20mmol/L PB (pH7.0) prior to loading the column.

After the end of the sample loading, 4 column volumes of the equilibration solution were used, followed by elution with a mixture I (vprst wash: vprst equilibration solution 20:80) (first wash (20mmol/L PB +1mol/L NaCl, pH 7; first equilibration solution 20mmol/L PB, pH7) of 8 column volumes, elution with a mixture II (vprst wash: vprst equilibration solution 38:62) of 10 volumes, collection of about 16L at 280nm was monitored with an ultraviolet detector, protein concentration was 0.51mg/ml, HPLC purity was 95.2%.

Treating 16L of the solution with a high-concentration tranexamic acid solution until the concentration of the tranexamic acid solution in the solution is 0.2mol/L, uniformly mixing, adding 26ml of 1mg/ml plasminogen activator for enzyme digestion, wherein the enzyme digestion temperature is 23 ℃, and carrying out enzyme digestion for 5 hours to obtain a primary enzyme digestion solution.

The primary enzyme-digested liquid treated with the high-concentration tranexamic acid solution was treated with (20mmol/LPB,0.2M tranexamic acid, 1mmol/L (NH)₄)₂SO₄) According to about 2: diluting with 1(V/V), filtering with 0.45um filter membrane, and storing with enzyme solution of 35kg weight and protein concentration of 0.23 g/L. The protein purity is unchanged within 4 days after detection for standby.

The above-mentioned cut solution was applied to a 2L hydrophobic chromatography column packed with Capto ButyL ImpRes, which had been loaded with 20mmol/PB, 0.2 mmol/tranexamic acid, 1 mol/(NH)₄)₂SO₄，pHThe second equilibrium liquid of 7 is well balanced. After the end of the sample loading, the column was equilibrated with 2 column volumes of the second equilibration solution, and then with 4 column volumes of 20% of the second eluent (Vsecond eluent: Vsecond equilibration solution: 20:80) (second equilibration solution: 20mmol/PB, 0.2 mmol/tranexamic acid, 1 mol/(NH)₄)₂SO₄pH 7; the second wash solution was washed with 20mmol/L PB,0.2 mol/L tranexamic acid, pH7) and then eluted with a linear gradient of 10-90% using the second eluent (Vsecond wash: and V (the second balance liquid and the second washing liquid) ═ 10-90%), monitoring 280nm by using an ultraviolet detector, respectively collecting each distillation peak, detecting the bioactivity and purity of the recombinant human oxk plasmin, and combining effective fractions of about 3.1L.

Adjusting pH to 3.0 with 2mol/L citric acid and 0.2M tranexamic acid, ultrafiltering with 5KDa membrane, replacing the solution with 20mmol/L citric acid monohydrate and 100mmol/L NaCl (pH3.0), and concentrating to obtain 1.3kg of high-purity high-activity recombinant human oxk plasmin I (shown in figure 1).

Table 1 shows the results of the determination of the biological activity of the recombinant human oxkerin I

The above results show that: by adopting the method of the embodiment 1 for purification, the recombinant human oxkerin with high purity and high protein yield can be obtained only by two-step chromatography.

Example 2

Preparation of high-purity recombinant human oxk plasmin II

A100 kg of the above described low purity recombinant human oxk plasminogen solution containing about 16.3g recombinant human oxk plasminogen, pH6, conductivity 15mS/cm was loaded onto a 4L cationic chromatography column packed with MMC Mustang packing, which had been well equilibrated with an equilibration solution of about 20mmol/L PB (pH6.8) prior to loading.

After the end of the sample loading, 4 column volumes of the equilibration solution were used, followed by elution with a mixture I (Vfirst wash: Vfirst equilibration solution 20:80) of 9 column volumes (20mmol/L PB +1mol/L NaCl, pH 6.8; first wash: 20mmol/L PB, pH6.8), elution with a mixture II (Vfirst wash: Vfirst equilibration solution 38:62) of 10 volumes, collection of about 31.5L at 280nm monitored by an ultraviolet detector, protein concentration of 0.51mg/ml, and HPLC purity of 95.3%.

Treating 31L of the solution with a high-concentration tranexamic acid solution until the concentration of the tranexamic acid solution in the solution is 0.2mol/L, uniformly mixing, adding 53ml of 1mg/ml plasminogen activator for enzyme digestion, wherein the enzyme digestion temperature is 23 ℃, and carrying out enzyme digestion for 4 hours to obtain a primary enzyme digestion solution.

