The High Throughput Preparation of N-Glycans Using Waters Technologies New Glycoworks Rapifluor-MS Automation Kit On The Freedom Evo Platform

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Danielle Cullen, Philip Lambert, Stephan Koza,Jennifer Fournier
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Glycan analysis remains a key area of research across many scientific disciplines. We have developed an automation compatible Glycoworks RapiFluor MS N-glycan kit in combination with a script developed on the Tecan Freedom EVO 100 for the high throughput analysis of N-glycans. The manual preparation of N-glycan samples using the current Glycoworks RapiFluor-MS N-glycan kit has been adapted for high throughput automation to provide a flexible and reproducible solution. This automation compatible kit coupled with a robust and validated script on the Tecan Freedom EVO 100 platform removes the bottle neck of large sample batch preparation allowing analysts to focus on other tasks.

The Glycoworks RapiFluor automation script contains further features in addition to the current sample preparation process including optional concentration normalisation. This feature allows for the N-glycan analysis of glycoprotein samples ranging in initial concentration from 1.5mg/ml to 20mg/ml. The script can run with this normalisation feature on a system with four fixed pipette tips preparing 96 samples in under three hours with a CV of <5% for N-glycan area % measurements. Further features include the option to track sample barcode IDs from initial preparation to final import of a sample list for sample analysis. The Glycoworks RapiFluor MS N-glycan automation kit was created to be amenable to multiple automation platforms (e.g. Tecan, Hamilton, Agilent Bravo). Excess volumes for reagents and solvents have been designed into the kit to allow for dead volumes required for liquid detection. All SPE solutions have been included so that minimal solvent preparation is required and these solutions can be added straight to the system.

While creating a script to capture the manual assay steps is somewhat straightforward due to the intuitive softwares available for automation platforms, the effort taken to optimise and develop critical parameters such as "Liquid classes" to obtain optimal results can be significant. This fully validated script provided on the Freedom EVO 100 platform will ensure there is consistency for high throughput testing such as quality control testing. The Glycoworks RapiFluor MS script developed in combination with the Glycoworks RapiFluor MS N-glycan kit provides a robust, reproducible and time efficient all in one solution for the high throughput preparation of a complex workflow.

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