Engineering a Human-Like Glycosylation to Produce Therapeutic Glycoproteins Based on 6-Linked Sialylation in CHO Cells

Abstract

When recombinant glycoproteins for therapeutic use are to be produced on an industrial scale, there is a crucial need for technologies that can engineer fast-growing stable cells secreting the protein drug at a high rate and with a defined and safe glycosylation profile. Current cell lines approved for drug production are essentially from rodent origin. Their glycosylation machinery often adds undesired carbohydrate determinants which may alter protein folding, induce immunogenicity, and reduce circulatory life span of the drug. Notably, sialic acid as N-acetylneuraminic acid is not efficiently added in most mammalian cells and the 6-linkage is missing in rodent cells. Engineering cells with the various enzymatic activities required for sialic acid transfer has not yet succeeded in providing a human-like pattern of glycoforms to protein drugs. To date, there is a need for engineering animal cells and get highly sialylated products that resemble as closely as possible to human proteins. We have designed ST6Gal minigenes to optimize the ST6GalI sialyltransferase activity and used them to engineer ST6(+)CHO cells. When stably transfected in cells expressing a protein of interest or not, these constructs have proven to equip cell clones with efficient transfer activity of 6-linked sialic acid. In this chapter, we describe a methodology for generating healthy stable adherent clones with hypersialylation activity and high secretion rate.

3.5 Manipulation of Cell Growth, Metabolism and Product Quality Attributes

“…Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/6/15 11:56 AM Rodent cell lines such as CHO are not able to attach terminal sialic acid to N-glycans via an α-2,6-linked glycosidic bond [164]. In order to achieve an efficient formation of α-2,6-linked sialylation, CHO host cells were transfected with ST6Gal [164].…”

“…In order to achieve an efficient formation of α-2,6-linked sialylation, CHO host cells were transfected with ST6Gal [164].…”

Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

“…Some of them regard the design of the expression vector and, for example, make use of inducible promoters and/or epigenetic regulators to increase and prolong transgene expression while decreasing toxicity of the expressed recombinant protein [13], [14], [15], [16]. Others approaches aim at manipulating pathways through cell engineering, in order to improve stress resistance, cell viability or to achieve better glycosylation profiles [7], [17]. Despite much progress has been made in this field, clonal variability and instability are still important issues that need to be addressed, particularly when production on large scales (1000's liters) is required.…”

“…Moreover, with the progress of systems biology, it will be possible to manipulate cells to introduce entire new molecular pathways ( e.g. human-like glycosylation) [17], [29]. The rational engineering of such robust and high-performing cells for specific applications can lead to a catalog of different cell lines, each optimized to tackle specific targets.…”

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

“…They reported altered glycan‐type ratios in the recombinant cell line. Maï et al reported a host CHO cell line with a St6gal1 minigene integrated to overexpress ST6GAL1, and they generated adherent clones secreting hypersialylated proteins …”