Diluting the primary enzyme solution treated with the high concentration tranexamic acid solution with diluent (20mmol/LPB,0.2M tranexamic acid, 1mmol/L (NH4)₂SO₄) According to about 2: diluting with 1(V/V), filtering with 0.45um filter membrane, and storing to obtain enzyme digestion solution II with weight of 67kg and protein concentration of 0.24 g/L.

The above-mentioned cut was applied to a 4L hydrophobic chromatography column packed with Capto ButyL ImpRes, previously loaded with 20mmol/PB, 0.2 mmol/tranexamic acid, 1 mol/(NH)₄)₂SO₄The second equilibrium liquid of pH6.8 is well balanced. After the end of the sample loading, the column was equilibrated with 2 column volumes of the second equilibration solution, and then with 3 column volumes of 20% of the second eluent (Vsecond eluent: Vsecond equilibration solution: 20:80) (second equilibration solution: 20mmol/PB, 0.2 mmol/tranexamic acid, 1 mol/(NH)₄)₂SO₄pH6.8; 20mmol/L PB of the second wash solution, 0.2mol/L tranexamic acid, pH6.8) and then a 10-90% linear gradient elution with the second eluent (V second wash solution: and V (the second balance liquid and the second washing liquid) ═ 10-90%), monitoring 280nm by using an ultraviolet detector, respectively collecting each distillation peak, detecting the bioactivity and purity of the recombinant human oxk plasmin, and combining effective fractions of about 6L.

Adjusting pH to 3.0 with 2mol/L citric acid and 0.2M tranexamic acid, ultrafiltering with an ultrafiltration membrane with a pore diameter of 5KDa, ultrafiltering with 20mmol/L citric acid monohydrate and 100mmol/L NaCl (pH3.0), and concentrating to obtain 2.5kg of solution, namely the high-purity high-activity recombinant human oxk plasmin II.

Table 2 shows the results of determination of biological activity and the like of the recombinant human oxkerin II

The results show that the method has good repeatability and can be used for scale-up production.

The quality (g) of the products obtained in the various operating phases of example 1 and example 2 is summarized in Table 3:

table 3 quality (g) of the product obtained in each operating phase of example 1 and example 2

Example 3

Recombinant human oxk fibrinolytic enzyme injection preparation

1000 bottles of recombinant human oxk plasmin injection preparations are manufactured, and each bottle contains 0.375mg of recombinant human oxk plasmin:

calculating the required amount (by mass) of the recombinant human oxkelin, measuring the amount of the recombinant human oxkelin I in example 1, dissolving the recombinant human oxkelin I in pyrogen-free water for injection, adjusting the amount to be isotonic by using an osmotic pressure regulator, adjusting the pH to be 5.5 by using a pH regulator, filtering by using a 0.22 mu m filter, aseptically packaging into penicillin bottles with 0.3mL of each bottle, and freezing and storing.

The obtained penicillin bottles contain 0.375mg of recombinant human oxk plasmin in each bottle.

Comparison of the high purity recombinant human oxtripsin obtained in example 1 with the commercial product purchased (JETREA, Lot 15088), SEC-HPLC is shown in FIG. 2.

TABLE 4 comparison of the quality of the recombinant human oxkerin solution obtained in example 1 with that of the commercial product

As can be seen from Table 4, the product of this example has better bioactivity and purity than the commercial product.

Example 4

In vivo analysis of high purity recombinant human okra plasmin solution induced post-vitreous separation

12 cynomolgus monkeys were used, randomized into 2 groups (3/sex/group), and administered intravitreally in one eye (left eye). Groups 1 and 2 were injected with the highly purified recombinant human oxkerin I of the invention (prepared as different concentrations of injection solution in example 3), at doses of 75 μ g/eye (1.25mg/mL, 60 μ L/eye) and 125 μ g/eye (1.25mg/mL, 100 μ L/eye), respectively, all given in a single dose. At 28 days post-dose (D29), OCT (optical coherence tomography) showed vitreal detachment at both 75 μ g/eye and 125 μ g/eye dose groups.

The results are shown in fig. 3, and it can be seen that 75 μ g/eye (a) and 125 μ g/eye (b) of the high purity recombinant human akkerplasmin I of the present invention can induce the posterior detachment of cynomolgus monkey vitreous (as indicated by white arrow) with substantially consistent effect by a single vitreous injection.

