Method for preparing high-purity and low-residue recombinant human thrombin and use thereof

Abstract

Provided are a method for preparing high-purity and low-residue recombinant human thrombin and a use thereof. Specifically, a process for using genetically engineered cells to produce recombinant human thrombin is optimized. By means of process optimization and quality control for rEcarin process enzyme, which is a key raw material, the expression efficiency and purity of a recombinant protein are improved, the amount of residual impurities is reduced, and the safety risk of a topical recombinant human thrombin product is reduced, thereby ensuring the medication safety and stable continuous production of topical recombinant human thrombin.

Description

Preparation method and application of high-purity and low-residue recombinant human thrombin Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing high-purity and low-residue recombinant human thrombin and applications thereof. Background Art

[0002] Thrombin is a key enzyme in the body's coagulation system. Produced near damaged vascular endothelial cells, it participates in every step of the coagulation process. During the human coagulation reaction, thrombin directly acts on fibrinogen, converting it into fibrous, insoluble fibrin. This entraps extravasated blood cells, ultimately forming a blood clot and completing the coagulation process.

[0003] Traditional industrial thrombin production technology primarily involves preparing prothrombin from animal and human plasma, which is then activated with an activator to produce thrombin. However, this production process is limited by blood resources and, as market demand increases, is far from meeting demand. Furthermore, since plasma often contains a large amount of pathogens and viruses, these pathogens and viruses can remain in the final product, increasing the risk of infection in patients. Furthermore, animal-produced thrombin is immunogenic, inducing the body to produce anti-drug antibodies. These antibodies can cross-react with human blood and often induce serious bleeding complications.

[0004] With the maturity and development of genetic engineering technology, the use of recombinant methods to mass-produce thrombin has become possible, and it can overcome the shortcomings of traditional industrial production of thrombin, with advantages such as high purity, pollution prevention, and mass production. In particular, the production of recombinant human thrombin using mammalian cell systems results in low protein concentrations, generally in the form of inactive zymogens or precursors, which require specific enzyme activation. The activated thrombin is unstable, resulting in high production costs and is not conducive to industrial mass production. In addition, the residues of the activated enzyme and other impurities during the production process pose safety risks to the production of human thrombin for external use.

[0005] Therefore, there is an urgent need in the art to develop a method for preparing high-purity and low-residue recombinant human thrombin and its application.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a method for preparing recombinant human thrombin with high purity and low residue and its application.

[0008] The present invention provides a method for preparing recombinant human thrombin, comprising the steps of:

[0009] (a) producing rEcarin process enzyme using genetically engineered cells, and purifying the rEcarin process enzyme to obtain purified rEcarin process enzyme;

[0010] (b) using the purified rEcarin process enzyme to enzymatically activate human prothrombin 2 to obtain a crude stock solution containing human thrombin; and

[0011] (c) purifying the crude stock solution containing human thrombin to obtain purified recombinant human thrombin.

[0012] In another preferred embodiment, in step (c), a stock solution containing purified recombinant human thrombin (or a recombinant human thrombin product stock solution) is obtained.

[0013] In another preferred embodiment, the recombinant human thrombin stock solution, as determined by high performance liquid chromatography, has a recombinant human thrombin protein content of 1.5 to 3.5 mg / ml, preferably 1.6 to 3.0 mg / ml.

[0014] In another preferred embodiment, the biological specific activity of the recombinant human thrombin in the recombinant human thrombin stock solution is 2500 to 5000 IU / mg, preferably 2600 to 4000 IU / mg, as determined by the fibrinogen coagulation method.

[0015] In another preferred embodiment, the main peak purity of the recombinant human thrombin stock solution is ≥92.0%, preferably ≥93.0%, and more preferably ≥94.0%.

[0016] In another preferred embodiment, the total content of related proteins in the recombinant human thrombin stock solution is ≤8%, preferably ≤7.0%, and more preferably ≤6.0%, wherein the related proteins refer to proteins corresponding to other peaks in FIG3 .

[0017] In another preferred embodiment, the content of impurity proteins in the recombinant human thrombin stock solution is ≤1.0%, preferably ≤0.5%, and more preferably ≤0.10%.

[0018] In another preferred embodiment, the impurity protein includes host cell protein, rEcarin process enzyme, or a combination thereof.

[0019] In another preferred embodiment, the residual amount of the rEcarin process enzyme in the recombinant human thrombin stock solution is ≤0.1%, preferably ≤0.05%, and more preferably ≤0.03%, based on the total weight of the protein in the recombinant human thrombin stock solution.

[0020] In another preferred embodiment, the residual amount of the host cell protein in the recombinant human thrombin stock solution is ≤0.1%, preferably ≤0.05%, more preferably ≤0.01%, based on the total weight of the protein in the recombinant human thrombin stock solution.

[0021] In another preferred embodiment, the residual amount of host cell DNA in the recombinant human thrombin stock solution is ≤18 pg / mg, preferably ≤16 pg / mg, and more preferably ≤15 pg / mg, as detected by real-time fluorescence quantitative PCR.

