High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated...

Area of Science:

• Biopharmaceutical Analysis
• Analytical Chemistry
• Glycobiology

Background:

• Protein glycosylation is critical for biotherapeutic efficacy and safety, necessitating robust monitoring throughout drug development.
• Traditional glycan analysis methods are often resource-intensive, limiting throughput and efficiency in characterizing glycoprofiles.
• Developing faster, more automated analytical platforms is crucial for timely biopharmaceutical characterization.

Purpose of the Study:

• To evaluate a comprehensive, automated liquid chromatography-mass spectrometry (LC-MS) platform for high-throughput released glycan analysis.
• To develop and optimize a rapid (5-minute) LC-FLR-MS method for N-glycan profiling.
• To demonstrate the platform's utility in biopharmaceutical comparability testing using infliximab samples.

Main Methods:

• Automated N-glycan release and fluorescent labeling using a robotic liquid handling system (2-hour process).
• Development of a 5-minute LC-FLR-MS method incorporating an integrated glycan library for identification.
• Application of the optimized workflow to analyze 48 released glycan samples from six infliximab batches.

Main Results:

• The automated workflow achieved N-glycan release and labeling in under 2 hours, minimizing user intervention.
• The 5-minute LC-FLR-MS method provided consistent relative abundance of critical glycan species (e.g., high mannose, sialylated glycans) with a mean %RSD of 5.3%.
• Data acquisition, processing, and reporting for 48 samples were completed within 6 hours, a 90% throughput improvement over conventional methods.

Conclusions:

• The developed comprehensive LC-MS workflow significantly enhances productivity and throughput for released glycan analysis.
• This automated, rapid glycan screening approach is suitable for various biopharmaceutical development stages, including process optimization and clone selection.
• The platform offers a robust solution for efficient and reliable glycan characterization, crucial for biotherapeutic quality control.