54 results about "Human Blood Coagulation Factor" patented technology

The invention discloses a method for preparing a freeze-dried human blood coagulation factor VIII. The method comprises the following process of: dissolving by taking water for injection, comprising 3-10 IU (International Unit) / ml of heparin, as a heparin sodium solution and cryoprecipitating; performing PEG (Polyethylene Glycol) precipitation and taking supernatant; performing centrifugal filtration; performing S / D (Solvent / Detergent) inactivation at the temperature of 24-26 DEG C; performing DEAE (Diethylaminoethyl) Sepharose Fast Flow chromatographic column balance, adsorption, washing andelution; performing molecular membrane ultrafiltration; preparing, removing bacteria, sub-packaging, freeze-drying and capping; and dry-heating at the temperature of 99.5-100.5 DEG C and inactivating. In the invention, the process method of combining the PEG precipitation and an ion exchange chromatography technology is adopted; the method is easy and convenient to operate; the F VIII active recovery rate is increased; miscellaneous proteins can be removed on a large scale; the product yield reaches over 60 percent; and the specific activity of the product reaches 5 IU / mg and is obviously greater than a value which is not less than 1 IU / mg stipulated in the pharmacopeia. Meanwhile, the PEG residue is 0.08g / L which is obviously less than the value which is less than or equal to 0.5 IU / mg stipulated in the pharmacopeia, so that Al<3+> residues in the final preparation of an Al(OH)3 gel method are avoided; the product has high purity and high safety; and the quality of the final product is obviously improved.

The invention provides a technology for extracting human blood coagulation factor VIII and human fibrinogen from plasma constituent precipitation. The preparation technology comprises the following steps: fresh plasma is subjected to primary sedimentation, so that blood coagulation factor VIII and fibrinogen precipitation can be jointly precipitated from the plasma; the primary precipitation is subjected to suspension; the suspension liquid is subjected to centrifugal separation to obtain supernatant; the centrifugally separated supernatant is subjected to virus inactivation and chromatography refining to respectively obtain human blood coagulation factor VIII refined liquid used for preparing human blood coagulation factor VIII products and chromatography effluent used for preparing human fibrinogen products. According to the invention, the human blood coagulation factor VIII and the human fibrinogen are precipitated through the one-step plasma constituent precipitation, and an ion-exchange column chromatography technology is adopted to perform purification preparation of the human blood coagulation factor VIII and the human fibrinogen, so that the deficiency that the human fibrinogen cannot be normally produced as the human blood coagulation factor VIII is prepared through cryoprecipitation is overcome.

The invention discloses a dry heat treatment stabilizer for human blood coagulation factor VIII, which comprises the following proportions of components: 0.005M-0.015M of sodium citrate, 0.001M-0.002M of calcium chloride, 0. 15M-0.25M of arginine hydrochloride, and 6-10g / L of human serum albumin. The invention also discloses a human blood coagulation factor VIII preparation and a preparation method of the human blood coagulation factor VIII preparation. The dry heat treatment stabilizer for human blood coagulation factor VIII provided by the invention can maintain the activity of the human blood coagulation factor VIII, and is low in cost, thus having good prospect in clinical application.

The invention discloses a method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma. The method comprises the following steps: 1, removing cold glue from blood plasma; 2, conducting strong anion-exchange column chromatography the first time; 3, conducting PEG sedimentation for removing impure protein; 4, conducting S / D viral inactivation; 5, conducting strong anion-exchange column chromatography the second time, and obtaining FVII eluent and FIX eluent; 6, conducting weak anion-exchange column chromatography, and concentration for purifying blood coagulation FVII; 7, conducting heparin affinity column chromatography for purifying blood coagulation FIX; 8, conducting ultrafiltration; 9, adding a stabilizing agent, and conducting adjustment; 10, conducting virus-removal filtration through nanofilms; 11, conducting sterilization, filtration and subpackage; 12, conducting freeze-drying; 13, conducting dry-hot viral inactivation. According to the method, PEG sedimentation is adopted for removing the impure protein, the target of preparing high-purity FVII and FIX simultaneously is achieved through combination of an ion-exchange column chromatography technology and an affinity chromatography technology, the process flow is simple, the production cycle is short, a product is subjected to three steps of virus eradicating measures, and use safety is high.

The invention provides a human blood coagulation factor VIII resisting monoclonal antibody as well as a preparation method and application thereof. The monoclonal antibody has the characteristic of strong specificity in combination with the human blood coagulation factor VIII antigen. The invention also provides a corresponding monoclonal antibody segment, immune conjugate and a detection kit for detecting the human blood coagulation factor VIII.

