52 results about "Human Blood Coagulation Factor" patented technology

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 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 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 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 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 an application of a mutant single-chain human blood coagulation factor VIII in preparation of a fusion protein of the human blood coagulation factor VIII; the fusion protein sequentially comprises a B-structural domain partially deleted mutant single-chain human coagulation factor VIII, a flexible peptide joint, at least one human chorionic gonadotropin beta subunit carboxyl terminal peptide rigid unit and a half-life prolonging part from an N terminal to a C terminal, and is used for preventing or treating hemorrhagic diseases.

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.

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 present invention relates to a method for mass producing human blood coagulation factor VII, and more specifically, to a method for mass producing human blood coagulation factor VII, and a cell line for mass production of a human blood coagulation factor VII derivative, the method comprising the steps of: a) preparing an expression vector comprising i) a base sequence of dihydrofolate reductase (DHFR) promoter in which at least one CCGCCC is removed in a GC-rich region and a base sequence encoding DHFR operably linked thereto, and ii) a base sequence of an early gene of cytomegalovirus (CMV) and a base sequence encoding a human blood coagulation factor VII derivative operably linked thereto; b) transforming an animal cell line by using the expression vector of step; c) selecting a cell line expressing a human blood coagulation factor VII derivative with high efficiency by culturing the transformed animal cell line of step b) in the presence of a DHFR inhibitor; and d) culturing the selected animal cell line of step c) by adding at least one selected from the group consisting of sodium butyrate, vitamin K and a culture medium additive. The present invention can express a human blood coagulation factor VII derivative with high efficiency and in a large quantity by using a vector in which GC-rich repeating sequences are deleted in a DHFR promoter region, and thus can be usefully applied to the preparation of a therapeutic agent for hemophilia.