CN111849945A - Method for purifying human blood coagulation factor VIIa - Google Patents
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6437—Coagulation factor VIIa (3.4.21.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
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Abstract
The invention provides a method for purifying human blood coagulation factor VIIa, which comprises the steps of purifying by adopting three steps of anion exchange chromatography, anion exchange chromatography and hydrophobic chromatography, inactivating viruses and removing viruses and filtering to prepare the human blood coagulation factor VIIa with purity, activity and safety meeting the medicinal requirements.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a method for purifying human blood coagulation factor VIIa.
Background
Human factor vii (fvii) is a vitamin K-dependent serine protease that can initiate the extrinsic pathway of coagulation or participate in the intrinsic pathway of coagulation by activating human factor IX. FVII is synthesized in the liver and secreted into the blood where it circulates as a single chain glycoprotein (zymogen) with a molecular weight of approximately 50,000 Da. FVII zymogen is hydrolyzed by proteases at positions R152-I153 to produce a two-chain molecule linked by a disulfide bond, which is converted to the active form FVIIa. Clinical studies have shown that FVIIa has a satisfactory therapeutic effect on thrombocytopenia and platelet dysfunction, severe trauma, hemostasis in extensive surgical procedures.
The early human coagulation factor VII preparation is prepared by taking fresh frozen plasma as a raw material, performing barium sulfate adsorption and multi-step chromatography purification, gradually changing to the preparation mainly based on A-50 gel adsorption along with the application of DEAE-Sephadex A-50 gel, and performing multi-step chromatography purification. The content of the human coagulation factor VII in the blood plasma is extremely low, generally 200-400 ng/ml, so that the preparation of the human coagulation factor VII is very difficult and the cost is extremely high; meanwhile, the plasma extraction and preparation process is complex, the difference between batches is large, and the stability is poor; in addition, the blood plasma is used as a raw material, so that the infection risk of exogenous viruses is greatly increased. In recent years, the development of genetic engineering techniques has made it possible to prepare FVII recombinantly in vitro. NovoSeven (NovoSeven) from Nonoh and Node, Denmark is a pharmaceutical recombinant human coagulation factor VIIa with biological activity, which is obtained by introducing cDNA gene of human coagulation factor VII into BHK cells (baby hamster kidney cells) to express and produce, activating and highly purifying through genetic engineering technology.
Tomokiyo et al report a purification process for the preparation of human coagulation Factor VIIa from plasma (Tomokiyo K, Yano H, Imamura M, et al, Large-scale production and properties of human plasma-derived activated Factor VII concentration [ J ]. Vox Sanguinis,2010,84(1):54-64.), by the specific steps of: FVII is captured from plasma by Q-Sepharose Fast Flow, then by immunoaffinity chromatography with anti-FVII antibodies, virus-free filtration and purification by DEAE-Sepharose to obtain a FVII-FVIIa mixture, then FVIIa is obtained by in-solution activation. Finally obtaining the FVIIa preparation through steps of dialysis, preparation filling, freeze-drying and the like.
Jurlander et al disclose downstream purification processes for FVIIa from Novonide (Jurlander B, Thiml, Klausen N K, et al. recombinant Activated Factor VII (rFVIIa): Characterification, Manufacturing, and Clinical Development [ J ]. Seminars in Thrombosis and Hemostasis,2001,27(4):373 384.): after cell culture fluid is harvested, a first step of anion exchange chromatography is carried out to capture FVII, and then virus inactivation and immunoaffinity chromatography are carried out, and then two steps of anion exchange chromatography are carried out to generate fully activated FVIIa.
CN1121723A discloses the addition of Zn during purification chromatography2+Controlled activation and degradation of FVII, chromatography steps: anion exchange, immunoaffinity, anion exchange.
CN101268185A discloses the addition of hydrophobic chromatographic processes during purification to control product related impurities in drugs, such as different levels of N-linked glycosylated sugar variants, oxidized forms, proteolytically degraded forms (heavy chain cleavage forms), aggregates, etc. contained in the late eluting peaks.
Existing downstream purification techniques for human coagulation factor FVIIa usually incorporate an affinity chromatography step to specifically capture FVIIa protein through a chromatography medium linked to FVIIa antibodies, but this technique has the following disadvantages: firstly, the industrialization cost is high, and secondly, the falling of the immune affinity chromatography ligand can cause related safety risks.
