Method for preparing human antithrombin-III product
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A technology of antithrombin and AT-III, which is applied in the field of separation and purification of human antithrombin-Ⅲ, which can solve the problems of limitation and low recovery rate
Active Publication Date: 2013-04-24
GUIZHOU TAIBANG BIOLOGICAL PROD +1
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[0003] In the 1960s and 1970s, people began to report the process of purifying AT-Ⅲ, but it was limited to laboratory development
In
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Embodiment 1
[0039] The loading chromatography conditions were optimized, and the pH value, sodium chloride concentration, and sodium citrate concentration of the component IV precipitation and extraction buffer were used as factors to design a three-factor three-level orthogonal experiment. The specific design scheme is shown in the table below . The average recovery activity of the three experiments was used as the investigation index, and the purpose of the experiment was to obtain the best combination of loading chromatographic conditions. The specific implementation process is as follows:
[0040] Weigh 50g of component IV precipitate and dissolve in different concentrations of extraction buffer, the ratio of extraction buffer to component IV is 4:1 (v / m) , adjust the pH value according to the design scheme, stir at room temperature for 1 hour, and then centrifuge it at 4°C and 4500 rpm for 30 minutes to remove the precipitate and collect the supernatant. Filter the supernatant, us...
Embodiment 2
[0044] 1. Basic requirements:
[0045] Debug the required equipment in the company's pilot workshop and mark it as a standby state;
[0046] Clean the required container pipelines and pilot plant and mark them as standby;
[0047] Prepare 100kg of component IV precipitate and store it in a -30°C freezer for future use.
[0048] 2. Main solution preparation method
[0049] The extraction buffer contains sodium chloride and sodium citrate, wherein the concentration of sodium chloride is 0.09-0.15M, the concentration of sodium citrate is 0.015-0.025M, the pH is 5.5-11, and the rest is water;
[0050] The balance solution contains sodium chloride and sodium citrate, wherein the concentration of sodium chloride is 0.09-0.15M, the concentration of sodium citrate is 0.015-0.025M, the pH is 6.5-7.5, and the rest is water;
[0051] The washing liquid contains sodium chloride and sodium citrate, wherein the concentration of sodium chloride is 0.3-0.5M, the concentration of sodium cit...
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Abstract
The invention relates to a method for preparing a human antithrombin-III product, which comprises the following steps of: separating a component IV in a plasma protein process by using a Cohn low-temperature ethanol method, precipitating the component IV serving as a raw material; and finally, preparing the high-purity antithrombin-III lyophilized product through extracting, filter pressing, carrying out heparin affinity chromatography, ultra-filtering, degerming, filtering, lyophilizing and the like and a necessary S/D and dry heat virus inactivating process. According to the invention, the process is adjusted, so that AT-III is dissolved as much as possible; the Pasteur virus inactivating process is replaced by the S/D virus inactivating process by adding proper protective agent; activity loss is reduced; the preparation time is shortened; furthermore, high-purity AT-III solution can be obtained only in the need of heparin affinity chromatography for one step; after carrying out the steps of ultra-filtering, freezing, drying, inactivating dry heat virus and the like, the purity of a final product is above 95%; and the specific activity is above 6.5 IU/mg and is 3.0 IU/mg more than specified limits in the European pharmacopoeia and united state pharmacopoeia.
Description
field of invention [0001] The invention relates to a process for separating and purifying human antithrombin-III from human Cohn plasma components, belonging to the field of biopharmaceuticals. The process steps are simple, and only one step of affinity chromatography is used, and after two different steps of virus inactivation, the obtained product is safe and reliable. It is mainly used to treat patients with antithrombin-Ⅲ deficiency caused by various reasons. Background technique [0002] Human antithrombin-Ⅲ (can be expressed as AT-Ⅲ) is a single peptide chain α-glycoprotein with a molecular weight of about 58kD (kilodaltons), consisting of 423 amino acids, and is a multifunctional serine protease inhibitor. The anticoagulant effect in human plasma accounts for about 70% of the whole anticoagulant effect, and its half-life is 2.69 days, and the half-life is shortened when sick, and it is mainly synthesized in the liver, so AT-Ⅲ has different levels when various liver ...
Claims
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