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Abstract

Disclosed is the preparation of eukaryotic cells which are capable of producing smaller and less heterogenic N-linked glycans, particularly glycans having the structure Man.sub.3 GlcNAc.sub.2. In particular embodiments, CHO cells are produced having two particular defects in enzymes involved in N-linked glycosylation. The first defect, PIR, leads to an accumulation of Man.sub.5 GlcNAc.sub.2 -P-P-dolichol and thus lends resistance to glycosylation processing inhibitors such as castanospermine and swainsonine. The second defect involves a defect in the enzyme N-acetylglucosaminyltransferase I activity, and thus prevents the attachment of N-acetylglucosamine residues to oligosaccharide structures. About 70 to 75% of the N-linked glycans produced by cells of the present invention are thus in the form of Man.sub.3 GlcNAc.sub.2, making these lines particularly well suited to the production of proteins, e.g., recombinant proteins such as EPO and t-PA, which one desires to have a smaller and more homogeneous N-linked glycan structure.

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First Claim