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Glycopegylated granulocyte colony stimulating factor

A colony-stimulating factor, granulocyte technology, applied in the direction of colony-stimulating factor, cytokines/lymphokines/interferons, chemical instruments and methods, etc., can solve the problems of reducing peptide biological or enzyme activity, lack of uniformity of finished products, etc.

Inactive Publication Date: 2007-01-17
蔚所番有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, the random incorporation of PEG molecules has its drawbacks, including lack of uniformity in the finished product, and the potential for reduced biological or enzymatic activity of the peptide

Method used

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  • Glycopegylated granulocyte colony stimulating factor
  • Glycopegylated granulocyte colony stimulating factor
  • Glycopegylated granulocyte colony stimulating factor

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Experimental program
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Embodiment approach

[0298] Particular embodiments according to the invention include:

[0299]

[0300] and carbonates and active esters of these substances, such as:

[0301]

[0302] Other reactive groups or leaving groups suitable for activating linear PEGs used to prepare the compounds described herein include, but are not limited to, the following:

[0303]

[0304] PEG molecules activated with these and other substances and methods of making activated PEGs are listed in WO04 / 083259.

[0305] Those skilled in the art will recognize that one or more of the m-PEG arms of a branched polymer may be replaced by a PEG moiety having a different end, e.g., OH, COOH, NH 2 、C 2 -C 10 - Alkyl etc. In addition, the above structures are easily modified by inserting an alkyl linker between the α-carbon atom and the side chain functional group or removing a carbon atom. Thus, "homo" derivatives and higher and lower homologues are within the core scope of the branched PEGs used in the present ...

Embodiment 1

[0541] GlycoPEGylation of G-CSF produced in CHO cells

[0542] a. Preparation of asialo (Asialo) granulocyte colony-stimulating factor (G-CSF)

[0543] Dissolve G-CSF produced in CHO cells at 2.5 mg / mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl 2 medium and concentrated to 500 μL in a Centricon Plus 20 centrifugal filter. The solution was incubated with 300 mU / mL neuraminidase II (Vibrio cholerae) at 32°C for 16 hours. To monitor the reaction, a small sample of the reaction was diluted with the appropriate buffer and subjected to an IEF gel. The reaction mixture was then added to prewashed N-(p-aminophenyl)oxamate-agarose conjugate (800 μL / mL reaction volume) and the washed beads were gently rotated at 4°C for 24 hours. The mixture was centrifuged at 10,000 rpm, and the supernatant was collected. The beads were washed 3 times with Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and once with 0.2 mL Tris-EDTA buffer, and all supernatants were pooled. I...

Embodiment 2

[0552] Recombinant GCSF - expression, refolding and purification

[0553] ●Collect cells by centrifugation and discard supernatant. Growth results on various media are shown in Figure 9 middle.

[0554] • Resuspend the cell pellet in 10 mM Tris pH 7.4, 75 mM NaCl, 5 mM EDTA using 10 ml / g (lysis buffer).

[0555] • Microlluidize cells (and French pressure processing).

[0556] ● Centrifuge at 5,000 RPM for 30 minutes at 4°C, -discard the supernatant.

[0557] • Resuspend pellet in lysis buffer and centrifuge as above.

[0558] • Wash IB's in 25mM Tris pH 8, 100mM NaCl, 1% TX-100, 1% NaDOC, 5mM EDTA. Resuspend the pellet by pipetting and vortexing. Centrifuge at 5,000 RPM for 15 minutes at 4°C. This step was repeated again (two washes in total).

[0559] • Wash the pellet twice in 25mM Tris pH 8, 100mM NaCl, 5mM EDTA to remove detergent and centrifuge as above.

[0560] ●Resuspend the pellet in dH 2 Aliquot in O and centrifuge as above. The pellet was fr...

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Abstract

The present invention provides a conjugate between granulocyte stimulating colony factor and a PEG moiety. The conjugate is attached via an intact glycosyl linking group interposed between the peptide and the modifying group and covalently linking the two. Conjugates are formed from glycosylated and aglycosylated peptides by the action of glycosyltransferases. Glycosyltransferases attach modified sugar moieties to amino acids or sugar groups on peptides. Pharmaceutical formulations including the conjugates are also provided. Methods of making the conjugates are also within the scope of the invention.

Description

[0001] Cross References to Related Applications [0002] This application is entitled to U.S. Provisional Patent Application No. 60 / 526,796 filed on December 3, 2003; U.S. Provisional Patent Application No. 60 / 555,813 filed on March 23, 2004; U.S. Provisional Patent Application No. 60 / 555,813 filed on May 11, 2004 Application No. 60 / 570,282; U.S. Provisional Patent Application No. 60 / 539,387 filed January 26, 2004; U.S. Provisional Patent Application No. 60 / 592,744 filed July 29, 2004; filed September 29, 2004 U.S. Provisional Patent Application No. 60 / 614,518; and U.S. Provisional Patent Application No. 60 / 623,387, filed October 29, 2004, each of which is hereby incorporated by reference in its entirety for all purposes. Background of the invention [0003] Granulocyte colony stimulating factor (G-CSF) is a glycoprotein that stimulates the survival, proliferation, differentiation and function of neutrophil progenitors and mature neutrophils....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N43/04A61K
CPCA61K38/193C07K14/535A61K47/48215A61K47/60A61P31/00A61P37/04A61P43/00C07K14/53
Inventor S·德弗里斯H·克劳森D·A·佐夫Z-G·王M·施瓦茨武兵元C·鲍
Owner 蔚所番有限公司
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