AUTOMATED SAMPLE PREPARATION FOR HYBRID LC-MS/MS PROTEIN QUANTIFICATION

Significance of the Topic

Quantitative analysis of therapeutic proteins in biological matrices is essential for drug development, pharmacokinetics and quality control. Hybrid LC-MS/MS methods using surrogate peptides require complex sample preparation steps including digestion, affinity capture and cleanup, which can be time consuming and prone to variability when performed manually.

Study Objectives and Overview

This work evaluated a fully automated sample preparation workflow for protein quantification, comparing its performance to traditional manual methods. Key goals included assessing digestion efficiency, affinity purification, SPE cleanup and reproducibility across multiple monoclonal antibodies in plasma.

Methodology and Instrumentation

Sample preparation employed Waters ProteinWorks Auto-eXpress Direct Digest Kit and µElution SPE Clean-up Kit on a Hamilton MicroLab STAR workstation. Affinity capture utilized streptavidin or anti-human IgG magnetic beads. Enzymatic digestion followed kit protocols at room temperature or low-level digest conditions. Peptides were analyzed on a Waters Xevo TQ-XS triple quadrupole MS coupled to an ACQUITY UPLC Peptide BEH C18 column (2.1×150 mm, 1.7 µm, 300 Å) on an I-Class PLUS system. MRM transitions for signature tryptic peptides were optimized for infliximab, etanercept, cetuximab and trastuzumab.

Main Results and Discussion

Automated digestion yielded raw peptide areas and %RSD values comparable to manual preparation, with normalized area ratios near 100 % and %RSD ≤5 %. Affinity capture of therapeutic antibodies demonstrated equivalent peak areas and reproducibility. Calibration curves were highly linear (r2 > 0.995) over dynamic ranges from low ng/mL to >250 000 ng/mL. QC samples showed accuracy between 89 and 107 % and precision ≤7 % across concentration levels down to 100 ng/mL.

Benefits and Practical Applications

Automation streamlined complex workflows, reduced hands-on time and minimized variability associated with manual handling. Standardized kits and protocols enhance throughput, reproducibility and facilitate method transfer in biopharma and clinical laboratories. This approach supports robust QA/QC operations and frees expert scientists for more strategic tasks.

Future Trends and Possible Applications

Further integration of automated sample prep into high-throughput environments will enable large-scale biomarker and therapeutic monitoring. Expansion to emerging biotherapeutics such as antibody-drug conjugates and biosimilars, combined immunoaffinity workflows and real-time data processing, will broaden applicability. Development of universal kits for diverse matrices and deeper multiplexing will drive the next generation of LC-MS/MS quantification.

Conclusion

Fully automated surrogate peptide workflows for protein quantification on the Hamilton STAR deliver performance equivalent to manual methods while significantly improving productivity, consistency and analytical robustness across multiple monoclonal antibodies.

Reference

Paula Orens and Mary Lame AUTOMATED SAMPLE PREPARATION FOR HYBRID LC-MS/MS PROTEIN QUANTIFICATION Waters Corporation