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Purification method of Ziconotide

A technology of ziconotide and purification method, which is applied in the field of purification of polypeptide drugs, can solve problems such as difficult separation, low product purity, and reduced yield of ziconotide, and achieve good polymer impurities, high yield, low cost effect

Active Publication Date: 2015-11-04
HANGZHOU HEZE PHARMA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the existence of many pairs of disulfide bonds, the purification of ziconotide is brought greater difficulty, which is reflected in: 1. when synthesizing ziconotide, various disulfide bond mismatched impurities are produced, and this type of impurity is related to The product is difficult to separate, resulting in low purity of the final product; 2. Due to the existence of multiple pairs of disulfide bonds, the three-dimensional structure of the ziconotide molecule is very different from that of general polypeptides, and it is difficult to obtain products with better purity with commonly used fillers; 3. .Many pairs of disulfide bonds tend to produce more polymers during the production process. This polymer is one of the most important impurities. It is extremely unstable and easy to change color, which seriously affects the color and content of ziconotide. and security
The present inventors found that it is difficult to control the polymer impurity content below 0.1% after research and use the prior art to purify, and at the same time, it is easy to cause the yield of ziconotide to decrease

Method used

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  • Purification method of Ziconotide
  • Purification method of Ziconotide
  • Purification method of Ziconotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: the solid-phase synthesis of ziconotide

[0082] Weigh 25g of Rink Amide AM resin with a degree of substitution of 0.40mmol / g, add it to the solid-phase reaction column, wash it once with DCM, and swell the resin with DCM for 30 minutes, then wash it with DMF:pyridine The mixed solution with a volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 17.57g Fmoc-Cys(Trt)-OH (30mmol) and 4.05g HOBt (30mmol) were added in a volume ratio of 1:1 DCM and DMF mixed solution, add 4.6ml DIC (30mmol) under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin method to detect and judge the reaction end point, if the resin has no If the color is transparent, it indicates that the reaction is complete; if the resin develops color, it indicates that the reaction is incomplete and needs to be reacted for another 1 hour. This criterion is applicable...

Embodiment 2

[0083] Embodiment 2: the splitting of ziconotide

[0084] Take by weighing 105.02g ziconotide linear peptide resin, join in the three-neck round bottom flask of 2000mL, be EDT, PhSMe, H in volume ratio 2.5:5:5:87.5 2 O and TFA were prepared with 1050ml of lysate, and the lysate was added to the above resin, and reacted at 20~30°C for 2 hours. The reaction solution was added to glacial ether for precipitation for 1 hour, centrifuged, washed with anhydrous ether for 6 times, and vacuum-dried to obtain 27.12 g of ziconotide linear peptide.

Embodiment 3

[0085] Embodiment 3: Cyclization of Ziconotide

[0086] Example 2 middle Add 27.12g of ziconotide linear peptide, 53.92g of ammonium acetate, 2.61g of disodium ethylenediaminetetraacetic acid and 0.847g of cysteine ​​into 7L of aqueous solution and stir to dissolve, use ammonia water to adjust the pH to 8, and stir at 5-10°C After reacting for 72 hours, it was used for later use. The purity of the crude product of ziconotide was 67.5% (detected by HPLC), the content of polymer impurities was 2.73% (detected by gel chromatography), and the synthesis yield was 62.2%.

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Abstract

The invention relates to a purification method of Ziconotide. The method is characterized by including the following steps that 1, the pH of a crude Ziconotide solution is regulated to 3-4 through trifluoroacetic acid; 2, the gradient is set according to the volume fraction, a reversed-phase filler column is washed for 10 min through a 50% mobile phase B, and then isocratic elution balancing is performed for 10 min through a 5% mobile phase B; 3, the solution in the step1 is loaded into the reversed-phase filler; 4, the gradient is set according to the volume fraction, the original state mobile phase B of the elution gradient is 5% and is maintained for 5 min, then the proportion of the mobile phase B is increased to 22% within 60 min, and elution fractions are collected; 5, the elution fractions are subjected to salt transfer, concentration and freeze-drying to obtain fine Ziconotide.

Description

technical field [0001] The invention relates to a purification method of polypeptide drugs, in particular to a purification method of ziconotide. Background technique [0002] Ziconotide is a synthetic ω-MVIIA conotoxin, which was first reported as a peptide component of hallucinogenic conotoxin. Its 25 amino acid sequences are: Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg- Ser-Gly-Lys-Cys-NH 2 (1-16), (8-20), (15-25)-tris(disulfide). [0003] Ziconotide can produce significant analgesic effect on patients with severe chronic pain who are ineffective with opioid analgesics, and no patients were found to be resistant to Ziconotide in clinical studies. At present, Ziconotide has become the first new type of neurotherapeutic drug --- N-type calcium ion channel blocker (NCCB). As a non-opioid analgesic, ziconotide is distinguished from other analgesic drugs by its safety and efficacy in clinical application, thus heralding its important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/20
CPCC07K14/43504Y02P20/55
Inventor 倪晟周亮姜维斌赵航朱明月
Owner HANGZHOU HEZE PHARMA TECH
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