Implementation of in vitro glycoengineering of monoclonal antibodies into downstream processing of industrial production

In vitro glycoengineering using exoenzymes for specific modification is recognized as appropriate method to tailor sugar moieties of glycan structures during the recombinant production of monoclonal antibodies (mAbs). This report describes enhanced in vitro glycoengineering approaches using β1,4-galactosyltransferase and α2,6-sialyltransferase to improve the efficiency of galactosylation and sialylation with the aim to implement in vitro glycoengineering into common mAb purification processes. Feasibility studies tested the potential of different in vitro glycoengineering protocols (two-step vs. one-step) to facilitate the overall procedure. Scalability of the reactions was demonstrated for mAb amounts ranging from 1 mg to 1 g. Additionally, the reactions of β1,4-galactosyltransferase and α2,6-sialyltransferase were shown to work on column during affinity chromatography using Protein A or KappaSelect, the latter providing more efficient galactosylation and sialylation of IgG1 and IgG4 mAbs. Performing in vitro glycoengineering on column enabled the use of cell culture harvest that yielded results comparable to those of purified bulk. Based thereon, an optimized two-step mixed mode approach was found most appropriate to integrate in vitro glycoengineering of the IgG1 mAb into the overall manufacturing process. Using harvest for on-column reaction of β1,4-galactosyltransferase combined with in-solution reaction of α2,6-sialyltransferase, this approach yielded 100% biantennary galactosylation and 61% biantennary sialylation. Moreover, the enzymes applied in in vitro glycoengineering could be separated, recycled and reused in further reactions to improve economic efficiency. Overall, the study provides a toolbox for in vitro glycoengineering and presents an optimized easy-to-handle workflow to implement this method into the downstream processing of industrial mAb production.