[0022] In another preferred embodiment, the residual amount of polysorbate 80 in the recombinant human thrombin stock solution is ≤120 μg / ml, preferably ≤110 μg / ml, and more preferably ≤100 μg / ml.

[0023] In another preferred embodiment, the residual amount of tributyl phosphate in the recombinant human thrombin stock solution is ≤3.5 μg / ml, preferably ≤3.0 μg / ml, and more preferably ≤2.5 μg / ml.

[0024] In another preferred embodiment, the bacterial endotoxin content in the recombinant human thrombin stock solution is ≤3.5 EU / mg, preferably ≤3.0 EU / mg, and more preferably ≤2.5 EU / mg.

[0025] In another preferred embodiment, in step (a), the process comprises:

[0026] (a1) providing a feed solution containing rEcarin process enzyme;

[0027] (a2) subjecting the feed solution containing the rEcarin process enzyme to DEAE column chromatography to obtain a first flow-through containing the rEcarin process enzyme;

[0028] (a3) clarifying the first flow-through containing the rEcarin process enzyme to obtain a clarified first filtrate containing the rEcarin process enzyme;

[0029] (a4) performing Phenyl column chromatography on the first filtrate to obtain a first eluate containing rEcarin process enzyme;

[0030] (a5) performing nanofiltration and / or sterilization filtration on the first eluate to obtain a stock solution containing the purified rEcarin process enzyme (or a stock solution of the rEcarin process enzyme product).

[0031] In another preferred embodiment, the liquid containing rEcarin process enzyme satisfies:

[0032] (t1) bacterial endotoxin ≤ 30 EU / ml, preferably ≤ 25 EU / ml, more preferably ≤ 20 EU / ml;

[0033] (t2) Mycoplasma test negative; and

[0034] (t3) rEcarin protein content ≥ 0.10 mg / mL, preferably ≥ 0.15 mg / mL, and more preferably ≥ 0.20 mg / mL.

[0035] In another preferred embodiment, the turbidity of the clarified first filtrate containing rEcarin process enzyme is ≤150NTU, preferably ≤120NTU, and more preferably ≤100NTU.

[0036] In another preferred embodiment, the protein content of the rEcarin process enzyme in the rEcarin process enzyme product stock solution is ≥0.2 mg / mL.

[0037] In another preferred embodiment, the biological activity of the rEcarin process enzyme is ≥150 U / mL.

[0038] In another preferred embodiment, the biological activity of the rEcarin process enzyme is ≥200 U / mL, more preferably, ≥500 U / mL.

[0039] In another preferred embodiment, the biological specific activity of the rEcarin process enzyme is ≥1000 U / mg, more preferably, ≥2000 U / mg.

[0040] In another preferred embodiment, the content of the purified rEcarin process enzyme per liter of culture supernatant is 16,000 U to 20,000 U.

[0041] In another preferred embodiment, the content of the purified rEcarin process enzyme per liter of culture supernatant is 18,000 U to 19,000 U.

[0042] In another preferred embodiment, the production scale of the process enzyme can reach 500L.

[0043] In another preferred embodiment, the host cell is a Chinese hamster ovary cell (CHO cell).

[0044] In another preferred embodiment, the host cell is CHO / dhFr - cells.

[0045] In another preferred embodiment, the DEAE column chromatography filler is DEAE Bestarose FF.

[0046] In another preferred embodiment, the DEAE column chromatography can remove viruses.

[0047] In another preferred embodiment, the pH of the DEAE column chromatography is 8.0±0.2, preferably 8.0±0.1.

[0048] In another preferred embodiment, the filler of the Phenyl column chromatography is Phenyl Bestarose FF.

[0049] In another preferred embodiment, the pH of the Phenyl column chromatography is 7.8±0.2, preferably 7.8±0.1.

[0050] In another preferred embodiment, the Phenyl column chromatography is not followed by ultrafiltration and concentration.

[0051] In another preferred embodiment, step (b) comprises:

[0052] (b1) activating recombinant human prothrombin 2 using rEcarin process enzyme; and

[0053] (b2) Virus inactivation by treatment with an organic solvent / detergent mixture (S / D).

[0054] In another preferred embodiment, the content of the rEcarin process enzyme required to activate 1 mg of the recombinant human prothrombin 2 is 1.5±0.5 U.

[0055] In another preferred embodiment, the temperature of the reaction system in step (b) is 25±3°C, preferably 25±1°C.

[0056] In another preferred embodiment, the reaction time of step (b) is 6.0±1 h, preferably 6.0±0.5 h.

[0057] In another preferred embodiment, the organic solvent / detergent mixture (S / D) in step (b2) comprises polysorbate 80, tributyl phosphate, or a combination thereof.

[0058] In another preferred embodiment, the concentration of polysorbate 80 is (1.0±0.1)%.

[0059] In another preferred embodiment, the concentration of tributyl phosphate is (0.3±0.03)%.

[0060] In another preferred embodiment, step (b2) includes stirring treatment.

[0061] In another preferred embodiment, the stirring speed is 50 to 150 rpm, preferably 100 to 120 rpm.

[0062] In another preferred embodiment, in step (b), the human prothrombin 2 is purified human prothrombin 2.