The present invention refers to a recombinant human blood coagulation factor VIII protein and a composition containing it. The present invention also refers to the use of the protein or composition of the invention for manufacturing a medicine for treating hemophilia A. Additionally, the present invention refers to the method of obtaining a recombinant human blood coagulation factor VIII protein. A further object of the present invention is a recombinant protein obtained by the method described herein, and its use in the preparation of a medicine for the treatment of hemophilia A.

The invention provides a novel recombinant human blood coagulation factor VIII and a production method thereof. Particularly, the invention relates to reconstruction of a human blood coagulation factor VIII gene. A novel expression vector containing a weakened internal ribosome entry site (IRES for short) sequence and double screening marks is constructed. The vector can be used for high-efficiency recombinant expression of the human blood coagulation factor VIII, so that the expression of the vector in a mammalian cell expression system is increased.

The invention provides a yeast cell which expresses a human coagulation factor seven; the yeast cell integrates a structure which expresses the human coagulation factor seven; the structure comprises two to fifteen human coagulation factor seven expression cassettes which are serially connected and lined; each expression cassette subsequently comprises the following elements from 5' to 3': (a) a start signal element AOX; (b) a human coagulation factor seven gene; (c) a stop signal element AOX (TT); and the yeast cell expresses the activated human coagulation factor seven under the induction of methanol. The invention also provides the structure and a preparation method thereof, a preparation method of the yeast cell and the human coagulation factor seven expressed by the yeast cell. The two to eighteen copied yeast cells structured by the invention can be used to improve yield and reduce cost, and are applicable in the mass production of human coagulation factor seven preparation.

The invention discloses a method for preparing a cryoprecipitate. The method comprises the steps of (1) thawing: selecting fresh frozen plasma, and heating to obtain thawed plasma of which the temperature is 0-5 DEG C; (2) filtering: filtering under the condition that the temperature of the thawed plasma is 0-5 DEG C to obtain filtrate and filter residues; (3) centrifuging: centrifuging under the condition that the temperature of the filtrate is 0-5 DEG C to obtain a precipitate; (4) combining the filter residues obtained in step (2) and the precipitate obtained in step (3) to obtain the cryoprecipitate. The preparation method disclosed by the invention is simple, and the prepared cryoprecipitate is high in yield and human blood coagulation factor VIII content, so the preparation method has a good industrial application prospect.

The invention discloses a method for preparing a freeze-dried human blood coagulation factor VIII through cryoprecipitation. The method comprises the following steps of (1) dissolving and carrying out filter pressing in cryoprecipitation; (2) deactivating S / D viruses; (3) removing a blood coagulation factor depended by vitamin K by using DEAE SephadexA-50 gel; (4) purifying FVIII through carrying out column chromatography on anion exchange resin; (5) carrying out ultrafiltration and dialysis, and concentrating an eluent; (6) adding a stabilizing agent in a concentrated solution, and regulating the titer and PH value of FVIII; (7) removing viruses on a nanofilm, and filtering; (8) degerming, filtering and subpackaging; (9) carrying out freeze drying; and (10) carrying out dry heat deactivation on the viruses. According to the method disclosed by the invention, the blood coagulation factor depended by vitamin K is removed through adsorption of the DEAE SephadexA-50 gel, the traditional aluminum hydroxide and PEG sedimentation way is replaced, and therefore, the method has the advantages of production stability, high yield and good quality; and the safety of the product is greatly improved due to the adoption of three-step virus removing measures.

The invention discloses a human blood coagulation factor IX mutant pichia pastoris expression vector and a construction method and application thereof. The procedures of the construction method of the human blood coagulation factor IX mutant pichia pastoris expression vector includes that according to a hFIX gene sequence, the reverse transcription-polymerase chain reaction (RT-PCR) technology is used for cloning hFIX overall length complementary deoxyribonucleic acid (cDNA) from human hepatic cells, signal peptide mixed with yeast Alpha -factor and yeast expression plasmids pPIC9K-hFIX are constructed to express pichia pastoris, and then based on that the yeast expression plasmids pPICZ Alpha A-hFIX are constructed, four saccharomycetes hFIX high-activity mutants are constructed. Cruor activity of the achieved hFIX yeast expression vector is apparently higher than that of standard hFIX. One of the mutants is through 50-litre pilot scale test of fermentation, protein purification product secretory expression quantity is 702 milligrams / litre, so that a good foundation is laid for producing efficient hFIX products used for treating hemophilia B in a low cost and industrialized mode.