Disclosure of Invention
The invention aims at providing an improved process for the purification of human factor VIIa.
In some embodiments, the present invention provides a method of purifying human factor VIIa comprising the steps of: anion exchange chromatography, hydrophobic chromatography.
In some embodiments, the present invention provides a method of purifying human factor VIIa comprising the steps of: anion exchange chromatography, hydrophobic chromatography, and each step is performed sequentially.
In some embodiments, the present invention provides a method of purifying human factor VIIa comprising the following steps performed sequentially:
1) anion exchange chromatography;
2) inactivating the virus;
3) anion exchange chromatography;
4) hydrophobic chromatography;
5) removing viruses and filtering;
6) ultrafiltration liquid exchange;
7) sterilizing and filtering to obtain a stock solution.
In some embodiments, the present invention provides a method of purifying human factor VIIa comprising the following steps performed sequentially:
1) anion exchange chromatography;
2) performing virus inactivation on the product obtained in the step 1);
3) further purifying the product obtained in step 2) by anion exchange chromatography;
4) further purifying the product obtained in step 3) by hydrophobic chromatography;
5) Performing virus removal and filtration on the product obtained in the step 4);
6) carrying out ultrafiltration liquid change on the product obtained in the step 5);
7) sterilizing and filtering the product obtained in the step 6) to obtain a stock solution.
In some embodiments, the invention first captures the protein of interest from the cell harvest using anion exchange chromatography, wherein the anion exchange chromatography optionally comprises (a) equilibrating the anion exchange material with an equilibration buffer, (b) rinsing the anion exchange material with a rinse buffer, and (c) eluting the anion exchange material with an elution buffer, wherein the equilibration buffer has a pH of 6 to 10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM, preferably a sodium chloride concentration of about 50 mM; the elution buffer has a pH of 6-10, preferably a pH of about 8, optionally comprising sodium chloride at a concentration of at least about 50 mM; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of 50-800mM, preferably at a concentration of about 500 mM.
In some aspects, the first step anion exchange chromatography of the invention comprises equilibrating the anion exchange material with an equilibration buffer, wherein the equilibration buffer has a pH of 6 to 10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM, preferably a sodium chloride concentration of about 50mM, and eluting the anion exchange material with an elution buffer; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of 50-800mM, preferably at a concentration of about 500 mM.
In some aspects, the first step of anion exchange chromatography of the invention comprises equilibrating the anion exchange material with an equilibration buffer and eluting the anion exchange material with an elution buffer, wherein the equilibration buffer and the elution buffer may each be independently selected from the group consisting of phosphate buffer, tris-hcl buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, N-tris (hydroxymethyl) methylglycine buffer, N-dihydroxyethylglycine buffer, preferably the equilibration buffer and the elution buffer are each phosphate buffer, most preferably the equilibration buffer and the elution buffer each comprise about 20mmol/L phosphate.
In some embodiments, the inactivation of the virus in the eluate from the first step of anion exchange chromatography is performed using an S/D (surfactant/detergent) method.
In some embodiments, the product after virus inactivation is subjected to a second anion exchange chromatography to remove Gla (γ -carboxyglutamic acid) deletion/partial deletion variants and other related impurities, wherein the anion exchange chromatography step optionally comprises (a) equilibrating the anion exchange material with an equilibration buffer, (b) rinsing the anion exchange material with a rinse buffer, and (c) eluting the anion exchange material with an elution buffer, wherein the equilibration buffer has a pH of 6 to 10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM, preferably about 100 mM; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM and/or calcium chloride at a concentration of at least about 1mM, preferably comprises about 100mM sodium chloride and about 2.25mM calcium chloride; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM and/or calcium chloride at a concentration of at least about 1mM, preferably comprises about 100mM sodium chloride and about 5mM calcium chloride.
In some aspects, the second step anion exchange chromatography of the invention comprises (a) equilibrating the anion exchange material with an equilibration buffer, (b) rinsing the anion exchange material with a rinse buffer, and (c) eluting the anion exchange material with an elution buffer, wherein the equilibration buffer has a pH of 6 to 10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM, preferably about 100 mM; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM and/or calcium chloride at a concentration of at least about 1mM, preferably comprises about 100mM sodium chloride and about 2.25mM calcium chloride; the elution buffer has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM and/or calcium chloride at a concentration of at least about 1mM, preferably comprises about 100mM sodium chloride and about 5mM calcium chloride.
