Reproducible IA-LC-MS/MS quantitation of human IgG from plasma with SMART Digest Fractionation Kits

Significance of the Topic

Human immunoglobulin G quantitation is essential for biotherapeutics development and clinical monitoring due to the rapid expansion of the biologics market. Combining immunoaffinity capture with LC-MS/MS addresses specificity and cross-reactivity limitations of traditional immunoassays in complex biological matrices like plasma.

Objectives and Study Overview

The primary goal was to establish a reproducible and efficient workflow for capturing and digesting human IgG in murine plasma using SMART Digest Bulk Fractionation kits. The study aimed to demonstrate linearity, sensitivity, and precision across a wide concentration range without requiring antibody biotinylation or linkage validation.

Methodology and Instrumentation

• Sample Preparation: Human IgG spiked into mouse plasma at 0.1–100 μg/mL forming calibration and QC samples at 0.1, 1, 10 and 100 μg/mL.
• Immunoaffinity Capture: Plasma samples were diluted and incubated with Protein G magnetic beads, washed to remove interfering proteins while retaining IgG.
• Enzymatic Digestion: Beads were treated with soluble SMART Digest trypsin at 70 °C for 90 minutes, then acidified prior to LC-MS analysis.
• LC-MS/MS Analysis: Separation on an Accucore C18 column using a rapid gradient with formic acid modified solvents and detection on a Velos Pro ion trap operated in positive HESI mode monitoring surrogate peptides m/z 603.4→805.4 and 937.7→836.5.

Main Results and Discussion

Calibration curves for the two signature peptides exhibited excellent linearity with correlation coefficients above 0.999 over four orders of magnitude. Limits of quantitation reached 0.1 μg/mL in plasma matrices. Method precision, evaluated by QC samples, showed coefficients of variation below 6.5% for both peptides, confirming high reproducibility. The targeted immunoaffinity approach combined with solid-phase digestion achieved robust sensitivity and sample cleanliness, reducing interference from abundant plasma proteins.

Benefits and Practical Applications

• The one-hour combined capture and digestion simplifies workflows compared to traditional 24-hour protocols.
• Protein G beads enable universal IgG capture across species without antibody biotinylation, streamlining method development.
• High sensitivity and reproducibility support low-level quantitation in preclinical and clinical research.
• Adaptable to various surrogate peptides for different biotherapeutic molecules.

Future Trends and Potential Uses

Advancements are expected in automating immunoaffinity-LC-MS workflows to increase throughput and reduce manual handling. Expanding the platform to multiplexed quantitation of multiple antibody subclasses or other biotherapeutic formats will address emerging screening and bioanalysis needs. Integration with high-resolution MS and data-independent acquisition may further enhance selectivity and throughput.

Conclusion

The SMART Digest Bulk Fractionation workflow delivers a rapid, robust, and reproducible solution for quantifying human IgG in plasma by LC-MS/MS. Its high sensitivity, broad dynamic range, and elimination of antibody linkage steps make it an attractive option for biopharmaceutical and clinical research laboratories.

References

1. Transparency Market Research Global Biologics Market report 2018
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4. Thermo Fisher Scientific Simpler Faster and More Reproducible Protein Digestion BR-21698-EN
5. Thermo Fisher Scientific Comparison of Antibody IgG Binding Proteins resource