Engineered CHO cells for production of diverse, homogeneous glycoproteins

Yang, Zifeng; Wang, Shengjun; Halim, Adnan; Schulz, Morten Alder; Frödin, Morten; Rahman, Shamim H.; Vester-Christensen, Malene Bech; Behrens, Carsten; Kristensen, Claus; Vakhrushev, Sergey Y.; Bennett, Eric; Wandall, Hans H.; Clausen, Henrik

doi:10.1038/nbt.3280

Cited by 212 publications

(197 citation statements)

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“…Although such cells produce properly sialylated glycoproteins, they have several limitations, like glycan heterogeneity and the incapability to produce distinct glycoforms on demand, that hamper further investigation on the biological impact of sialylation and the development of optimized therapeutic proteins. Substantial efforts have been devoted to genetic engineering of mammalian cells and other organisms to expand their glycosylation capacity and to improve or alter glycosylation, including sialylation (9,10). Despite impressive recent achievements, designed sialylation strategies are largely elusive.…”

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confidence: 99%

Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

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confidence: 99%

Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Comprehensive glycomic characterization of clinical isolates may lead to identification of sites and structures important for proteinprotein interaction or raising potent immune responses. Using our expanding library of glycoengineered cell lines would enable production of designer viruses presenting defined glycostructures on envelope glycoproteins for antiviral vaccine development (125).…”

Section: Discussionmentioning

confidence: 99%

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

Bagdonaite

Nordén

Joshi

et al. 2016

Journal of Biological Chemistry

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Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a "bottom up" mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.

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“…Because of its large glycome however, CHO cellderived glycoproteins exhibit substantial glycan heterogeneity, precluding the ability to generate distinct glycoforms that could be used in comparative studies of specific biological effects (4). Furthermore, N-glycan processing in CHO cells is prone to environmental variation and is difficult to control during bioprocessing (4). CHO cells also add sialic acid in a different linkage and do not produce bisecting GlcNAc structures that are common in human glycoproteins (1).…”

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confidence: 99%

“…manipulation of the N-glycosylation pathway in mammalian cells (4). Plants also exhibit a remarkable tolerance toward various glycan manipulations and in response display no major phenotypic changes in growth or development (1).…”

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confidence: 99%

“…The majority of pharmaceutical companies currently use Chinese hamster ovary (CHO) cells to produce human biologics. CHO cells generally produce N-glycans with structures similar to their human counterparts (4). Because of its large glycome however, CHO cellderived glycoproteins exhibit substantial glycan heterogeneity, precluding the ability to generate distinct glycoforms that could be used in comparative studies of specific biological effects (4).…”

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confidence: 99%

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