Fast Track to Certainty: Confident Biopharma Decisions with LC-Single Quadrupole Mass Detection

Importance of the topic

In the rapidly evolving biopharmaceutical and pharmaceutical industries, timely and accurate analytical characterization of therapeutic biomolecules and oligonucleotides is critical to ensure product identity, purity, efficacy, and safety. Single quadrupole LC-MS techniques enable consolidation of multiple QC workflows into a single run, reducing analysis time, instrument footprint, and sample consumption. Simplified LC-MS platforms with enhanced sensitivity, mass range, and spectral quality facilitate glycosylation profiling, oligonucleotide confirmation, peptide degradant monitoring, and improve preparative purification efficiency.

Objectives and overview of the study/article

This compendium presents a collection of application examples demonstrating the capabilities of a next-generation single quadrupole LC-MS system coupled with high-performance LC and intuitive software. Key objectives are to

• Show consolidation of biopharma QC workflows (identity confirmation, purity assessment, impurity profiling) with single quadrupole LC/UV/MS
• Illustrate intact and subunit-level glycosylation profiling of monoclonal antibodies
• Demonstrate ion-pairing-free methods for oligonucleotide analysis
• Profile stress-induced degradants in therapeutic peptides (e.g., GLP-1 analogues such as tirzepatide)
• Assess antisense oligonucleotide (ASO) purity and impurity content via mass detection
• Enhance preparative purification workflows through mass-directed fraction collection

Methodology and instrumentation

A unified approach employs:

• High-performance LC systems with dedicated bio or oligonucleotide columns
• Single quadrupole mass detectors featuring broad mass range (m/z 2–3000), fast scan speeds, and superior sensitivity
• Integrated software with spectral deconvolution, target compound editors, and mass-directed fraction collection logic (including multi-threshold triggers and exclusion logic)
• Sample preparation protocols for intact and reduced monoclonal antibodies, synthetic oligonucleotides, stress-test peptide solutions, and crude mixtures for preparative purification

Key results and discussion

• Intact and subunit mAb glycoform profiling achieved reliable relative abundance measurements consistent with high-resolution MS, identifying five glycoforms at the intact level and four in the heavy chain subunit.
• Non-ion-pairing reversed-phase LC using ammonium bicarbonate/methanol provided robust retention and sensitivity for phosphorothioate ASOs and siRNA conjugates, with reproducible injections and mass errors <1 ppm.
• Oxidative degradants of tirzepatide under pH 5–9 and refrigerated storage were detected at sub-ppm levels, enabling monitoring of +O₂ and O₂–CO modifications, as well as unknown impurities.
• ASO purity assessment with full-scan MS quantified over twenty species (n−1, n+1, abasic, adducts), delivering MS purity complementary to UV results.
• Preparative HPLC workflows benefited from customizable mass triggers for adducts or charge states, multi-threshold settings, and exclusion logic to avoid coeluting impurities, improving fraction purity (e.g., caffeine purity raised from 93 % to 99 %).

Benefits and practical applications of the method

• Consolidation of multiple QC assays into a single LC/UV/MS run accelerates throughput and reduces sample consumption.
• Guided software intelligence and intuitive user interfaces minimize method development time and operator training.
• Wide mass range and enhanced ion transmission support analysis of small molecules, large biologics, and complex oligonucleotides without dedicated hardware.
• Flexible fraction collection triggers and override features streamline isolation of target compounds from complex matrices.
• Automated deconvolution and reporting shorten data review times and support regulatory compliance.

Future trends and potential applications

Emerging directions include:

• Integration of automated sample preparation and real-time fraction mapping for biotherapeutic development.
• Application of machine-learning algorithms for more robust impurity detection and spectral deconvolution.
• Expansion to novel modalities such as mRNA, lipid nanoparticles, and protein conjugates requiring even broader m/z coverage.
• Enhanced data integrity, compliance features, and real-time reporting for GMP environments.

Conclusion

The next-generation single quadrupole LC/MS system combined with best-in-class LC and streamlined software provides a robust, cost-effective solution for biopharma and oligonucleotide analytics. It simplifies workflows for glycan mapping, oligonucleotide confirmation, peptide impurity profiling, and preparative purification, delivering high confidence results with minimal method complexity.

Reference

• Pack, B. W.; Manheim, J.; Chahrour, O.; et al. Therapeutic Peptides Control Strategy: Org. Process Res. Dev. 2025, 29(1).
• Higel, F.; Seidl, A.; Sörgel, F.; Friess, W. N-glycosylation Heterogeneity in Monoclonal Antibodies. Eur. J. Pharm. Biopharm. 2016, 100.
• Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Analysis of Oligonucleotides by HPLC–ESI–MS. Anal. Chem. 1997, 69(7).
• Guimaraes, G. J.; Bartlett, M. G. The Critical Role of Mobile Phase pH in Oligonucleotide Ion-Pair LC–MS. Future Sci. OA 2021, 7(10).
• Badgujar, D.; Bawake, S.; Sharma, N. Identification of Liraglutide Degradation Products. J. Pept. Sci. 2025, 31.
• Rentel, C.; Gaus, H.; Bradley, K.; et al. Purity and Impurity Profile of Phosphorothioate Oligonucleotides by IP-HPLC–MS. Nucleic Acid Ther. 2022, 32(3).