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Methods for producing recombinant glycoproteins with modified glycosylation

A glycoprotein and glycosylation technology, applied in biochemical equipment and methods, glycosylase, chemical instruments and methods, etc., can solve the problem of high cost of recombinant protein

Active Publication Date: 2017-05-24
ONENESS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, currently available methods for glycosylation remodeling often require multiple enzymes and / or multiple steps, resulting in high cost of manufacturing glycoengineered recombinant proteins

Method used

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  • Methods for producing recombinant glycoproteins with modified glycosylation
  • Methods for producing recombinant glycoproteins with modified glycosylation
  • Methods for producing recombinant glycoproteins with modified glycosylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0087] method

[0088] i. Construction of expression vectors for production of fucosidase or endoglycosidase

[0089] In order to construct the expression vector of fucosidase or endoglycosidase, the fucosidase or endoglycosidase gene was isolated by conventional techniques, and codon optimization was performed based on the codon usage of hamster cells. The synthetic gene was prepared by GeneArt Corp, and inserted into the pcDNA3.1B(-)Myc-His vector (Invitrogen, US) at the restriction enzyme site Bgl II / EcoR I ( figure 2 Middle A). The expression cassette for α-fucosidase, from 5' to 3', comprises Kozak sequence, Igk leader sequence, fucosidase coding sequence and His-tag coding sequence. figure 2 Middle B. The endoglycosidase expression cassette, from 5' to 3', comprises a Kozak sequence, an Igk leader sequence, an endoglycosidase coding sequence, and a His-tag coding sequence. figure 2 Middle B.

[0090] ii. Preparation of afucosylated antibody

[0091] Antibody-pro...

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Abstract

Genetically engineered host animal cells capable of producing glycoproteins having modified glycosylation patterns, e.g., defucosylation and / or monoglycosylation. Such host animal cells can be engineered to express fucosidase, endoglycosidase or both.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 954,337, filed March 17, 2014, which is hereby incorporated by reference in its entirety. Background technique [0003] Glycosylation is critical to the structure and function of glycoproteins. For example, glycosylation is thought to affect protein folding (and thus stability) and / or biological activity of glycoproteins. The demand for therapeutic recombinant glycoproteins, especially monoclonal antibodies, has grown strongly in the last two decades. Previous studies have shown that small differences in the structure of recombinant glycoprotein glycans may affect the biological activity and pharmacokinetics of glycoproteins. For example, darbepoetin alfa is a highly glycosylated analog of recombinant human erythropoietin (EPO) with two additional N-linked glycosylation sites. Additional glycosylation increased the molecular mass percentage of ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/56C12N15/85C12P21/00
CPCC07K16/2887C07K16/4291C12P21/005C12N9/2405C07K2317/14C07K2317/41C12Y302/01096C12Y302/01051C12N9/2402C07K16/00C12N15/85Y02P20/52
Inventor 陈念宜吴哲豪陈虹琦汤竣钧
Owner ONENESS BIOTECH
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