title: Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins.
abstract: A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.
authors: Analytical Chemistry, Inhibitex, Alpharetta, GA 30004, USA. bprater@amgen.com
authors: Bradley D
authors: BD
authors: Prater
authors: Kalyan R
authors: KR
authors: Anumula
authors: Jeff T
authors: JT
authors: Hutchins
mesh_terms: D001331:Automation; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry; D009757:Nylons / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D012117:Resins, Synthetic / Q000737:chemistry; D013954:Thyroglobulin / Q000032:analysis / Q000737:chemistry; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D009757:Nylons; D009844:Oligosaccharides; D012117:Resins, Synthetic; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D013954:Thyroglobulin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase