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Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product

A colony-stimulating factor and granulocyte technology, which is used in PEGylated recombinant human granulocyte colony-stimulating factor conjugated compounds, the application of long-acting preparations of protein compounds, and the use of column chromatography PEGylated protein nitrogen-terminal point-specific couplings. It can solve problems affecting application value and significance, etc.

Active Publication Date: 2009-11-25
TIANJIN PAIGE BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, many previous patents on PEG-coupling proteins or polypeptides, including Kinstler et al. The actual application value and significance of these patents; let alone the use of column chromatography involved in the application of the present invention to directly affect the pharmaceutical and pharmacological properties of rhG-CSF in a specific and controllable manner, and Developed a new long-acting formulation with long-acting, high potency and low immunogenicity compared with Neulasta (pegfilgrastim)

Method used

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  • Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product
  • Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product
  • Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] Example 1 Expression and preparation of rhG-CSF and rhG-CSFm

[0174] Expression of rhG-CSF and rhG-CSFm:

[0175] Fresh human bone marrow cells were cultured and activated with pokeweed kinogen (Sigma, USA). Human bone marrow cell mRNA was extracted, and the hG-CSF gene was amplified by RT-PCR method, which was confirmed to be hG-CSF gene through sequencing (completed by Beijing Boshang Biotechnology Co., Ltd.), and its cDNA sequence is shown in SEQID NO.2. The amino acid sequence is shown in SEQID NO.1. DNA sequence analysis of hG-CSF gene was analyzed by DNA Strider software; PCR (polymerase chain reaction)) and site-directed mutagenesis were used to modify the human natural G-CSF gene sequence to obtain a new modified human granulocyte colony-stimulating factor ( rhG-CSF) gene coding sequence, its nucleotide sequence is shown in SEQID NO.3, this gene is constructed on the pMD18-T vector, and the vector is transformed into Top10 bacterial strain and preserved.

[...

Embodiment 2

[0198] Example 2 Preparation of PEG-rhG-CSF by site-directed coupling of PEG-propionaldehyde and rhG-CSF N-terminus

[0199] Load MacroCap SP strong cation exchange chromatographic column (2.6cmX20cm), and connect it to AKTAExplorer 100 liquid chromatography system (Amersham Bioscience, Sweden), use 20mM sodium acetate buffer pH 5.5 (buffer solution A) and 0.005% poly Sorbitol ester 80 500ml fully equilibrates the chromatographic column; using a feed pump, add 0.5mg / ml 1500ml of rhG-CSF stock solution to the chromatographic column at a flow rate of 10ml / min; Solution A washes the column to remove substances that do not electrostatically adsorb with the chromatographic medium; at a flow rate of 3ml / min, add 5mg / ml 30kDa PEG-propionaldehyde (PEG-propionaldehyde) and 0.1mg / mlNaBH 3 CN solution 1500ml, continuous sample loading for about 240 minutes; the pH of the reaction system is 5.5; then wash the column with about 500ml buffer A at a flow rate of 15ml / min to remove substances...

Embodiment 3

[0203] Example 3 Analysis of the Coupling Site of PEG-ALD and rh-CSF Using Trypsin Digestion Peptide Mapping by Liquid-MS

[0204] 1) Measurement method

[0205] Select liquid chromatograph Agilent 1100 and mass spectrometer LCQ Deca XP MS to analyze, and specific method is as follows:

[0206] Liquid chromatograph operating conditions:

[0207] The mobile phase is: A: 0.1% A: 0.1% TFA in water, B: 0.1% TFA in ACN;

[0208] Chromatographic conditions: the chromatographic column is Zorbax SBC18 (2.1 × 150mm, Agilent), the flow rate is 0.2ml / ml, the gradient is 0-60min, 5%B-60B, 60-120min, 60%B-90%B, and the detection wavelength is 214nm;

[0209] Mass spectrometer operating conditions:

[0210] Sheath gas: 60arb; Aux gas: 0; Spray voltage: 4.5kV; Detection method: Triple play; Primary mass spectrometry: Mass range: m / z300-2000; Zoom scan: Data dependent mode; Data processing software: Bioworks3.1; mass spectrometry data analysis software Turbosequest3.1.

[0211] The enzy...

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Abstract

The invention discloses a method for the fixed-point coupling of polyethylene glycol on a nitrogen-terminal of a colony stimulating factor of a column chromatography granulocyte, wherein the coupling method comprises the following steps that: 1) a chromatography medium is preferred to be an exchange medium; 2) the height of a column bed of a chromatography column is preferred to be the upper limit of the recommended height of the column; 3) the sample volume of rhG-CSF protein is preferred to be 30 to 50 percent of the actually measured maximum loading capacity of the medium; 4) the activated mPEG is sampled under conditions of lower sample flow rate, longer sample time (3 to 5 hours) and higher pH value of reaction rate; 5) the sample volume of the activated mPEG is made according to the reaction volume ratio of the activated mPEG to the rhG-CSF of 1-50mol:1mol; and 6) 0.5 to 1M of salt solution of sodium chloride is used for gradient elution. The invention also discloses a coupling compound prepared by the method, which is a pegylation recombination human granulocyte colony stimulating factor, of which the structural formula is CH3-(CH2CH2-O)n-(CH2)r-NH-rhG-CSF or CH3-(CH2-CH2-O)n-(CH2)r-NH-rhG-CSFm, wherein n is between 570 and 2,200, and r is between 1 and 3; moreover, the invention also discloses application of the coupling compound in the preparation of a preparation preventing and treating the granulocytopenia.

Description

technical field [0001] In a broad sense, the present invention belongs to the field of protein modification and water-soluble multimer coupling protein, polypeptide and its mutants or analogues; more specifically, the present invention relates to a column chromatography polyethylene glycol protein nitrogen terminal A new method for point-specific coupling, and a class of pegylated recombinant human granulocyte colony-stimulating factor conjugated compound (PEG-rhG-CSF) obtained by this method and its long-acting, long-acting, Application of long-acting preparations of protein compounds with high efficiency and low immunogenicity characteristics. Background technique [0002] Recombinant human granulocyte colony stimulating factor (rhG-CSF) can promote the proliferation and differentiation of bone marrow granulocyte progenitor cells, increase the number and activity of mature neutrophils in peripheral blood, and large doses of rhG-CSF can still mobilize The progenitor cells ...

Claims

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Application Information

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IPC IPC(8): C07K1/10C07K1/18C07K14/535A61K38/19A61P7/00
Inventor 张旋马光辉苏志国祁庆生雷建都
Owner TIANJIN PAIGE BIOTECHNOLOGY CO LTD
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