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Method for removing endotoxin of biological product

A technology for biological products and endotoxins, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of poor removal effect and high cost, and achieve clinical application prospects, low cost, and treatment methods simple effect

Active Publication Date: 2015-06-17
信立泰(苏州)药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the use of adsorption methods to remove endotoxins is not only ineffective, but also expensive, and needs to be improved urgently.

Method used

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  • Method for removing endotoxin of biological product
  • Method for removing endotoxin of biological product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 endotoxin removal method of the present invention

[0031] 1. Test material

[0032] Antibody protein sample acquisition:

[0033] Step 1. Preparation of a CHO cell capable of expressing an anti-CD20 monoclonal antibody:

[0034] Preparation of CHO cells expressing anti-CD20 monoclonal antibody: The dhfr (dihydrofolate reductase) expression unit in the pSV2-dhfr vector (ATCC product) was cloned into the pCDNA3.1 (+) vector (Invitrogen Company product), the mammalian cell expression vector pBF01 capable of expressing DHFR was constructed.

[0035] According to literature reports (US Patent, US6399061), the light chain and heavy chain gene fragments of the recombinant anti-CD20 monoclonal antibody were synthesized by chemical synthesis technology; the synthetic gene fragments were respectively cloned into pBF01 vector by DNA recombination technology, and express The recombinant expression vectors pBF01-CD20L and pBF01-CD20H of the light chain and heavy cha...

Embodiment 2

[0070] Embodiment 2 endotoxin removal method of the present invention

[0071] 1. Test material

[0072] With embodiment 1.

[0073] 2. Endotoxin removal method of the present invention

[0074] Except for the parameters changed in the following Tables 1 to 2, other conditions are the same as in Example 1.

[0075] Table 1 Changes of anion resin type and hydroxyapatite type

[0076] Numbering

Anion resin type

Hydroxyapatite type

1

Q Sepharose High Performance

CHT Ceramic hydroxyapatite type I,40um

2

DEAE Sepharose Fast Flow

CHT Ceramic hydroxyapatite type II,40um

3

Nuvia Q

CHT Ceramic hydroxyapatite type I,80um

[0077] Table 2 Changes in the buffer system

[0078]

[0079] 3. Detection and analysis

[0080] With embodiment 1.

[0081] 4. Experimental results

[0082] Table 3 Test results

[0083]

[0084] As can be seen from Table 3, before adopting the method of the present invention to proce...

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Abstract

The invention discloses a method for removing endotoxin of a biological product. The method comprises the following steps: (1) performing anion exchange chromatography on a biological product to be treated; (2) performing hydroxyapatite chromatography, thereby removing endotoxin of the biological product. The invention further provides a biological product prepared by using the method. Due to the adoption of the method disclosed by the invention, endotoxin can be effectively removed, the protein recycling rate is high, no special antibody is needed to adsorb target protein, the treatment method is simple, the cost is low, the content of endotoxin of the biological product treated by using the method disclosed by the invention is smaller than 0.05EU / mg of protein, and the method is good in security and has relatively good application prospect.

Description

technical field [0001] The invention relates to a method for removing endotoxin from biological products. Background technique [0002] Endotoxin is lipopolysaccharide (LPS), which comes from the outer membrane of Gram-negative bacteria. Its structure includes three regions, namely lipid A region, core polysaccharide region and specific polysaccharide region, and is a kind of pyrogen. A very small amount of endotoxin entering the animal or human body will trigger a strong inflammatory response, leading to serious consequences such as fever and death. [0003] In biological products, endotoxin contamination is widespread. For example, monoclonal antibodies, as biomacromolecular drugs, have complicated production and purification processes, and there are many links that may introduce endotoxins. Therefore, endotoxins need to be removed to ensure the safety of biological products. sex. The drug regulatory authorities of various countries also have strict regulations on the en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K1/18
Inventor 罗天学罗培文荣艳珍
Owner 信立泰(苏州)药业有限公司
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