[0063] In another preferred embodiment, the purification process of human prothrombin 2 adopts a purification route that is substantially the same as the purification route of the rEcarin process enzyme.

[0064] In another preferred embodiment, the purification route includes:

[0065] (z1) providing a feed solution containing rEcarin process enzyme or a feed solution containing human prothrombin 2;

[0066] (z2) performing DEAE column chromatography on the feed solution to obtain a first flow-through containing rEcarin process enzyme or a first flow-through containing human prothrombin 2;

[0067] (z3) clarifying the first flow-through liquid to obtain a clarified first filtrate containing rEcarin process enzyme or a clarified first filtrate containing human prothrombin 2;

[0068] (z4) performing Phenyl column chromatography on the first filtrate to obtain a first eluate containing rEcarin process enzyme or a first eluate containing human prothrombin 2;

[0069] (z5) performing nanofiltration and / or sterilization filtration on the first eluate to obtain a stock solution containing purified rEcarin process enzyme (or rEcarin process enzyme product stock solution) or a stock solution containing purified human prothrombin 2.

[0070] In another preferred embodiment, step (c) comprises: performing Ni column chromatography on the crude stock solution containing human thrombin to remove and / or inactivate the rEcarin process enzyme to obtain purified recombinant human thrombin.

[0071] In another preferred embodiment, the rEcarin process enzyme carries a 6×His tag.

[0072] In another preferred embodiment, in Ni column chromatography, rEcarin is combined with Ni Sepharose 6FF chromatography medium, and recombinant human thrombin flows through, thereby obtaining a flow-through containing recombinant human thrombin.

[0073] In another preferred embodiment, the Ni column chromatography can inactivate the residual rEcarin process enzyme.

[0074] In another preferred embodiment, the Ni column chromatography loading capacity is 4 to 15 mg / ml chromatography medium.

[0075] In another preferred embodiment, the flow rate of the Ni column chromatography is 120±30 cm / h.

[0076] In another preferred embodiment, the pH of the equilibrium buffer for Ni column chromatography is 6.0±0.1.

[0077] In another preferred embodiment, the residual amount of rEcarin is detected by ELISA.

[0078] In another preferred embodiment, the detection antibody used in the ELISA method is an anti-rEcarin antibody.

[0079] In another preferred embodiment, the detection antibody used in the ELISA method is mouse anti-rEcarin antibody.

[0080] In another preferred embodiment, the detection antibody used in the ELISA method is HRP-labeled mouse anti-rEcarin antibody.

[0081] In another preferred embodiment, the residual amount of rEcarin is ≤0.05%, preferably ≤0.04%, and more preferably ≤0.03%, based on the total weight of the protein in the recombinant human thrombin stock solution.

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG1 shows a schematic flow diagram of the process for producing recombinant human thrombin of the present invention.

[0084] Figure 2 shows the results of identifying the recombinant human thrombin stock solution and the reference substance of the present invention using Tricine-SDS-PAGE electrophoresis; wherein lanes 1 and 4 are recombinant human thrombin stock solutions, and lanes 2 and 3 are recombinant human thrombin physical and chemical reference substances.

[0085] FIG3 shows a graph showing the purity of recombinant human thrombin stock solution. DETAILED DESCRIPTION

[0086] After extensive and in-depth research, the inventors optimized the process for producing recombinant human thrombin using genetically engineered cells. In particular, through process optimization and quality control of the key raw material rEcarin process enzyme, the expression efficiency and purity of the recombinant protein were significantly improved, the amount of residual impurities was reduced, and the safety risk of topical recombinant human thrombin products was greatly reduced, thereby ensuring the safe, stable and continuous production of topical recombinant human thrombin.

[0087] Specifically, the inventors used genetically engineered cells to produce recombinant human prothrombin 2 and rEcarin process enzyme, respectively, and optimized the purification process of the rEcarin process enzyme. Through optimized DEAE column chromatography, filtration, and Phenyl column chromatography, the yield, purity, and activity of the rEcarin process enzyme were significantly improved. The highly active rEcarin process enzyme was used to effectively activate human prothrombin 2, thereby obtaining a crude human thrombin stock solution. The crude stock solution was subjected to Ni column chromatography to specifically remove and inactivate residual rEcarin process enzyme, ultimately obtaining a high-purity, low-residue recombinant human thrombin stock solution. The present invention was completed on this basis.

[0088] rEcarin

[0089] rEcarin process enzyme is a key raw material in the production of recombinant human thrombin stock solution. Its function is to activate recombinant human prethrombin 2 (rhPT2) to form active recombinant human thrombin (recombinant human thrombin).

[0090] Ecarin is a snake-derived serine protease. rEcarin refers to recombinant Ecarin. In the present invention, rEcarin is produced by genetically engineered cells. Specifically, rEcarin is produced by culturing, harvesting, concentrating, and purifying Chinese hamster ovary (CHO) cells that efficiently express the rEcarin gene.

