Rapid quantitation of therapeutic antibodies in animal plasma

Importance of the topic

Precise quantitation of therapeutic antibodies in animal plasma is a critical component in preclinical development and bioanalysis. Traditional immunoaffinity-LC/MS workflows are laborious, spanning up to 24 hours with multiple steps prone to variability. The SMART Digest Immunoaffinity (IA) kits streamline this process by integrating antibody capture and enzymatic digestion in a single well, reducing sample preparation time by over 80% while improving reproducibility and sensitivity.

Objectives and Study Overview

This study demonstrates rapid quantification of low-level human IgG in mouse plasma using three SMART Digest IA kits (Streptavidin, Protein A, Protein G). Key objectives include:

• Combining immunoaffinity enrichment and digestion in one workflow
• Assessing assay linearity, precision, and sensitivity across a wide dynamic range
• Comparing performance of magnetic and non-magnetic kit formats

Methodology and Instrumentation

Plasma samples spiked with human IgG (range 5–6 000 ng/mL) underwent capture on SMART Digest IA beads, followed by on-bead digestion at 70 °C for 60 min. Three capture strategies were evaluated:

• Biotinylated anti-IgG Fc with Streptavidin beads
• Anti-IgG cross-linked to Protein A beads
• Anti-IgG cross-linked to Protein G beads

Instrumentation

Liquid chromatography was performed on an UltiMate 3000 RS system with an Accucore C18 column (2.1 × 50 mm, 2.6 µm). Detection used a Velos Pro ion-trap mass spectrometer operating in positive SRM mode targeting surrogate Fc peptides (m/z 603.4→805.4 and 937.7→836.5).

Main Results and Discussion

Digestion and capture kinetics optimized at 60 min each yielded complete peptide generation and antibody enrichment. All three kits delivered linear calibration (R²>0.995) over four orders of magnitude. The Streptavidin format achieved an LLOQ of 5 ng/mL with 6 % CV, and ULOQ of 500 ng/mL with 2.3 % CV. Protein A and G kits extended quantitation to 6 000 ng/mL with comparable precision. Magnetic bead formats facilitated automation without loss of performance.

Benefits and Practical Applications

The integrated SMART Digest IA workflow offers:

• Sample preparation time of 3–4 h versus ~24 h
• Over ten-fold sensitivity improvement via immunoaffinity capture
• Single-method quantitation of diverse monoclonal antibodies using universal Fc peptides
• Compatibility with automation platforms (e.g., KingFisher)

Future Trends and Potential Applications

Emerging opportunities include expanding to other biotherapeutics, multiplexed antibody assays, and full automation of sample-to-result workflows. Additional capture reagents and alternative digestion chemistries could further broaden applicability in proteomics and pharmacokinetics.

Conclusion

The SMART Digest IA kits drastically accelerate and simplify antibody quantitation in complex matrices, delivering robust, sensitive, and automatable workflows suitable for preclinical and bioanalytical applications.

References

1. Ewles et al., Bioanalysis 2016, 8(24), 2565–2579.
2. Bardsley et al., Thermo Fisher Scientific Application Note AN21198.
3. SMART Digest IA User Manual, Thermo Fisher Scientific.
4. Thermo Fisher Scientific IgG Binding Proteins Comparison.
5. Berna & Ackermann, Anal. Chem. 2009, 81, 3950–3956.