Immobilized enzyme cascade for targeted glycosylation.

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method.

CHOGlycoNET: Comprehensive glycosylation reaction network for CHO cells.

Chinese hamster ovary (CHO) cells are extensively used for the production of glycoprotein therapeutics proteins, for which N-linked glycans are a critical quality attribute due to their influence on activity and immunogenicity. Manipulation of protein glycosylation is commonly achieved through cell or process engineering, which are often guided by mathematical models. However, each study considers a unique glycosylation reaction network that is tailored around the cell line and product at hand.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies.

Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics.

The impact of the glycan distribution on the in vivo function and half-life of monoclonal antibodies has long motivated the genetic engineering of producer cells to achieve structures that enhance efficacy, safety and stability. To facilitate glycoengineering of IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis. We apply this methodology to the Fut8 gene, responsible for the addition of core fucose, which is a typical target for increasing antibody-dependent cellular cytotoxicity.

Glycoengineering Chinese hamster ovary cells: a short history.

Biotherapeutic glycoproteins have revolutionised the field of pharmaceuticals, with new discoveries and continuous improvements underpinning the rapid growth of this industry. N-glycosylation is a critical quality attribute of biotherapeutic glycoproteins that influences the efficacy, half-life and immunogenicity of these drugs. This review will focus on the advances and future directions of remodelling N-glycosylation in Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production, with particular emphasis on antibody products, using strategies such as cell line and protein backbone engineering.