Method for in vitro glycoengineering of antibodies

A method for producing a glycosylation modified antibody comprising:

• applying a solution comprising an antibody with a glycosylation at an N-glycosylation site to an antibody affinity ligand bound to a solid phase,
• enzymatically modifying a glycosylation at an N-glycosylation site of the antibody by applying a solution comprising a first glycosylation modifying enzyme and a first activated sugar residue for a time sufficient and under conditions suitable for enzymatic modification to the ligand-bound antibody thereby producing a first modified antibody,
• recovering the first modified antibody from the antibody affinity ligand, and
• incubating the recovered first modified antibody in solution with a second enzyme and a second activated sugar residue for a time sufficient and under conditions, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

A method for the enzymatic production of an antibody with a modified glycosylation at an N-glycosylation site, comprising

• a) incubating the antibody light chain affinity ligand-bound monoclonal antibody with a glycosylation at an N-glycosylation site with a first enzyme and a first activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site thereby producing a first modified antibody,
• b) recovering the first enzyme and producing thereby a recycled first enzyme,
• c) recovering the first modified antibody from the antibody light chain affinity ligand,
• d) incubating the recovered first modified antibody in solution with a second enzyme and a second activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site of the first modified antibody thereby producing the glycosylation modified antibody;
• e) separating the glycosylation modified antibody from the second enzyme in a cation exchange chromatography, optionally recovering the second enzyme and producing thereby a recycled second enzyme, and
• f) repeating step a) with the first recycled enzyme of step b) at least once and repeating step d) with the second recycled enzyme of step e) at least once, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.