Automated Preparation of MS-Sensitive Fluorescently Labeled N-Glycans with a Commercial Pipetting Robot

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• N-glycan analysis is crucial for characterizing biotherapeutic proteins.
• Existing methods like reductive amination are time-consuming.
• A new kit using RapiFluor-MS (RFMS) offers improved speed and detection.

Purpose of the Study:

• To demonstrate the semiautomation of the RFMS N-glycan analysis kit.
• To evaluate the productivity gains and reproducibility of the automated protocol.
• To adapt the RFMS method for use with automated liquid handling systems.

Main Methods:

• Semiautomation of the RFMS N-glycan analysis kit using an Andrew Alliance pipetting robot.
• Optimization of denaturation and de-N-glycosylation temperatures for the automated platform.
• Analysis of RFMS-labeled glycan profiles using hydrophilic interaction liquid chromatography (HILIC).

Main Results:

• The automated protocol achieved percent relative standard deviations (RSDs) of 9%-19% for total glycan area.
• Reproducibility for major and minor glycoforms ranged from 0.8%-20% RSD.
• The semiautomated method demonstrated convenience and reliability for N-glycan preparation.

Conclusions:

• Semiautomation of the RFMS N-glycan analysis kit using a pipetting robot significantly enhances productivity.
• The optimized automated protocol provides reproducible N-glycan profiles suitable for biotherapeutic characterization.
• This robotic platform offers a convenient and reliable solution for high-throughput N-glycan analysis.