Accurate and Sensitive LC-MS/MS Quantification of Adalimumab in Serum/Plasma: Impact of Sample Preparation on Method Performance

Importance of the topic

The accurate quantification of therapeutic monoclonal antibodies in biological matrices is critical for drug development, biosimilar research, and clinical dose optimization. Adalimumab (HUMIRA®), a leading anti–TNF-α antibody, presents analytical challenges due to its sequence homology with endogenous IgG and the broad concentration range encountered in pharmacokinetic and clinical studies.

Objectives and study overview

This application note evaluates a generic, kit-based LC-MS/MS workflow for adalimumab quantification in serum/plasma. Three sample preparation strategies of increasing complexity—direct digestion, generic protein-level immunoaffinity, and specific immunocapture—are compared to assess their impact on sensitivity, accuracy, and reproducibility.

Methodology and instrumentation

• Sample preparation workflows:
  • Method 1: Direct digestion with ProteinWorks eXpress Direct Digest Kit followed by peptide-level clean-up using the µElution SPE Clean-Up Kit.
  • Method 2: Generic Protein A immunopurification, digestion with ProteinWorks eXpress Digest Kit, and SPE clean-up.
  • Method 3: Specific anti–human IgG Fc capture, digestion with ProteinWorks eXpress Digest Kit, without further SPE.
• LC-MS/MS conditions:
  • UPLC: ACQUITY UPLC Peptide BEH C18, 300 Å, 1.7 µm, 2.1×150 mm column at 55 °C; gradient from 5% to 35% acetonitrile over 8 min at 0.3 mL/min.
  • MS: Xevo TQ-XS triple quadrupole, positive ESI, MRM detection of three signature tryptic peptides and a 15N13C-labeled SILuMAb internal standard, controlled by MassLynx 4.1 and TargetLynx software.

Main results and discussion

• Peptide selection: Three unique adalimumab tryptic peptides (APYTFGQGTK, NYLAWYQQKPGK, GLEWVSAITWNSGHIDYADSVEGR) were identified via in silico digestion and confirmed for specificity and signal intensity.
• Chromatography: The optimized gradient provided baseline separation of all peptides with peak widths under 5 s.
• Sensitivity and dynamic range:
  • Method 1 LLOQ: 1,000–40,000 ng/mL
  • Method 2 LLOQ: 100–10,000 ng/mL
  • Method 3 LLOQ: 5–25 ng/mL
• Linearity and precision: Calibration curves were linear (R²>0.99) across 1–5 orders of magnitude. QC precision (%RSD) was below 15% and accuracy ranged from 88% to 111% in all workflows.
• Sample clean-up via SPE and immunoaffinity significantly improved signal-to-noise ratios and lowered quantification limits.

Benefits and practical applications

• Modular, kit-based sample preparation simplifies method development for protein quantification, even for scientists accustomed to small-molecule workflows.
• Flexible sensitivity tuning—from high-throughput, high-concentration assays to low-level clinical monitoring—by selecting the appropriate enrichment strategy.
• Applicable platform for biosimilar development, PK/PD studies, and therapeutic drug monitoring in research and QC laboratories.

Future trends and applications

Emerging developments in high-throughput immunoenrichment, microflow LC, and advanced automation are expected to further boost sensitivity, throughput, and reproducibility. Integration with AI-driven peptide selection and real-time data analysis could accelerate method optimization for a broad range of biotherapeutics.

Conclusion

A standardized, kit-based LC-MS/MS approach, combined with tailored sample enrichment, achieves robust, accurate, and sensitive quantification of adalimumab in serum/plasma. Up-front immunoaffinity capture delivers the lowest detection limits, while simpler workflows remain suitable for higher concentration ranges, offering a versatile bioanalytical solution.

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