In some aspects, the second step of anion exchange chromatography of the invention comprises equilibrating the anion exchange material with an equilibration buffer, eluting the anion exchange material with an elution buffer, and eluting the anion exchange material with an elution buffer, wherein the equilibration buffer, elution buffer, and elution buffer can each be independently selected from the group consisting of phosphate buffer, tris-hcl buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, N-tris (hydroxymethyl) methylglycine buffer, N-dihydroxyethylglycine buffer, preferably the equilibration buffer, elution buffer, and elution buffer are each tris-buffer, and most preferably the equilibration buffer, elution buffer, and elution buffer each comprise about 10mmol/L tris-hcl.
In some embodiments, the eluate from the second anion exchange chromatography is further subjected to hydrophobic chromatography to remove product-related impurities such as polymers, oxides, and degradants, said hydrophobic chromatography step optionally comprising (a) equilibrating the hydrophobic interaction chromatography material with an equilibration buffer, (b) rinsing the hydrophobic interaction chromatography material with a rinse buffer, and (c) eluting the hydrophobic interaction chromatography material with an elution buffer, wherein the loading solution of the step contains salts and/or zwitterions, said salts may be selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride, and potassium sulfate, and the zwitterion may be selected from the group consisting of: glycine, alanine, leucine and isoleucine, preferably ammonium acetate.
In some embodiments, the hydrophobic chromatography of the invention comprises equilibrating the hydrophobic interaction chromatography material with an equilibration buffer and eluting the hydrophobic interaction chromatography material with an elution buffer, wherein the equilibration buffer comprises 10-30mmol/L glycylglycine, preferably about 10mmol/L glycylglycine, and 1-20mol/L ammonium acetate, preferably about 1.8mol/L ammonium acetate, and the equilibration buffer has a pH of 6-10, preferably a pH of about 6; the elution buffer comprises 10-30mmol/L glycylglycine, preferably about 10mmol/L glycylglycine, and 10-100mmol/L trisodium citrate dihydrate, preferably about 30mmol/L trisodium citrate dihydrate, and the pH of the elution buffer is 6-10, preferably about pH 6.
In some embodiments, the hydrophobic chromatography eluate product is subjected to virus removal filtration, followed by ultrafiltration and sterile filtration to obtain a stock solution.
In some embodiments, the methods of purifying human factor VIIa provided herein do not comprise an affinity chromatography step.
In some embodiments, the methods of purifying human factor VIIa provided herein do not comprise an immunoaffinity chromatography step in which an antibody to factor VIIa is coupled to an affinity chromatography medium.
In some embodiments, the invention provides methods of purifying human factor VIIa, which is recombinant human factor VIIa produced under cell culture conditions. In some embodiments, the human factor VIIa of the invention is not plasma-derived human factor VIIa.
It is also an object of the present invention to provide a method of reducing Gla variants in human factor vila, said method of reducing Gla variants comprising the sequential steps of: anion exchange chromatography, hydrophobic chromatography, in some embodiments the human factor VIIa is recombinant human factor VIIa, in other embodiments the method does not comprise affinity chromatography.
It is also an object of the present invention to provide a method for reducing the content of polymers, degradants and oxides in a recombinant protein, which in some embodiments is recombinant human coagulation factor VIIa.
In some embodiments of the invention, the purification of recombinant human factor VIIa excludes the step of affinity chromatography. In the immunoaffinity chromatography step, the affinity filler-coupled antibody protein may contaminate or interfere with the purified product. The invention eliminates affinity chromatography, and can avoid the risk of anti-FVIIa antibody shedding, thereby obtaining a recombinant protein product with higher purity and smaller immunogenicity.
The purification method of human blood coagulation factor VIIa provided by the invention has the following beneficial effects: the human blood coagulation factor VIIa with purity, activity and safety meeting the medicinal requirements can be prepared by adopting two-step anion exchange and one-step hydrophobic chromatography, and the risk of virus pollution of a final product can be guaranteed by adopting the other two-step virus inactivation and virus removal filtration processes. The method does not contain an immunoaffinity chromatography step, has low industrialization cost and avoids the risk of falling of the anti-FVIIa antibody; in addition, the three-step chromatography process is simple and is easy for large-scale production.
