Separation and purification process for synthetic thymosin alpha 1

A technology for separation and purification of thymosin, applied in the field of separation and purification of thymosin α1, which can solve the problems of large volume of liquid medicine, variability and inactivation, and long time required for concentration

Inactive Publication Date: 2012-05-30
BEIJING KAWIN TECH SHARE HLDG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification method of separation and solid-phase synthesis of Tα1 mostly adopts two-step separation method, that is, firstly, an ion-exchange chromatographic column is used to remove most of the impurities in the crude product, and then, reverse-phase high-performance liquid chromatography is used for final purification, and then the high-efficiency The purified Tα1 in the liquid phase is concentrated under reduced pressure in a vacuum to remove organic solvents, such as acetonitrile, which is often used, and due to the large volume of the prepared medicinal liquid in the industrial production process, it needs to be concentrated by rotary vacuum. It is carried out at a certain temperature, but it is not suitable for peptide or protein drugs to be concentrated at a higher temperature. Only a relatively low temperature can be controlled for vacuum concentration. The time required for concentration is long, and peptide or protein drugs are heated for a long time. In the case of easy denaturation and inactivation
[0006] Although nanofiltration technology has been widely used, when separating or concentrating oligopeptides, it is concentrated by nanofiltration to remove some small molecular amino acids or short peptides that can be fractionated by nanofiltration membranes, or for monovalent or polyvalent salts. For organic solvents mixed in peptides or proteins, vacuum concentration technology is usually used to remove the organic solvents, and there is no application of nanofiltration technology to remove organic solvents in peptides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Ion Exchange Chromatography Purification of Solid-Phase Synthesized Crude Tα1

[0032]Purification preparation: Prepare the container, dry heat at 250°C to depyrogenate or wash with water for injection; prepare column equilibration buffer A (20mmol / L PB, pH 5.8) and column elution buffer B (20mmol / L PB, 500mmol / L Sodium chloride, pH5.8), stand-by; take an appropriate amount of Tα1 crude peptide, add column equilibration buffer to dissolve to about 10 mg / ml, filter at 0.45 μm to make crude peptide solution, stand-by; prepare AKTABASIC10 preparative liquid chromatography system, The medium is DEAEsepharose Fast Flow (anion exchange resin, preferably weak anion exchange resin). Put the liquid inlet of pump A into column equilibration buffer and the liquid inlet of pump B into column elution buffer. Power on, turn on the drive, equilibrate the chromatographic column with column equilibration buffer, set the UV detector to 220nm, adjust the flow rate to 100ml / min,...

Embodiment 2

[0035] Embodiment 2 reversed-phase chromatography is purified

[0036] Preparation: Prepare the container, dry heat at 250°C to depyrogenate or wash with water for injection; prepare mobile phase A (12% acetonitrile aqueous solution, containing 0.1% TFA) and mobile phase B (25% acetonitrile aqueous solution, containing 0.1% TFA) , filtered at 0.45 μm, and ready for use; take Tα1 collection solution I, add acetonitrile, so that the final concentration of acetonitrile is 12%, and the content of TFA is 0.1%, and filter at 0.45 μm to make peptide solution, ready for use; prepare Shimadzu LC-20A Liquid chromatographic system, stuffing C18, power on, turn on the drive, enter mobile phase A, set the UV detector to 220nm, adjust the flow rate to 50ml / min, reach the baseline equilibrium, and set it aside.

[0037] Purification and collection of C18 reverse-phase chromatography: take 100ml of the treated Tα1 collection solution I and inject the sample, the flow rate remains unchanged, a...

Embodiment 3

[0039] The concentration of embodiment 3 refining Tα1 solution

[0040] 1 Spin Vacuum Concentrate

[0041] Take 20L of the purified Tα1 solution obtained by reverse-phase high performance liquid chromatography in the example, and concentrate it in a rotary vacuum at about 40°C. More than 10 hours.

[0042] 2 nanofiltration concentration

[0043] Prepare the nanofiltration machine, the pore size of the nanofiltration membrane is 100 Daltons; start the machine, adjust the pump flow rate, keep the membrane inlet pressure at 1.2Mpa, and the membrane outlet pressure at 1.0Mpa, so that the feed liquid is concentrated to 1 / 3 of the original volume, and then add purified water to the original volume. volume. Continue to concentrate to 1 / 3 of the original volume, and the concentration is completed. The time required for concentration is about 30 minutes, which is much shorter than the time for rotary vacuum concentration, which is very suitable for industrial production.

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Abstract

The invention relates to a purification process for thymosin alpha 1 after chemical synthesis. Specifically speaking, the conventional manner of combined ion exchange and vacuum condensation for desalination and condensation of thymosin alpha 1 obtained through chemical synthesis is changed into one-shot nanofiltration in the invention. The process provided in the invention has the advantages of simpleness, high efficiency, low energy consumption and suitability for industrial production.

Description

technical field [0001] The invention relates to a method for separating and purifying synthetic thymosin α1, which belongs to the field of biopharmaceutical preparation technology. Background technique [0002] Thymosin α1 (Tα1) is the main bioactive component of thymosin and an important immunoregulatory substance in the human body. Studies have shown that thymosin α1 promotes the development and differentiation of bone marrow and liver cells into pro-lymphocytes and pro-lymphocytes, and induces the differentiation and maturation of T lymphocytes. Make mature T cells further differentiate into pro-lymphocytes and pro-lymphocytes; induce T lymphocyte differentiation and maturation, make mature T cells further differentiate into several different subgroups, such as killer cells, memory cells, effector cells , helper T lymphocytes, etc., and produce various soluble media; enhance the response of lymphocytes to mitosis, and increase the synthesis of protein and nucleic acid in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/36C07K1/34C07K1/20C07K1/18
Inventor 许晓吉春熊国裕
Owner BEIJING KAWIN TECH SHARE HLDG
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