Comprehensive analysis of low abundant mannose glycopeptides in peptide mapping of adalimumab

Significance of the Topic

Glycosylation profoundly influences the stability, bioactivity and immunogenicity of monoclonal antibodies (mAbs). Monitoring low‐abundance glycoforms, particularly high mannose species, is essential for ensuring consistent product quality throughout development and manufacturing of biotherapeutics.

Objectives and Overview of the Study

This study demonstrates the use of capillary electrophoresis–electrospray ionization mass spectrometry (CESI-MS) on the SCIEX CESI 8000 Plus coupled to a TripleTOF 6600+ System for bottom-up peptide mapping of adalimumab. The goal is to achieve high‐resolution separation and confident identification of low‐abundance mannose and fucosylated glycopeptides, alongside routine peptide mapping and post-translational modification (PTM) analysis.

Methodology and Instrumentation

Sample Preparation and Digestion:

• Adalimumab (1 mg/mL) reduced with DTT, alkylated with iodoacetamide, then digested with trypsin (1:20 enzyme:protein, 2 h at 37 °C).

CESI-MS Conditions:

• Bare fused‐silica OptiMS cartridge (44 nL injection, ~39 ng protein).
• Background electrolyte: 100 mM ammonium acetate, pH 4.
• Hydrodynamic injection, voltage and pressure rinse steps (NaOH, HCl, water, acetic acid).
• Separation by capillary zone electrophoresis at ~20 nL/min flow.

MS Acquisition:

• TripleTOF 6600+ with NanoSpray III source, Analyst 1.8.1 control.
• Data‐dependent acquisition (DDA) with 10 MS/MS scans (MS:150 ms; MS/MS:50 ms).
• m/z range 100–2250, charge states +1 to +5, intensity threshold 100 cps.
• Rolling collision energy with charge‐dependent slopes for optimized glycopeptide fragmentation.

LC-MS Comparison:

• Waters CSH C18 (2.1×150 mm, 1.7 µm) on ExionLC AD with X500B QTOF.
• Gradient from 1% to 90% acetonitrile (0.1% formic acid) over 75 min.

Main Results and Discussion

High-Mannose Glycopeptides:

• CESI-MS baseline‐separated Man5, Man6, Man7 and Man8 glycopeptides, whereas RP-LC-MS showed coelution.
• MS/MS spectra exhibited specific y-ions (e.g., y-ion at m/z 793 for loss of five mannose units) and diagnostic glycan fragments (m/z 204, 366, 528).

Fucosylated and Minor Glycoforms:

• Clear separation of G0F, G1F, G2F by CESI, with LC-MS missing low‐abundance G2F.
• Identification of minor species such as G0, G0F-GlcNAc and G1F-GlcNAc, for a total of 11 distinct glycopeptides.

Peptide Mapping and PTMs:

• Sequence coverage >95% from <40 ng sample.
• Separation of deamidated vs unmodified peptides (~6% deamidation in VSVLTVLHQDWLNGK).
• Resolution of methionine‐oxidized peptides (~1.2% oxidation in NSLYLQMNSLR).
• Detection of short hydrophilic peptides (e.g., VSNK) often overlooked by LC-MS.

Benefits and Practical Applications of the Method

CESI-MS offers an orthogonal approach to LC-MS with ultra-low flow rates that enhance ionization efficiency and sensitivity. It enables comprehensive mapping of glycosylation patterns and PTMs in a single run with minimal sample consumption, supporting critical quality attribute (CQA) monitoring in mAb development and QC workflows.

Future Trends and Possibilities for Use

Integration of CESI-MS into routine biopharma QC for real-time monitoring of glycoforms and PTMs
Coupling with data‐independent acquisition (DIA) methods for deeper glycoproteome coverage
Automation and high-throughput CE-MS platforms for broader applications in biosimilar comparison, viral vector characterization, and glycosylation profiling of novel protein therapeutics

Conclusion

CESI 8000 Plus coupled to the TripleTOF 6600+ System provides high-resolution separation and reliable identification of low-abundance mannose and fucosylated glycopeptides in adalimumab peptide mapping. The approach delivers >95% sequence coverage, resolves challenging PTMs, and complements LC-MS for comprehensive characterization of therapeutic antibodies.

References

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