4.1 Control of Biotheraputics Glycosylation

Bennun, Sandra V.; Heffner, Kelley; Blake, Emily; Chung, Andrew; Betenbaugh, Michael J.

doi:10.1515/9783110278965.247

Cited by 3 publications

(5 citation statements)

References 81 publications

(169 reference statements)

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“…1,2 Protein fucosylation can be decreased by various means including (i) manipulation of cell growth and production conditions, (ii) use of engineered cell lines in which a key enzyme involved in protein fucosylation has been knocked out, and (iii) addition of chemical inhibitors of one or more of these enzymes. 3,4 Chemical inhibition is an attractive approach to reduce fucosylation during antibody manufacture as other protein or cell growth attributes remain undisturbed. Additionally, this method allows fucose levels to be controlled based on inhibitor dosage using already optimized cell lines.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Improved antibody-dependent cellular cytotoxicity (ADCC) has been observed using IgG1 antibodies having reduced levels of Asn297 glycan fucosylation due to more effective binding of the Fc region of these proteins to the FcγRIIIa receptors of effector T cells. Increased in vivo efficacy in animal models and oncology clinical trials has been associated with improved ADCC. , Protein fucosylation can be decreased by various means including (i) manipulation of cell growth and production conditions, (ii) use of engineered cell lines in which a key enzyme involved in protein fucosylation has been knocked out, and (iii) addition of chemical inhibitors of one or more of these enzymes. , Chemical inhibition is an attractive approach to reduce fucosylation during antibody manufacture as other protein or cell growth attributes remain undisturbed. Additionally, this method allows fucose levels to be controlled based on inhibitor dosage using already optimized cell lines.…”

Section: Introductionmentioning

confidence: 99%

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Telescoped Process to Manufacture 6,6,6-Trifluorofucose via Diastereoselective Transfer Hydrogenation: Scalable Access to an Inhibitor of Fucosylation Utilized in Monoclonal Antibody Production

et al. 2016

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IgG1 monoclonal antibodies with reduced glycan fucosylation have been shown to improve antibody-dependent cellular cytotoxicity (ADCC) by allowing more effective binding of the Fc region of these proteins to T cells receptors. Increased in vivo efficacy in animal models and oncology clinical trials has been associated with the enhanced ADCC provided by these engineered mAbs. 6,6,6-Trifluorofucose (1) is a new inhibitor of fucosylation that has been demonstrated to allow the preparation of IgG1 monoclonal antibodies with lower fucosylation levels and thus improve the ADCC of these proteins. A new process has been developed to support the preparation of 1 on large-scale for wide mAb manufacture applications. The target fucosylation inhibitor (1) was synthesized from readily available d-arabinose in 11% overall yield and >99.5/0.5 dr (diastereomeric ratio). The heavily telescoped process includes seven steps, two crystallizations as purification handles, and no chromatography. The key transformation of the sequence involves the diastereoselective preparation of the desired trifluoromethyl-bearing alcohol in >9/1 dr from a trimethylsilylketal intermediate via a ruthenium-catalyzed tandem ketal hydrolysis-transfer hydrogenation process.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Telescoped Process to Manufacture 6,6,6-Trifluorofucose via Diastereoselective Transfer Hydrogenation: Scalable Access to an Inhibitor of Fucosylation Utilized in Monoclonal Antibody Production

et al. 2016

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“…use of engineered cell lines in which key enzymes involved in protein fucosylation are knocked out, or by inclusion of chemical inhibitors of fucosylation. 4,5 Some glycosidase inhibitors (including castanospermine, 6 swainsonine, 7 and N-butyldeoxynojirimycin 8 ) can result in significant changes in protein glycans, but their effects are multiple and extensive. The α-mannosidase inhibitor kifunensine can reduce antibody fucosylation more specifically in cell culture but also increases high mannose glycans, an undesirable side effect since this often results in faster clearance of antibodies in vivo.…”

mentioning

confidence: 99%

“…Incorporation of the 6,6,6-trifluorofucose moiety of 1 in recombinantly expressed antibodies occurred at low (<1%) levels. Such incorporation of a non-native sugar in antibody glycans was completely eliminated through the rational design of the phosphonate analog, fucostatin II (5). In the case of 1, the per-O-acetylated sugar is proposed to diffuse into cells as a prodrug that, once deacetylated, is then utilized in the fucose salvage pathway to form the C-1 phosphate 2C and corresponding GDP sugar 2D (Figure 2).…”

mentioning

confidence: 99%

“…Increased ADCC has been associated with increased in vivo efficacy in animal models and in the clinic. , Fucosylation levels are therefore an important product attribute to control in the production of therapeutic antibodies . Antibody fucosylation may be modulated to an extent by changing cellular growth conditions, by selection of clones that express antibodies with lower fucosylation levels, by use of engineered cell lines in which key enzymes involved in protein fucosylation are knocked out, or by inclusion of chemical inhibitors of fucosylation. , Some glycosidase inhibitors (including castanospermine, swainsonine, and N -butyldeoxynojirimycin) can result in significant changes in protein glycans, but their effects are multiple and extensive. The α-mannosidase inhibitor kifunensine can reduce antibody fucosylation more specifically in cell culture but also increases high mannose glycans, an undesirable side effect since this often results in faster clearance of antibodies in vivo .…”

mentioning

confidence: 99%

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Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase

et al. 2016

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The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

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A global RNA‐seq‐driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation

Könitzer

Müller

Leparc

et al. 2015

Biotechnology Journal

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Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality.

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4.1 Control of Biotheraputics Glycosylation

Cited by 3 publications

References 81 publications

Telescoped Process to Manufacture 6,6,6-Trifluorofucose via Diastereoselective Transfer Hydrogenation: Scalable Access to an Inhibitor of Fucosylation Utilized in Monoclonal Antibody Production

Telescoped Process to Manufacture 6,6,6-Trifluorofucose via Diastereoselective Transfer Hydrogenation: Scalable Access to an Inhibitor of Fucosylation Utilized in Monoclonal Antibody Production

Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase

A global RNA‐seq‐driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation

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