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Pegylated long-acting growth hormone as well as preparation method and medical application thereof

A polyethylene glycol, growth hormone technology, applied in growth hormone, chemical instruments and methods, hormone peptides, etc., to achieve the effect of improving pharmacokinetic properties, better stability, and good intermolecular consistency

Pending Publication Date: 2022-05-27
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor tried to modify the growth hormone with the same modifier, but through repeated exploration of the modification and purification process conditions, the obtained modification always contained a variety of modified isomers

Method used

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  • Pegylated long-acting growth hormone as well as preparation method and medical application thereof
  • Pegylated long-acting growth hormone as well as preparation method and medical application thereof
  • Pegylated long-acting growth hormone as well as preparation method and medical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation and purification of rhGH samples modified with different branched polyethylene glycol propionaldehyde

[0068] 1. Sample preparation

[0069] (1) Preprocessing:

[0070] Take the original rhGh protein (secreted and expressed in the periplasm of Escherichia coli, the sequence is consistent with the natural sequence), treated with a 10kDa ultrafiltration membrane bag, the replacement buffer is sodium dihydrogen phosphate / disodium hydrogen phosphate buffer pH 6.0, and concentrated to the protein Concentration 10mg / mL.

[0071] (2) Feeding modification

[0072] PEG30K-rhGH modification preparation: add PEG to the pretreated rhGH protein solution according to the ratio of recombinant human growth hormone (hereinafter referred to as rhGH) and Y-PALD-PEG 30K molar ratio of 1:2, press PEG and reducing agent (cyano group) Sodium borohydride) in a molar ratio of 1:50, add a reducing agent to the mixed solution of PEG30K and protein, stir slowly until the ...

Embodiment 2

[0091] Example 2 Preparation and purification of Y-PEG-NHS modified rhGH samples

[0092] 1. Sample preparation

[0093] With reference to Chinese invention patent CN101385858A, the preparation of Y-PEG-NHS 40K random single-site modified rhGH samples was carried out. The specific plans are as follows:

[0094] The rhGh original protein was taken and treated with a 10kDa ultrafiltration membrane bag. The replacement buffer was 50mM sodium dihydrogen phosphate / disodium hydrogen phosphate buffer pH 6.5-6.8, and at the same time, it was concentrated to a protein concentration of 10 mg / mL.

[0095]The PEG modifier is polyethylene glycol-N-hydroxysuccinimidyl ester (trade name Y-PEG-NHS 40K, purchased from Beijing Jiankai Technology Co., Ltd.), and PEG is carried out according to the protein:PEG mass ratio of 1:6 Feeding, stirring was continued until the Y-PEG-NHS was completely dissolved, and the reaction was carried out at 4° C. for 16 hours to obtain a reaction mixture.

[00...

Embodiment 3

[0111] Example 3 Determination of the binding number of rhGH PEG modified with different PEGs

[0112] In the present invention, the following method can be used to compare the number of PEG-binding molecules of GH in different PEG-modified rhGH.

[0113] Take PEG60K-rhGH as an example:

[0114] 1. Detection method

[0115] Use liquid chromatography differential-UV combined detection method.

[0116] The detection column is BEH SEC 3.5um, 7.8*300mm. The mobile phase was 20mM pb7.0+5% isopropanol, the column temperature was set to 35°C, and 10ul of sample was loaded. The collection time was set to 30min; the temperature of the flow cell was set to 35°C; the flow rate of the liquid phase was set to 0.5ml / min. The detector used RI+UV (collection wavelength 280 nm).

[0117] The sample processing method is as follows: PEG60K-rhGH and GH proprotein are respectively diluted to 1.0mg / ml with pH7.0 phosphate buffer; 5mg of PEG60K is accurately weighed, accurately dissolved with...

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Abstract

The invention relates to pegylated long-acting growth hormone as well as a preparation method and medical application thereof. The PEG-rhGH molecule modified by the branched PEG provided by the invention is modified by a single site, the intermolecular consistency is good, the modification and purification processes are more controllable, and the yield of a modifier is higher; on the basis of retaining the activity of the growth hormone to a greater extent, the pharmacokinetic property of the growth hormone is well improved, and an unexpected technical effect is achieved.

Description

technical field [0001] The present invention relates to pegylated long-acting growth hormone and its preparation method and medical application. Background technique [0002] Human Growth Hormone (hGH) is a protein hormone with a single peptide chain secreted by eosinophils in the anterior pituitary gland. . There are currently 11 FDA-approved rhGH indications: GHD (1985), chronic renal insufficiency before kidney transplantation (1993), HIV infection-related wasting syndrome (1996), Turner syndrome (1996) years), adult growth hormone deficiency (1997), Prader-Willi syndrome (Prader-Willi syndrome, 2000), small for gestational age (SGA, 2001), idiopathic short stature ( idiopathic short stature, ISS, 2003), short bowel syndrome (2003), SHOX gene deficiency without GHD (2006), Noonan syndrome (2007). With the deepening of clinical research, growth hormone also has a good effect in the treatment of anti-aging, osteoporosis and cardiovascular disease. [0003] At present, t...

Claims

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Application Information

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IPC IPC(8): C07K14/61A61K38/27A61K47/60A61P5/06A61P17/02A61P31/18
CPCC07K14/61A61K47/60A61P5/06A61P31/18A61P17/02A61K38/00
Inventor 马永王俊王和江辰阳
Owner ZONHON BIOPHARMA INST
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