[0091] The rEcarin precursor contains 622 amino acids, including a signal peptide, a precursor peptide, and mature rEcarin, with a continuous six-histidine His tag at the C-terminus. The theoretical molecular formula of unmodified mature rEcarin is: C 2113 H 3255 N 593 O 648 S 44 The theoretical molecular weight of unmodified mature rEcarin protein is 48744.3Da.

[0092] rEcarin has the following basic properties:

[0093] (1) rEcarin process enzyme is a colorless or light yellow clear liquid.

[0094] (2) The theoretical isoelectric point of mature rEcarin is 6.16, and the theoretical extinction coefficient is 1.14.

[0095] (3) Mature rEcarin can specifically cleave prothrombin and recombinant human prethrombin-2 (rhPT2) to produce biologically active recombinant human thrombin (rhT).

[0096] (4) The rEcarin process enzyme contains a 6×His tag that can specifically bind to the Ni column chromatography medium.

[0097] (5) rEcarin process enzyme is a metalloproteinase whose activity can be inhibited by reducing agents, EDTA and phosphate. Trace metal ions in the solution significantly affect the biological activity, resulting in the inactivation of rEcarin process enzyme.

[0098] The production process of rEcarin process enzyme mainly includes cell culture and purification stages. In the present invention, the cell purification process of rEcarin is optimized to obtain rEcarin process enzyme liquid with higher protein content and higher activity.

[0099] The rEcarin produced by the method of the present invention has improved specific activity and uniformity, as well as further improved human prothrombin activation ability.

[0100] In the present invention, the optimized rEcarin process enzyme purification process is as follows:

[0101] (1) Providing a feed solution containing rEcarin process enzyme; wherein the bacterial endotoxin of the feed solution is ≤30EU / ml; the mycoplasma test is negative; and the rEcarin protein content is ≥0.10mg / mL, more preferably ≥0.20mg / mL.

[0102] (2) performing DEAE column chromatography on the feed solution containing the rEcarin process enzyme to obtain a first flow-through containing the rEcarin process enzyme;

[0103] (3) Clarifying the first flow-through containing the rEcarin process enzyme to obtain a clarified first filtrate containing the rEcarin process enzyme; the turbidity of the first filtrate is ≤150NTU, more preferably ≤100NTU.

[0104] (4) performing Phenyl column chromatography on the first filtrate to obtain a first eluate containing rEcarin process enzyme;

[0105] (5) Nanofiltration and / or sterilization filtration are performed on the first eluate to obtain a stock solution containing the purified rEcarin process enzyme (or a stock solution of the rEcarin process enzyme product).

[0106] The rEcarin process enzyme product obtained by the purification method of the present invention has a protein content of ≥0.2 mg / mL, a biological activity of greater than 150 U / mL, and a biological specific activity of ≥500 U / mL. Preferably, the biological specific activity of the rEcarin process enzyme is ≥1000 U / mg, more preferably, ≥2000 U / mg.

[0107] Production and purification method of recombinant human thrombin

[0108] The purified rEcarin process enzyme stock solution is used as a raw material for production and added to the activation and S / D treatment steps of the recombinant human thrombin purification process to activate (enzymatically digest) the recombinant human prethrombin 2 to form active recombinant human thrombin.

[0109] The production processes of rEcarin and recombinant human thrombin are highly similar: the upstream and downstream process steps are the same, and the same host cells (CHO / dhFr - ), the same expression vector (pGN1), the same basal culture medium (BalanCDCHO Growth A), the same chromatography medium, and especially in the purification process, after the rEcarin process enzyme was introduced, the purification processes of rEcarin and recombinant human thrombin were integrated into one (see Figure 1).

[0110] In the quality standards of recombinant human thrombin, the residual amount of rEcarin and its possible residual activity are critical to the safety of the final product. Therefore, in the present invention, the removal and inactivation steps of the rEcarin process enzyme are optimized to effectively reduce the residual amount of rEcarin in the product.

[0111] The rEcarin process enzyme contains a 6×His tag. The inventors employed a Ni column chromatography step to specifically and effectively remove residual rEcarin from the recombinant human thrombin stock solution. Furthermore, because metal ions significantly affect the biological activity of rEcarin, the activity of the rEcarin process enzyme is essentially and irreversibly lost after Ni column chromatography. Therefore, even if trace amounts of rEcarin remain in the recombinant human thrombin stock solution, it is no longer biologically active.

[0112] In the recombinant human thrombin stock solution purified by the method of the present invention, the residual amount of rEcarin is ≤0.05%. In a preferred embodiment of the present invention, the residual amount of rEcarin is ≤0.03%.

[0113] In one embodiment of the present invention, the inventors used a highly sensitive sandwich ELISA method (LOQ = 70 ppm) to detect the residual amount of rEcarin, and the detection results of the residual rEcarin in the recombinant human thrombin stock solution were all "not detected".

[0114] The main advantages of the present invention include:

[0115] 1. The present invention improves the efficiency of rEcarin in activating human prothrombin-2 by optimizing the production process of rEcarin process enzyme. The rEcarin content required to activate 1 mg of recombinant human prothrombin is reduced by about 12 times, saving costs and significantly reducing the impurity load brought by the rEcarin process enzyme.

