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Method for purifying human immunoglobulin from separated component I+III of blood plasma

A human immunoglobulin and plasma separation technology, applied in the field of separation and purification of human immunoglobulin, can solve the problems of retaining the physicochemical properties and biological activity of the immunoglobulin, unable to completely inactivate/remove the virus, and the purification effect is difficult to meet the requirements. , to achieve the effect of shortening the production process time, good economic and social value, and considerable economic benefits

Active Publication Date: 2011-11-23
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process needs to purify component II by heating it to 51-53°C for 3 hours. Since the heat resistance of immunoglobulin is related to its purity, generally the higher the purity, the worse its heat resistance, so this method cannot Retain the natural physicochemical properties and biological activities of human immunoglobulins to the greatest extent
At the same time, since Component III is the most concentrated component of the virus in plasma separation, virus inactivation is particularly important, and this process only uses one virus inactivation method, which cannot completely inactivate / remove the virus
[0007]3. In recent years, chromatography has been more and more widely used in the separation and purification of plasma proteins. In the preparation of human immunoglobulins, ion Exchange chromatography, but the combination of cations and anions or two-step anion exchange column chromatography is often used, otherwise the purification effect is difficult to meet the requirements
[0008]4. The technology of using octanoic acid or caprylate to precipitate non-IgG miscellaneous proteins to achieve the separation of IgG is not yet mature. It is currently in the research stage and has not been used at home and abroad. A report on the purification of immunoglobulin by octanoic acid precipitation fraction Ⅰ+Ⅲ

Method used

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  • Method for purifying human immunoglobulin from separated component I+III of blood plasma
  • Method for purifying human immunoglobulin from separated component I+III of blood plasma
  • Method for purifying human immunoglobulin from separated component I+III of blood plasma

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The present invention is realized by the following steps:

[0050] a. Dissolution of component I+III precipitate:

[0051] Preparation of acetic acid sodium acetate buffer: Take 6.9 mL of glacial acetic acid and 10.88 g of sodium acetate to prepare 10 L of acetic acid sodium acetate buffer with a content of 0.02 mol / L, and adjust the pH to 4.5;

[0052] Fully dissolve the component I+III precipitate with 3 times the weight of acetic acid sodium acetate buffer at 7°C;

[0053] b. After the sample is fully dissolved, heat up to 20°C, add 0.3mol / L NaOH solution at a flow rate of 0.6L / min, adjust the pH to 4.5±0.05, stir for 1 hour, and then add positive Octanoic acid, so that the n-octanoic acid in the solution can be evenly distributed until the final concentration of octanoic acid in the solution is 100mmol / L, stop adding octanoic acid, stir vigorously to facilitate the thorough mixing of octanoic acid, stir for 2 hours, precipitate non-IgG miscellaneous proteins, and...

Embodiment 2

[0065] The specific implementation steps of the process of the present invention are as follows:

[0066] a. Component I + III precipitates were dissolved in 0.02mol / L acetic acid sodium acetate buffer at 7°C with 3 times the amount of precipitation, and fully dissolved at room temperature;

[0067] b. After fully dissolving the sample, do not adjust the pH, and raise the temperature to 20°C. After fully stirring and mixing, slowly add n-octanoic acid to make the final concentration of octanoic acid in the solution 110mmol / L, and vigorously stir to facilitate the thorough mixing of octanoic acid. Stir for 2 Hour;

[0068] c. Rinse the filter plate of the filter press with 600 kg of water for injection at 20°C at a flow rate of 20 kg / min, then dry the filter plate with compressed air at normal temperature, press filter after balancing, and obtain a clarified filtrate, in which the IgG purity is 65%. The above; adjust the pH of the above filtrate to 5.8 with 0.3mol / L NaOH, fi...

Embodiment 3

[0075] The specific implementation steps of the process of the present invention are as follows:

[0076] In step a, the precipitate of component I+III is dissolved in 3 times the amount of precipitate, pH 4.5, concentration of 0.02mol / L acetic acid sodium acetate buffer, fully dissolved at room temperature;

[0077] In step b, n-octanoic acid is slowly added to make the final concentration of octanoic acid in the solution 90mmol / L. Through this step, the IgG purity in the solution reaches 65%, and the lipid-enveloped virus is removed;

[0078] In step c, use 600kg of water for injection at 20°C to wash the filter plate of the filter press at a flow rate of 20kg / min, then dry the filter plate with compressed air at normal temperature, press filter after balancing, and obtain a clarified filtrate; use 0.3mol / min of the filtrate Adjust the pH to 5.4 with L NaOH; filter with a 0.45um filter, adjust the conductivity of the solution to 1.5mS / cm, and purify on an anion exchange chro...

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Abstract

The invention relates to a method for separating and purifying human immunoglobulin from a component I+III of blood plasma, and aims to provide a high-efficiency method for recovering high-purity human immunoglobulin. According to the technical scheme provided by the invention, the method comprises the following steps of: a, fully dissolving component I+III precipitate; b, precipitating with octylic acid and removing lipid and a part of impurity protein to prepare IgG (Immunoglobulin G); c, purifying through anion exchange column chromatography; and d, collecting flow-through liquid, performing membrane nanofiltration, ultrafiltration and concentration, preparing the human immunoglobulin, sterilizing and packaging. The method has the beneficial effects of capability of being operated at the room temperature, simple and short steps, high yield, low energy consumption and high output and is suitable for mass production; comprehensive utilization of the blood plasma is fully realized; the time of the entire production process is shortened; the cost is reduced; extremely considerable economic benefit can be produced; the safety of a product is guaranteed by using two virus inactivation / elimination methods of different mechanisms; the environmental pollution is avoided; and the method has high economic and social values.

Description

technical field [0001] The invention belongs to the field of biopharmaceutical blood products, and specifically relates to a method for separating and purifying human immunoglobulin from plasma production waste components I+III by using n-octanoic acid precipitation combined with one-step anion exchange chromatography technology. Background technique [0002] Immunoglobulin (Immunoglobulin, Ig) is the main substance of the human body's immune response to foreign antigens (such as bacteria, viruses and other toxins or foreign bodies). Plasma-derived human immunoglobulin products are divided according to the route of injection: intravenous injection of human immunoglobulin (Intravenous Immunoglobulin G, IVIG) and intramuscular injection of human immunoglobulin, the main component of which is immunoglobulin G (IgG), which is the most One of the important plasma proteins, the molecular weight is 150kDa, and the content in plasma is about 6.6~14.5g / L. Since its introduction in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K1/36C07K1/30C07K1/18
CPCC07K16/065
Inventor 马山邵玉娟师秀梅菅长永李斌高亚朋高建锋高超巩艳艳朱孟沼
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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