Direct quality control of glycoengineered erythropoietin variants

Čaval, Tomislav; Tian, Weihua; Yang, Zifeng; Clausen, Henrik; Heck, Albert J. R.

doi:10.1038/s41467-018-05536-3

Cited by 81 publications

(87 citation statements)

References 34 publications

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“…After overexpression of EPO, glycans were cleaved using PNGase, and then assayed by mass spectrometry. Upon retrieval of these data from the study, we picked 16 glycoprofiles that are used again in their following up study 11 and further processed the data as follows. All measurements were taken from distinct samples.…”

Section: Methodsmentioning

confidence: 99%

“…We demonstrate the functionality and performance of this approach with both protein-conjugated and unconjugated glycomic analysis, using recombinant erythropoietin (EPO) N-glycosylation and human milk oligosaccharides (HMOs). Specifically, we analyzed sixteen MALDI-TOF glycoprofiles of EPO, where each EPO glycoprofile was produced in a different glycoengineered CHO cell line 9,11 . We also analyzed forty-eight HPLC glycoprofiles of HMO from six mothers 22 .…”

Section: Introductionmentioning

confidence: 99%

“…New tools that aid in the acquisition and aggregation of glycoprofiles are emerging, making large-scale comparisons of glycoprofiles possible. Advances in mass spectrometry now enable the rapid generation of large volumes of glycoprofiles with detailed glycan composition 7–10 , exposing the complex and heterogeneous glycosylation patterns on lipids and proteins 11,12 . Large glycoprofile datasets and supporting databases are also emerging, including GlyTouCan 13 , UnicarbDB 14 , Glygen and UniCarbKB 15 .…”

Section: Introductionmentioning

confidence: 99%

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Correcting for sparsity and non-independence in glycomic data through a systems biology framework

Bao

Kellman

Chiang

et al. 2019

Preprint

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23Glycans are fundamental cellular building blocks, involved in many organismal functions. 24Advances in glycomics are elucidating the roles of glycans, but it remains challenging to 25 properly analyze large glycomics datasets, since the data are sparse (each sample often has only a 26 few measured glycans) and detected glycans are non-independent (sharing many intermediate 27 biosynthetic steps). We address these challenges with GlyCompare, a glycomic data analysis 28 approach that leverages shared biosynthetic pathway intermediates to correct for sparsity and 29 non-independence in glycomics. Specifically, quantities of measured glycans are propagated to 30 intermediate glycan substructures, which enables direct comparison of different glycoprofiles 31 and increases statistical power. Using GlyCompare, we studied diverse N-glycan profiles from 32 glycoengineered erythropoietin. We obtained biologically meaningful clustering of mutant cell 33 glycoprofiles and identified knockout-specific effects of fucosyltransferase mutants on tetra-34 antennary structures. We further analyzed human milk oligosaccharide profiles and identified 35 novel impacts that the mother's secretor-status on fucosylation and sialylation. Our substructure-36 oriented approach will enable researchers to take full advantage of the growing power and size of 37 glycomics data. 38 39 40 the rapid generation of many glycoprofiles with detailed glycan composition 7-10 , exposing the 54 complex and heterogeneous glycosylation patterns on lipids and proteins 11,12 . Large glycoprofile 55 datasets and supporting databases are also emerging, including GlyTouCan 13 , UnicarbDB 14 , 56GlyGen and UniCarbKB 15 . 57These new technologies and databases provide opportunities to examine global trends in 58 glycan function and their association with disease. However, the rapid and accurate comparison 59 of glycoprofiles can be challenging with the size, sparsity and heterogeneity of such datasets. 60Indeed, in any one glycoprofile, only a few glycans may be detected among the thousands of 61 possible glycans 16 . Thus, if there is a major perturbation to glycosylation in a dataset, few 62 glycans, if any, may overlap between samples. However, these non-overlapping glycans may 63 4 only differ in their synthesis by as few as one enzymatic step. Thus, it can be difficult to know 64 which glycans to compare. Furthermore, since glycans often share substantial portions of their 65 biosynthetic pathways with each other, statistical methods that assume independence (e.g., t-66 tests, ANOVA, etc) are inappropriate for glycomics. Here we address these challenges by 67 proposing glycan substructures, or intermediates, as the appropriate functional units for 68 meaningful glycoprofile comparisons, since each substructure can capture one step in the 69 complex process of glycan synthesis. Thus, using substructures for comparison, we account for 70 the shared dependencies across glycans. 71Previous work has investigated the similarity across glycans using glycan motifs, ...

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Correcting for sparsity and non-independence in glycomic data through a systems biology framework

Bao

Kellman

Chiang

et al. 2019

Preprint

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“…To assess sortase-mediated conjugation a small nucleophilic peptide (GGGCK(FAM)) was Orbitrap instrument with extend mass range (EMR) as described before (42). Intact Mass software was used for zero-charge deconvolution of the spectra to determine the antibody mass (43).…”

Section: Sortase-mediated Ligationmentioning

confidence: 99%

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Schoot

Fennemann

Valente

et al. 2019

Preprint

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These authors jointly supervised this work and are corresponding authors. Email: Martijn.Verdoes@radboudumc.nl (M.V.); f.a.scheeren@lumc.nl (F.A.S.). One Sentence Summary:We demonstrate a universal CRISPR/HDR based platform for rapid genetic engineering of hybridomas to obtain functionally diverse antibody isotype panels in the species and format of choice. Abstract:Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas which secrete antibodies in the preferred format, species and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemo-enzymatic modification. We believe this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

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“…3,[6][7][8][9] Glycan is a major structural moiety that has important effects on the pharmacokinetics and pharmacodynamics of EPO analogues such as darbepoetin alfa and epoetin alpha and beta. [10][11][12][13] The structure of glycan in EPO plays an important role in various biological activities. First, several studies have shown that sialic acid capping of the terminal galactose residue prevents hepatic clearance of EPO.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Characterization of N‐Glycosylation in Darbepoetin Alfa

Lee

Choi²,

Choi³

et al. 2019

Bulletin Korean Chem Soc

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EPO analogues are representative glycoprotein drugs composed of heterogeneous, multiple antennary N‐glycans. NESP® and Aranesp® are hyperglycosylated EPO, marketed under the same international nonproprietary name as darbepoetin alfa. The multiple antennary N‐glycans are a key factor that increases the half‐life of the drug in the body, which is closely related to efficacy. Thus, control of N‐glycosylation is important in providing patients with consistent efficacy. This paper reports on the comprehensive characterization of N‐glycosylation of darbepoetin alfa products. The results of N‐glycan analysis of NESP® and Aranesp® showed difference in the ratio of N‐glycans containing N‐acetyllactosamine and O‐acetylated sialic acid. Although there were some differences, the overall N‐glycan structures and profiles were comparable. In particular, darbepoetin alfa products accounted for the largest proportion of tetra‐sialo N‐glycans, greater than 75%. This study increases understanding of N‐glycan profiles and structural information of darbepoetin alfa.

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Direct quality control of glycoengineered erythropoietin variants

Cited by 81 publications

References 34 publications

Correcting for sparsity and non-independence in glycomic data through a systems biology framework

Correcting for sparsity and non-independence in glycomic data through a systems biology framework

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Comprehensive Characterization of N‐Glycosylation in Darbepoetin Alfa

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