[0116] 2. The present invention improves the activity of rEcarin process enzyme by optimizing the production process of rEcarin process enzyme, and the content of rEcarin process enzyme obtained by purification from each liter of culture supernatant is increased by about 2.7 times.

[0117] 3. The production process of the optimized rEcarin process enzyme of the present invention is applicable to large-scale production, and the culture scale can reach 500L.

[0118] 4. The rEcarin process enzyme of the present invention contains a 6×His tag, and the Ni column chromatography step can specifically and effectively remove the residual rEcarin in the recombinant human thrombin stock solution. The residual amount was detected using a highly sensitive method, and the results were all "not detected".

[0119] 5. The detection results of host cell protein (HCP) residues and host DNA residues in multiple batches of recombinant human thrombin stock solutions proved that the recombinant human thrombin production process can effectively control impurity residues; the purification step has sufficient virus clearance ability, which can effectively control potential endogenous and exogenous viral contamination and ensure the safety of the product.

[0120] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are by weight.

[0121] Example 1: rEcarin culture process

[0122] The working cells were revived in the culture dish. When the culture volume reached 100 mL and the viable cell density reached (5.0-7.0)×10 5 When the viable cell density reaches (7.0-12.0)×10 5 / mL, with a survival rate of >90%, and transferred to a 5L reactor for culture.

[0123] In a 5L reactor, the culture temperature was 36.0-37.0℃, the pH value was 6.80-7.40, the dissolved oxygen was automatically controlled to >20%, the rotation speed was 100±20rpm, and when the viable cell density reached (7.0-10.0)×10 6 When the concentration of 100 μg / mL was reached, the cells were transferred to a 30 L reactor.

[0124] The initial volume in a 30L reactor is 13-16L, the initial culture temperature is 36.0-37.0°C, the pH is 6.80-7.40, the dissolved oxygen is automatically controlled to >20%, the rotation speed is 70±20rpm, and the culture volume is gradually increased. When the viable cell density reaches (2.0-3.0)×106 When the viable cell density reaches 3.0-6.0×10 6 When the cell viability reaches 60% to 70%, the culture is terminated.

[0125] During the culture process, glucose was added as needed to maintain the glucose concentration in the culture medium at 0.5-3.0 g / L. If the pH of the culture medium fell below the set range, 8% NaHCO₃ solution was added; if the pH of the culture medium rose above the set range, CO₂ gas was introduced.

[0126] The feed solution containing rEcarin protein is obtained by culturing using the above method.

[0127] Example 2: Production Process 1.0 of rEcarin

[0128] The rEcarin protein-containing solution obtained in Example 1 was purified using the following method:

[0129] DEAE column chromatography: After ultrafiltration and centrifugation, the cell culture supernatant was diluted with dilution buffer (0.1% PEG3350, 20 mM Tris-HCl, pH 8.0) to a final NaCl concentration of 0.05 M before loading. The DEAE column was packed with DEAE Bestarose FF. The anion chromatography column was fully equilibrated with equilibration buffer (0.05 M NaCl, 0.1% PEG3350, 20 mM Tris-HCl, pH 8.0). After loading, the column was fully equilibrated with equilibration buffer (0.4 M NaCl, 0.1% PEG3350, 20 mM Tris-HCl, pH 8.0). The elution peak was collected.

[0130] Phenyl column chromatography: Adjust the sample salt concentration to 1.25 M (NH4)2SO4 by adding a diluent (3.75 M (NH4)2SO4) equivalent to half the volume of the DEAE flow-through. The hydrophobic chromatography medium is Phenyl Bestarose FF. The column is fully equilibrated with an equilibration buffer (1.25 M (NH4)2SO4, 0.1% PEG3350, 20 mM Tris-HCl, pH 7.8). After sample loading, the column is fully equilibrated with the equilibration buffer again. Elution is performed with an eluent (0.1 M NaCl, 0.1% PEG3350, 20 mM Tris-HCl, pH 7.8), and the eluted peak is collected.

[0131] Ultrafiltration and concentration: The Phenyl column chromatography eluate was concentrated by ultrafiltration, and then an equal volume of glycerol was added to the ultrafiltration concentrate, mixed thoroughly, and stored at -70±5℃.

[0132] The product obtained by the above method was tested, and the results showed that the content of the purified rEcarin process enzyme per liter of culture supernatant was 6713 U.

[0133] Example 3: rEcarin Production Process 2.0

[0134] To further enhance the biological activity of the target protein, expand production capacity, and simplify operations, the inventors optimized purification process 1.0 by optimizing the DEAE column chromatography, Phenyl column chromatography, and ultrafiltration steps. Furthermore, the purification scale was expanded from 30 L to 100 L of culture volume corresponding to the harvested liquid, resulting in purification process 2.0. Compared to purification process 1.0 in Example 2, purification process 2.0 has the following major changes:

[0135] (1) The DEAE column chromatography step was optimized, and the original binding and elution mode was changed to flow-through mode, thereby simplifying the operation.

