Roche Diagnostics Operations, Inc.

Hoffmann-la Roche Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Marco Thomann

Sebastian Malik

Harald Sobek

Michael Greif

Roberto Falkenstein

Rainer Mueller

Alfred Engel

Bernhard Suppmann

Heiko Walch

Helmut Schwab

Dietmar Reusch

Christine Jung

Ingrid Grunert

Matthias Freiherr Von Roman

Bernd Nidetzky

Thomas Dams

Max L Tejada

Christiane Luley

Maria Maier

Tibor Czabany

Doris Ribitsch

Katharina Schmoelzer

Sabine Zitzenbacher

Roland Dorn

Michael Hingar

Alfred Schnueriger

Herein is reported a method for the enzymatic preparation/production of an antibody with a modified glycosylation in the Fc-region comprises the steps of incubating an antibody that has a glycosylation in the Fc-region with one or more enzymes for a time sufficient and under conditions suitable to modify the glycosylation of the Fc-region to a defined form, separating in one or more chromatography steps the antibody with a modified glycosylation in the Fc-region from the one or more enzymes and thereby producing the antibody with a modified glycosylation in the Fc-region and one or more recycled enzymes, and repeating the incubating step with the one or more recycled enzymes at least once.

Re-use of enzymes in in vitro glycoengineering of antibodies

The properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions are disclosed. Particularly, mutants that exhibit &#x3b1;-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site, are disclosed. A fundamental finding documented in the present disclosure is that enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

CMP-dependent sialidase activity

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.

Method for in vitro glycoengineering of antibodies

The present invention relates to a method for the production of in vitro glycoengineered erythropoiesis stimulating protein, comprising the steps of providing sialic acid free erythropoiesis stimulating protein, treating the erythropoiesis stimulating protein with N-Acetyl-Glucosamin-transferase B3GNT2, treating the erythropoiesis stimulating protein with galactosyltransferase, and treating the erythropoiesis stimulating protein with sialyltransferase.

Method for in vitro glycoengineering of an erythropoiesis stimulating protein

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit alpha-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that these enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

Aqueous composition

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human &#x3b2;-galactoside-&#x3b1;-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant &#x394;89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant &#x394;108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation and specific mono-sialylation

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Penzberg, Germany

Marco Thomann