Rapid quantitation of therapeutic antibodies in animal plasma

Significance of the Topic

Quantitative analysis of therapeutic monoclonal antibodies at low concentrations in animal plasma is critical for preclinical pharmacokinetic and bioanalytical studies. Conventional immunoaffinity–LC–MS/MS methods require multiple steps for enrichment, digestion, and cleanup, often spanning over 24 hours with high variability.

The SMART Digest IA workflow integrates immunocapture and proteolysis in a single well, reducing sample preparation time by over 80% while improving reproducibility and sensitivity. This advancement is particularly valuable for laboratories engaged in biopharmaceutical development, quality control, and high-throughput screening.

Objectives and Study Overview

The primary goal was to demonstrate rapid and robust quantification of low-level human IgG spiked into mouse plasma using Thermo Scientific SMART Digest Immunoaffinity kits (Streptavidin, Protein A, and Protein G). Key objectives included shortening overall sample preparation time, achieving a broad dynamic range down to single-digit nanograms per milliliter, and validating linearity, precision, and accuracy of the assay.

Methodology

• Immunocapture: Anti-human IgG Fc fragment was biotinylated and immobilized on streptavidin beads, or cross-linked to Protein A/G beads, to selectively enrich human IgG from complex plasma matrices.
• Simultaneous Denaturation and Digestion: Captured antibodies were subjected to 70 °C for 60 minutes in SMART Digest Buffer, activating heat-stable trypsin and completing proteolysis directly on the bead surface.
• Sample Cleanup: Post-digestion, peptides were acidified with 1% trifluoroacetic acid and transferred to a 96-well plate for LC–MS/MS analysis without additional desalting steps.

Used Instrumentation

• Liquid Chromatography: Thermo Scientific UltiMate 3000 Rapid Separation Dual System with SRD-3600 degasser, DGP-3600RS rapid separation pump, WPS-3000TRS thermostatted autosampler, and TCC-3000RS column compartment.
• Column: Thermo Scientific Accucore C18 (2.1 × 50 mm, 2.6 μm) maintained at 50 °C.
• Mass Spectrometry: Thermo Scientific Velos Pro ion trap equipped with a HESI source, operating in positive mode with selected reaction monitoring of surrogate peptides VVSVLTVLHQDWLNGK and TTPPVLDSDGSFFLYSK.
• Consumables: 96-well deep-well plates, Sepraseal caps, SMART Digest IA magnetic and non-magnetic bead kits.

Main Results and Discussion

Optimization experiments established that complete digestion of human IgG occurs after 60 minutes at 70 °C and full immunocapture is achieved within 60 minutes at room temperature. The SMART Digest IA Streptavidin kit delivered linear quantitation from 5 to 1580 ng/mL (R² > 0.995) with precision (CV) ≤ 6% at the LLOQ and ≤ 2.3% at the ULOQ. Protein A and G kits extended the dynamic range to 6000 ng/mL, with an LLOQ of 6 ng/mL and R² > 0.99.

The integrated workflow reduced total sample preparation time to 3–4 hours compared to ~24 hours for traditional protocols, representing an >80% time savings. Magnetic bead formats further simplified washing steps and enabled compatibility with automated platforms without compromising sensitivity or reproducibility.

Benefits and Practical Applications

• Substantially reduced hands-on time and overall processing duration.
• Improved sensitivity for low-abundance monoclonal antibodies down to single-digit ng/mL concentrations.
• High reproducibility and accuracy across a wide concentration range.
• Versatile capture reagents (Streptavidin, Protein A, Protein G) for different antibody subclasses.
• Scalable and automation-friendly for routine preclinical and bioanalytical laboratories.

Future Trends and Possibilities of Use

• Full integration with automated bead-handling platforms (e.g., KingFisher) for ultra-high throughput.
• Multiplexed quantitation of multiple antibody candidates in a single LC–MS/MS run.
• Extension of the single-well immunocapture/digestion approach to other protein biotherapeutics and biomarker classes.
• Development of enhanced immobilized enzyme chemistries for faster digestion and higher peptide yield.

Conclusion

The SMART Digest Immunoaffinity kits provide a streamlined, single-well workflow that seamlessly combines enrichment and digestion for rapid, sensitive quantitation of therapeutic antibodies in animal plasma. By reducing sample preparation time by over 80% and delivering robust LC–MS/MS performance, this approach accelerates preclinical studies and supports routine bioanalysis with minimal method development.

References

1. Ewles M, Mannu R, Fox C, Stanta J, Evans G, Goodwin L, Duffy J, Bell L, Estdale S, Firth D. LC–MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug antibodies. Bioanalysis. 2016;8(24):2565–2579.
2. Bardsley J, Jones J, Humphryes P. Improvement in Speed and Reproducibility of Protein Digestion and Peptide Quantitation Using SMART Digest Technology in a Full Solution Workflow. Thermo Fisher Scientific Application Note AN21198; 2016.
3. Thermo Scientific. SMART Digest Immunoaffinity (IA) User Manual; 2017.
4. Thermo Fisher Scientific. Comparison of Antibody IgG Binding Proteins. Technical Resource Document.
5. Berna M, Ackermann B. Increased Throughput for Low-Abundance Protein Biomarker Verification by Liquid Chromatography/Tandem Mass Spectrometry. Anal Chem. 2009;81(10):3950–3956.