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Method for separating and purifying cobra venom neurotoxin with combination of ion exchange and reverse phase chromatography

An ion-exchange chromatography, separation and purification technology, applied in the field of biopharmaceuticals, can solve the problems of long separation and purification cycle and complicated operation steps, and achieve the effect of simplifying the separation and purification steps, high purity and high yield

Inactive Publication Date: 2015-10-14
SUZHOU RENBEN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this method is owing to need to use the column chromatography methods such as gel column chromatography, ion exchange chromatography, hydrophobic chromatography and reverse phase chromatography in combination, not only operation steps are complicated, and separation and purification period is long (only the crude of king cobra venom It takes 576 hours to separate this step), so that its application range is limited to the laboratory

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  • Method for separating and purifying cobra venom neurotoxin with combination of ion exchange and reverse phase chromatography
  • Method for separating and purifying cobra venom neurotoxin with combination of ion exchange and reverse phase chromatography
  • Method for separating and purifying cobra venom neurotoxin with combination of ion exchange and reverse phase chromatography

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Embodiment 1

[0036] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0037] (1) The cobra venom crude product solution of 0.10g / mL is centrifuged, and a 0.45 μm microporous membrane is used to filter to obtain the test solution A, which is set aside.

[0038] (2) said need testing solution A is purified by ion exchange chromatography, the medium of said ion exchange chromatography is CM Sepharose Fast Flow, with 10mM NaH 2 PO 3 The buffer was mobile phase A to include 10 mM NaH with 1 M NaCl 2 PO 3 The buffer is mobile phase B. Before gradient elution, use 2 column bed volumes of mobile phase B for activation elution, then use 3 column bed volumes of mobile phase A for equilibrium elution; then perform gradient elution, the steps of gradient elution are: A : B volume ratio is 100%: 0%, A: B volume ratio is 90%: 10%, A: B volume ratio is 85%: 15%, A: B volume ratio is 40%: 60%, HPLC detection, Collect, get need ...

Embodiment 2

[0042] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0043] (1) The cobra venom crude product solution of 0.05g / mL is centrifuged, and a 0.45 μm microporous membrane is used to filter to obtain the test solution A, which is set aside.

[0044] (2) said need testing solution A is purified by ion exchange chromatography, the medium of said ion exchange chromatography is CM Sepharose Fast Flow, with 8mM NaH 2 PO 3 The buffer was mobile phase A to include 1M NaCl in 8mM NaH 2 PO 3 The buffer is mobile phase B. Before gradient elution, use 1 column bed volume of mobile phase B for activation elution, then use 2 column bed volumes of mobile phase A for equilibrium elution, and then perform gradient elution. The steps of gradient elution are: A : B volume ratio is 100%: 0%, A: B volume ratio is 90%: 10%, A: B volume ratio is 85%: 15%, A: B volume ratio is 40%: 60%, HPLC detection, Collected to obtain the...

Embodiment 3

[0048] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0049] (1) The cobra venom crude product solution of 0.15g / mL is centrifuged, and a 0.45 μm microporous membrane is used to filter to obtain the test solution A, which is set aside.

[0050] (2) said need testing solution A is purified by ion exchange chromatography, the medium of said ion exchange chromatography is CM Sepharose Fast Flow, with 12mM NaH 2 PO 3 The buffer was mobile phase A to include 12 mM NaH with 1M NaCl 2 PO 3 The buffer is mobile phase B. Before gradient elution, use 3 column bed volumes of mobile phase B for activation elution, then use 4 column bed volumes of mobile phase A for equilibrium elution, gradient elution, the steps of gradient elution are: A:B The volume ratio is 100%:0%, A:B volume ratio is 90%:10%, A:B volume ratio is 85%:15%, A:B volume ratio is 40%:60%, HPLC detection, collection, Need testing solution B, ...

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Abstract

The invention relates to the field of bio-pharmacy, and specifically relates to a method for separating and purifying cobra venom neurotoxin with a combination of ion exchange and reverse phase chromatography. The separation and purification method comprises the following steps: (1) a 0.05-0.15g / mL cobra venom crude product solution is centrifuged and filtered, such that a test sample solution A is obtained; (2) the test sample solution A is processed through ion exchange chromatography purification, gradient elution, HPLC detection and collection, such that a test sample solution B is obtained; (3) the test sample solution B is processed through reverse phase column chromatography purification, gradient elution, HPLC detection, collection, concentration and drying, such that a cobra venom neurotoxin pure product is obtained. According to the method, on a basis that component loss and damage are avoided, the operation steps are simplified, separation and purification period is shortened, yield is improved, and product purity is relatively high. The method is suitable for industrialized production of cobra venom neurotoxin.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for separating and purifying cobra venom neurotoxin by combining ion exchange and reverse phase chromatography. Background technique [0002] Cobra venom is the venom secreted by the venom glands of cobras. Its chemical composition is complex, containing a variety of proteins, polypeptides, enzymes and other small molecular substances, and has a wide range of biological activities. Since Monaelessert and Taguet first reported that cobra venom had a significant effect on pain caused by cancerous tissue compression of nerves in 1933, the analgesic effect of cobra venom has been studied in depth, and the cobra venom neurotoxin has shown a unique analgesic effect. Cobra venom neurotoxins can be divided into long chain and short chain. The short chain contains 60-62 amino acids and 4 disulfide bonds; the long chain contains 65-72 amino acids and 5 disulfide bonds...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/20C07K1/18
CPCC07K14/46
Inventor 秦正红李国庆
Owner SUZHOU RENBEN PHARMA
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