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Method for purifying human parathyroid hormone (1 to 34)

A purification method and solution technology, which are applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., to achieve the effects of simple operation, good yield and high purity

Inactive Publication Date: 2010-07-21
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the published literature and patents, no purification method suitable for large-scale production and high yield has been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] 1. Sample treatment: Dissolve the crude peptide in water, make the sample completely dissolved, filter it with a filter membrane, and collect the filtrate for later use.

[0013] 2. Purification:

[0014] Purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 50mm×300mm. Mobile phase: A phase: the substance concentration is 0.2% mol / L phosphoric acid solution, adjust the pH to 2.5-3.5 with triethylamine; B phase: acetonitrile, flow rate: 70-80ml / min, gradient: B%: 17%-32 %, detection wavelength: 230nm. The injection volume is 1.3-1.5g.

[0015] Purification process: Rinse the chromatographic column with more than 50% acetonitrile, then equilibrate the sample, and the sample volume is 1.3-1.5g. After linear gradient elution for 45 minutes, the target peak was collected, and the collected lepirudin solution was concentrated by rotary evapora...

Embodiment 2

[0019] 1. Sample treatment: Dissolve the crude peptide in water, make the sample completely dissolved, filter it with a filter membrane, and collect the filtrate for later use.

[0020] 2. Purification for the first time: purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 150mm×300mm. Mobile phase: Phase A: 0.2% mol / L phosphoric acid solution with triethylamine to adjust pH to 2.5-3.5; Phase B: acetonitrile, flow rate: 450-550ml / min, gradient: B%: 17%-32%, detection wavelength : 230nm. The injection volume is 13-15g.

[0021] Purification process: wash the chromatographic column with more than 50% acetonitrile, and then equilibrate the sample, and the sample volume is 13-15g. After linear gradient elution for 60 minutes, the target peak was collected, and the collected lepirudin solution was concentrated by rotary evaporation under reduced pre...

Embodiment 3

[0025] 1. Sample treatment: Dissolve the crude peptide in water, make the sample completely dissolved, filter it with a filter membrane, and collect the filtrate for later use.

[0026] 2. The first purification: purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 300mm×300mm. Mobile phase: Phase A: 0.2% mol / L phosphoric acid solution with triethylamine to adjust pH to 2.5-3.5; Phase B: acetonitrile, flow rate: 1900-2200ml / min, gradient: B%: 17%-32%, detection wavelength : 230nm. The injection volume is 55-75g.

[0027] Purification process: wash the chromatographic column with more than 50% acetonitrile, then balance the sample, and the sample volume is 55-75g. After linear gradient elution for 120 minutes, the target peak was collected, and the collected lepirudin solution was concentrated to about 70-80 mg / ml by rotary evaporation under redu...

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PUM

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Abstract

The invention relates to a method for purifying human parathyroid hormone (1 to 34). The invention provides a method suitable to industrially purify lepirudin, which comprises the following steps: (1) using octadecylsilane chemically bonded silica as a solid phase, using a phosphate buffered solution the pH value of which is between 2.5 and 3.5 as an A phase, using acetonitrile as a B phase and carrying out gradient elution on crude peptide solution, wherein the gradient B percent is between 17 and 32 percent; (2) and adopting the reversed phase high performance liquid chromatography to convert lepirudin into acetate. The method for purifying lepirudin, which is provided by the invention, has simple operation, high product purity and good yield and reaches the industrialization requirements.

Description

technical field [0001] The invention relates to a method for purifying polypeptides, in particular to a method for purifying lepirudin. Background technique [0002] Lepirudin is a polypeptide drug used to treat thromboembolic diseases, cerebral hematoma, subcutaneous hematoma and other diseases. It has good effects and few side effects, and has a good market prospect. In the published literature and patents, no purification method suitable for large-scale production and high yield has been reported. Contents of the invention [0003] The invention provides a method for purifying lepirudin suitable for industrialization, which has high purity and good yield. [0004] To achieve the above object, the technical solution provided by the invention comprises the following steps: [0005] 1) Using octadecylsilane bonded silica gel as the stationary phase, using a phosphate buffer solution with a pH value of 2.5-3.5 as the A phase, and acetonitrile as the B phase, gradient: B%:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/815C07K1/20
Inventor 付信覃亮政马亚平袁建成
Owner HYBIO PHARMA
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