US 8,932,825 B2 · Method to engineer mammalian-type carbohydrate structures

Abstract

The present invention relates to host cells having modified lipid-linked oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells have a GlcNAcMan3GlcNAc2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

Claims31

Dependent claims

A method for producing a recombinant glycoprotein in a Pichia pastoris host cell that lacks OCH1 activity, the method comprising the steps of diminishing or depleting the activity of one or more enzymes-selected from the group consisting of:

(a) an enzyme having dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity;
(b) an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity;
and
(c) an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity,
said host cell further expressing:
(i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and (iii) the recombinant glycoprotein,
wherein said method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAcManxGlcNAc2 core structures, wherein X is 3 or 4.

4 more elements

A method for producing a recombinant glycoprotein in a Pichia pastoris host cell, the method comprising:

(a) providing a Pichia pastoris host cell that lacks OCH1 activity and that has diminished or depleted activity of one or more enzymes selected from the group consisting of:
(a) an enzyme having dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity,
(b) an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity, and
(c) an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity,
said host cell further expressing:
(i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell,
(ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and
(iii) a recombinant glycoprotein;
and
(b) growing the host cell under conditions to produce the recombinant glycoprotein, wherein the recombinant glycoprotein has N-glycans attached thereto comprising GlcNAcManxGlcNAc2 core structures, wherein X is 3 or 4.

7 more elements

Dependencies are inferred from the claim text.

Method to engineer mammalian-type carbohydrate structures

Life of this patent

Method to engineer mammalian-type carbohydrate structures

Life of this patent

Abstract

Claims31