The present invention relates to a process for the enzymatic modification of a glycoprotein. The process comprises the step of contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc-moiety, in the presence of glycosyltransferase that is, or is derived from, a β-(1,4)-N-acetylgalactosaminyltransferase, with a non-natural sugar-derivative nucleotide. The non-natural sugar-derivative nucleotide is according to formula (3): ##STR00001##
wherein A is selected from the group consisting of —N.sub.3, —C(O)R.sup.3, —(CH.sub.2).sub.iC≡C—R.sup.4, —SH, —SC(O)R.sup.8, —SC(O)OR.sup.8, —SC(S)OR.sup.8, —F, —Cl, —Br —I, —OS(O).sub.2R.sup.5, terminal C.sub.2-C.sub.24 alkenyl groups, C.sub.3-C.sub.5 cycloalkenyl groups, C.sub.4-C.sub.8 alkadienyl groups, terminal C.sub.3-C.sub.24 allenyl groups and amino groups. The invention further relates to a glycoprotein obtainable by the process according to the invention, to a bioconjugate that can be obtained by conjugating the glycoprotein with a linker-conjugate, and to β-(1,4)-N-acetylgalactosaminyltransferases that can be used in preparing the glycoprotein according to the invention.

Modified Fc regions of antibodies and antibody fragments, both human and humanized, and having enhanced stability and efficacy, are provided. Fc regions with core fucose residues removed, and attached to oligosaccharides comprising terminal sialyl residues, are provided. Antibodies comprising homogeneous glycosylation of Fc regions with specific oligosaccharides are provided. Fc regions conjugated with homogeneous glycoforms of monosaccharides and trisaccharides, are provided. Methods of preparing human antibodies with modified Fc using glycan engineering, are provided.

Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.

The present invention relates to a cell, wherein the cell is modified to: reduce O-GalNAc galactosylation activity in the cell by reduction of functional COSMC molecular chaperone in the cell and/or by reduction of functional T-synthase in the cell; and overexpress β1,4-galactosyltransferase in the cell; and associated methods, kits and uses.

Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.

The present invention relates to a process for the modification of a glycoprotein, using a β-(1,4)-N-acetylgalactosaminyltransferase or a mutant thereof. The process comprises the step of contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc-moiety, in the presence of a β-(1,4)-N-acetylgalactosaminyltransferase or a mutant thereof, with anon-natural sugar-derivative nucleotide. The non-natural sugar-derivative nucleotideis according to formula (3), wherein A is selected from the group consisting of —N.sub.3; —C(O)R.sup.3; —C═C—R.sup.4; —SH; —SC(O)R.sup.8; —SC(V)OR.sup.8, wherein V is O or S; —X wherein X is selected from the group consisting of F, Cl, Br and I; —OS(O).sub.2R.sup.5; an optionally substituted C.sub.2-C.sub.24 alkyl group; an optionally substituted terminal C.sub.2-C.sub.24 alkenyl group; and an optionally substituted terminal C.sub.3-C.sub.24 alkenyl group. ##STR00001##

This disclosure relates to glycoengineering, and methods of utilizing glycoengineering for various therapeutic purposes.

The present invention provides means and methods for the production in eukaryotic cells of homogeneous forms of small glycan structures which carry terminal galactose residues. In addition, the invention provides glycan-conjugates based on specific coupling with galactose residues present on the recombinant glycoproteins.

Herein is reported a method for the enzymatic production of an antibody with a modified glycosylation in the Fc-region comprising the steps of incubating the antibody light chain affinity ligand-bound monoclonal antibody with a glycosylation in the Fc-region with a first enzyme for a time sufficient and under conditions suitable to modify the glycosylation of the Fc-region, recovering the antibody from the antibody light chain affinity ligand, incubating the recovered antibody in solution with a second enzyme for a time sufficient and under conditions suitable to modify the glycosylation of the Fc-region to a defined form, separating the antibody with the modified glycosylation in the Fc-region from the second enzyme in a cation exchange chromatography, and thereby producing the antibody with a modified glycosylation in the Fc-region.

Disclosed are a construction method and application of a microorganism capable of realizing high production of lacto-N-neotetraose, belonging to the field of microbial genetic engineering. Coding genes of -1,3-acetyl glucosamine transferase, -1,4-galactosyl transferase and/or UDP-glucose 4 epimerase are over-expressed on the basis of a strain which is previously constructed by the team and is subjected to related-gene knockout, thus enabling the strain to have a synthesis capability of producing the lacto-N-neotetraose. The present disclosure accurately regulates the carbon flux of a metabolic pathway and relieves the metabolic stress by screening the high-efficiency -1,4-galactosyl transferase gene and regulating the expression of IgtA, Aa--1,4-GalT and galE in a lacto-N-neotetraose synthesis pathway in a combined manner. In a shake flask experiment, the lacto-N-neotetraose production capacity of Escherichia coli is 0.91 g/L. The lacto-N-neotetraose yield in a 3 L fermentation tank reaches 12.14 g/L. Therefore, the microorganism has an industrial application prospect.