Ultrahigh Throughput, Ultrafiltration-Based <i>N</i>‑Glycomics Platform for Ultraperformance Liquid Chromatography (ULTRA³)

Accurate, reproducible, and fast quantification of <i>N</i>-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker discovery. Several methods exist for fluorescent labeling of <i>N</i>-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy, and expensive. Here we report an automated ultrafiltration-based <i>N-</i>glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 μg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform

<i>N</i>‑Glycosylation of Serum IgG and Total Glycoproteins in MAN1B1 Deficiency

MAN1B1-CDG has recently been characterized as a type II congenital disorder of glycosylation (CDG), disrupting not only protein <i>N</i>-glycosylation but also general Golgi morphology. Using our high-throughput, quantitative ultra-performance liquid chromatography assay, we achieved a detailed characterization of the glycosylation changes in both total serum glycoproteins and isolated serum IgG from ten previously reported MAN1B1-CDG patients. We have identified and quantified novel hybrid high-mannosylated MAN1B1-CDG-specific IgG glycans and found an increase of sialyl Lewis <i>x</i> (sLe<i>x</i>) glycans on serum proteins of all patients. This increase in sLex has not been previously reported in any CDG. These findings may provide insight into the pathophysiology of this CDG

Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow

The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from <i>Rhodococcus triatomea</i>, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. <i>R. triatomea</i> EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)­Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery

Aminoquinoline Fluorescent Labels Obstruct Efficient Removal of <i>N</i>‑Glycan Core α(1–6) Fucose by Bovine Kidney α‑l‑Fucosidase (BKF)

Here we report evidence that new aminoquinoline <i>N</i>-glycan fluorescent labels interfere with the release of core α(1–6) fucose from <i>N</i>-glycans by bovine kidney α-l-fucosidase (BKF). BKF is a commonly employed exoglycosidase for core α(1–6) fucose determination. Molecular simulations of the bound and unbound Fuc-α(1–6)-GlcNAc, where GlcNAc is situated at the reducing end for all <i>N</i>-glycans, suggest that the reduced BKF activity may be due to a nonoptimal fit of the highest populated conformers in the BKF active binding site at room temperature. Population analysis and free energy estimates suggest that an enhanced flexibility of the labeled sugar, which facilitates recognition and binding, can be achievable with extended reaction conditions. We provide these experimental conditions using a sequential exoglycosidase digestion array using high concentrations of BKF

Glycan composition of TGA and HRP.

<p>Glycan composition of TGA and HRP.</p

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems—The taliglucerase alfa story

<div><p>Plants are a promising alternative for the production of biotherapeutics. Manufacturing <i>in-planta</i> adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies.</p></div

Schematic representation of the assay.

<p>A stepwise format (1–5) was developed for the binding of serum antibodies to TGA (A) with HRP and (B) without prior HRP incubation, showing a response reduction in the presence of HRP.</p

Total ADA and anti-plant glycan antibodies prevalence found in GD patient population treated with TGA.

<p>(A) Data evaluated from ERT-naïve patient samples and (B) ERT-experienced patient samples. *One patient of the ERT-experienced group, was counted for both baseline ADA and treatment induced ADA, since he was ADA positive at baseline and had a treatment-boosted following treatment with TGA (had ≥6-fold higher titer after TGA treatment).</p

Assay cut-point determination.

<p>Percent (%) Immunodepletion by HRP of healthy individual serum samples. The data show three independent runs and their mean± standard deviation. Individuals 5 and 40 (highlighted) were identified as outliers, and were excluded from the calculation.</p

A Consort flowchart of the clinical studies included in the study.

<p>Consort flowchart shows all three studies with original enrolment, number of excluded subjects and the final amount of subjects included in the immunogenicity tests.</p