Interpretation and definition
Unless otherwise indicated, the following terms used in the specification and claims shall have the following meanings for the purposes of this application.
"blood coagulation factor": refers to various protein components involved in the blood coagulation process, including coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII.
"recombinant human coagulation factor": is human blood coagulation factor produced by recombinant DNA technology or recombinant RNA technology. The recombinant protein is obtained by applying gene recombination technology and connected with a recombinant vector which can be translated into a gene segment of a target protein, and then the recombinant vector is transferred into a host cell which can express the target protein so as to express a specific recombinant protein molecule. The production of recombinant proteins currently mainly involves four major systems: prokaryotic expression systems, mammalian cell expression systems, eukaryotic expression systems, and insect cell expression systems.
"antibody": in its broadest sense, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments are specifically contemplated so long as they possess the desired biological activity.
"Gla (γ -carboxyglutamic acid) deletion/partial deletion variant": native human factor VII undergoes post-translational modifications, including microbe-K dependent carboxylation, resulting in the production of 10 Gla (γ -carboxyglutamic acid) residues at the N-terminus of FVII or FVIIa. FVIIa produced by gene recombination technology is easy to form variants with different Gla degrees in the expression process, which are called Gla (gamma-carboxyglutamic acid) deletion/partial deletion variants, wherein the higher the modification ratio of Gla (gamma-carboxyglutamic acid), the higher the specific activity of protein.
"affinity chromatography" refers to a purification and extraction method that combines chromatographic techniques with antigen-antibody specific reactions. It is a common practice to chemically bind an antibody (or antigen) to a column having a molecular sieve function such as agarose or dextran, and when the extract passes through an affinity column, bind to the corresponding antigen (antibody) on the column.
The words "comprise" or "comprise" and variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "including but not limited to.
Unless otherwise stated, "about" in the context of the present invention means within + -5%, preferably within + -2%, more preferably within + -1% of the specified numerical range given. For example, a pH of about 5.5 means a pH of 5.5. + -. 5%, preferably a pH of 5.5. + -. 2%, more preferably a pH of 5.5. + -. 1%.
As used herein, "polymer" refers to a polymer produced by aggregation of recombinant proteins due to interactions between amino acid residues in the peptide chain or intermolecular interactions, such as hydrophobic interactions or electrostatic interactions.
The term "degradation product" as used herein refers to a product having a lower molecular weight than that of recombinant proteins produced by deamidation or peptide chain cleavage or the like in an aqueous solution.
Drawings
FIG. 1 is a graph showing the effect of removing a GLa (gamma-carboxyglutamic acid) deletion/partial deletion variant by the second anion exchange chromatography (determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)). Lane 1 is the loading solution, lane 2 is the flow-through, lane 3 is the elution peak 1, lane 4 is the elution peak 2, lane 5 is the elution peak, and M is the molecular weight marker.
Detailed Description
EXAMPLE 1 purification of FVIIa
Downstream purification of FVIIa is performed by the following seven steps:
in the first step, anion exchange chromatography.
The clarified cell culture broth containing FVIIa was loaded onto a TMAE anion chromatography column that had been pre-equilibrated with buffer A1(20mmol/L disodium hydrogenphosphate hydrate, 50mmol/L sodium chloride, pH 8.0-8.2). After equilibration after loading with the same buffer, FVIIa was eluted with buffer B1(20mmol/L disodium hydrogenphosphate hydrate, 500mmol/L sodium chloride, pH 8.0-8.2).
And secondly, inactivating S/D virus.
Triton X-100 (polyethylene glycol octylphenyl ether) and TNBP (tributyl phosphate) were added to the TMAE eluate to give final concentrations of 2% and 0.45%, respectively, and the mixture was maintained at 25 ℃ for 45min to inactivate lipid-enveloped viruses.
And thirdly, anion exchange chromatography.
The conductance of the S/D virus inactivation product was adjusted to below 12mS/cm with buffer D (10mmol/L Tris, pH 8.0-8.2). The sample was loaded onto a Q Sepharose High Performance anion chromatography column which had been pre-equilibrated with buffer A2(10mmol/L Tris-hydroxymethyl aminomethane, 100mmol/L NaCl, pH 8.0-8.2). The equilibration after loading was carried out with the same buffer as the pre-equilibration, followed by elution with buffer B2(10mmol/L Tris +100mmol/L NaCl +2.25mM calcium chloride solution, pH8.0-8.2), followed by elution with eluent C1(10mmol/L Tris +100mmol/L NaCl +5mM calcium chloride solution, pH8.0-8.2), and the eluate was collected.