[0136] Specifically, the cell culture supernatant after ultrafiltration and centrifugation was diluted with dilution buffer (0.1% PEG3350, 20 mM Tris-HCl, pH 8.0) to a final NaCl concentration of 0.1 M before loading. The anion chromatography column was fully equilibrated with equilibration buffer (0.1 M NaCl, 0.1% PEG3350, 20 mM Tris-HCl, pH 8.0). After loading, the column was fully equilibrated with equilibration buffer again. The flow-through was collected.

[0137] (2) The Phenyl column chromatography step was optimized by adding a filtration step before loading the sample onto the Phenyl column to reduce sample turbidity and increase the service life of the chromatography medium.

[0138] Specifically, in purification process 1.0, after adding about 1 / 2 volume of 3.75M (NH4)2SO4 to the DEAE flowthrough, the intermediate becomes turbid, resulting in an increase in column pressure during Phenyl chromatography, affecting the life of the chromatography medium and the purification effect. Therefore, adding a filtration operation before Phenyl chromatography in purification process 2.0 can reduce the sample turbidity and increase the service life of the chromatography medium.

[0139] (3) Due to the increased activity of the rEcarin culture supernatant, the ultrafiltration process was eliminated, simplifying the steps.

[0140] Since the activity of the rEcarin culture supernatant is improved, the biological activity of the rEcarin process enzyme finally purified is improved, and ultrafiltration concentration is no longer required to improve the biological activity of the rEcarin process enzyme, so the ultrafiltration step is removed.

[0141] The comparison of purification results of process 1.0 and 2.0 is shown in Table 1.

[0142] Table 1 Comparison of purification results between process 1.0 and 2.0

[0143] The results show that compared with production process 1.0, the activity of rEcarin process enzyme purified per liter of culture supernatant in production process 2.0 is increased by 2.7 times. Relatively speaking, the amount of corresponding cell culture supernatant added to the recombinant human thrombin process is reduced to about 1 / 3.

[0144] Example 4: rEcarin Production Process 3.0

[0145] In order to further expand production capacity and achieve large-scale mass production, based on the culture process 2.0, the culture volume was gradually increased from 100L to 500L, and the clarification and filtration method was optimized to form the production process 3.0. Compared with the production process 2.0 in Example 2, the main changes in the production process 3.0 are as follows:

[0146] (1) The working volume was changed from 100L to 500L to expand production capacity; the cell clarification filtration method was changed from centrifugation to deep filtration to simplify the operation.

[0147] (2) In the purification process, the loading capacity of the DEAE chromatography column and the loading capacity of the Phenyl chromatography column were both doubled.

[0148] A 500 L scale culture and purification was carried out using Production Process 3.0 and compared with a batch of rEcarin process enzyme (100 L culture scale) using Production Process 2.0. The results are shown in Table 2.

[0149] Table 2 Results of enzyme activity and specific activity test of rEcarin process in production processes 2.0 and 3.0

[0150] The results show that the protein content, biological activity and biological specific activity of the batch using production process 3.0 were significantly increased, and the scale was expanded to 500L, which is conducive to industrial mass production.

[0151] Example 5: Activation of recombinant human prothrombin-2 by rEcarin process enzyme

[0152] After recombinant human prothrombin-2 and rEcarin process enzyme were produced and purified, recombinant human prothrombin-2 was activated and processed using the following method:

[0153] Activation: Add rEcarin process enzyme according to the ratio of 1.5±0.5U of rEcarin process enzyme to 1.0±0.1mg of recombinant human prethrombin 2 (rhPT2), and dilute the protein content of the Phenyl elution collection solution to 1.0±0.1mg / ml with buffer (0.1M NaCl, 0.1% PEG3350, 20mM Tris-HCl, pH7.8).

[0154] S / D treatment: Add buffer solution (10% polysorbate 80, 3% tributyl phosphate) to the activation system to a final concentration of polysorbate 80 and tributyl phosphate of 1.0% and 0.3%, respectively, and stir at a speed of 50-150 rpm.

[0155] Activation and S / D treatment conditions: 25±1℃, time 6.0±0.5h.

[0156] After the above treatment, the S / D collected solution is obtained, which is the crude stock solution of recombinant human thrombin of the present invention, and can be stored at 2-8° C. for 72 hours, or at room temperature for 24 hours.

[0157] Example 6: Purification of recombinant human thrombin crude stock solution

[0158] The residual amount of rEcarin process enzyme in the crude recombinant human thrombin solution will affect the purity of the product. Therefore, the crude recombinant human thrombin solution obtained in Example 4 was purified by Ni column chromatography to remove the rEcarin process enzyme with His tag:

[0159] Ni column chromatography uses Ni Sepharose 6FF filler to complete the column packing and complete the column efficiency test (column efficiency test pass criteria: column efficiency: ≥3000 Nm, symmetry: 0.8-1.8). The filler cycle life does not exceed 65 times.

[0160] The intermediate solutions after the two cycles of Heparin purification were combined, and the conductivity was adjusted to 12.5±1 mS / cm, pH 6.0±0.2 using a buffer (0.1% PEG3350, 10 mM PB, pH 6.0) or a 2 M NaCl solution.

