A cost-effective plate-based sample preparation for antibody N-glycan analysis.
During early cell line and process development of therapeutic antibodies, a cost-effective high-throughput approach to characterize the N-linked glycans is highly desired given that a large number of samples need to be analyzed. Using commercially available, low cost 96-well plates, we developed a practical procedure to prepare fluorescently labeled N-linked glycans for both qualitative and quantitative analysis by mass spectrometry (MS) and ultrahigh performance liquid chromatography (UPLC). Antibody samples were continuously denatured, reduced, and deglycosylated in a single 96-well hydrophobic membrane filter plate. Subsequently, released glycans were fluorescently labeled in a collection plate, and cleaned-up using a hydrophilic membrane filter plate. Carried out entirely in ready-to-use 96-well plates with simple buffer systems, this procedure requires less than 90min to finish. We applied the optimized procedure to examine the N-linked glycosylation of trastuzumab and were able to quantify ten major N-linked glycans. The results from different amounts of starting materials (10-200μg) were highly similar and showed the robustness of this procedure. Compared to other methods, this new procedure is simple to implement, economically more affordable, and could be very valuable for early screenings of antibody development.
GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH, 03766, USA.
Irina
I
Burnina
Erik
E
Hoyt
Heather
H
Lynaugh
Huijuan
H
Li
Bing
B
Gong
D000906:Antibodies / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D000068878:Trastuzumab
D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000068878:Trastuzumab
Antibody glycosylation; Fluorescence labeling; High-throughput; Plate-based