The invention relates to a preparation method of a clinical blood coagulation inspection preparation and particularly relates to a preparation method of a blood coagulation factor IX quality control product. The preparation method comprises the following steps: carrying out affinity chromatography on the mixed blood plasma of multiple persons by using an anti-human blood coagulation factor IX monoclonal antibody immunoaffinity chromatography column, removing a blood coagulation factor IX in the mixed blood plasma of multiple persons to obtain a blood plasma in shortage of the blood coagulation factor IX; mixing the mixed blood plasma of multiple persons with the blood plasma in shortage of the blood coagulation factor IX according to a certain proportion to prepare a blood coagulation factor IX quality control product with the content of the blood coagulation factor IX at different concentration levels, adding a freeze-drying protective additive, carrying out sub-packaging, and carrying out freeze drying so as to obtain the blood coagulation factor IX quality control product. According to the blood coagulation factor IX quality control product prepared by the preparation method, the uniformity, the stability and the stability of freeze-dried aquatic product subjected to re-melting are good, and the quality control product can replace an imported product to be used for quality control on detection of blood coagulation factor IX, so that the reduction of the detection cost is facilitated and the capability of detecting the blood coagulation factor IX in China can be promoted.

The present invention discloses a preparation method of a highly purified human blood coagulation factor IX. The human blood coagulation factor IX is prepared by performing anion exchange chromatography, cation exchange chromatography, heparin affinity chromatography to the material containing human blood coagulation factor IX (taken from human blood serum or recombined cell culture medium), which includes the period of passivating virus through S / D treatment (Solvent / Detergent treatment) or removing virus through nanofiltration. The highly purified safe coagulation factor IX preparation having a specific activity of above 150 IU / mg, with substantially undoped proteins can be prepared by the preparation method of the invention.

The invention discloses a method for preparing a freeze-dried human blood coagulation factor VIII. The method comprises the following process of: dissolving by taking water for injection, comprising 3-10 IU (International Unit) / ml of heparin, as a heparin sodium solution and cryoprecipitating; performing PEG (Polyethylene Glycol) precipitation and taking supernatant; performing centrifugal filtration; performing S / D (Solvent / Detergent) inactivation at the temperature of 24-26 DEG C; performing DEAE (Diethylaminoethyl) Sepharose Fast Flow chromatographic column balance, adsorption, washing andelution; performing molecular membrane ultrafiltration; preparing, removing bacteria, sub-packaging, freeze-drying and capping; and dry-heating at the temperature of 99.5-100.5 DEG C and inactivating. In the invention, the process method of combining the PEG precipitation and an ion exchange chromatography technology is adopted; the method is easy and convenient to operate; the F VIII active recovery rate is increased; miscellaneous proteins can be removed on a large scale; the product yield reaches over 60 percent; and the specific activity of the product reaches 5 IU / mg and is obviously greater than a value which is not less than 1 IU / mg stipulated in the pharmacopeia. Meanwhile, the PEG residue is 0.08g / L which is obviously less than the value which is less than or equal to 0.5 IU / mg stipulated in the pharmacopeia, so that Al<3+> residues in the final preparation of an Al(OH)3 gel method are avoided; the product has high purity and high safety; and the quality of the final product is obviously improved.

The invention provides a method for expressing and producing recombinant human blood coagulation factors VII in animal cells, in particular an expression vector for expressing exogenous protein in the animal cells. The expression vector contains an expression cassette for expressing the exogenous protein, wherein the expression cassette comprises the following elements from 5' to 3': (a) a first promoter; (b) a polyclone locus and an optional coding sequence of the exogenous protein; (c) a first polyA sequence; (d) a second promoter; (e) a selected marker gene; and (f) a second polyA signal sequence. The expression vector is particularly suitable for expressing the FVII efficiently in the animal cells.

The invention discloses a human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method. The method constructs specific in-situ remediation treatment and can specifically repair the type of F8 mutation, and the in-situ remediation strategy is verified in hemophilia patient specific iPSCs by combination with a TALEN technology. The in-situ remediation strategy is a first remediation strategy for remediation of intron 22 inversion as common HA mutation. The in-situ remediation strategy utilizes a sequence accurate fixed-point introduction method, is relatively safe and has no nondeterminacy of the prior art.

The invention discloses a freeze-drying and dry heat treatment protecting agent for human coagulation factor VIII. The freeze-drying and dry heat treatment protecting agent for human coagulation factor VIII does not contain human serum albumin or other animal-source proteins, sugar or sugar alcohol, is composed of soluble salt, amino acid and a surface active agent, is free from the risk of spreading other viruses or causative agents, and is applicable to a wide crowd range, including diabetics; after being freeze-dried, a product of the human coagulation factor VIII using the protecting agent is quick in redissolving and has a good redissolving effect, still keeps high potency and high specific activity, and has the potency higher than 80 percent and the specific activity higher than 40 IU / mg; in addition, the freeze-drying and dry heat treatment protecting agent for human coagulation factor VIII is low in cost, simple to prepare, has an obvious protecting effect, is safe and effective, and has an excellent industrial application prospect.

