Method for purifying liraglutide

A technology of liraglutide and carbon dioxide, applied in the field of purification, can solve problems such as uneconomical stationary phase, and achieve the effects of reducing operation, increasing recovery rate, and improving quality and yield

Active Publication Date: 2021-06-25
HYBIO PHARMA WUHAN CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The average purity of the pooled fractions was 97%, so not as high as desired
Also, it is not economical to use two different stationary phases

Method used

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  • Method for purifying liraglutide
  • Method for purifying liraglutide
  • Method for purifying liraglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of Liraglutide

[0044] The first step, liraglutide purification

[0045] Take 100.0 g of liraglutide crude peptide, containing 76.8 g of liraglutide, dissolve it in 4.5 L of ammonium bicarbonate (100 mM) solution, add 0.5 L of acetonitrile, filter at 0.45 μm, and divide the filtrate twice with 10 μm of C18 Preparative column (inner diameter 15cm, column length 30cm, filler pore diameter is ) purification, that is, 50.0 g per loading. First set mobile phase A as A1, 200mM ammonium bicarbonate + 50mM sodium bicarbonate, carbon dioxide to adjust the pH to 7.5, mobile phase A1 is in a sealed pressure tank, control the pressure at 0.1-0.5Mpa; mobile phase B is acetonitrile. The flow rate is 620mL / min, the elution gradient is 40%B-55%B (100min), the mobile phase A1 and B are controlled at 25°C, the column temperature is 25°C, and when the elution reaches 100min, stop the flow rate and change the elution conditions .

[0046] At this time, the mobi...

Embodiment 2

[0049] Example 2: Preparation of Liraglutide

[0050] The first step, liraglutide purification

[0051] Take 100.0 g of liraglutide crude peptide, containing 76.8 g of liraglutide, dissolve it in 4.5 L of sodium bicarbonate (80 mM) solution, add 0.5 L of acetonitrile, filter at 0.45 μm, and divide the filtrate twice with 10 μm of C8 Preparative column (inner diameter 15cm, column length 30cm, filler pore diameter is ) purification, that is, 50.0 g per loading. First set mobile phase A as A1, 100mM ammonium bicarbonate + 100mM potassium bicarbonate, carbon dioxide to adjust the pH to 7.5, mobile phase A1 is in a sealed pressure tank, control the pressure at 0.1-0.5Mpa; mobile phase B is acetonitrile. The flow rate is 620mL / min, the elution gradient is 40%B-55%B (100min), the mobile phase A1 and B are controlled at 25°C, the column temperature is 25°C, and when the elution reaches 100min, stop the flow rate and change the elution conditions .

[0052] At this time, switch m...

Embodiment 3

[0055] Example 3: Preparation of Liraglutide

[0056] The first step, liraglutide purification

[0057] Take 100.0 g of liraglutide crude peptide, containing 76.8 g of liraglutide, dissolve it in 4.5 L of potassium bicarbonate (200 mM) solution, add 0.5 L of acetonitrile, filter at 0.45 μm, and prepare a column with 10 μm of C4 (inner diameter 15cm, the column length is 30cm, and the packing pore diameter is ) was purified in two steps. First set mobile phase A as A1, 100mM ammonium bicarbonate + 100mM potassium bicarbonate, carbon dioxide to adjust pH to 7.50, mobile phase A1 is in a sealed pressure tank, control pressure at 0.1-0.5Mpa; mobile phase B is acetonitrile. The flow rate is 620mL / min, the elution gradient is 37%B-45%B (100min), the mobile phase A1 and B are controlled at 25°C, the column temperature is 25°C, and when the elution reaches 100min, stop the flow rate and change the elution conditions .

[0058] At this time, switch mobile phase A to A2, 50mM ammon...

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Abstract

The invention discloses a method for purifying liraglutide, which comprises the following steps: dissolving and filtering crude peptide, then purifying with reversed-phase HPLC, in the purification process, washing with buffer salts A1 and B with low pH values as mobile phases, strictly controlling the temperature of a preparative column and the mobile phases at the moment, carrying out gradient elution for a certain time, and keeping the sample not eluted on the preparation column; then replacing a mobile phase with A2 and B with high pH values for washing, simultaneously changing the temperature and gradient of the preparative column, strictly controlling the temperature of the preparative column and the temperature of the mobile phase at the moment, carrying out gradient elution with B, and collecting qualified fractions; and desalting the collected qualified fractions, concentrating, and freeze-drying. According to the method, the maximum impurity removal effect can be achieved through one-step purification, qualified samples can be collected only through one-step purification, the quality and yield of products are improved, meanwhile, waste liquid can be reduced, and the method is environmentally friendly and beneficial to industrial production.

Description

technical field [0001] The invention belongs to the technical field of purification methods, and in particular relates to a method for purifying liraglutide. Background technique [0002] Liraglutide is a long-acting glucagon-like peptide 1 (GLP-1) analogue for the treatment of type II diabetes. It belongs to the GLP-1 receptor agonist and is the first human high Glucagon-like polypeptide-1 (GLP-1) analogs. Developed by Novo Nordisk, it was approved by the FDA for marketing on January 25, 2010, and was approved by the SFDA for marketing in China on March 4, 2011. Liraglutide, as a new generation of incretin-based hypoglycemic drugs, not only has a long duration of action, but also fully retains multiple physiological activities of natural GLP-1, which can safely and effectively lower blood sugar and may prevent various cardiovascular risks. Factors have a protective effect, bringing new options for the treatment of type 2 diabetes. The clinical treatment effect is encoura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/20
CPCC07K14/605
Inventor 姜绪邦黄嘉成尹传龙陶安进余品香
Owner HYBIO PHARMA WUHAN CO LTD
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