Combination of Bottom-Up and Top-Down Characterization of Biologics Using a High Throughput Capable Workflow in Proteome Discoverer Software

Significance of the Topic

Monoclonal antibodies are critical therapeutics and require thorough structural characterization to ensure safety and efficacy. Integrating bottom-up and top-down mass spectrometry methods enables comprehensive sequence coverage, post-translational modification profiling, and variant quantitation in a single workflow.

Study Objectives and Overview

This study evaluates a combined bottom-up and top-down protein analysis workflow implemented in Proteome Discoverer 2.0. Using the model antibody Rituximab, the goal was to achieve full sequence coverage, detect glycoforms and common modifications, and quantify proteoform variants.

Methodology

Bottom-up workflow

• Denaturation in 7 molar urea and 50 millimolar Tris buffer at pH 8
• Reduction with dithiothreitol, alkylation with iodoacetamide, tryptic digestion overnight
• Quenching with trifluoroacetic acid, cleanup and injection into UHPLC
• Separation on reversed-phase column with water/acetonitrile gradient containing formic acid

Top-down workflow

• Buffer exchange into ammonium acetate, reduction with tris(2-carboxyethyl)phosphine
• Direct infusion via static nanospray for intact protein analysis

Instrumentation

• UHPLC system with 2.1 by 250 millimeter C18 column
• Thermo Scientific Q Exactive Plus and Q Exactive HF mass spectrometers in protein mode
• Heated electrospray ionization source for peptides; nanoFlex static nanospray for intact proteins

Data Analysis

Proteome Discoverer 2.0 platform with Sequest HT search algorithm for peptides, ProsightPD node for top-down deconvolution, and Byonic node for glycopeptide and variant identification.

Results and Discussion

Complete sequence coverage was obtained by combining bottom-up and top-down data. Bottom-up alone achieved 98.6 percent coverage of the light chain and full coverage of the heavy chain. Top-down analysis provided complementary confirmation of sequence and highlighted proteoform variants. Glycopeptide analysis identified two major glycoforms of the heavy chain peptide EEQYNSTYR and its extended form TKPREEQYNSTYR. Common modifications detected included methionine oxidation, asparagine deamidation, glutamine conversion to pyroglutamate, and C-terminal lysine truncation. Relative quantitation of C-terminal lysine removal yielded approximately four point three percent truncated form.

Benefits and Practical Applications

• Unified platform simplifies data processing for combined approaches
• Robust detection and quantitation of glycoforms and modifications
• Enhanced confidence in monoclonal antibody quality control and comparability studies

Future Trends and Applications

Advances may include higher throughput chromatography, real-time data processing with machine learning, expansion to other biologics and antibody formats, and integration of alternative fragmentation techniques for deeper proteoform resolution.

Conclusion

The combined bottom-up and top-down workflow in Proteome Discoverer 2.0 provides a comprehensive, efficient solution for monoclonal antibody characterization, delivering full sequence coverage, detailed modification mapping, and variant quantitation on a single software platform.

References

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