[0161] Flow-through component collection range: When UV 280 Start collecting the readings within the range of 20 ± 10 mAU and end collecting the readings within the range of 20 ± 10 mAU. Mix the sample at a speed of 150 ± 50 rpm for 15 ± 5 minutes to ensure uniform mixing of the intermediates. The target protein flows through, while rEcarin and other impurities bind to the Ni Sepharose 6FF chromatography medium, achieving separation and purification.

[0162] After purification through the above steps, the recombinant human thrombin stock solution product of the present invention is obtained.

[0163] Example 7: Testing of recombinant human thrombin

[0164] 7.1 Identification of recombinant human thrombin stock solution by electrophoresis

[0165] Tricine-SDS-PAGE electrophoresis was used to identify proteins. The principle is that most proteins can combine with SDS to form complexes based on weight ratio, so that the negative charge carried by the protein molecules far exceeds the net charge of the natural protein molecules, eliminating the charge effect of different protein molecules and separating the proteins according to molecular size. Tricine gel was introduced into the method, and Tricine was used as the trailing ion in the electrophoresis buffer to separate and reduce the A and B chains. The recombinant human thrombin stock solution obtained in Example 6 was used as a test sample to detect and identify the proteins therein. The specific operation is as follows:

[0166] Loading: Add the test sample and recombinant human thrombin physical and chemical reference substance to the sample wells, loading 10 mg of the test sample and recombinant human thrombin physical and chemical reference substance into each well. After adding the sample, connect the positive and negative electrodes and perform electrophoresis at a constant voltage. The electrophoresis voltage for 1.0 mm gel is 30 V for the stacking gel and 100 V for the gap gel and separation gel. When the bromophenol blue has migrated to the bottom edge of the gel, cut off the power supply to end the electrophoresis. Remove the gel after electrophoresis, remove the stacking gel, and place the gel in the fixative for 20 minutes. Staining: Place the gel in Coomassie Brilliant Blue staining solution and stain for approximately 1 to 2 hours. Destaining: Remove the gel from the staining solution and immerse it in destaining solution to destain until the base color of the gel is colorless. Store the gel in water.

[0167] The results are shown in Figure 2. The main bands of the test and control samples were consistent, and the relative mobility deviation of the main bands was less than 5%.

[0168] 7.2 Isoelectric Point and Purity Testing of Recombinant Human Thrombin Stock Solution

[0169] Capillary isoelectric focusing (IEF) is used to determine the isoelectric point of proteins. IEF uses IEF technology to record the final position of proteins in the capillary tube using full-column imaging to identify the isoelectric point of proteins. The specific steps are as follows:

[0170] Place an appropriate amount of the test sample in an ultrafiltration tube, concentrate by ultrafiltration to approximately 100 μl, and then replace the solution three times with 400 μl of 0.2 mol / L sodium chloride solution. After the solution replacement, use a micro-UV spectrophotometer to detect the protein content in the test sample, and adjust the protein content of the test sample to approximately 16 mg / ml with 0.2 mol / L sodium chloride solution. Shake the above solution and centrifuge it at 12000 rpm for 5 minutes. Transfer 80 μl of the supernatant to a 96-well plate and centrifuge it at 1200 × g for 10 minutes. Determine the isoelectric point using a capillary isoelectric focusing electrophoresis instrument. According to the area normalization method, the software calculates the percentage of the peak area of ​​each peak of the test sample, excluding the pI marker, to the total peak area, which is the purity.

[0171] The purity of the recombinant human thrombin stock solution is shown in Figure 3. The results show that the total area percentage of the main peaks 1, 2, and 3 of the test sample is greater than 80%, indicating that the recombinant human thrombin stock solution of the present invention has a high purity.

[0172] Example 8: Detection of rEcarin Residue in Recombinant Human Thrombin Stock Solution

[0173] In the quality standard of recombinant human thrombin, special attention is paid to the residue of rEcarin and its possible residual activity. The enzyme-linked immunosorbent assay (ELISA) is used to determine the residual amount of rEcarin in the recombinant human thrombin stock solution. The anti-rEcarin capture antibody is coated onto an enzyme-labeled plate, and then combined with the rEcarin content control substance or the residual rEcarin in the test sample, and then combined with the HRP-labeled anti-rEcarin detection antibody. After incubation, the difference in the color intensity of the enzymatic reaction is used to calculate the residual amount of rEcarin in the test sample using a standard curve. The anti-rEcarin capture antibody and HRP-labeled anti-rEcarin detection antibody used in this embodiment are mouse anti-rEcarin monoclonal antibodies. The specific operations are as follows:

[0174] Coating: Add 100 μl / well (duplicate) of the capture antibody working solution to the ELISA plate and coat overnight at 2-8°C. Washing: Discard the supernatant and wash the plate three times with PBST, using a 200 μl volume per wash. Blocking: Add 150 μl of blocking solution to each well and incubate at 25±4°C for 1 hour. Sample Addition: Discard the blocking solution, tap off any remaining blocking solution, and add 100 μl / well (duplicate) of the standard curve solution, test solution, and quality control solution of varying concentrations. Cover the plate and incubate at 25±4°C for 1 hour. Washing: Discard the supernatant and wash the plate three times with PBST, using a 200 μl volume per wash. Enzyme-labeled Antibody: Tap off any remaining liquid and add 100 μl of detection antibody working solution to each well. Incubate at 25±4°C for 1 hour. Washing: Discard the supernatant and wash the plate five times with PBST, using a 200 μl volume per wash. Color development: Add 100 μl of color development solution to each well and incubate at 25 ± 4°C in the dark for 15–30 minutes. Stop color development: Add 100 μl of stop solution to each well. Reading: Detect at 450 nm using a microplate reader and record the OD value.