And fourthly, hydrophobic chromatography.
The conductivity of the eluate of the previous step of anion exchange chromatography is adjusted to 100mS/cm with saturated ammonium acetate. The sample was loaded onto a phenyl Sepharose High Performance hydrophobic chromatography column that had been pre-equilibrated with buffer A3(10mmol/L glycylglycine, 1.8mol/L ammonium acetate, pH 6.0). And (3) carrying out equilibrium after loading by using the same buffer solution as the pre-equilibrium, eluting by using 35% B3 buffer solution (B3: 10mmol/L glycylglycine +30mmol/L trisodium citrate dihydrate, pH6.0), then eluting by using 10 column volume gradients of 35% -100% B3, collecting eluent in sections, and merging qualified components after RP-HPLC and SEC-HPLC purity detection.
And fifthly, removing viruses and filtering.
Washing the washed 20nm virus-removing filter membrane with hydrophobic eluent (10mmol/L glycylglycine +30mmol/L trisodium citrate dihydrate, pH6.0), and then performing virus-removing filtration on the product obtained by hydrophobic chromatography, wherein the filtration pressure is controlled at 3 bar.
Sixthly, ultrafiltration is carried out for liquid exchange.
The ultrafiltration membrane (10kDa) was washed with water and equilibrated with displacement buffer (10mmol/L glycylglycine, 40mmol/L sodium chloride, 10mmol/L calcium chloride, 0.5g/L methionine, pH 6.0). And then concentrating and replacing the product after virus removal and filtration, replacing the product into a replacement buffer solution, and then adding polysorbate 80, sucrose and mannitol mother liquor to protect the target protein.
And seventhly, sterilizing and filtering.
Sterile filtration was performed using a 0.22 μm filter.
The purity of the stock solution finally obtained by the above purification process is shown in table 1 below.
TABLE 1 Final stock solution purity chart
EXAMPLE 2 determination of Polymer content
Detecting the molecular weight distribution of the recombinant human coagulation factor VIIa by using a molecular exclusion chromatographic column, eluting by using a phosphate-sodium chloride mobile phase system, separating according to different retention capacities of components with different molecular weights in a stationary phase matrix, and determining the purity of the components by using a peak area normalization method through an ultraviolet detector. Using a ThermoUltimate 3000 high performance liquid chromatograph with an ultraviolet detector to balance the chromatographic column until the baseline is stable, and using a mobile phase: 50mmol/L disodium hydrogen phosphate, 300mmol/L sodium chloride, pH 7.5. The parameters are shown in Table 2 below.
TABLE 2 gel chromatography liquid analysis parameters
The results show that the method of the invention can obviously remove the polymer in the rFVIIa by using the size exclusion chromatography-high performance liquid chromatography.
EXAMPLE 3 determination of oxide and degradant content
And (3) adopting a reversed-phase chromatographic column to detect the purity of the recombinant human coagulation factor VIIa for injection, eluting components with different retention capacities along with the increase of an organic phase to achieve separation, and adopting a peak area normalization method to determine the purity of the recombinant human coagulation factor VIIa through an ultraviolet detector. Using a Thermo Ultimate 3000 high performance liquid chromatograph with an ultraviolet detector to balance the chromatographic column until the baseline is stable, and using a mobile phase A: 0.1% aqueous trifluoroacetic acid, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. The parameters are shown in Table 3 below.
TABLE 3 reversed phase chromatography liquid phase analysis parameters
The result shows that the method can obviously remove the oxides and degradation products in the rFVIIa by adopting the reversed phase chromatography-high performance liquid chromatography.
Example 4 assay of Gla (gamma-carboxyglutamic acid) variants
The Gla (gamma-carboxyglutamic acid) -deleted/partially deleted variants of rFVIIa were determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is a denaturing polyacrylamide gel electrophoresis method, the principle is based on that most proteins can be combined with anionic surfactant Sodium Dodecyl Sulfate (SDS) according to weight ratio to form a complex, so that the negative charge of protein molecules is far higher than the net charge of natural protein molecules, the charge effect of different protein molecules is eliminated, and the proteins are separated according to the molecular size.
