Biomolecule Purification, Characterization, and Analysis

Significance of Topic

Advances in genomics, proteomics, metabolomics and system biology demand robust methods for purification, characterization and quantitative analysis of biomolecules. Accurate profiling of peptides, proteins, glycans, oligonucleotides and amino acids is essential for drug discovery, biomarker research, clinical diagnostics and quality control of biotherapeutics.

Objectives and Overview

This document surveys integrated solutions and specialized chromatographic consumables from Waters Corporation. It covers:

• Pre-column chemistry for amino acid analysis (AccQ•Tag, Pico•Tag, RapiFluor-MS)
• Reversed-phase, HILIC and ion-exchange columns for peptides, proteins and monoclonal antibodies
• Glycan and glycoprotein workflows from sample preparation to LC-MS analysis
• Size-exclusion chromatography for aggregate profiling
• Oligonucleotide separation by ion-pair RPLC

Instrumentation and Methodology

Key components include ACQUITY UPLC, UPLC H-Class Bio, Arc and Alliance HPLC systems, coupled to fluorescence or mass spectrometry detectors (Xevo G2-XS QTof, SYNAPT G2-Si, ACQUITY QDa). Methods combine novel derivatization reagents (AQC, RapiFluor-MS), quality-controlled columns, and pre-configured software workflows (Empower™, UNIFI™). Samples are processed using dedicated kits for glycan release, amino acid derivatization or peptide desalting and automated on liquid-handling platforms.

Main Results and Discussion

• Amino acid analysis by UPLC AccQ•Tag Ultra yields baseline resolution of 20+ amino acids in <8 minutes, with 100-fold sensitivity increase versus HPLC.
• Glycan workflows (GlycoWorks RapiFluor-MS) deliver rapid release, labeling and purification of N-glycans with high fluorescence and MS sensitivity.
• BioResolve RP mAb Polyphenyl columns resolve intact antibodies and subunits in MS-friendly eluents, improving quantitation of glycoforms.
• Peptide BEH and CSH C18 columns offer complementary selectivity, high peak capacity and peptide loadability for reversed-phase LC and LC-MS.
• SEC BEH and XBridge columns enable precise aggregate sizing of proteins down to 1–2% level.
• Oligonucleotide BEH C18 columns achieve base-resolution of 15–60 mer ladders in <4 minutes.

Practical Benefits

• Seamless method transfer between HPLC, UHPLC and UPLC platforms
• Consistent column performance via rigorous QC standards and Protein/Pepide-Pak test mixtures
• Automation-ready kits minimize pipetting steps and sample loss
• MS-compatible mobile phases reduce ion suppression

Future Trends and Opportunities

■ Integration of high-throughput automation with advanced LC-MS for large clinical and biomanufacturing datasets
■ Expansion of novel stationary phases to target challenging biomolecule classes (glycopeptides, intact glycoproteins)
■ Application of machine-learning-guided method development to accelerate workflows
■ Growth of microflow and nano-LC-MS to increase sensitivity for low-abundance analytes

Conclusion

Waters’ comprehensive portfolio of derivatization chemistries, stationary phases and integrated software solutions supports high-performance analysis of biomolecules across multiple modalities. These streamlined workflows deliver reproducible, high-resolution separations adaptable to research, quality control and regulated environments.

References

• Application Note 720005369EN
• Application Note 720005202EN
• Application Note 720005409EN