High-Throughput Monoclonal Antibody Peptide Mapping Using 15-s HPLC Gradients Coupled with Cyclic Ion Mobility-Mass...

Area of Science:

• Biochemistry and Biophysics
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Accurate characterization of critical quality attributes (CQAs) like sequence variants and post-translational modifications (PTMs) is vital for monoclonal antibody (mAb) safety and efficacy.
• Traditional peptide mapping with liquid chromatography-mass spectrometry (LC-MS) offers simultaneous CQA monitoring but lacks the throughput for rapid analysis.

Purpose of the Study:

• To develop a high-throughput peptide mapping workflow for comprehensive mAb analysis.
• To enable rapid and high-resolution characterization of CQAs in mAb therapeutics.

Main Methods:

• Implementation of a multidimensional, high-throughput peptide mapping workflow combining fast liquid chromatography (LC) with cyclic ion mobility-mass spectrometry (cIM-MS).
• Utilized a 15-s LC gradient coupled with single-pass cIM-MS for analyzing tryptic digests of mAbs.
• Employed mobility-aligned collision-induced dissociation for precise localization of PTMs.

Main Results:

• Achieved a peak capacity of 490 and analyzed a 96-well plate in 37 minutes, demonstrating high throughput.
• Obtained 97% sequence coverage with robust reproducibility in retention time, arrival time, and peak intensity.
• Demonstrated a linear dynamic range across nearly two orders of magnitude and successfully tracked PTM kinetics during forced degradation.

Conclusions:

• The fast LC-cIM-MS platform provides a robust and efficient approach for assessing CQAs in high-throughput applications like stability studies and process monitoring.
• This method has the potential to significantly accelerate mAb development and manufacturing by enabling faster analysis.
• Highlights the utility of fast multidimensional separations for complex biological sample analysis.