The method is carried out by adopting Bio-Rad/PowerPac Basic equipment, and comprises the following steps: the gel was placed in an electrophoresis tank and electrode buffer was added. Adding 5 μ l of molecular weight standard into the sample well of the gel, adding the control solution and the sample solution into the sample well adjacent to the sample well, and covering the electrophoresis tank cover. And adjusting the power voltage to 140V in a voltage stabilizing mode, and stopping electrophoresis when the bromophenol blue migrates to the position of the gel bottom. And pouring out the electrode buffer solution, taking out the gel, and dyeing and decoloring.
The results of the measurements according to the method are shown in FIG. 1. The results show that anion exchange chromatography, the third step in example 1, can elute the Gla deleted/partially deleted variant.
While the method of the present invention has been described in terms of preferred embodiments in light of the present disclosure, it will be apparent to those of skill in the art that variations may be applied to the method and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.
The disclosures of all documents cited herein are incorporated by reference herein, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.
Claims (10)
1. A method of purifying human factor VIIa comprising the steps of, in order:
1) anion exchange chromatography;
2) inactivating the virus;
3) anion exchange chromatography;
4) hydrophobic chromatography;
5) removing viruses and filtering;
6) ultrafiltration liquid exchange;
7) sterilizing and filtering to obtain a stock solution.
2. The method according to claim 1, characterized in that it comprises the following steps carried out in sequence:
1) anion exchange chromatography;
2) performing virus inactivation on the product obtained in the step 1);
3) Further purifying the product obtained in step 2) by anion exchange chromatography;
4) further purifying the product obtained in step 3) by hydrophobic chromatography;
5) performing virus removal and filtration on the product obtained in the step 4);
6) carrying out ultrafiltration liquid change on the product obtained in the step 5);
7) sterilizing and filtering the product obtained in the step 6) to obtain a stock solution.
3. The method of any one of claims 1-2, wherein step 1) captures the protein of interest from the cell harvest using anion exchange chromatography, which optionally comprises (a) equilibrating anion exchange material, (b) rinsing the anion exchange material, and (c) eluting the anion exchange material.
4. The method according to any one of claims 1 to 3, wherein in step 2) virus inactivation is performed by S/D method.
5. The method according to any one of claims 1 to 4, wherein step 3) optionally comprises (a) equilibrating the anion exchange material, (b) rinsing the anion exchange material, and (c) eluting the anion exchange material, wherein the buffer used for equilibration has a pH of 6 to 10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM, preferably about 100 mM; the buffer used for rinsing has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least about 50mM and/or calcium chloride at a concentration of at least about 1mM, preferably comprises about 100mM sodium chloride and about 2.25mM calcium chloride; the elution buffer used has a pH of 6-10, preferably a pH of about 8, and comprises sodium chloride at a concentration of at least 50mM and/or calcium chloride at a concentration of at least 1mM, preferably the elution buffer comprises about 100mM sodium chloride and about 5mM calcium chloride.
6. The method according to any one of claims 1 to 5, wherein step 4) optionally comprises (a) equilibrating the hydrophobic interaction chromatography material, (b) rinsing the hydrophobic interaction chromatography material, and (c) eluting the hydrophobic interaction chromatography material, wherein the sample solution of the step contains salts and/or zwitterions, wherein the salts may be selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride, and potassium sulfate, and the zwitterion may be selected from the group consisting of: glycine, alanine, leucine, and isoleucine.
7. The method of claim 6, wherein the salt and/or zwitterion is preferably ammonium acetate.
8. The method of any one of claims 1-7, wherein the method does not comprise an affinity chromatography step.
9. The method of claim 8, wherein the affinity chromatography is immunoaffinity chromatography with an antibody to factor VIIa coupled to an affinity chromatography medium.
10. The method according to any one of claims 1-9, wherein: the human coagulation factor VIIa is recombinant human coagulation factor VIIa produced under cell culture conditions.
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| CN113337493A (en) * | 2021-06-30 | 2021-09-03 | 武汉禾元生物科技股份有限公司 | Method for expressing and preparing recombinant reteplase by using genetically engineered rice |
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