A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Abstract

N-linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N-linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N-glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis-tris, and 1-amino-1-methyl-1,3-propanediol), contain one similar molecular structure: 2-amino-1,3-propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost-effective, and stable at room temperature. In addition, tris and bis-tris provide multiple low-cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.

A novel approach to residence time distribution characterization in a mAb continuous process

“…The mannosidase I inhibitor, Tris, was selected as the tracer to provide insight into the flow characteristics of a small molecule through each unit operation. The addition of Tris alters the high mannose glycan content excreted from the CHO cells and the increase in high mannose glycans was used as a secondary tracer to provide insights into the impact each unit operation has on the residence time of the target protein (Brantley et al, 2021). Additionally, Tris mimics a process disruption and demonstrates the duration in which the product is out‐of‐specification for multiple unit operations.…”

“…Cell consumption and production can be excluded from the model as no metabolic pathway for Tris production or consumption exist in mammalian cells and Tris concentration is below detectable limits in the control process (Orsi et al, 2009; Walther et al, 2019). Tris alters the high mannose glycan content secreted from Chinese hamster ovary (CHO) cells and the increase in high mannose glycans was used as a secondary tracer to provide insights into the impact each unit operation has on the residence time of the target protein (Brantley et al, 2021). Additionally, Tris mimics a process disruption and demonstrates the duration in which the product is out‐of‐specification for multiple unit operations.…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…Mannosylation modification: The content of a high-mannose structure should be minimized as much as possible during the production of recombinant antibodies due to the high immunogenicity of high-mannose structures. In addition, the content of mannose in the Fc fragment of the antibody varies greatly between different cultured cells and different batches ( Brantley et al, 2021 ). Recombinant antibodies have two clearance pathways in organisms; one is the asialoglycoprotein receptor in the liver that binds and mediates the clearance, and the other is to bind to mannose receptors on the surface of macrophages in the liver.…”

Development and validation of a platform reduced intact mass method for process monitoring of monoclonal antibody glycosylation during routine manufacturing

“…High-mannose glycans have been implicated in shortened in vivo half-life of recombinant monoclonal antibodies[ 12 ]. In addition, trace impurities in the source material used to supplement media have also been shown to modify the N-linked glycosylation profile of recombinant monoclonal antibodies [ 13 , 14 ]. The lack of significant change in the N-glycosylation profile of monoclonal antibodies from downstream purification processes allows a control strategy to be implemented much earlier in the production process.…”