Glycans, the carbohydrate units in glycoconjugates, are important ingredients in today’s biopharmaceutical and biotherapeutics industries. Adding these multi-unit polysaccharide chains to the drug backbone makes recombinant therapeutic proteins more effective, with the effect tailored according to the conjugate’s structure. Until now full characterization of glycans has been a complex procedure due to multiple chain length and branching variations. However, a recent development from Thermo Scientific introduces a fully integrated workflow that couples novel column technology with mass spectrometric (MS) analysis for enhanced structural analysis1.
Glycans, found naturally within the body, are responsible for post-translational protein folding and degradation events, cell-to- cell interactions and blood antigen determination among other roles. When used as a post-translational modification in therapeutic protein manufacture they confer stability and bioactivity, prolonging half-life by reducing renal and immune-mediated clearance. Glycans are essential to drug performance, safety and efficacy in recombinant human proteins and monoclonal antibodies. As such, they are also subject to much scrutiny; regulatory processes require full structural characterization.
Before release onto the therapeutic marketplace, sugar sequence, linkage sites, isomers, charge and branching variations in conjugated glycan products must be fully defined. Currently scientists use hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry to determine these parameters. The liquid chromatography (LC) component separates the glycans according to hydrogen bonding, resulting in differentiation according to size and composition. Although subsequent MS analysis will characterize most glycans, it cannot identify them according to different charge states as the different species intermingle in the separation envelope.
Scientists at Thermo Scientific have recently developed a new addition to the LC tools for separation prior to MS analysis. The Glycan Pac AXH-1 column (Thermo Scientific), a dedicated high performance/ultra high performance liquid chromatography column, is specifically designed to separate glycans prior to MS or fluorescence analysis. It is based on mixed-mode surface chemistry that combines weak-anion exchange (WAX) with hydrophilic interaction liquid chromatography (HILIC) methodology for efficient glycan sorting. WAX is selective for neutral charge glycans whereas HILIC accomplishes separation based on charge, polarity and size, delivering a properly prepared sample for accurate MS analysis.
In a detailed application note released recently, Thermo Scientific scientists describe a step-by-step method for the release, fluorescence labeling, separation, and structural elucidation of N-glycans from proteins by Q Exactive hybrid quadrupole Orbitrap MS analysis (Thermo Scientific). Their method is equally applicable to native N-glycan species although the authors note that analysis of unlabeled glycans requires larger sample volumes.
In the protocol, N-glycans are released from the protein samples with an overnight incubation with PNGase F (for validation, bovine fetuin was used as the glycoprotein source) then purified by Hypercarb (Thermo Scientific) column chromatography. Following fluorescent labeling using a 2-aminobenzamide (2-AB) labeling protocol, scientists used a two-step process to clean up the glycan mixture prior to MS analysis. This involved an initial separation through a GlykoClean G cartridge before size exclusion chromatography coupled with UV detection.
A large number of different glycan species are released following glycoprotein digestion so MS analysis generates vast, complex data sets. In their workflow, the scientists used SimGlycan, a bioinformatics package (Thermo Scientific) specifically designed for glycan data analysis and structural elucidation, to process the MS spectra. The software integrated smoothly into the data processing pipeline alongside data acquisition program, Xcalibur v 2.2 SP1.48 (Thermo Scientific).
The high scan speeds and sensitivity of the Q Exactive MS and excellent resolution at 140,000 FWHM with an m/z of 200 produced high quality MS spectra regardless of glycan species abundance. Minor glycan species were easily detected. Furthermore, the higher energy collisional dissociation produces high resolution/accurate-mass fragment ions allowing better differentiation of similarly sized products. This is useful for characterizing branching and linkage properties accurately.
In addition to demonstrating accuracy in fully characterizing N-glycan species released from glycoproteins, the scientists also report that their method can analyze O-linked glycans, glycosaminoglycans and glycolipids. This new protocol successfully combines a specialized liquid chromatography (LC) column and software analysis package with sensitive Q Exactive MS technology for efficient regulatory testing.
1. Aich, U. et al “Integrated LC/MS Workflow for the Analysis of Labeled and Native N-Glycans from Proteins Using a Novel Mixed-Mode Column and a Q Exactive Mass Spectrometer”, Thermo Scientific Application Note 595 – available here http://planetorbitrap.com/data/uploads/52d6e636ee70f.pdf
- Essentials of Glycobiology from http://www.ncbi.nlm.nih.gov/books/NBK1895/
- GlycanPak AXH-1 product notes http://www.dionex.com/en-us/webdocs/114170-PS-GlycanPac-AXH1-Column-PS20695_E.pdf
- Q Exactive quadrupole MS product notes http://planetorbitrap.com/q-exactive#.UwwWy_ldWAs
- SimGlycan product notes http://www.thermoscientific.com/en/product/simglycan-software.html and online tutorials http://www.premierbiosoft.com/glycan/
- Xcalibur software for instrument control and data analysis product notes http://www.thermoscientific.com/en/product/xcalibur-software.html
For more details why not visit Thermo Scientific at booth 2441, Pittcon 2014? Full details and registration here http://event1.thermoscientific.com/content/pittcon?ca=pittcon
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer; SAHM and expat trailing spouse.
A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.