Tackling Non-Specific Binding of Biotherapeutics Using LC-MS Compatible QuanRecovery Sample Plates with MaxPeak High Performance Surfaces

Importance of the Topic

Large hydrophobic biotherapeutic molecules such as monoclonal antibodies are susceptible to non-specific binding (NSB) to labware surfaces, leading to analyte loss, reduced sensitivity, and poor assay reproducibility. Mitigating NSB is crucial to achieve the low detection limits required for biotherapeutic development and quality control.

Objectives and Study Overview

This study evaluated QuanRecovery sample plates featuring MaxPeak High Performance Surfaces (HPS) for their ability to reduce NSB of monoclonal antibodies in LC-MS/MS assays. Recovery of three model biotherapeutics—adalimumab, cetuximab, and NISTmAb—was assessed under various storage and sample preparation conditions.

Methodology and Instrumentation

• Sample Plates: QuanRecovery with MaxPeak HPS vs standard polypropylene
• Sample Preparation: Neat solutions (100 ng/mL) and carrier protein benchmarks (100% recovery reference)
• Immunopurification: Human Fc-targeted magnetic beads from rat plasma (10 μL sample volume)
• Reduction and Alkylation: ProteinWorks reduction and alkylation kit with dithiothreitol and iodoacetamide
• Chromatography: ACQUITY UPLC I-Class PLUS system, BioResolve RP mAb Polyphenyl column (2.1×50 mm, 2.7 μm, 450 Å), gradient 25–30% B over 2.5 min, flow rate 0.4 mL/min
• Mass Spectrometry: Xevo TQ-XS triple quadrupole, monitoring mAb light chain fragment at m/z 1329.85

Main Results and Discussion

• Recovery in QuanRecovery Plates after 24 h: 84–94% for intact mAbs vs 23–60% in standard plates
• Calibration and Sensitivity: Linear range 25–100,000 ng/mL for adalimumab and cetuximab, 50–100,000 ng/mL for NISTmAb
• Analytical Performance: Correlation coefficients (r²) >0.996, accuracy within ±15%, precision with CV <10%
• Lower Limits of Quantification (LLOQs): 25 ng/mL for adalimumab and cetuximab; 50 ng/mL for NISTmAb

Benefits and Practical Applications

• Enhanced analyte recovery and assay reproducibility at low concentrations
• Reduced reliance on carrier proteins, simplifying sample matrices
• Compatibility with LC-MS/MS workflows for biotherapeutic quantification
• Suitable for limited volume samples such as preclinical plasma studies

Future Trends and Potential Applications

As biotherapeutic complexity grows and detection requirements tighten, surface-engineered labware will become increasingly important. Future developments may include tailored high-performance coatings for specific molecule classes, integration with automated sample processing, and expansion to other bioanalytical platforms beyond LC-MS/MS.

Conclusion

QuanRecovery sample plates with MaxPeak HPS effectively mitigate non-specific binding of hydrophobic biotherapeutics, enabling robust LC-MS/MS quantification at sub-µg/mL levels. This technology streamlines workflows by improving recovery, accuracy, and precision without additional matrix additives.

References

• Lagassé HAD, Alexaki A, Simhadri VL, et al. Recent Advances in (Therapeutic Protein) Drug Development. F100Res. 2017;6:113.
• Rabe M, Verdes D, Seeger S. Understanding Protein Adsorption Phenomena at Solid Surfaces. Adv Colloid Interface Sci. 2011;162(1-2):87–106.