At-line high throughput site-specific glycan profiling using targeted mass spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protein post-translational modifications (PTMs), particularly glycosylation, are critical for biotherapeutic protein safety and efficacy.
• Mass spectrometry (MS) is a powerful tool for characterizing protein glycosylation but often faces limitations in throughput and sample requirements.
• Existing MS methods can be time-consuming and require purified samples, hindering rapid screening.

Purpose of the Study:

• To develop a high-throughput, site-specific mass spectrometry method for analyzing protein glycosylation.
• To enable rapid screening and ranking of candidate production cell lines and culture conditions.
• To facilitate direct analysis of glycosylation profiles from conditioned culture media.

Main Methods:

• A targeted mass spectrometric peptide mapping approach was employed.
• The method combines aspects of 'top-down' and 'bottom-up' mass spectrometry for speed and site-specificity.
• Analysis was performed directly on conditioned culture media for an Fc-fusion protein.

Main Results:

• The developed method offers high throughput and site-specific characterization of protein glycosylation.
• It allows for rapid screening of cell lines and culture conditions based on glycosylation profiles.
• The approach is compatible with automation and can be integrated into process development.

Conclusions:

• This novel MS strategy provides an efficient means to assess and optimize glycosylation for biotherapeutic proteins.
• It accelerates cell line selection and process development by enabling direct analysis of culture media.
• The method supports multi-attribute product quality screening and monitoring.