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

[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 ...

[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.

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[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...