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19 protocols using glycoworks rapifluor ms n glycan kit

1

Enzymatic Release and RFMS Labeling of N-Glycans

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The enzymatic release and RFMS labelling of N-glycans was performed according to the Waters Glycoworks RapiFluor-MS N-Glycan Kit Care and Use Manual [28 ]. The protocol was carried out by introducing a glycoprotein quantity of 15 µg for each antibody product. The obtained RFMS derivatized N-glycans were directly analysed after labelling. For the RFMS label, approximately 5 pmol of labelled glycan material was loaded onto the column.
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2

IgG N-Glycan Analysis by RapiFluor-MS

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Deglycosylation, RapiFluor-MS labelling and purification of IgG N-glycans were performed using the GlycoWorks RapiFluor-MS N-Glycan Kit obtained from Waters Corporation (USA). The entire procedure was done following Waters’ protocol (Waters Corporation, 2017, https://www.waters.com/webassets/cms/support/docs/715004793en.pdf), which was further adapted to suit the high-throughput analysis in the 96-well PCR plate format (Deriš et al., 2021 (link)). Isolated and dried IgG was first resuspended in 10.8 μL ultrapure water, swiftly denatured with 3 μL 5 % RapiGest SF solution in a 3 min reaction at 99°C and enzymatically deglycosylated with 1.2 μL GlycoWorks Rapid PNGase F in a 5 min reaction at 50°C. Released N-glycans were then labeled with the RapiFluor-MS label. RapiFluor-MS dye contains a rapid tagging function group, N-hydroxysuccinimide carbamate, which rapidly modifies glycosylamine-bearing N-glycans after their enzymatic release, yielding a stable urea linkage (Lauber et al., 2015 (link)). Labeled N-glycans were purified by hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE) clean-up. In the end, the samples of released and labelled IgG glycans were stored at -20 °C until further use.
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3

N-Glycan Profiling of Plasma Membrane

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Glycoworks RapiFluor-MS N-Glycan kit was purchased from Waters Corporation (MA, USA). Minute™ plasma membrane protein isolation kit was purchased from Invent Biotechnologies (MN, USA). IgG from porcine serum, fetuin from fetal bovine serum, lactoferrin from bovine milk and ribonuclease B from bovine pancreas were all purchased from Sigma-Aldich (St. Louis, MO USA). Acetonitrile of LC–MS grade was purchased from Merck (Darmstadt, Germany). Ultra-pure water was prepared by ELGA LabWater (resistivity ≥18.2 MΩ×cm, 25 °C).
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4

N-Glycan Oligosaccharide Analysis by HPLC-MS

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N-Glycan oligosaccharide analysis was performed30 using the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters Corporation, Milford, MA). Fluor-MS N-glycan analysis was performed using an Agilent 1260 Infinity II HPLC system equipped with a 1260 FLD detector and an Agilent 6230 electrospray ionization Time-of-Flight mass spectrometer (Agilent, Santa Clara, CA). A HILIC AdvanceBio Glycan Mapping column (120 Å, 2.1 × 150 mm, 2.7 μm), operated at 45°C, was used to separate various N-glycans. Fluorescence was obtained using excitation and emission wavelengths of 265 and 425 nm, respectively. MS was acquired simultaneously from 400 to 2000 m/z at a constant scan rate of one spectrum per second. N-glycans were assigned based on m/z values using a N-glycan database (Water/NIBRT Glycan 3+) and N-glycan quantification was calculated on integration of the fluorescence chromatogram.
Site-specific glycosylation analysis was also performed as described previously.52 (link)
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5

IgG N-Glycan Analysis by RapiFluor-MS

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Deglycosylation, RapiFluor-MS labelling and purification step of IgG N-glycans was performed using GlycoWorks RapiFluor-MS N-Glycan Kit obtained from Waters Corporation (Milford, USA), in compliance with the manufacturer’s protocol [13 ]. All samples containing eluted and labelled glycans were stored at -20 °C until further use. RapiFluor-MS labelled IgG N-glycans were analysed using HILIC-UPLC-FLR on Water Acquity UPLC H-class instruments. Detailed description of the analysis is available in the Supplementary Material (Supplementary Material: EXPERIMENTAL PROTOCOL DESCRIPTION).
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6