Example 5

Incidence of posterior vitreal detachment and effects of posterior limiting membranes and fibers thereof following single intravitreal injection of cynomolgus monkeys at different dosages

48 cynomolgus monkeys, male and female halves, were used, and randomly divided into 4 groups, 6/sex/group, first 3/sex/group animals were administered intravitreally with one eye (left eye) and second 3/sex/group animals were administered intravitreally with both eyes. Group 1 vehicle control injections; groups 2, 3 and 4 are injected with the high-purity recombinant human oxkerin I (different concentrations of injection solution prepared in example 3), the dosage is respectively 62.5 mu g/eye (1.25mg/mL), 75 mu g/eye (1.5mg/mL) and 125 mu g/eye (2.5mg/mL), and the administration volume is 50 mu L/eye/time in single administration. Optical Coherence Tomography (OCT) was performed 2 days after dosing (D3), 4 weeks during recovery (D29) and at the end of recovery (D57) in groups 1-4 animals before transfer to the test. The detection parts are optic disc, retina vascular arch and retina posterior pole.

The results are shown in fig. 4 and 5, and show that the single-eye administration, the separation after the vitreous body is not seen in the vehicle control group, and the separation after the vitreous body is seen in each dose group of the high-purity recombinant human oxkerplasmin I of the invention. After single-eye administration of the high-purity recombinant human oxkerin I62.5 mu g/eye, 75 mu g/eye and 125 mu g/eye dose group for 2 days (D3), the incidence rates of posterior vitreous detachment of animals are 6/6 eyes, 6/6 eyes and 6/6 eyes respectively.

After single-eye administration of the high-purity recombinant human oxkerin I62.5 mu g/eye, 75 mu g/eye and 125 mu g/eye dose groups for 4 weeks (D29), the incidence rates of posterior vitreous detachment of animals are 6/6 eyes, 6/6 eyes and 6/6 eyes respectively.

After single-eye administration of the high-purity recombinant human oxkerin I62.5 mu g/eye, 75 mu g/eye and 125 mu g/eye dose group for 8 weeks (D57), the incidence rates of posterior vitreous detachment of animals are 5/6 eyes, 6/6 eyes and 6/6 eyes respectively.

The detailed results are shown in Table 5. Detachment after 2 days of administration (D3) of the vitreous is shown in OCT in figures 4 and 5.

Table 5 dissociation of the summary table (male 3, female 3) after 2 days (D3), 4 weeks (D29) and 8 weeks (D57) of monocular administration after vitreous humor

And (3) electron microscope inspection: the degradation condition of the posterior vitreous limiting membrane of the macular area is checked by an electron microscope for animals in a vehicle control group, 62.5 mu g/eye dose group and 125 mu g/eye dose group, and the eyes are photographed with a field with the magnification of 1500 times of the scanning electron microscope.

The scanning electron microscope picture is shown in FIG. 6. The surface of the inner limiting membrane of the control animal of the solvent (a) is covered with dense reticular collagen fibers; 62.5 mu g/eye dose group (b) animal inner limiting membrane surface covering sparse distribution collagen fiber; the 125. mu.g/eye dose group (c) showed almost no distribution of collagen fibers on the limiting membrane surface in the animals. The results show that the recombinant human oxkelysin prepared by the method can effectively degrade or loosen the collagenous fibers of the posterior limiting membrane of the cynomolgus monkey vitreous body.

Comparative example 1

Substantially the same as in example 1, except that, in the hydrophobic chromatography, when elution is performed with the second eluent, isocratic elution is performed using 30%, 40%, 50%, 60%, 70%, and 80% of the second eluent (Vsecond eluent: V (second equilibrium solution + second eluent)), respectively.

The results show that the yield of isocratic elution protein is not high, and the protein purity is lower than 99 percent. Compared with gradient elution, the purity of the protein distilled by the gradient elution is obviously superior to that of isocratic elution through detection, and specific results are shown in table 6.

TABLE 6 protein purity by gradient elution and isocratic elution

In conclusion, compared with the plasmin purification method in 2003801087731, the method creatively simplifies the purification steps before enzyme digestion, and removes part of impurities in the hydrophobic chromatography after enzyme digestion, thereby reducing the operation steps, greatly improving the protein yield (more than 95%), and successfully obtaining the recombinant human oxk plasmin with high purity (more than or equal to 99.7%) by optimizing the elution method in the hydrophobic chromatography. The recombinant human oxkerin plasmin obtained by the invention can be directly used for preparing intravitreal injection preparations, and has high activity and good treatment effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> JINGZE biomedical corporation of Jiangsu

SHANGHAI JINGZE BIOLOGICAL TECHNOLOGY Co.,Ltd.

Chengdu Zeyan Biotechnology Co.,Ltd.

