Hypersil GOLD Peptide column for reliable monitoring of glycopeptide variants in monoclonal antibodies by LC-UV-MS analysis

Significance of the Topic

Monitoring glycopeptide variants in mAb therapeutics is critical for ensuring product safety efficacy and consistency during development and manufacturing. This approach addresses the need for precise characterization of post translational modifications that impact biological activity and immunogenicity.

Aims and Study Overview

This study evaluates Thermo Scientific Hypersil GOLD Peptide column performance in LC UV SQMS analysis of tryptic digest of adalimumab. The aim is to demonstrate column to column and lot to lot reproducibility sensitivity and robustness for glycopeptide monitoring in a routine QC setting.

Methodology and Instrumentation

Sample preparation utilized SMART Digest Trypsin Kit combined with TCEP reduction and formic acid quench. Digests were pooled and stored at 6 C prior to analysis.
Chromatography employed a 150 by 2.1 mm 1.9 µm Hypersil GOLD Peptide column with water and acetonitrile mobile phases containing 0.1% formic acid under a gradient from 2 to 90 B over 27 minutes at 0.25 mL/min flow and 50 C.
Detection combined a Vanquish Flex UHPLC system with variable wavelength detector at 214 nm and an ISQ EM single quadrupole mass spectrometer in positive HESI mode using SIM scans for glycopeptide variants.

Main Results and Discussion

Column lot to lot reproducibility of retention time achieved %RSD ≤ 0.6 peak width %RSD ≤ 3.9 and peak area %RSD ≤ 6.2 across the chromatographic window. Glycopeptide variant quantification of A2G0F A2G1F and A2G2F showed relative abundance %RSD ≤ 5.2 between lots and ≤ 3.0 across columns. These metrics demonstrate stable peak shape retention and quantitation performance.

Benefits and Practical Applications

Hypersil GOLD Peptide columns offer the consistent reproducibility required for QC workflows in biopharma. Sharp symmetric peaks enable enhanced sensitivity and lower limits of quantification for glycoform profiling. The robust column performance supports high throughput analysis and method transferability.

Future Trends and Opportunities

Integration with high resolution MS architectures and automated sample handling will further increase throughput and depth of glycopeptide analysis. Expanded applications may include multiplexed PTM monitoring and real time process control in biomanufacturing.

Conclusion

Hypersil GOLD Peptide columns paired with LC UV SQMS deliver reliable reproducibility and sensitivity for monitoring mAb glycopeptide variants supporting routine QC and development studies in biopharmaceutical analysis.

References

• Gucinski AC Boyne MT Evaluation of intact mass spectrometry for the quantitative analysis of protein therapeutics Analytical Chemistry 2012 84 18 8045 8051
• Jefferis R Posttranslational modifications and the immunogenicity of biotherapeutics Journal of Immunology Research 2016 1 15