A Single Injection LC-MS Analysis Scheme for Simultaneous Analysis of Biotherapeutics and Host-Cell Impurities via Online Digestion LC-MS/MS

Significance of the Topic

In biopharmaceutical research and quality control, reliable detection of therapeutic proteins and their host-cell impurities is essential for safety and regulatory compliance. Traditional workflows often separate intact mass analysis from peptide mapping and require offline digestion steps, which can be time-consuming and prone to variability. An integrated, fully automated online digestion LC-MS/MS approach can streamline these analyses, reduce hands-on time and improve reproducibility.

Objectives and Study Overview

The primary goal of this study was to develop a single-injection LC-MS scheme that combines automated online protein digestion, affinity capture or depletion of target proteins, intact mass measurement and peptide mapping in one run. The method was demonstrated on mixtures of human immunoglobulin G (IgG) and HEK293 host-cell lysate to profile both the biotherapeutic and co-existing impurities without multiple separate assays.

Methodology and Instrumentation

The workflow employed a Perfinity Workstation integrated with a Shimadzu LCMS-9030 QTOF. Key components and conditions included:

• Affinity capture column: Protein A/G for selective IgG binding and depletion
• Digestion reactor: Four-minute online trypsin digestion at 50 °C using proprietary Perfinity buffers
• Separation columns: Restek Ultra C4 for intact proteins and C18 Aeris peptide wide-pore for peptide mapping
• Mobile phases: 0.1 % formic acid in water (A) and acetonitrile (B); 90 min gradient from 2 % to 35 % B at 0.15 mL/min
• Mass spectrometer: Shimadzu 9030 QTOF for high-resolution MS and MS/MS data acquisition

Main Results and Discussion

Online digestion of undiluted (20 µg) and four-fold diluted HEK293 lysate identified dozens of abundant host-cell proteins via summed extracted ion chromatogram (XIC) areas. In IgG/cell-lysate mixtures, affinity depletion removed IgG effectively, enabling targeted profiling of unbound host-cell proteins. Conversely, affinity capture enriched IgG, yielding peptide maps dominated by IgG-derived fragments. Intact mass analysis of a NIST monoclonal antibody standard on the same instrument produced high-quality deconvoluted mass spectra, confirming molecular mass with minimal sample handling.

Benefits and Practical Applications

The integrated single-injection workflow delivers several advantages:

• Reduced sample preparation time through automated online digestion and capture steps
• Improved reproducibility and fewer manual interventions
• Concurrent acquisition of intact mass and peptide mapping data from one injection
• Flexible affinity strategies to selectively deplete or enrich therapeutic proteins

Future Trends and Potential Applications

Further developments may include the adaptation of this scheme to other antibody formats and fusion proteins, implementation of multi-enzyme digestion protocols for deeper sequence coverage, miniaturized reactor designs to lower sample consumption and integration with advanced data analysis tools for fully automated impurity profiling.

Conclusion

The presented online digestion LC-MS/MS platform simplifies the simultaneous analysis of biotherapeutic candidates and host-cell impurities. By merging affinity capture, rapid digestion and high-resolution mass spectrometry into a single injection, this approach enhances throughput and consistency, supporting accelerated biopharmaceutical development and robust quality control.