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Method for removing endotoxin out of polypeptide

An endotoxin and corticotropin technology, which is applied in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of long time consumption, inactivation of polypeptides, and inability to completely remove them, so as to improve production efficiency. , the effect of reducing the operating volume

Inactive Publication Date: 2014-09-10
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The technical problem to be solved by the present invention is to provide a method for removing endotoxins in polypeptides in order to overcome the defects in the prior art that endotoxins in polypeptides cannot be completely removed, easily cause polypeptide inactivation, and take a long time

Method used

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  • Method for removing endotoxin out of polypeptide
  • Method for removing endotoxin out of polypeptide
  • Method for removing endotoxin out of polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The crude hyaluronidase 1220g, batch number 090122, was purchased from Lixin Biochemical Products Factory in Tianshan District, Urumqi. The crude hyaluronidase was obtained from fresh bovine testis by separating, mincing into pulp, brewing in acid solution, pulling pulp, and salting out. Below 250U / mg. Above-mentioned crude product is put into stainless steel barrel, according to raw material (total titer): purified water (volume)=100 million U: 800ml dissolve, namely add 4 ℃ of purified water 2800ml, stir for 1 hour to completely dissolve, the solution after dissolving is filtered, filtrate is used A 10kD ultrafiltration membrane (model: Hydrosart, Germany Sartorius) was used for ultrafiltration concentration, and the solution volume after ultrafiltration was 530ml. Slowly add 60ml 0.4mol / L 4°C sodium phosphate solution, stir, then slowly add 36ml 1mol / L 4°C calcium acetate solution, stir, adjust pH to 8.40 with 2.5M NaOH, stir for 10 minutes and freeze at 3000 rpm Ce...

Embodiment 2

[0044]Crude hyaluronidase 1400g, batch number 090123, purchased from Lixin Biochemical Products Factory in Tianshan District, Urumqi. The crude hyaluronidase is obtained from fresh bovine testis by separating, mincing into pulp, brewing in acid solution, pulling pulp, and salting out. Below 250U / mg. The above-mentioned crude product was put into a stainless steel barrel, and dissolved according to the raw material (total titer): purified water (volume)=100 million U: 800ml, namely, 3300ml of purified water at 4° C. was added, stirred for 1 hour to completely dissolve, the dissolved solution was filtered, and the filtrate was used A 5kD ultrafiltration membrane (model: Hydrosart, Germany Sartorius) was used for ultrafiltration concentration, and the solution volume after ultrafiltration was 650 ml. Slowly add 70ml 0.4mol / L 4°C sodium phosphate solution, stir, then slowly add 42ml 1mol / L 4°C calcium acetate solution, stir, adjust pH to 8.60 with 2.5M NaOH, stir for 10 minutes an...

Embodiment 3

[0050] The crude hyaluronidase batch No. 100501 was purchased from Lixin Biochemical Products Factory in Tianshan District, Urumu. The crude hyaluronidase was obtained from fresh bovine testis by separating, mincing into pulp, soaking in acid liquid, pulling pulp, and salting out. Its specific activity should not be lower than 250U / mg; 500g of hyaluronidase crude product was charged, and dissolved according to raw material (total titer): purified water (volume) = 100 million U: 800ml, that is, 1120ml of purified water at 4°C was added, and stirred for 1 hour to completely dissolve. The solution was filtered, 100 ml of samples were reserved for testing, and the rest were subjected to one-step ultrafiltration to obtain an ultrafiltrate (refer to Example 1 for specific operations). The ultrafiltrate is evenly divided into several parts, and sodium phosphate and calcium acetate solutions of 0.1 times the volume of ultrafiltrate are added in turn to achieve the final molar ratio of ...

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Abstract

The invention discloses a method for removing endotoxin out of polypeptide. The method comprises the steps of dissolving the crude polypeptide in water, and carrying out ultrafiltration concentration to obtain a concentrated solution, sequentially adding an aqueous solution of calcium salt and an aqueous solution of phosphate to adjust the pH value to 8-9; and centrifuging, wherein the crude polypeptide is crude hyaluronidase, crude chorionic gonadotrophin, crude menotrophin or crude corticotropin. The method for removing endotoxin in the production of polypeptide disclosed by the invention; under the premise that the activity and yield of the polypeptide are not affected, endotoxin contaminants contained in the production of the polypeptide are effectively removed; at present, the method has been used in the manufacture of the polypeptide crude drugs, and the content of endotoxin is in line with the requirements of Chinese Pharmacopoeia (2010 Edition).

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for removing endotoxin in polypeptides. Background technique [0002] Endotoxin is produced in the cell outer wall of Gram-negative bacteria, and its main components are lipopolysaccharide (LPS) and lipid A, which are the active parts of pyrogen. Their relative molecular mass is generally 10,000 to 25,000, and they form aggregates in aqueous solution, and their relative molecular mass can reach 500,000 to 1 million. It has heat resistance and chemical stability, and is not easy to be eliminated. The amount of pyrogen injected into the human body can cause adverse reactions when the amount of pyrogen reaches 1 μg / kg. The fever reaction usually occurs 1 hour after the injection, which can cause the body to produce symptoms such as chills, chills, fever, sweating, nausea, and vomiting. Sometimes The body temperature can rise to above 40 ℃, and even in severe cases, coma ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C07K14/59C07K14/695C07K14/575C07K1/36C07K1/34C07K1/30C07K1/16
CPCC12N9/2474C07K14/59C07K14/695C12N9/6424C12N9/6427C12Y302/01035C12Y304/21004C12Y304/21039
Inventor 黄臻辉周建华周伟洁琚姝王克平孙源远
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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