Disclosed are a recombinant fusion protein of human blood coagulation factor FVII-Fc, preparation method therefor and use thereof. The fusion protein comprises a human FVII, flexible peptide linker, and IgG2 Fc variant sequentially from the N-terminal to the C-terminal. The Fc variant has on lytic activity, and shows minimal Fc-mediated adverse side-effects. The fusion protein has a bioactivity similar to or higher than that of human FVII and a greatly prolonged plasma half-life, thereby improving the pharmacokinetics and pharmaceutical effect.

The invention belongs to the field of biological medicines, and in particular relates to a gene recombinant human blood coagulation factor VIII for treating hemophilia A. By performing zone B partially-deleted type mutation on a full-length blood coagulation factor VIII, the obtained novel gene recombinant human blood coagulation factor VIII product has a characteristic that the structure is more stable.

The invention belongs to the technical field of genetic engineering, relating to a target multifunctional anti-embolism fusion protein an amino acid sequence of which is as shown in SEQ ID NO.1. The invention also relates to a gene for encoding the fusion protein, a recombinant expression vector containing the gene, a transformant containing the recombinant expression vector and a method for preparing the fusion protein. The fusion protein can reasonably splice human antibacterial peptide LL-37, leech peptide Hirudin-12, Agkistrodon acutus peptide (AAP), arginine-glycin-aspartate (RGD) and human blood coagulation factor Xa identification sites, so that the functions among a target component, an antibacterial component, a thrombin resisting component and a platelet aggregation resisting component are complementary and in synergistic effect; and the fusion protein can well exert target anti-inflammatory and anticoagulation activities at a thrombus position, and simultaneously can repair a vessel endothelial cell, can commonly inhibit the formation and development of thrombus by virtue of multiple ways, and can be used for preparing a medicament for preventing and treating thrombotic diseases.

The invention belongs to the technical field of biological engineering, and relates to a method for preparing a recombinant human blood coagulation factor VIII, in particular to a method for culturing cells for expressing a recombinant human blood coagulation factor VIII by using a wave type bioreactor and separating and purifying the recombinant human blood coagulation factor VIII from a cell culture liquid. The WAVE bioreactor is high in mixing efficiency, sufficient in gas-liquid exchange, small in foam amount and low in shearing force, damage of paddles of a stirring type stainless steel reactor and foams to cells is avoided, and thus the cell state, the cell survival rate and the protein activity of the WAVE bioreactor are all superior to those of the stirring type stainless steel reactor.

The invention discloses a preparation method of a human blood coagulation factor VIII and a human blood coagulation factor VIII product and relates to the field of blood products. The preparation method of the human blood coagulation factor VIII comprises the following steps: dissolving a cryoprecipitate with a 0.015-0.025mol / L tromethamine solution in a mass ratio of (3-5): 1 by reasonably processing the cryoprecipitate; and carrying out separation and purification by means of a polyethylene glycol precipitating method combined with ion exchange chromatography, wherein the recovery ratio of the human blood coagulation factor VIII and the specific activity of a final product can be improved effectively, the yield reaches 180-240IU / L plasma, and the specific activity is not lower than 100IU / mg proteins. The prepared human blood coagulation factor VIII product is rich in vWF factors and the proportion of the vWF factors and the human blood coagulation factor VIII is close to 1: 1. Besides treating hemophiliac A, the human blood coagulation factor VIII can be also used for treating patients with angiohemophilia, and the human blood coagulation factor VIII has good stability and heat resistance.

The invention relates to a preparation method of a freeze-dried human blood coagulation factor VIII. The preparation method comprises the following steps of collecting plasma and quick-frozen plasma;performing plasma melting and separating cryoprecipitate; dissolving cryoprecipitate; performing acid precipitation; performing gel adsorption; inactivating S / D virus; performing anion exchange chromatography; performing ultrafiltration; preparing a semi-finished product; carrying out sterilizing and sub-packaging; performing freeze-drying; and carrying out dry heat virus inactivation. The preparation method disclosed by the invention is short in product remelting time, high in specific activity, stable in quality and simple to operate, and after industrialization, the utilization rate of plasma resources can be greatly improved, market supply is increased, and the situation of FVIII medication tension in the Chinese market is relieved.

