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Purified PEG human growth hormone conjugates and preparation thereof

A technology of human growth hormone and conjugates, which is applied in the field of pharmaceutical preparations containing the conjugates, high-purity PEGylated human growth hormone conjugates, and can solve problems such as unpredictable purity and masking of process details

Active Publication Date: 2009-03-18
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCT application WO2003044056A and Chinese patent application CN1477126A respectively disclose PEG-modified conjugates at the N-terminus of hGH. However, the preparation steps disclosed therein can only make about 90% of the PEG in the final conjugated product conjugated to the N-terminal of hGH. On the other hand, even with the further purification steps disclosed therein, the resulting PEGylated hGH conjugate can only reach a purity of about 95%, including unconjugated PEG, hGH and their degradation products
Although the Chinese patent application CN1565624A claims that a conjugate with a purity greater than 98% can be obtained, the details of its preparation (purification) process are deliberately concealed, and there is no such thing as the disclosure of PCT application WO2003044056A and Chinese patent application CN1477126A to achieve a purity of about 95%. The technical plan is detailed, and the existence of the purity product has not been confirmed by recognized detection methods, and it is impossible to expect to achieve the corresponding purity effect

Method used

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  • Purified PEG human growth hormone conjugates and preparation thereof
  • Purified PEG human growth hormone conjugates and preparation thereof
  • Purified PEG human growth hormone conjugates and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] [Example 1] Conjugation of Human Growth Hormone and Polyethylene Glycol

[0029] 1. Put human growth hormone solution (human growth hormone concentration is 10 mg / ml, dissolved in 50 mM phosphate buffer, pH to 6.5) in a sterile Erlenmeyer flask, and stir at a speed of 50-100 rpm. Under the condition of continuous stirring, add bismethoxy PEG-N-hydroxysuccinimidyl ester (mPEG2-NHS, molecular weight is 42.5kDa, purchased from Shearwater Company) to the reaction vessel in batches, bismethoxy The total weight of PEG N-hydroxysuccinimidyl ester added is 4 times that of human growth hormone (that is, its weight ratio to rhGH is 4:1), and the added bismethoxy PEG N-hydroxysuccinimide All the base esters were dissolved, and stirred at 50-100 rpm for 16-18 hours at 4°C to allow a conjugation reaction between human growth hormone and polyethylene glycol to obtain a reaction mixture.

[0030] 2. Put the human growth hormone solution (the concentration of human growth hormone is 1...

Embodiment 2

[0031] [Example 2] Purification of PEGylated hGH conjugates

[0032] The Sephadex G-25 chromatographic column (purchased from Huamei Company) was washed with 10 mM sodium hydroxide, then washed with water for injection to neutrality, and then the chromatographic column was equilibrated with 20 mM Tris-HCl buffer solution of pH 7.4. After equilibration, add 5 times the volume of water for injection to the reaction mixture obtained in the embodiment 1-1 recorded as 1 volume to dilute, and then load the sample, wherein the loading volume does not exceed 15% of the column volume, and use 20mM Tris-Hcl The pH7.4 buffer is eluted, the flow rate of sample loading and elution is between 150-250cm / h, the light absorption value of the eluted product is detected at a wavelength of 280nm, and the eluate of the first elution peak is collected .

[0033] Wash the Q-Sepharose chromatographic column (purchased from Huamei Company) with 10mM sodium hydroxide, then rinse with water for injecti...

Embodiment 3

[0034] [Example 3] Identification of PEGylated hGH conjugates

[0035] The PEGylated hGH conjugate solution obtained in Example 2 was taken for the following identification:

[0036] 1. Size exclusion high performance liquid chromatography (SEC-HPLC)

[0037] The product obtained in Example 2 was evaluated by SEC-HPLC. According to manufacturer's instruction, adopt Protein Pak300SW column (7.8mm * 300mm, purchased from Waters company) with the 50mM phosphate buffer of pH6.5 as mobile phase, in 0.1% SDS, with flow rate 0.6mL / min, carry out SEC-HPLC analysis, The detection wavelength is 214nm. As shown in Figure 1, the purity is 99.14%.

[0038] 2. Reversed phase high performance liquid chromatography (RP-HPLC)

[0039]The product obtained in Example 2 was evaluated by RP-HPLC. According to the manufacturer's instructions, Vydac (250 mm×4.6 mm) RP-HPLC column (purchased from GRACE VYDAC, USA) was used for RP-HPLC. The experiment was performed at room temperature, and the t...

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Abstract

The invention relates to a high-purity pegylation human growth hormone (HGH) conjugate which is especially suitable for drug uses and has a rate of purity no less than 96 percent. In addition, the invention also relates to a drug preparation containing the conjugate, as well as a preparation method and drug uses the conjugate.

Description

field of invention [0001] The invention belongs to the field of biopharmaceuticals, in particular, the invention relates to a high-purity PEGylated human growth hormone conjugate especially suitable for pharmaceutical application, and simultaneously relates to a pharmaceutical preparation containing the conjugate. In addition, the present invention also relates to the preparation method of the conjugate and its pharmaceutical application. technical background [0002] Natural human growth hormone (hGH) is a protein secreted by the human pituitary gland, which consists of 191 amino acid residues and has a molecular weight of 22 kDa. The main biological function of hGH is to promote the growth of body tissues in young mammals and maintain the tissues of older mammals, including bones, connective tissues, muscles, and tissues of organs such as liver, intestines, and kidneys . Therefore, hGH can be used to treat dwarfism or Turner syndrome caused by pituitary insufficiency, an...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K38/27A61K47/34A61P43/00A61K47/10A61K47/60
Inventor 金磊裴瑾王俊才牛俊
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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