<120> preparation method of recombinant human oxk plasmin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Claims

1. A preparation method of recombinant human oxk plasmin comprises the following steps:

(a) providing a recombinant human oxk plasminogen solution;

(d) passing the primary enzyme digestion solution through a hydrophobic chromatographic column to obtain a purified recombinant human oxk plasmin solution;

wherein the packing of the cationic chromatographic column is selected from the group consisting of: a Diamond MMC Mustang, Diamond MMC, Capto MMC, or combinations thereof;

in the step (c), the adding amount of the tranexamic acid is that the final concentration of the tranexamic acid in the eluent is 0.10-0.50 mol/L;

the packing of the hydrophobic chromatography column is selected from the group consisting of: capto ButyL imprs, Capto ButyL, Capto phenyl imprs, or combinations thereof; and is

The recombinant human oxk plasminogen solution is pichia pastoris or saccharomyces cerevisiae fermentation supernatant.

2. The method of claim 1, further comprising, prior to step (d), the steps of:

(d1) diluting the primary enzyme digestion solution with a diluent; and/or

(d2) Filtering with a filter membrane to obtain filtrate;

the resulting liquid is then subjected to step (d).

3. The method of claim 1, wherein the recombinant human oxk plasminogen solution is a pichia pastoris fermentation supernatant.

4. The method of claim 1, wherein the recombinant human oxk plasminogen comprises a peptide fragment having the amino acid sequence set forth in SEQ ID No.: 1.

5. The method of claim 1, wherein the packing material of the cation chromatography column is Diamond MMC Mustang.

6. The method of claim 1, wherein the ratio of the packing of the cationic chromatography column to the amount of oxk plasminogen sample is 1L: 0.1-100 g.

7. The method of claim 1, wherein in step (b), the elution method for the cationic chromatography column comprises the steps of:

i) balancing with 2-6 times of column volume of the first balancing solution;

ii) leaching with a mixture I of 3-10 times of the column volume of the first washing solution and the first balancing solution; then the

iii) eluting with a mixture II of 5-25 column volumes of a first wash solution and a first equilibration solution;

wherein the first equilibrium solution is a buffer solution with the pH value of 7 +/-1;

the first washing solution is an inorganic salt solution prepared from a buffer solution, and the concentration of the inorganic salt is 0.5-1.5 mol/L; the pH value of the buffer solution is 7 +/-1;

in the mixed solution I, the volume ratio of the first washing solution to the first balance solution is 10-30: 70-90;

in the mixed solution II, the volume ratio of the first washing solution to the first balance solution is 20-50: 50-70.

8. The method of claim 1, wherein in step (c), the enzymatic reaction has one or more of the following characteristics:

1) the weight ratio of the recombinant human Oakkerin plasminogen activator to the recombinant human Oakkerin plasminogen activator is 1: 120-500;

2) the temperature of the enzyme digestion reaction is 15-30 ℃;

3) the temperature of the enzyme digestion reaction is 2-12 h; and/or

4) The recombinant human oxk plasminogen activator is streptokinase, staphylokinase, urokinase, or a combination thereof.

9. The method of claim 1, wherein the ratio of packing to oxk plasminogen loading of the hydrophobic chromatography column is 1L: 0.1-100 g.

10. The method of claim 1, wherein the packing of the hydrophobic chromatography column is Capto ButyL ImpRes; and is

The packing of the cation chromatographic column is Diamond MMC Mustang.

11. The method of claim 1, wherein in step (d), the method of eluting the hydrophobic chromatography column comprises the steps of:

I) balancing with a second balancing solution with 1-4 times of column volume;

II) leaching with a mixed solution III of a second washing solution and a second equilibrium solution in 2-6 times of the column volume; then the

III) gradient elution with a second eluent;

wherein the second equilibrium solution is tranexamic acid and (NH) prepared by buffer solution₄)₂SO₄The concentration of the tranexamic acid in the solution is 0.1-0.3 mol/L; and (NH)₄)₂SO₄The concentration of (A) is 0.1-5 mmol/L;

the second washing solution is a tranexamic acid solution prepared from a buffer solution;

in the mixed solution III, the volume ratio of the second washing solution to the second equilibrium solution is 10-30: 70-90; and

wherein the gradient elution is that the B phase is eluted by a linear gradient with V/V of (10-20)% to (80-90)% based on the total volume of the second eluent;

each buffer is independently a buffer at pH7 ± 1.

12. The method of claim 1, wherein the method further comprises the steps of:

13. The method of claim 1, wherein the purified recombinant human oxkerin has a purity of 98% or more.