Method for producing recombinant glycoprotein 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: 2021-03-16
ONENESS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Method for producing recombinant glycoprotein with modified glycosylation
  • Method for producing recombinant glycoprotein with modified glycosylation
  • Method for producing recombinant glycoprotein with modified glycosylation

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Experimental program
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Embodiment

[0087] method

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

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

[0090] ii. Preparation of defucosylated antibodies

[0091] Antibody-producin...

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Abstract

Genetically engineered host animal cells are capable of producing glycoproteins with modified glycosylation patterns, such as defucosylation and / or monoglycosylation. Such host animal cells can be engineered to express fucosidases, endoglycosidases, or both.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of US Provisional Application No. 61 / 954,337, filed March 17, 2014, the entire contents of which are incorporated herein by reference. Background technique [0003] Glycosylation is essential for the structure and function of glycoproteins. For example, glycosylation is believed to affect protein folding (and thus stability) and / or the biological activity of glycoproteins. 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 glycoproteins may affect the bioactivity 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. Compared with endogenous or recombinant EPO, additional glycosylation incre...

Claims

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Application Information

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Patent Type & Authority Patents(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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