Insect Cell Glycosylation Patterns in the Context of Biopharmaceuticals

Geisler, Christoph; Jarvis, Donald L.

doi:10.1002/9783527626601.ch7

Cited by 19 publications

(30 citation statements)

References 116 publications

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“…1A). This problem has been addressed through glycoengineering efforts, in which mammalian genes have been incorporated into baculovirus expression vectors and/or lepidopteran insect cell lines (reviewed by Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; Jarvis, 2009; Shi and Jarvis, 2007). These efforts have yielded modified baculovirus/insect cell systems with more extensive protein N -glycosylation pathways and the capacity to sialylate recombinant glycoproteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This requirement restricts the utility of the unmodified baculovirus/insect cell system as a glycoprotein biologics production platform, which does not support recombinant glycoprotein sialylation (reviewed by Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; Jarvis, 2009; Marchal et al, 2001; Shi and Jarvis, 2007). The inability of the unmodified baculovirus/insect cell platform to support recombinant glycoprotein sialylation is due to the fact that lepidopteran insect cell lines lack functional levels of late acting glycosyltransferases, as well as the pathways needed for sialic acid biosynthesis and utilization (reviewed by Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; Jarvis, 2009; Marchal et al, 2001; März et al, 1995; Shi and Jarvis, 2007). Previous studies have shown that these deficiencies can be corrected by metabolically engineering baculovirus vectors and/or their lepidopteran insect cell hosts with mammalian genes encoding these functions (reviewed by Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; Jarvis, 2009; Shi and Jarvis, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…This limitation has been addressed at the basic research level by metabolic engineering, which has been used to extend the endogenous processing capabilities of the insect cell protein N -glycosylation pathway (reviewed by Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; Jarvis, 2009; Shi and Jarvis, 2007). These glycoengineering efforts have yielded genetically modified baculovirus/insect cell systems capable of producing N -glycoproteins with complex, mammalian-type carbohydrate side chains ( N -glycans) terminated with the most common sialic acid, N -acetylneuraminic acid (Neu5Ac; Aumiller et al, 2003; Aumiller et al, 2012; Hill et al, 2006; Hollister et al, 2002; Hollister and Jarvis, 2001; Jarvis et al, 2001; Seo et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Geisler

Jarvis

2012

Metabolic Engineering

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The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Geisler

Jarvis

2012

Metabolic Engineering

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“…However, the production of this intermediate represents a key branch point because it is subsequently elongated to produce hybrid and complex N-glycans in mammalian cells, whereas it is subsequently trimmed to produce paucimannosidic N-glycans in most insect cells [28][29][30][31] . An unusual ß-N-acetylglucosaminidase, designated fused lobes (FDL) for its phenotype in mutant flies 36 , catalyzes the trimming reaction that yields paucimannosidic N-glycan products in insect cells [37][38][39][40] .…”

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confidence: 99%

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology

et al. 2015

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Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.

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“…We then purified the recombinant hEPO from the cell-free media and analyzed samples of each by SDS-PAGE. Previous studies have shown that Sf9 cells produce recombinant glycoproteins with mainly paucimannose-type N -glycans plus a small subpopulation of hybrid structures with a single terminal N -acetylglucosamine residue (Altmann et al, 1999; Geisler and Jarvis, 2009; Harrison and Jarvis, 2006; März et al, 1995). In addition, previous studies have shown that Sf9 cells expressing bovine B4GALT1 can produce mono-antennary, terminally galactosylated N -glycans (Hollister et al, 1998; Jarvis and Finn, 1996).…”

Section: Resultsmentioning

confidence: 99%

Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells

Geisler

Mabashi-Asazuma

Kuo

et al. 2015

Journal of Biotechnology

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β1,4-galactosyltransferase (B4GALT1) is a Golgi-resident enzyme that elongates glycoprotein glycans, but a subpopulation of this enzyme is secreted following proteolytic cleavage in its stem domain. We hypothesized that engineering B4GALT1 to block cleavage and secretion would enhance its retention and, therefore, its function. To test this hypothesis, we replaced the cytoplasmic/transmembrane/stem (CTS) domains of B4GALT1 with those from human α1,3-fucosyltransferase 7 (FUT7), which is not cleaved and secreted. Expression of FUT7-CTS-B4GALT1 in insect cells produced lower levels of secreted and higher levels of intracellular B4GALT1 activity than the native enzyme. We also noted that the B4GALT1 used in our study had a leucine at position 282, whereas all other animal B4GALT1 sequences have an aromatic amino acid at this position. Thus, we examined the combined impact of changing the CTS domains and the amino acid at position 282 on intracellular B4GALT1 activity levels and N-glycan processing in insect cells. The results demonstrated a correlation between the levels of intracellular B4GALT1 activity and terminally galactosylated N-glycans, N-glycan branching, the appearance of hybrid structures, and reduced core fucosylation. Thus, engineering B4GALT1 to reduce its cleavage and secretion is an approach that can be used to enhance N-glycan elongation in insect cells.

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Insect Cell Glycosylation Patterns in the Context of Biopharmaceuticals

Cited by 19 publications

References 116 publications

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology

Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells

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