A multi-method approach toward de novo glycan characterization: a Man-5 case study

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Biotherapeutic product characterization is increasingly stringent, requiring identification of minor glycan species down to 0.1%.
• Structural isomers of Man(5)GlcNAc(2) (M(5)) have been previously reported, posing a challenge for detailed glycan analysis.
• Accurate characterization of glycan structures is critical for biotherapeutic safety and efficacy.

Purpose of the Study:

• To demonstrate a multi-method approach for de novo glycan characterization and quantitation, including minor species.
• To establish a workflow capable of identifying and quantifying glycan isomers at or approaching the 0.1% benchmark.
• To provide a comprehensive solution for biotherapeutic glycan profiling.

Main Methods:

• Development of a workflow integrating fluorescence derivatization, online rapid resolution reversed-phase separation with negative-mode sequential mass spectrometry (RRRP-(-)-MS(n)).
• Utilizing permethylation derivatization coupled with nanospray sequential mass spectrometry (NSI-MS(n)) for glycan structural determination.
• Employing a commercially available stable isotope variant of 2-aminobenzoic acid for enhanced detection and resolution of M(5) isomers in bovine ribonuclease B.

Main Results:

• Quantitative analysis confirmed low abundance (<1%) of M(5) isomers in commercial standards, validating their use as a 'litmus test' for minor species characterization.
• The multi-method workflow successfully achieved comprehensive glycan structural determination, mitigating individual method limitations.
• Demonstrated the first quantitative chromatographic separation of M(5) isomers and their detection using a stable isotope labeled reagent.

Conclusions:

• A multi-method workflow provides a robust solution for comprehensive de novo glycan characterization, addressing limitations of single analytical techniques.
• This approach enables the characterization of biotherapeutic glycan arrays, including structural isomers at levels of 0.1% or higher.
• The developed workflow enhances the ability to meet stringent regulatory expectations for biotherapeutic product quality.