Large-scale separation and purification method of teriparatide

A technology for separation and purification of teriparatide, which is applied in the field of large-scale separation and purification of teriparatide by reversed-phase high performance liquid chromatography, can solve problems such as unfavorable industrial production, unstable yield, peptide degradation, etc. The effect of large-scale industrial production, high production yield and high product purity

Inactive Publication Date: 2016-11-30
ZHEJIANG PEPTITES BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the product is dissolved in 60% phosphoric acid aqueous solution in the salting-out step, it is easy to cause peptide degradation, resulting in unstable yield
[0007] Patent CN102731643A discloses a purification method of teriparatide, using water as solvent, then using 0.2% TFA/acetonitrile as mobile phase to use C18 column to purify, and then using 0.2% acetic acid/acetonitrile...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Sample treatment: Dissolve each gram of crude teriparatide in 50ml of acetonitrile aqueous solution with a volume ratio: acetonitrile: water = 30:70, sonicate, and after the sample is completely dissolved, filter it with a filter membrane with a pore size of 0.45 μm and collect the filtrate stand-by.

[0032] 2. Purification: Purification conditions: Chromatographic column: DAC-CXTH200 dynamic axial pressurized column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20*25cm. Mobile phase A: 0.2% ammonium acetate aqueous solution by mass percentage, adjust the pH value to 5.0 with acetic acid; phase B: acetonitrile. The flow rate is 700~1000ml / min. The detection wavelength is 220nm. Gradient: B%: 20~70% (60min). The injection volume is 60g.

[0033] Purification process: Equilibrate the chromatographic column with mobile phase A and load the sample, with a sample volume of 3L sample solution. The linear gradient ...

Embodiment 2

[0036] 1. Sample treatment: Dissolve each gram of crude teriparatide in 50ml of acetonitrile aqueous solution with a volume ratio: acetonitrile: water = 30:70, sonicate, and after the sample is completely dissolved, filter it with a filter membrane with a pore size of 0.45 μm and collect the filtrate stand-by.

[0037] 2. Purification: Purification conditions: Chromatographic column: DAC-CXTH200 dynamic axial pressurized column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20*25cm. Mobile phase A: 0.4% ammonium acetate aqueous solution by mass percentage, adjust the pH value to 5.5 with acetic acid; phase B: acetonitrile. The flow rate is 700~1000ml / min. The detection wavelength is 220nm. Gradient: B%: 25~75% (60min). The injection volume is 80g.

[0038] Purification process: equilibrate the chromatographic column with mobile phase A and then load the sample, with a sample volume of 4L sample solution. The linear grad...

Embodiment 3

[0041] 1. Sample treatment: Dissolve each gram of crude teriparatide in 50ml of acetonitrile aqueous solution with a volume ratio: acetonitrile: water = 30:70, sonicate, and after the sample is completely dissolved, filter it with a filter membrane with a pore size of 0.45 μm and collect the filtrate stand-by.

[0042] 2. Purification: Purification conditions: Chromatographic column: DAC-CXTH200 dynamic axial pressurized column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20*25cm. Mobile phase A: 0.6% ammonium acetate aqueous solution by mass percentage, adjust the pH value to 6.0 with acetic acid; phase B: acetonitrile. The flow rate is 700~1000ml / min. The detection wavelength is 220nm. Gradient: B%: 30~80% (60min). The injection volume is 100g.

[0043] Purification process: equilibrate the chromatographic column with mobile phase A and load the sample, with a sample volume of 5L sample solution. After linear gradie...

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PUM

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Abstract

The invention relates to a large-scale separation and purification method of teriparatide by reversed high performance liquid chromatography. The method comprises the following steps: dissolving a teriparatide crude product in 30% acetonitrile aqueous solution, carrying out ultrasonic treatment, filtering with a filter membrane, and fetching a filtrate for later use; separating and purifying the filtered crude product by a high performance liquid chromatography instrument; letting a concentrated solution pass through a reversed-phase column for conversion to acetate; carrying out vacuum decompression rotary-evaporation concentration on a product with 99% purity after conversion to acetate, freeze-drying to obtain a powdery product, and detecting qualified so as to obtain the final product. The method is convenient to operate and excessive generating equipment is not required. Product purity is high and yield is high. Product purity reaches 99% and above, purification recovery rate reaches 60% and above. The technology can meet requirements of large-scale industrial production.

Description

technical field [0001] The invention relates to a method for large-scale separation and purification of teriparatide, in particular to a method for large-scale separation and purification of teriparatide by reversed-phase high-performance liquid chromatography. Background technique [0002] Teriparatide is a synthetic 34-peptide, which is a 1-34 amino acid fragment of human parathyroid hormone PTH. The amino acid sequence is as follows: [0003] H-Ser1-Val2-Ser3-Glu4-Ile5-Gln6-Leu7-Met8-His9-Asn10-Leu11-Gly12-Lys13-His14-Leu15-Asn16-Ser17-Met18-Glu19-Arg20-Val21-Glu22-Trp23-Leu24- Arg25-Lys26-Lys27-Leu28-Gln29-Asp30-Val31-His32-Asn33-Phe34-OH [0004] This fragment is the biologically active N-terminal region of endogenous parathyroid hormone PTH containing 84 amino acids. The immunological and biological characteristics of this product are exactly the same as those of endogenous parathyroid hormone PTH and bovine parathyroid hormone PTH (bPTH). [0005] Teriparatide is t...

Claims

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Application Information

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IPC IPC(8): C07K14/635C07K1/20C07K1/18C07K1/16
CPCC07K14/635
Inventor 刘志国李雪豪纪东亮秦德志
Owner ZHEJIANG PEPTITES BIOTECH CO LTD
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