Granular leukocyte colony-stimulating factors coupling agent with chemical modification and production thereof

A technology of chemical modification and conjugate, applied in the field of granulocyte colony-stimulating factor conjugate and its preparation, which can solve the problems of not giving experimental plan, experimental result identification data, etc.

Inactive Publication Date: 2006-05-31
BEIJING KAIZHENG BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN: 1404401 mentions branched polyethylene glycol modified GCSF, the polyethylene glycol structure used is different from the structure of the patented compound, and no specific experimental scheme, experimental results, and identification data are given

Method used

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  • Granular leukocyte colony-stimulating factors coupling agent with chemical modification and production thereof
  • Granular leukocyte colony-stimulating factors coupling agent with chemical modification and production thereof
  • Granular leukocyte colony-stimulating factors coupling agent with chemical modification and production thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, Preparation and Identification of U-PEG Modified GCSF

[0063] U-PEG reagent:

[0064]

[0065]

[0066] III

IV

V

VI

code name

IIIa

IIIb

IIIc

IVa

IVb

IVc

Va

Vb

Vc

VIa

VIb

VIc

molecular weight

10k

20k

40k

10k

20k

40k

10k

20k

40k

10k

20k

40k

[0067] *Molecular weight unit is Dalton Da

[0068]The molecular weights of various U-PEG reagents are shown in Table 1, provided by Beijing Kaizheng Bioengineering Development Co., Ltd., and the synthesis methods refer to US patents US: 5932462 and US: 5643575.

[0069] GCSF was provided by North China Pharmaceutical Group New Drug Research and Development Co., Ltd.

[0070] U-PEG modification reaction:

[0071] Factors affecting the modification reaction include: 1) pH value, 2) temperature, 3) reaction time, 4) molar ratio of GCSF to U-PEG, and 5) protein concentration. B...

Embodiment 2、II

[0088] Example 2, Preparation and identification of IIIb (molecular weight is 20kDa) modified rhG-CSF conjugate

[0089] a, preparation of multi-U-PEGylated rhG-CSF composition

[0090] Recombinant human granulocyte colony-stimulating factor (rhG-CSF) samples were provided by Hangzhou Jiuyuan Genetic Engineering Co., Ltd.

[0091] 1ml (1.53mg / ml) rhG-CSF solution was put into a dialysis bag (3500Da), and dialyzed overnight with 20mM boric acid buffer (pH=9.0). Take it out and add it to 8.09mg IIIb (MW20KDa) solid, IIIb is 4 moles more than rhG-CSF, shake at room temperature for two hours, add 25% acetic acid to the mixture to stop, and adjust the pH to 4.0. At this time, the reaction mixture is mainly composed of single U-PEGylated rhG-CSF, partly double U-PEGylated rhG-CSF, unmodified rhG-CSF and reaction by-products (NHS). like figure 1 shown.

[0092] b. Purification of single U-PEGylated rhG-CSF

[0093] The above mixture was loaded on a PHarmacia Biotech SP SepHaros...

Embodiment 3

[0121] Example 3. In vivo activity detection of multi-U-PEGylated rhG-CSF compositions prepared under different pH conditions

[0122] Preparation of multi-U-PEGylated rhG-CSF composition

[0123] rhG-CSF samples were purchased from Hangzhou Jiuyuan Gene Engineering Co., Ltd.

[0124] Put 1ml (1.53mg / ml) rhG-CSF solution into a dialysis bag (3500Da), dialyze overnight with 20mM borate buffer solution with pH 6.2, take it out and add it to 8.09mg IIIb (MW 20KDa) solid, IIIb ratio rhG-CSF 4 more moles, after shaking at room temperature for two hours, 25% acetic acid was added to the mixture to terminate, and the pH was adjusted to 4.0. At this time, the reaction mixture is mainly composed of single U-PEGylated rhG-CSF, partly double U-PEGylated rhG-CSF, unmodified rhG-CSF and reaction by-product (NHS).

[0125] Preparation of multi-U-PEGylated rhG-CSF composition under different pH reaction conditions

[0126] The modified product with pH 7.0 was prepared by dialysis with 20 ...

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Abstract

A grain cell colony stimulating factor coupler with chemical modification and its production are disclosed. Its advantages include more special combining site, low immunity, less activity loss and more output.

Description

technical field [0001] The invention relates to a granulocyte colony-stimulating factor conjugate chemically modified by a branched water-soluble polymer and a preparation method thereof. Background technique [0002] Granulocyte colony-stimulating factor (GCSF) is a hematopoietic growth factor, which acts on bone marrow neutrophil hematopoietic precursor cells, promotes their proliferation, differentiation, and promotes the maturation and release of neutrophils, enhancing the maturity of neutrophils. Granulocyte function. The earliest indications of CSF were to promote the recovery of neutrophils after cancer chemotherapy and bone marrow transplantation, and then gradually expanded to neutropenia caused by various etiologies (Metcalf, Blood 67: 257 (1986); Yan, et al ., Blood84(3):795-799(1994); Bensinger, et al., Blood 81(11):3158-3163(1993); Neben, et al., Blood 81(7):1960-1967(1993) ). [0003] GCSF has a short half-life in the body and is easily degraded by proteases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/475A61K38/18C07K1/02
Inventor 刘谦严玖凤张晓伟李晋峰侯广才许可李先钟刘佳王蕊陈婷申晓阳
Owner BEIJING KAIZHENG BIOTECH DEV
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