A fast and simple immuno-mass spectrometry method for preclinical bioanalysis for IgG1 mAb

Significance of the topic

Bioanalysis of monoclonal antibodies is critical in preclinical drug development for accurate pharmacokinetic, pharmacodynamic and toxicokinetic assessment. Traditional ligand binding assays deliver sensitivity and throughput but can be limited by reagent development time, matrix interferences and linear range. Integrating immunoaffinity capture with high-resolution mass spectrometry offers both selectivity and sensitivity, along with the ability to monitor structural integrity.

Study objectives and overview

This work describes a universal, high-throughput immuno-mass spectrometry method for quantifying human IgG1 monoclonal antibodies in preclinical animal serum. The goals were to shorten sample preparation, apply a single workflow across multiple species and instruments, and combine quantitation with structural monitoring via signature peptides.

Methodology and workflow

The assay uses a four-step on-bead protocol:

• Immobilize biotinylated anti-human IgG-Fc ligand on streptavidin magnetic beads at room temperature.
• Capture target IgG1 mAb from 50 µL serum along with a heavy-isotope-labeled universal mAb internal standard.
• Wash to remove nonspecific proteins.
• Activate immobilized heat-stable trypsin at 70 °C for simultaneous denaturation and digestion, eliminating denaturants and SPE.

Resulting peptides from conserved constant regions are quantified by parallel reaction monitoring on Orbitrap mass spectrometers.

Used instrumentation

• SMART Digest IA Streptavidin Kit
• CaptureSelect Human IgG-Fc PK Biotin Conjugate
• SILuMAB K1 heavy-isotope-labeled mAb standard
• Thermo Scientific Orbitrap ID-X Tribrid and Q Exactive Plus mass spectrometers
• Vanquish UHPLC system with Accucore C18 column
• KingFisher Duo Prime/Flex magnetic bead processors and thermal mixers

Main results and discussion

• Capture recovery exceeded 95% for up to 40 µg mAb per sample across mouse, rat, dog and monkey sera.
• On-bead digestion achieved 90–100% recovery for peptides in CL, CH1 and CH3 domains relative to solution digestion; peptides in the CH2 domain were avoided due to epitope binding.
• Four universal signature peptides were selected for quantitation and structural monitoring.
• Linearity from 39 to 10,000 ng/mL (LLOQ 20–50 ng/mL) using 50 µL serum.
• Precision and accuracy within 10% CV for QC samples at 100, 800 and 6,000 ng/mL.
• Successful method transfer from Orbitrap ID-X to Q Exactive Plus with equivalent performance.

Benefits and practical applications of the method

• Complete sample preparation in under 5 hours.
• Automatable in 12- to 96-well formats for high throughput.
• Universal applicability to all human IgG1 mAbs and preclinical species without re-optimization.
• Combined quantitation and structural integrity assessment via multiple peptides.

Future trends and potential uses

Further expansions could include multiplexed analyses of antibody panels, increased sensitivity with larger sample volumes, extension to other isotypes and engineered formats, and adoption in regulated PK/PD and biotherapeutic comparability studies.

Conclusion

A streamlined immuno-MS workflow employing SMART Digest IA beads, a universal Fc ligand and an isotope-labeled mAb standard delivers rapid, sensitive and robust quantitation of IgG1 monoclonals in preclinical sera. This approach minimizes development time and supports high-throughput pharmacokinetic and structural monitoring needs in biopharmaceutical research.

References

1. Jenkins R et al. AAPS J. 2015;17(1):1–16.
2. Qu M et al. Mass Spectrom Rev. 2017;36(6):734–754.
3. Heudi O et al. Anal Chem. 2008;80(11):4200–4207.
4. Li F et al. Bioanalysis. 2011;3(21):2459–2480.
5. Ouyang Z et al. Bioanalysis. 2012;4(1):17–28.
6. Yuan L et al. Bioanalysis. 2012;4(24):2887–2896.
7. Wisniewski JR et al. Nat Methods. 2009;6(5):359–362.
8. Ewles M et al. Bioanalysis. 2016;8(24):2565–2579.
9. Whiteaker JR et al. Anal Biochem. 2007;362(1):44–54.
10. Kaur S. Bioanalysis. 2013;5(9):981–983.
11. Li H et al. Anal Chem. 2012;84(3):1267–1273.
12. An B et al. Anal Chem. 2015;87(7):4023–4029.