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Method of modifying protein alpha-amido by carbowax

A polyethylene glycol and protein technology, which is applied in the field of modification and transformation of protein molecules, can solve problems such as slow reaction, paradox, and different molecular structures, and achieve a significant increase in ANC, uniform molecular weight and structure, and sustained drug efficacy. long-lasting effect

Inactive Publication Date: 2005-10-26
山东格兰百克生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still disadvantages in this method: ①The reaction is slow and the cycle is long, generally 10-16 hours; Prolonging the proportion of PEGylation on lysine increases, because this method only slows down the reaction speed by lowering the pH value of the reaction system, thereby widening the reaction speed difference between the two different amino groups, making the reaction easy to control, but it cannot be eliminated Further PEGylation of amino groups on lysine
[0009] At present, the cross-linking reactants generated under the commonly used PEG reaction conditions are heterogeneous PEG-protein molecules (different molecular weights, different molecular structures, and cross-linking PEG on different lysine residues of protein molecules), which is contrary to the review of new drugs in my country. The relevant principles cannot be applied clinically. In order to develop an acceptable and uniform PEGylated protein molecule, it is necessary to seek a condition for the site-specific cross-linking reaction between the PEG macromolecule and the N-terminus of the protein.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] mPEG 20000 - The preparation process of rhG-CSF:

[0039] ①Concentrate the GC solution by ultrafiltration, replace the buffer system with 0.1M phosphate buffer (pH8.5), and obtain 1000ml of GC solution with a concentration of 5mg / ml.

[0040] ②Add 1.66g of DMMAn, keep the temperature of the reaction system at 0°C in an ice bath, and maintain the reaction for 30min.

[0041] ③Add 52.6g mPEG to the reaction system 20000 -acetaldehyde, stir to dissolve, heat up to 37°C, add 0.16g NaBH 3 CN, reacted for 1 hour. The above reaction mixture was adjusted to pH 6.0 with 0.1M HCl at 37°C for 30 minutes.

[0042] ④ Load the reaction product and elute with a linear gradient of 10mM sodium acetate buffer (pH4.0) and 0-0.45M sodium chloride on SPSepharose Big Beads ion column equilibrated with 10mM sodium acetate buffer (pH4.0) 6 column volumes, the main peak is collected as mPEG 20000 -rhG-CSF. Go through the Superdex 75 column to desalt, and replace the buffer with 10mM sodi...

Embodiment 2

[0046] mPEG 30000 - The preparation process of rhG-CSF:

[0047] ①Concentrate the GC solution by ultrafiltration, replace the buffer system with 0.1M phosphate buffer (pH8.5), and obtain 1000ml of GC solution with a concentration of 5mg / ml.

[0048] ② Add 1.83g DMMAn, keep the temperature of the reaction system at 0°C in an ice bath, and keep the reaction for 30min.

[0049] ③Add 86.8gmPEG to the reaction system 20000 -acetaldehyde, stir to dissolve, heat up to 39°C, add 0.176g NaBH 3 CN, reacted for 1.5 hours. The above reaction mixture was adjusted to pH 6.2 with 0.1M HCl at 35°C for 40 minutes.

[0050] ④The reaction product was loaded on the SPSepharose Big Beads ion column equilibrated with 10mM sodium acetate buffer (pH4.0), and eluted with a linear gradient of 10mM sodium acetate buffer (pH4.0) and 0-0.6M sodium chloride 10 column volumes, collect the main peak as mPEG 20000 -rhG-CSF. Go through the Superdex 75 column to desalt, and replace the buffer with 10mM s...

Embodiment 3

[0054] mPEG 20000 -The preparation technology of IFNα-2b (interferon):

[0055] ① Dissolve interferon IFNα-2b in 0.1M phosphate buffer (pH8.5) to obtain concentrated

[0056] 1000ml of IFNα-2b solution with a concentration of 2mg / ml.

[0057] ② Keep the temperature of the above solution at 0°C in an ice bath, add 0.664g DMMAn under the condition of slow stirring, and keep the reaction for 30min after dissolving.

[0058] ③The temperature of the above reaction system was raised to 37°C, and 26.3gmPEG was added under the condition of slow stirring 20000 -acetaldehyde, add 0.032g NaBH after dissolution 3 CN, keep reacting for 0.5 hours after dissolving. The above reaction mixture was adjusted to pH 6.0 with 0.1M HCl at 37°C for 30 minutes.

[0059] ④ The reaction mixture was determined by electrophoresis (SDS-PAGE) and reverse phase HPLC (C18) to determine that ~91% of IFNα-2b had been modified.

[0060] ⑤Equilibrate the SP Sepharose Big Beads ion column with 20mM sodium ac...

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Abstract

The present invention provides a method for modifying protein alpha-amino group by using polyethuylene glycol. Said method includes the following steps: utilizing amino protective agent to close lysine epsilon amino group capable of reacting with PEG in the protein, using activated PEG with dominant response with alpha amino group to make amino modification, then removing amino protective agent so as to obtain nitrogen end specific PEG modified protein derivative. The conversion rate of product obtained by said method is high, can be up to 90%-95%.

Description

Technical field: [0001] The invention relates to a modification and transformation of protein molecules, in particular to a method for point-directed modification and transformation of protein molecules. Background technique: [0002] Polypeptide and protein drugs are mainly cleared in the body through degradation, excretion, receptor-mediated endocytosis, etc. Among them, polypeptide factors with a molecular weight of less than 20 kDa are easily filtered by the glomerulus during the metabolic process. Polypeptide factors are partially degraded by protease and excreted from urine, so the half-life is short. The elimination half-life of intravenous recombinant human granulocyte colony-stimulating factor (rhG-CSF) is 1 to 2 hours, and that of subcutaneous injection is 2 to 3 hours. In order to maintain a certain curative effect, it needs to be applied every day, which not only increases the suffering of patients but also easily causes A series of side effects. [0003] Chemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107
Inventor 张雪山徐宜铁
Owner 山东格兰百克生物制药有限公司
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