[0175] The OD value of the test sample was substituted into the linear regression equation to calculate the rEcarin concentration C (ng / ml), and the residual rEcarin content (%) of the test sample was calculated according to the following formula: rEcarin residual content (%) = C (ng / ml) / concentration of test sample solution (mg / ml) × 10 -6 The results showed that the residual amount of rEcarin in each batch produced by the process of the present invention was less than 0.03%.

[0176] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing recombinant human thrombin, characterized in that: Including steps: (a) producing rEcarin process enzyme using genetically engineered cells, and purifying the rEcarin process enzyme to obtain purified rEcarin process enzyme; (b) using the purified rEcarin process enzyme to enzymatically activate human prothrombin 2 to obtain a crude stock solution containing human thrombin; and (c) purifying the crude stock solution containing human thrombin to obtain purified recombinant human thrombin.

2. The method according to claim 1, wherein The recombinant human thrombin stock solution is detected by high performance liquid chromatography, and the recombinant human thrombin protein content is 1.5-3.5 mg / ml.

3. The method according to claim 1, wherein In the recombinant human thrombin stock solution, the biological specific activity of the recombinant human thrombin is 2500-5000 IU / mg as determined by the fibrinogen coagulation method.

4. The method according to claim 1, wherein The content of impurity proteins in the recombinant human thrombin stock solution is ≤1.0%; the impurity proteins include host cell proteins, rEcarin process enzymes, or a combination thereof.

5. The method according to claim 1, wherein In step (a), comprising: (a1) providing a feed solution containing rEcarin process enzyme; (a2) subjecting the feed solution containing the rEcarin process enzyme to DEAE column chromatography to obtain a first flow-through containing the rEcarin process enzyme; (a3) clarifying the first flow-through containing the rEcarin process enzyme to obtain a clarified first filtrate containing the rEcarin process enzyme; (a4) performing Phenyl column chromatography on the first filtrate to obtain a first eluate containing rEcarin process enzyme; (a5) performing nanofiltration and / or sterilization filtration on the first eluate to obtain a stock solution containing the purified rEcarin process enzyme (or a stock solution of the rEcarin process enzyme product).

6. The method according to claim 5, wherein The liquid containing rEcarin process enzyme satisfies: (t1) bacterial endotoxin ≤ 30 EU / ml; (t2) Mycoplasma test negative; and (t3) rEcarin protein content ≥ 0.10 mg / mL.

7. The method according to claim 6, wherein In the rEcarin process enzyme product stock solution, the rEcarin protein content is ≥0.2 mg / mL.

8. The method according to claim 1, wherein Step (b) comprises: (b1) activating recombinant human prothrombin 2 using rEcarin process enzyme; and (b2) Virus inactivation by treatment with an organic solvent / detergent mixture (S / D).

9. The method according to claim 8, wherein The content of the rEcarin process enzyme required to activate 1 mg of the recombinant human prothrombin 2 is 1.5±0.5 U.

10. The method according to claim 1, wherein In step (b), the human prothrombin 2 is purified human prothrombin 2.

11. The method according to claim 10, wherein The purification process of the human prothrombin 2 adopts a purification route that is substantially the same as the purification route of the rEcarin process enzyme.

12. The method according to claim 11, wherein The purification route comprises: (z1) providing a feed solution containing rEcarin process enzyme or a feed solution containing human prothrombin 2; (z2) performing DEAE column chromatography on the feed solution to obtain a first flow-through containing rEcarin process enzyme or a first flow-through containing human prothrombin 2; (z3) clarifying the first flow-through liquid to obtain a clarified first filtrate containing rEcarin process enzyme or a clarified first filtrate containing human prothrombin 2; (z4) performing Phenyl column chromatography on the first filtrate to obtain a first eluate containing rEcarin process enzyme or a first eluate containing human prothrombin 2; (z5) performing nanofiltration and / or sterilization filtration on the first eluate to obtain a stock solution containing purified rEcarin process enzyme (or rEcarin process enzyme product stock solution) or a stock solution containing purified human prothrombin 2.

13. The method according to claim 1, wherein Step (c) comprises: performing Ni column chromatography on the crude stock solution containing human thrombin to remove and / or inactivate the rEcarin process enzyme to obtain purified recombinant human thrombin.

14. The method according to claim 13, wherein The residual amount of the rEcarin is ≤0.05%, calculated based on the total weight of the protein in the recombinant human thrombin stock solution.

15. The method according to claim 13, wherein The residual amount of the rEcarin is ≤0.03%, calculated based on the total weight of the protein in the recombinant human thrombin stock solution.

Images