Rapid, High-Resolution N-linked Oligosaccharide Analysis with SCIEX CE* for Comprehensive Glycan Characterization
Therapeutic protein glycosylation is an important post-translational modification that can affect protein function, clearance, and stability. Variations in glycosylation have been implicated in the regulation of immune responses like antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
Due to the microheterogeneity of therapeutic proteins, an analytical method capable of resolving a large number of these glycan species is needed to quickly and confidently enable their identification. Using a fluorophore derivatization strategy, glycans can be provided charge and fluorescent properties enabling separation and detection by CE. SCIEX offers standard glycan analysis technology as well as a new high-speed methodology.
Key benefits to carbohydrate profiling using capillary electrophoresis include:
Quantitation of N-linked oligosaccharides
High-resolution separation methods capable of differentiating critical glycan species including positional isomers
Validated methodology developed for routine use environments
Rapid sample preparation and analysis of monoclonal antibody N-glycans by magnetic bead technology and CE-LIF
Results: Using a novel sample preparation protocol in which magnetic bead technology is utilized for clean-up, N-linked oligosaccharide sample preparation and workflow takes less than four hours to complete. The resulting benefit of this sample prep is the omission of centrifugation and/or vacuum centrifugation steps, allowing compatibility with fully automated liquid handling systems like Beckman Coulter’s Biomek platform. Additionally, this approach includes unbiased glycan labeling and electrokinetic sample injection for unambiguous detection of highly sialylated carbohydrates that otherwise may co-migrate with excess APTS dye.
Fully automated sample preparation with ultrafast N-glycosylation analysis of therapeutic antibodies
Standard Method: Oligosaccharide Analysis Simplified
The PA 800 Plus Carbohydrate Labeling and Analysis Assay contain reagents, buffers, and separation capillaries for the analysis of N-linked oligosaccharides. These characteristics are required for labeling, separation and quantification of oligosaccharides released from glycoproteins. Following release (enzymatic or chemical), glycans are labeled with 1-amino 3, 6, 8 - pyrene trisulfonic acid (APTS) at the reducing terminus using a reductive amination reaction. The stoichiometry of labeling is such that only one APTS molecule is attached to each glycan. The highly charged and fluorescent oligosaccharides can then be easily resolved in an electric field and detected by laser-induced fluorescence. APTS derivatized glycans moving past the laser induced fluorescence detector yield an equal detector response so relative quantities can be directly compared. Oligosaccharide populations associated with a protein yield a fingerprint that can be used for protein identification or interpretation of quality or function. The PA 800 Plus provides an easy, quantitative and robust determination of protein micro-heterogeneity with a typical analysis time of fewer than 15 minutes.
Carbohydrate Labeling and Analysis Include:
Carbohydrate Separation Buffer, 56 mL
N-CHO Coated Capillary, 2 pieces
Labeling Dye (APTS), 4 x 5 mg
Labeling Dye Solvent, 1 mL
Glucose Ladder Standard, 50 mg
Quantitation/Mobility Marker (Maltose), 0.18 mg
APTS-M (monosaccharides grade), 20 mg
Carbohydrate Labeling and Analysis Guide, 1 piece
System and Chemistry Features:
Unique Capillary Cooling
Unique High Resolution Separation Methods
Ability to Separate Positional Isomers of Glycoproteins
Unique Sample Cooling
Incorporation of separation buffer modifications enables increased separation of co-migrating species thus enhancing the assay's identification and quantitation capability. In the figure above, up to eighteen different glycans were resolved from one another using this optimized carbohydrate separation method.
Other examples of Glycoprotein Analysis
Analysis of Ribonuclease B high mannose glycoforms
Separation of the 1-aminopyrene-3, 6, 8-trisulfonate-labeled high mannose type oligosaccharides released from bovine ribonuclease B and the individual standard structures. Capillary: 50 cm (to detector) x 50 um eCAP NCHO coated capillary. Buffer: 25 mM acetate (pH 4.75) with 0.4% polyethylene oxide. Field Strength: 500 V/cm; Temperature 20 °C; Detection: Laser-induced fluorescence (excitation at 488 nm, emission at 520 nm).
The analysis of monosaccharide composition can provide valuable information during the initial characterization of a glycoprotein. A clear advantage of our CE-based approach is in the specificity of the assay. The reductive amination and open-tube format allow this analysis to be performed directly from the glycoprotein hydrolysate--no need for post hydrolysis cleanup. With LIF and APTS derivatization, each sugar yields the same detector response, such that their relative quantities can be directly compared.
CE separation of (A) the mild acid hydrolysis product of fetuin after aldolase treatment. (B) The strong acid hydrolysis product after reacetylation and (C) monosaccharide standards. Capillary: 27 cm x 25 um bare fused silica; Buffer: 240 mM borate (pH 9.0); Field Strength: 740 Volts/cm; Injection: 0.5 psi for 5; Detection: Laser-induced fluorescence (excitation at 488 nm, emission at 520 nm).
The solution-based approach of CE allows one to easily create an environment to assess differences in a protein's glycosylation state--directly from the protein. Through the addition of salts like borate, complexes which amplify the charge state of the glycovariants can be formed. This is the simplest approach to assessing the heterogeneity of a glycoprotein.