Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Abstract

Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

“…This network is affected by multiple factors, including the availability of precursor, co-factors and enzyme activities levels, all of which give rise to the variable final glycan structure. 17 Research on glycosylation variations is important to better understand glycoproteins. In recent developments, mass spectrometry has become an indispensable analytical tool for PTMs analysis due to its superior resolution over other analytical techniques.…”

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

“…Previous reports on the bio-process development of mammalian cell culture discussed site-occupancy, antennary structure, and sialylation of oligosaccharides attached to proteins, and the major physical factors shown to affect these properties included ammonia, pH, dissolved oxygen, temperature, harvest-criteria (Butler 2006;Goochee et al 1991;Hossler et al 2009), and dCO 2 (Kimura and Miller 1997;Oliveira et al 2008;Zanghi et al 1999). However, no reports have yet analyzed the effect of these physical conditions on deFuc%.…”

“…Approximately 70% of therapeutic proteins and biologic candidates are glycoproteins (Sethuraman and Stadheim 2006). Variation in glycoforms can be seen in site-occupancy (macroheterogeneity) and in the structure of attached glycans (microheterogeneity) (Butler 2006). Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein (Hossler et al 2009).…”

A simultaneous assessment metric for MAb quantity and glycan quality

“…Glycosylation, which has been identified as a Critical Quality Attribute (CQA), has become a research focus. Importantly, CHO cell lines possess the cellular machinery required to produce glycosylated MAbs (Butler 2006;Hossler et al 2009). However, many different glycoforms exist, each exhibiting different functional properties.…”