A Fast and Simple Immuno-mass Spectrometry Based Method Enables Universal Preclinical Bioanalysis for IgG-1 Type mAb

Significance of the topic

A reliable and universal preclinical bioanalysis method for IgG1-type monoclonal antibodies is critical in early drug development to support pharmacokinetic studies and ensure structural integrity. Traditional ligand-binding assays often face matrix interferences and lengthy development times. Combining immuno-capture with high-resolution mass spectrometry offers enhanced sensitivity, selectivity and speed for quantitative and structural monitoring of therapeutic antibodies.

Objectives and study overview

The primary goal was to establish a fast, simple and generic immuno-mass spectrometry workflow that can be applied to any IgG1 type monoclonal antibody in serum from various animal species at the preclinical stage. The method also aimed to monitor antibody structural integrity in vivo while achieving a sensitive lower limit of quantification without assay reconfiguration.

Methodology and instrumentation

Sample preparation and capture were performed using streptavidin magnetic beads co-immobilized with heat-activated trypsin and a biotinylated CaptureSelect human IgG-Fc PK reagent. Key steps included ultrafast on-bead digestion at 70 °C and direct transfer of peptide supernatant to LC/MS:

• Bead capture reagent : SMART Digest IA streptavidin magnetic beads coupled with CaptureSelect human IgG-Fc PK biotin conjugate
• Digestion conditions : On-bead trypsin digestion at 70 °C for 60 minutes without detergents or denaturants
• Automation platform : KingFisher magnetic bead purification system
• Chromatography : Thermo Scientific Vanquish Flex UHPLC with Accucore 150 C18 column (2.1 × 50 mm, 2.6 µm)
• Mass spectrometry : Orbitrap IDX in parallel reaction monitoring mode (PRM), MS/MS resolution 30 000, isolation width 1.4 Th

Main results and discussion

Eight candidate signature peptides conserved across humanized IgG1 molecules were initially evaluated for digestion efficiency, signal strength and matrix interference. Four peptides located in distinct constant domains (CL, CH1, CH2-CH3 junction and CH3) met all criteria. Key performance metrics included:

• LLOQ of 20 ng/mL in 50 µL serum for two peptides; 30–50 ng/mL for the remaining two
• Linear dynamic range up to 10 000 ng/mL with correlation coefficients > 0.993
• Intra- and inter-day precision and accuracy within standard validation limits across multiple animal serum matrices
• Comparable or improved on-bead digestion recovery relative to in-solution controls

The workflow enabled quantification and simultaneous structural monitoring of the target antibody, demonstrating robust performance and applicability across species without method adjustments.

Benefits and practical applications

This immuno-mass spectrometry approach offers:

• Rapid 4–5 hour sample preparation suitable for routine preclinical studies
• Full automation potential to increase throughput and reduce variability
• Universal applicability to any IgG1 monoclonal antibody without reoptimization
• High sensitivity and selectivity, enabling low-volume serum analysis
• Capability to monitor in vivo structural integrity alongside quantification

Future trends and potential applications

Further developments may include multiplexed analysis of multiple antibodies in a single run, expansion to other therapeutic protein classes, integration with real-time data processing for rapid decision making, and refinement of bead chemistries for enhanced digestion kinetics. Advances in high-throughput automation and microfluidic platforms could further streamline preclinical bioanalysis workflows.

Conclusion

A generic immuno-mass spectrometry workflow based on SMART Digest IA beads and PRM detection provides a fast, sensitive and universal solution for preclinical quantification and structural monitoring of IgG1 monoclonal antibodies. The method’s simplicity, automation readiness and robust performance make it an attractive option for biopharmaceutical research and development support.