8 results about "Human Blood Coagulation Factor" patented technology

The invention discloses a human blood coagulation factor VIII and a preparation method thereof, and the preparation method comprises the following steps: S1, carrying out refrigerated centrifugation on cryoprecipitate to obtain an extracting solution; s2, mixing the extracting solution and the heparin sodium solution to obtain a dissolving solution; s3, adjusting the pH value of the dissolving solution to 6.2-6.4 by using hydrochloric acid, and controlling the temperature to be 20-30 DEG C to obtain an acid precipitate; s4, mixing the acid precipitate with polyethylene glycol to obtain a polyethylene glycol precipitate; s5, adding the carboxylated ferroferric oxide nano-particles into the polyethylene glycol precipitate, incubating, and carrying out magnetic separation to obtain a first separation solution; s6, performing centrifugal separation on the first separation liquid to obtain a second separation liquid; s7, clarifying and filtering the second separation liquid, and adjusting the pH value to 6.4-7.4 to obtain a clarified liquid; and S8, adding a stabilizer and an inactivator into the clear liquid, and carrying out water bath heat preservation to obtain a human blood coagulation factor VIII stock solution. The method has the effect of improving the recovery activity and purity of the human blood coagulation factor VIII.

The invention belongs to the technical field of medicine, and particularly relates to a preparation method and application of a recombinant human blood coagulation factor VII, and the preparation method comprises the following steps: transforming a nucleotide sequence of the recombinant human blood coagulation factor VII into a vector, and constructing a recombinant expression plasmid; transfecting the recombinant expression plasmid into a eukaryotic cell to obtain a recombinant expression cell; screening the recombinant expression cells to obtain stable transfected cells; domesticating the stably transfected cells to obtain monoclonal suspension engineered cells; and performing amplification culture on the monoclonal suspension engineered cells, collecting supernate, and extracting target protein in the supernate to obtain the recombinant human blood coagulation factor VII. The recombinant human blood coagulation factor VII produced by using the method disclosed by the invention is high in yield and good in procoagulant activity, so that the production efficiency and the application effect of the recombinant human blood coagulation factor VII can be improved by using the preparation method disclosed by the invention.

The present application belongs to the technical field of blood product preparation, and particularly relates to a method for separating blood coagulation factor VIII and vWF factor from blood plasma. The method comprises the following steps in sequence: after cellulose is balanced, the cellulose is mixed with raw material blood plasma, filtered after stirring, and cellulose precipitate is obtained; after the cellulose precipitate is eluted, the cellulose precipitate is soaked in precipitate dissolving solution, and filtrate is obtained by filtering; the filtrate is subjected to three times of ion exchange chromatography, and factor VIII and / or vWF factor product is obtained. The technical scheme solves the technical problems of the prior art, such as complicated process steps for purifying and enriching human blood coagulation factor VIII and vWF from blood plasma cold precipitate, and unsatisfactory production efficiency and product quality. The cold precipitate protein solution prepared by the method can separate factor VIII, vWF factor and fibrinogen product at the same time through chromatography, improve the utilization rate of plasma protein, and has an ideal application prospect.

The application provides a completely humanized anti-human blood coagulation factor IX monoclonal antibody or an antigen binding fragment thereof and application thereof. The antibody provided by the application has high affinity to human blood coagulation factor IX and significant inhibitory activity, and belongs to a neutralizing antibody, and has a good treatment prospect for thrombus diseases. The functional antibody is directly screened from blood plasma of hemophilia B patients by a mass spectrometry + de novo sequencing technology, and has the advantages of complete human origin, high safety and the like.

The invention discloses a preparation method of a human blood coagulation factor IX stock solution, which comprises the following steps: S1, combining plasma, centrifugally filtering to obtain a supernatant, adjusting the pH value, and carrying out A-50 gel adsorption; s2, washing with an A-50 washing solution, retaining the washing solution, adding a protective agent and an S / D inactivator, and stirring for inactivation; s3, adding the A-50 washing solution again for washing, then adding the A-50 eluant for elution, and combining the eluant; and S4, performing chromatography washing and elution, and performing ultrafiltration to prepare a human blood coagulation factor IX stock solution. According to the preparation method of the human blood coagulation factor IX stock solution, the production steps are obviously reduced, the production cost is reduced, and the production efficiency is improved. By adopting the preparation method disclosed by the invention, tributyl phosphate in the stock solution is not detected, the residual quantity of polysorbate 80 is not higher than 5mu g / ml, the safety of the product is greatly improved, and meanwhile, the specific activity of the human blood coagulation factor IX is not lower than 600IU / mg and is obviously higher than that of the traditional method.

The invention provides a completely humanized anti-human coagulation factor IX monoclonal antibody or an antigen binding fragment thereof and application thereof. The antibody provided by the invention has high affinity and remarkable inhibitory activity on the human coagulation factor IX, belongs to a neutralizing antibody, and has a good treatment prospect on thrombosis diseases. The functional antibody is directly screened from the blood plasma of the hemophilia B patient through the'mass spectrum + de novo sequencing 'technology, and the method has the advantages of being completely humanized, high in safety and the like.

The invention discloses an HPLC-FLD (high performance liquid chromatography-flash laser diode) detection method for the residual quantity of polysorbate 80 in a recombinant human blood coagulation factor VIII for injection. According to the invention, a high performance liquid chromatography-fluorescence detector method (HPLC-FLD) for detecting the residual amount of polysorbate 80 in the recombinant human blood coagulation factor VIII for injection is established, N-phenyl-1-naphthylamine (NPN) is used for dyeing groups of polysorbate 80, and a fluorescence detector is used for detecting the content of polysorbate 80. The method is simple and convenient to operate, can be used for treating multiple batches of samples, is superior to a current standard method in the aspects of repeatability, accuracy and sensitivity, and provides reference for improvement of a polysorbate 80 determination method.

Provided is a polynucleotide that enables genome editing to address a plurality of genetic mutations in hemophilia B.　The polynucleotide includes a sequence that induces a change in the amino acid at the R338 site in a mature protein of human blood coagulation factor IX (FIX). The sequence may induce a C > T mutation. The sequence may be a guide RNA sequence for genome editing. The sequence may be any one or any combination of SEQ ID NOS: 1-3 in the Sequence Listing. In addition, a vector or a lipid nanoparticle, each containing the polynucleotide and a nucleotide editing tool, may be used. A composition for treating hemophilia B containing the vector or the lipid nanoparticle may also be provided.