Shimadzu Solutions for Biopharmaceutical - Application Notebook

Importance of the topic

Biopharmaceuticals such as monoclonal antibodies require advanced analytical methods to ensure safety, efficacy and regulatory compliance. Key quality attributes include accurate quantitation of antibody levels, characterization of post-translational modifications such as glycosylation, evaluation of protein aggregation, and monitoring of cell culture media components.

Goals and overview

• Develop robust LC-MS/MS bioanalysis workflows for monoclonal antibodies using Fab-selective proteolysis (nSMOL).
• Accelerate MRM method development and multiplexed quantitation of antibody drugs.
• Simultaneously profile cell culture media metabolites by triple quadrupole LC/MS/MS.
• Map peptide digests for primary structure confirmation.
• Analyze glycans with fluorescence and mass spectrometry.
• Detect high-mass proteins and protein aggregates by MALDI-TOF MS.
• Evaluate formulation additives and surfactant quality.

Methodology and instrumentation

• nSMOL Antibody BA Kit: IgG capture resin and trypsin-immobilized nanoparticles for selective Fab digestion, combined with Shimadzu LCMS-8050/8060 triple quadrupole systems.
• Skyline software for design and optimization of MRM transitions targeting complementarity-determining region peptides.
• Nexera-i UHPLC for high-resolution peptide mapping with core-shell C18 columns.
• RF-20Axs fluorescence detection of 2-AB labeled N-glycans by HILIC separation.
• PPSQ-51A/53A Protein Sequencer for automated Edman degradation and N-terminal sequencing of antibodies.
• Benchtop MALDI-8020 and MALDI-7090 TOF instruments with high mass detectors for intact protein and aggregation analysis.
• Aggregates Sizer and Aggregates Sizer TC for quantitative laser-diffraction measurement of subvisible particles under controlled temperature and stress conditions.
• Shim-pack MAYI restricted access SPE columns coupled with Kinetex and Shim-pack GISS C18 columns for rapid online deproteinization and surfactant analysis.

Main results and discussion

• nSMOL bioanalysis achieved lower limits of quantitation down to 0.06 μg/mL for Trastuzumab and validated MRM assays for Bevacizumab, Nivolumab and multiplex panels with high accuracy (<15% CV) and precision.
• MRM methods developed in Skyline exhibited linear dynamic range over four orders of magnitude for CDR peptides in complex matrices.
• Simultaneous LC/MS/MS profiling of 95 cell culture medium components in 17 minutes enabled rapid monitoring of metabolite consumption and secretion during hybridoma growth.
• Reproducible peptide mapping of tryptic digests of IgG was demonstrated on Nexera-i with excellent retention time precision (RSD <0.3%).
• Fluorescence detection of 2-AB glycans provided femtomole-level sensitivity with robust linearity; one-pot PMP labeling reduced peeling artifacts for O-glycan analysis.
• MALDI-TOF MS with high mass detectors detected intact antibodies, protein multimers and aggregates at low femtomole levels.
• Quantitative laser-diffraction with Aggregates Sizer TC enabled real-time monitoring of aggregate formation under heat and stirring stress and evaluation of surfactant and additive effects on aggregation.
• Online SPE deproteinization with Shim-pack MAYI columns provided rapid quantitation of polysorbate 80 and its degradation products by LC–MS/MS with excellent repeatability.

Benefits and practical applications

• Streamlined workflows from sample prep to data analysis reduce development time for bioanalytical assays.
• High sensitivity, specificity and robustness support preclinical and clinical pharmacokinetics and toxicokinetics of antibodies.
• Multiplex capability allows simultaneous quantitation of multiple biotherapeutics in a single run.
• Comprehensive cell culture profiling aids optimization of bioprocess conditions and media formulations.
• Aggregation analysis under controlled stress offers rapid formulation screening for improved stability.
• Automated software tools facilitate method creation, data processing and report generation.

Future trends and possibilities

Integration of automation, artificial intelligence and high-throughput platforms will enable real-time in-process monitoring and adaptive control of biomanufacturing. Expanded multiplexed nSMOL assays may cover diverse antibody formats. Advanced software and informatics will support deeper characterization of glycoforms, structural variants and aggregate populations for next-generation biotherapeutics.

Conclusion

The suite of analytical methods and instruments presented provides end-to-end solutions for characterizing monoclonal antibodies, glycans, peptides, aggregates and formulation components. Together they enhance productivity, data quality and regulatory compliance across biopharmaceutical development, manufacturing and quality control.

Reference

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• Iwamoto N et al. J Chromatogr B 2016; DOI:10.1016/j.jchromb.2016.04.038
• Iwamoto N et al. Clin Pharmacology & Biopharmaceutics 2016; DOI:10.4172/2167-065X.1000164
• Wang C et al. Proteomics 2011;11:4229–4242
• Totoki T et al. J Pharm Sci 2015;104(2):618–626