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Method of labelling interferons with peg

A labeling method, interferon technology, applied in the direction of interferon, cytokines/lymphokines/interferons, chemical instruments and methods, etc., can solve problems such as the influence of protein folding activity, and achieve the effect of enhancing activity

Inactive Publication Date: 2012-04-18
ALMAC SCI SCOTLAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although there are a large number of methods known in the art, each method still has its disadvantages in terms of, for example, the need to introduce additional chemical moieties, the mimicry of the modification site, the effect on protein folding and the effect on activity.

Method used

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  • Method of labelling interferons with peg

Examples

Experimental program
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Effect test

Embodiment 1

[0135] Example 1: Generation of Pyruvyl PEG

[0136] Such as figure 1 As shown in Scheme 1, a 10 kDa PEG target compound (4) containing an N-terminal pyruvyl functional group was prepared. This was achieved by overnight acylation of a commercially available PEG amine (3) with preformed pyruvic chloride (2). PEG amine was obtained from Nektar {MeO-PEG-NH2Nektar / 2M2U0101 / PT03F24].

[0137] The acid chloride (2) is formed by treating pyruvate (1) with α,α-dichloromethyl methyl ether. Briefly, pyruvic acid (5 g) was charged under nitrogen to a 50 ml 3-neck RB flask equipped with a reflux condenser, dropping funnel and connected to a dreschel bottle containing 2N NaOH(aq). α,α-Dichloromethyl methyl ether (5.16 ml) was added dropwise, the reaction mixture was heated to 50 °C for 30 minutes, the methyl formate by-product was removed by evaporation under reduced pressure, and in 82% yield (4.96 g) The crude acid chloride was obtained as a yellow oil.

[0138] The crude acid ch...

Embodiment 2

[0140] Example 2: Generation of site-specific C-terminal PEGylated IFNα2b hydrazide

Embodiment 21

[0141] Example 2.1: Cloning, expression and purification of soluble IFNα2b hydrazide

[0142] IFNα2b cDNA (IMAGE clone 30915269) was purchased from Gene Service Ltd. The IFNα2b coding sequence was amplified by PCR using the following primers:

[0143] The forward primer was designed to include an NdeI site immediately before the 5' IFNα2b sequence:

[0144] 5'-GGTGGTCATATGTGTGATCTGCCTCAAACCC-3'

[0145] A reverse primer was designed to eliminate the stop codon at the end of the IFNα2b coding sequence and replace it with a glycine codon that was immediately followed by a SapI site:

[0146] 5'-GGTGGTTGCTCTTCCGCACCCTTCCTTACTTCTTAAACTTTCTTGC-3'

[0147] The resulting PCR product was cloned into the NdeI SapI site of the pTXB1 vector (NEB). The pTXB1 IFNα2b GLY construct encodes a fusion protein whereby IFNα2b is linked via glycine to the N-terminus of the GyrA intein which in turn is fused to the N-terminus of the chitin binding domain (CBD). It was transformed into E. col...

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Abstract

A method of site specific labelling of an interferon molecule is provided. The method comprises the steps: a) providing a label molecule comprising a PEG moiety having an aldehyde or ketone moiety; b) providing an interferon molecule having a C terminal hydrazide moiety; and c) allowing the aldehyde or ketone moiety of the PEG moiety to react with the C terminal hydrazide of the interferon molecule to form a labelled interferon molecule, which comprises a PEG moiety attached to the C terminus of the interferon molecule via a hydrazone bond. Interferon molecules labelled using such a method are also described.

Description

field of invention [0001] The present application relates to methods for site-specific modification of peptides, proteins and the like. In particular, it relates to methods of labeling proteins such as interferons with PEG. Background of the invention [0002] Recombinant protein therapeutics have emerged as effective treatments for a variety of conditions ranging from cancer to metabolic disorders and autoimmune diseases, but they are often limited by their pharmacokinetics and immunogenicity. Accordingly, a number of strategies have been developed to overcome these, including glycosylation, albumin attachment, cyclization and PEGylation. Protein glycosylation is the attachment of one or more sugars, which may contribute to protein stability and confer some protection against proteolysis and immune recognition (Doores et al., 2006). Human albumin is the most prevalent serum protein in circulation and has an unusually long half-life of approximately 19 days. Thus, genetic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/21C07K14/555
CPCA61K38/21A61K47/48215C07K14/56C07K14/555A61K47/00A61K38/00A61K47/60A61P1/16A61P3/10A61P25/00A61P31/12A61P35/00A61P35/02A61P37/02
Inventor 詹妮弗·罗伯茨格雷厄姆·科顿
Owner ALMAC SCI SCOTLAND
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