engleski

AUTOMATION OF IgG N-GLYCAN SAMPLE PREPARATION METHOD FOR HIGH THROUGHPUT ANALYSIS

Role of protein N-glycosylation and especially changes in IgG Nglycosylation pattern has repeatedly been confirmed as crucial in different physiological and pathological processes. Expanding knowledge requires more affordable, reliable, and higher throughput methods. New approaches to glycosylation analysis would allow its optimization and application in the biopharmaceutical industry, epidemiology as well as advance clinical diagnostics that would rely on glycan biomarkers. Though there were successful attempts to develop new automated strategies for glycan research, many laboratories still leverage manual sample preparation protocols that limit their throughput. To help address this issue, automated methods for glycosylation analysis should be developed further. This research proposes one possible approach to automation of IgG glycan sample preparation using the Tecan Freedom Evo liquid handling automated platform. To improve throughput and robustness, the manual method was substituted by the automated system that was equipped with a liquid handling arm (LiHa) and a robotic manipulator arm (RoMa), while the vacuum manifold operations were replaced with A200, a positive pressure unit which allows for higher pressure and flow-through. The adapted automated protocol features IgG isolation on a protein G plate, IgG deglycosylation, and glycan 2-AB labeling. This automated protocol allows the samples to be analyzed using ultra-high- performance liquid chromatography (UPLC). UPLC analysis showed that peaks' number, retention time, and peak area corresponded with the manual method. Peaks with the largest area showed small variation, while the smaller peaks exhibited larger variation. Method has yielded promising results and could encourage additional automation efforts in other protocols with development.

liquid handling, lab automation, glycomics, UPLC

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