The invention relates to the preparation of recombinant human coagulation factor IX and an application thereof. The recombinant hFIX protein and hFIX of human blood source have the same molecular composition and biological activity and can be eased by being used for hemophilia B mouse model. The recombinant hFIX protein can be used as a standard in the tests for clinically detecting hFIX and the like, and can also cure diseases requiring supplementing hFIX, such as hemophilia B, etc.

The invention provides a method for expressing and producing recombinant human blood coagulation factors VIII in animal cells, in particular an expression vector for expressing exogenous protein in the animal cells. The expression vector contains an expression cassette for expressing the exogenous protein, wherein the expression cassette comprises the following elements from 5' to 3': (a) a first promoter; (b) a polyclone locus and an optional coding sequence of the exogenous protein; (c) a first polyA sequence; (d) a second promoter; (e) a selected marker gene; and (f) a second polyA signal sequence. The expression vector is particularly suitable for expressing the VIII efficiently in the animal cells.

Peptides derived from amino acids 307 to 356 of the human blood coagulation factor Va are provided. Such peptides comprise: i) a length of between 3 and 50 amino acids, ii) a minimum of 3 contiguous amino acids from the 307–356 heavy chain region of factor Va, excluding peptide segments comprising amino acids 311 to 325 and amino acids 321 to 335, iii) optional additional amino acids at one or both ends of the contiguous amino acids such that the entire peptide is at least 60% identical to a sequence within 307 to 356 of factor Va, and iv) have an IC50 of between 50 nM to 500 μM for inhibition of prothrombinase. The present invention also provides a pharmaceutical composition comprising one or more prothrombinase-inhibiting peptide segments. The present invention also provides administration of the pharmaceutical composition to human subjects for the purpose of preventing thrombotic disorders.

The invention relates to the technical field of blood products, in particular to a blood-source human blood coagulation factor VIII / von willebrand factor compound production process which comprises the following steps: (1) preparing cryoprecipitate from plasma, dissolving the cryoprecipitate and removing crude impurities; (2) product amino acid protection incubation; (3) S / D virus inactivation; (4) ion exchange chromatography: incubating a chromatographic filler by using a solution containing protective components before loading; (5) ultrafiltration preparation; and (6) sub-packaging, freeze-drying and dry-heating. According to the production process, only two steps of centrifugation, one step of chromatography and one step of ultrafiltration are needed from plasma to a finished product, the process steps are simple, and aluminum hydroxide, PEG and other allogenic substances which are not contained in a human body are not introduced; the purity of the product is effectively improved through amino acid protection in the incubation step and the chromatography step after cryoprecipitation dissolution and crude impurity removal.

The invention relates to a preparation method of a clinical blood coagulation inspection preparation and particularly relates to a preparation method of a blood coagulation factor IX quality control product. The preparation method comprises the following steps: carrying out affinity chromatography on the mixed blood plasma of multiple persons by using an anti-human blood coagulation factor IX monoclonal antibody immunoaffinity chromatography column, removing a blood coagulation factor IX in the mixed blood plasma of multiple persons to obtain a blood plasma in shortage of the blood coagulation factor IX; mixing the mixed blood plasma of multiple persons with the blood plasma in shortage of the blood coagulation factor IX according to a certain proportion to prepare a blood coagulation factor IX quality control product with the content of the blood coagulation factor IX at different concentration levels, adding a freeze-drying protective additive, carrying out sub-packaging, and carrying out freeze drying so as to obtain the blood coagulation factor IX quality control product. According to the blood coagulation factor IX quality control product prepared by the preparation method, the uniformity, the stability and the stability of freeze-dried aquatic product subjected to re-melting are good, and the quality control product can replace an imported product to be used for quality control on detection of blood coagulation factor IX, so that the reduction of the detection cost is facilitated and the capability of detecting the blood coagulation factor IX in China can be promoted.

The invention belongs to the field of genetic engineering and in particular relates to a production method of a recombinant human blood coagulation factor XIIa. The production method of the recombinant human blood coagulation factor XIIa comprises the following steps of: acquiring DNA (deoxyribonucleic acid) coding an active region of a human blood coagulation factor XIIa, amplifying by virtue of pichia pastoris, centrifuging and dialyzing a yeast culture solution and carrying out filter pressing by adopting a filter membrane on the yeast culture solution, and then carrying out column chromatographic purification by taking Ni-NTA (Ni-nitrilotriacetic acid) as medium, so that the recombinant human blood coagulation factor XIIa is obtained. By adopting the production method of the recombinant human blood coagulation factor XIIa, production cost of the human blood coagulation factor XIIa is reduced, and product activity is higher than that of blood coagulation factor XIIa extracted from blood.