How to Maximize Bioanalytical Performance of Fc-Fusion Proteins: Practical Sample Preparation and LC-MS/MS Workflows for Dulaglutide Quantification from Plasma

Significance of the Topic

Quantitative analysis of Fc-fusion proteins in plasma is critical for preclinical pharmacokinetic (PK) studies and therapeutic monitoring. Fc-fusion constructs, such as dulaglutide (a GLP-1 analogue linked to an IgG4 Fc), combine the bioactivity of peptide hormones with extended half-life conferred by the Fc domain. Robust bioanalytical workflows that deliver high sensitivity, reproducibility, and throughput are essential to support drug development and regulatory requirements.

Objectives and Study Overview

This application note presents a streamlined sample preparation and LC-MS/MS workflow to quantify dulaglutide in rat plasma with a lower limit of quantification (LLOQ) of 1 ng/mL. Key aims include: optimizing immunoaffinity capture of the Fc-fusion protein, developing a fast tryptic digest protocol, refining peptide-level SPE, and establishing a sensitive LC-MS/MS assay using a triple quadrupole instrument.

Methodology and Instrumentation

A multistep workflow was implemented:

• Immunoaffinity capture: Biotinylated anti-human Fc antibody on streptavidin magnetic beads enriches dulaglutide from 50 µL plasma, with blocking buffers to minimize nonspecific binding and a 0.1% TFA elution.
• Tryptic digestion: Waters ProteinWorks Auto-eXpress Low Digest Kit denatures with RapiGest, digests at 45 °C for 2 h, and quenches with TFA; reduction/alkylation steps were omitted without impact on peptide yield.
• Peptide SPE cleanup: Oasis PRiME MCX µElution plate removes matrix interferences using a 40% acetonitrile wash and 1% ammonium hydroxide in 40% acetonitrile elution, achieving high recovery and low background.
• LC-MS/MS analysis: Waters ACQUITY UPLC I-Class PLUS with Peptide BEH C18, 300 Å, 1.7 µm, 2.1×100 mm column at 80 °C, coupled to a Xevo TQ-XS Triple Quadrupole. Skyline software optimized MRM transitions for two surrogate peptides from the GLP-1 (HGEG…) and Fc (GLPS…) regions.

Key Results and Discussion

• Surrogate peptide selection: HGEGTFTSDVSSYLEEQAAK (GLP-1 N-terminal) and GLPSSIEK (Fc region) provided representative quantification.
• MRM optimization: Two transitions per peptide were summed to improve signal-to-noise and reproducibility at low ng/mL levels.
• Chromatography: A 300 Å pore-size column at high temperature minimized peptide carryover and matrix interferences, outperforming 130 Å and CSH chemistries.
• Assay performance: Linear range 1–10,000 ng/mL (r²>0.99), LLOQ = 1 ng/mL, intra- and inter-day accuracy within ±15%, CVs <11% across QC levels.

Benefits and Practical Applications

This workflow delivers:

• High sensitivity enabling preclinical PK studies using only 50 µL plasma.
• Streamlined sample prep with minimal steps and reduced potential for error.
• Compatibility with high-throughput formats via 96-well plates and automated digestion.
• Broad applicability to other Fc-fusion and monoclonal antibody therapeutics.

Future Trends and Opportunities

Advances in immunocapture reagents, digestion chemistries, and microflow LC-MS platforms promise further gains in sensitivity and throughput. Emerging data-independent acquisition (DIA) and high-resolution MS methodologies may enable multiplexed quantification of multiple biotherapeutics in a single run. Integration with automated sample handling and cloud-based data processing will enhance scalability for large toxicology and clinical studies.

Conclusion

An optimized combination of Fc-specific immunoaffinity capture, rapid trypsin digestion, selective SPE, and UPLC-MS/MS achieves robust quantification of dulaglutide in rat plasma with an LLOQ of 1 ng/mL. The workflow is adaptable to other Fc-fusion proteins, providing a reproducible, high-throughput solution for bioanalytical support of drug development.

Reference

• Glaesner W et al. Engineering and Characterization of the Long-Acting GLP-1 Analogue LY2189265. Diabetes Metab Rev. 2010;26(4):287–296.
• Kang L et al. Simultaneous Catabolite Identification and Quantitation of Large Therapeutic Protein at the Intact Level by IAC-LC-HRMS. Anal Chem. 2017;89(11):6065–6075.
• Dulaglutide protein sequence. DrugBank DB09045. Retrieved August 2019.
• MacLean B et al. Skyline: An Open Source Document Editor for Targeted Proteomics. Bioinformatics. 2010;26(7):966–968.