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Method of preparing high-purity polypeptide or analogue thereof

An analog and high-purity technology, which is applied in the field of preparing high-purity polypeptides or their analogs, can solve the problems of the mobile phase system for preparation, the method is not described in detail, the polarity of AMG416 is large, and the purification is difficult, and the effective preparation and purification can be achieved. , Good prospects for industrial application, and the effect of improving purification yield

Active Publication Date: 2018-06-01
SICHUAN KELUN PHARMA RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to the large polarity and instability of AMG416, it is very difficult to purify it, and the yield of purification by some conventional purification methods is very low
The patent applications of CN201080045024 and WO2015154031 disclose that AMG416 can be purified by reverse-phase high-performance liquid phase (RP-HPLC), but the mobile phase system and method for specific preparation are not described in detail

Method used

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  • Method of preparing high-purity polypeptide or analogue thereof
  • Method of preparing high-purity polypeptide or analogue thereof
  • Method of preparing high-purity polypeptide or analogue thereof

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Embodiment 1

[0060] Chromatographic conditions: Agilent 1260 preparative system, YMC 5μm C18 filler prepacked 20*250mm preparative column. Prepare an aqueous ammonium phosphate solution with a concentration of 50mM, use phosphoric acid to adjust pH=3.0, use it as phase A, methanol as phase B, the volume ratio of phase A to phase B is 97:3, the elution method is isocratic elution, the flow rate is 10ml / min.

[0061] Take 50 mg of AMG416 crude product (purity of the crude product is 69%) dissolved in 1 ml of purified water and filter the sample. The peak of purpose is collected, and the peak fraction of purpose is combined to obtain AMG416 solution, and its RP-HPLC purity is 99.75% (such as figure 1 shown); the solution is converted to hydrochloride by chemical method after using an ion exchange packing chromatographic column or lyophilized, and lyophilized to obtain 31.5mg AMG416 hydrochloride refined peptide, and its RP-HPLC purity is 99.52% (such as figure 2 shown). It can be seen fr...

Embodiment 2

[0067] step one:

[0068] Chromatographic conditions: Agilent 1260 preparative system, 20*250mm preparative column prepacked with Daiso 10μm C18 filler. Prepare an aqueous solution of ammonium formate with a concentration of 30mM, use formic acid to adjust the pH to 3.5, use as phase A, methanol as phase B, the volume ratio of phase A to phase B is 95:5, the elution method is isocratic elution, the flow rate is 10ml / min.

[0069] Take 50 mg of AMG416 crude product (purity of the crude product is 69%) dissolved in 1 ml of purified water and filter the sample. Collect the target peak, combine the target fractions and freeze-dry the solution to obtain the refined product of AMG416.

[0070] Step two:

[0071] The Agilent 1260 preparation system was used, and the 20*250mm preparation column prepacked with Suzhou Nanowell 10μm C18 filler was used. Prepare 0.1% TFA aqueous solution as phase A and methanol as phase B. Phase B was increased from 5% to 20% within 25 minutes at a ...

Embodiment 3

[0074] Chromatographic conditions: Hanbang DAC80 preparation system, Hanbang SP 10μm C18 packing DAC 80*250mm preparative column. Prepare an aqueous ammonium phosphate solution with a concentration of 50mM, use phosphoric acid to adjust the pH to 3.0, use it as phase A, methanol as phase B, the volume ratio of phase A to phase B is 95:5, the elution method is isocratic elution, and the flow rate is 160ml / min.

[0075] Take 810 mg of AMG416 crude product (purity of the crude product is 69%) and dissolve it in 20 ml of purified water, use a small amount of acetic acid / ammonia water to adjust the pH to 6.80, and filter the sample. Collect the purpose peak, and the purpose peak cuts are combined to obtain the AMG416 solution, and its RP-HPLC purity is 99.39% (such as image 3 Shown), the solution is converted to acetate, and freeze-dried to obtain the AMG416 acetate essence peptide of 572mg. Calculation shows that the weight yield of the method is 70.6%.

[0076] Table 3: Chro...

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Abstract

The invention discloses a method of preparing a high-purity polypeptide or an analogue thereof. The method includes the steps of: preparing a purified AMP416 water solution through high performance liquid chromatography, and collecting a fraction of a target peak. Chromatographic conditions are described as follows: a stationary phase is C18, a mobile phase A is a phosphate, a formate or an acetate water solution and a mobile B is methanol or acetonitrile, wherein volume ratio of the mobile phase A to the mobile phase B is (80-100) : (0-20). The method can high-effectively separate and purifythe AMG416 at high yield and high product purity, so that the method has excellent industrial application potential.

Description

technical field [0001] The invention relates to a method for preparing high-purity polypeptide or its analogue. Background technique [0002] Secondary hyperparathyroidism (SHPT, referred to as secondary hyperparathyroidism) refers to chronic renal insufficiency, renal tubular acidosis, vitamin D deficiency, etc., where the parathyroid glands suffer from long-term hypocalcemia, low A chronic compensatory clinical manifestation of excessive secretion of PTH stimulated by serum magnesium or hyperphosphatemia. Long-term parathyroid hyperplasia eventually leads to the formation of functionally autonomous adenomas. At present, the commonly used drug for the treatment of secondary hyperparathyroidism is cinacalcet produced by Amgen, which has grown fairly steadily since it was launched more than ten years ago. In 2014, the sales in the United States alone reached as high as $1 billion. [0003] AMG416 (also known as vecalcetide, etelcalcetide) is a new calcimimetic agent (Calcim...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/20
CPCC07K7/06
Inventor 李长兵庹世川杨燕苹薛宏祥王利春王晶翼
Owner SICHUAN KELUN PHARMA RES INST CO LTD
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