IgG Isolation and N-Glycan Analysis

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IgG was isolated using a 96-well protein G monolithic plate (BIA Separations, Slovenia, Cat No. 120.1012-2)19 using a protocol described by Trbojević-Akmačić and colleagues.20 (link) After IgG isolation, IgG eluates were heated at 65°C for 30 minutes to reduce the risk of any potential residual virus in the IgG eluate. An appropriate volume of IgG was aliquoted in a PCR plate (Thermo Scientific, UK, Cat. No. AB1300) and dried in a vacuum centrifuge, if needed. Deglycosylation, released N-glycan labeling and clean-up were performed using two different kits, GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, USA, Cat. No. 176003910) and AdvanceBio Gly-X N-glycan Prep with InstantPC Kit (Agilent, USA, Cat. No. GX96-IPC) according to the manufacturer's instructions.
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7

N-Glycan Profiling of Secretome Proteins

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After centrifugation for cell and debris removal, the supernatant (~3 mL) was concentrated using Amicon® Ultra-4 Centrifugal Filter Units per manufacturer’ instructions. Secretome proteins were fluorescently labeled with GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, MA) per manufacturer’s protocol. N-linked glycan analysis was performed by LC-MS using a Thermo Ultimate 3000 HPLC with the fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS (run in positive mode). Separation gradient was 30% to 43% buffer. The amount of N-glycan was measured by integrating the areas under the normalized fluorescence spectrum peaks with Thermo Xcalibur software (Thermo Fisher Scientific, Waltham, MA) giving a normalized, relative glycan quantities.
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8

N-Glycan Analysis of Secretome

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Example 3

N-glycan analysis was performed with GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass.) according to the manufacturer's instruction. In this case 12 μl of 10× concentrated (MWCO filtered, Amicon Ultra-15, Merck, Darmstadt, Germany) secretome or purified protein sample were used for each. Labeled N-Glycans were analyzed by a LC-MS system using a Thermo Ultimate 3000 HPLC with fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS. Separation gradient 30% to 43% buffer and MS was run in positive mode.

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9

N-Glycan Profiling of Therapeutic Proteins

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For Rituximab, Enbrel, and alpha-1-antitripsin, 12 μL of concentrated protein solutions (concentrations varying between 0.1 and 1 mg/mL) were subjected to N-glycan labeling using the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters). Labeled N-glycans were analyzed by LC-MS as described previously [53 (link)]. Initial conditions 25% 50 mM ammonium formate buffer 75% Acetonitrile, separation gradient from 30% to 43% buffer. MS were run in positive mode, no source fragmentation. The normalized, relative amount of the N-glycans is calculated from the area under the peak with Thermo Xcalibur software (Thermo Fisher Scientific).
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10

Automated N-glycan Release and Labeling for Mass Spectrometry

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The N-glycans were prepared using the GlycoWorks RapiFluor-MS N-glycan kit (Waters) on an
Andrew Alliance pipetting robot (Geneva, Switzerland) as described previously.19 (link) Briefly, 10 µL of diluted infliximab sample was loaded to a 96-well sample plate.
Reagents including RapiGest surfactant, Rapid PNGase F enzyme, and RFMS
labeling reagent were used for protein denaturation, N-glycan release, and labeling,
respectively, by following a modified protocol in the GlycoWorks RapiFluor-MS
N-Glycan Kit Care and Use Manual
(p/n 715004793).19 (link) A total of 48 released N-glycan samples were prepared, including 12 samples from
batch 1 of innovator infliximab, 6 samples from each of the other three batches of
innovator infliximab, 6 samples for each of two batches of biosimilar infliximab, and 6
samples from a blank control solution. After the glycan labeling protocol was completed,
0.25 pmol of a high mannose glycan standard (Waters) was spiked into each of six released
glycan samples prepared from innovator batch 1. This group of samples was used to evaluate
the capability of the workflow to capture the relative abundance changes in glycans across
samples.
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