Development of a novel, label-free N-glycan method using charged aerosol detection

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Monoclonal antibodies (mAbs) are crucial biotherapeutics, with N-linked glycans on their Fc region significantly impacting efficacy and half-life.
• Current methods for N-glycan analysis involve denaturation, enzymatic release (e.g., PNGase F), and fluorescent labeling, which are time-consuming and use hazardous reagents.
• These glycans are critical quality attributes requiring robust analytical methods for biopharmaceutical release testing.

Purpose of the Study:

• To develop and validate a novel, label-free method for the detection and quantification of N-glycans from monoclonal antibodies.
• To reduce sample preparation time, costs, and the use of toxic reagents in N-glycan analysis.
• To provide an alternative analytical approach for routine biopharmaceutical quality control.

Main Methods:

• N-glycans were analyzed using a label-free approach employing mixed-mode PGC chromatography.
• Detection of non-derivatized glycans was achieved using a charged aerosol detector (CAD).
• The results were compared quantitatively against the conventional fluorescent-labeled method using HILIC.

Main Results:

• The label-free method successfully detected and quantified N-glycans without fluorescent labeling.
• The developed method demonstrated significantly reduced sample preparation time and associated costs.
• Quantitative results obtained from the label-free method were comparable to those from the conventional fluorescent-labeled approach.

Conclusions:

• A novel, label-free method using mixed-mode PGC and CAD offers a streamlined and cost-effective alternative for N-glycan analysis of mAbs.
• This method is validated and suitable for routine use in biopharmaceutical product release testing.
• The label-free approach addresses limitations of traditional methods, improving efficiency and safety in glycan analysis.