Orbitrap Ascend BioPharma Tribrid mass spectrometer

Importance of the Topic

The characterization of biotherapeutic molecules demands instruments with high resolution sensitivity and throughput to ensure product safety efficacy and quality. Advanced mass spectrometry platforms enable detailed analysis of intact proteins peptides oligonucleotides and complex biomolecules meeting the growing requirements of biopharma research and production.

Objectives and Study Overview

This summary reviews the key innovations and performance of the Thermo Scientific Orbitrap Ascend BioPharma Tribrid mass spectrometer for scaling up biotherapeutic characterization workflows. The study focuses on intact mass analysis peptide mapping post translational modification profiling antibody drug conjugate evaluation native mass spectrometry proteoform analysis oligonucleotide characterization and guide RNA sequencing.

Instrumentation

This mass spectrometer integrates several hardware and software advances

• Dual ion routing multipoles for parallel MS2 and MS3 fragmentation
• Advanced active ion beam guide to prevent neutrals and clusters
• EASY IC ETD and PTCR ion source based on Townsend discharge
• Electrodynamic ion funnel for efficient ion transfer
• QR5 segmented quadrupole with 0.4 m z precursor isolation
• Ultra high field Orbitrap analyzer with resolution above 480000 FWHM and acquisition rates up to 45 Hz
• Auto ready ion source enabling automated remote calibration
• High capacity ion transfer tube to increase ion flux
• Fragmentation options CID HCD ETD EThcD ETciD and UVPD
• Optional modules Native MS PTCR UVPD and FAIMS

Key Results and Discussion

Peptide Mapping and PTM Analysis

• Achieved 100 sequence coverage of both heavy and light chains of trastuzumab at 300 microliters per minute flow rate
• Detected low abundance deamidation at 0.9 relative intensity

Host Cell Protein Profiling

• Identified over 260 host cell protein peptides down to 7 parts per billion using analytical flow rates

Antibody Drug Conjugate Characterization

• Combined middle down digestion with EThcD and UVPD to map drug to antibody ratio and conjugation sites in a single workflow

Native Mass Spectrometry

• Used data independent acquisition DIA windows native MS option and PTCR to simplify spectra extend m z detection to 16 000 and identify low abundance proteoforms

Oligonucleotide Analysis

• Separated modified tRNA variants by ion pairing reversed phase chromatography and located under modified species using MS3 CID and UVPD

Top Down RNA Sequencing

• Obtained near 100 base pair coverage of a 100 mer sgRNA by applying low frequency CID to reduce spectral complexity and enhance sequence ion signals

Benefits and Practical Applications

The instrument delivers high throughput and versatility enabling accelerated development and quality control of biopharmaceuticals. Simplified spectra and advanced fragmentation improve confidence in PTM localization and proteoform identification. Automation features enhance reproducibility and ease of operation in research manufacturing and QA QC environments.

Future Trends and Opportunities

Emerging directions include integration of real time database search and library directed acquisition AI driven data analysis for deeper interpretation expansion of native mass workflows to larger assemblies development of novel fragmentation schemes single cell proteomics coupling high throughput glycoform profiling and further automation of sample handling and instrument calibration.

Conclusion

The Orbitrap Ascend BioPharma Tribrid mass spectrometer presents a comprehensive platform for scaling up biotherapeutic characterization. Its combination of hardware innovations optional modules and automated features meets the demanding needs of modern biopharma research enabling robust analysis of complex molecules with high confidence and speed.

References

• Beaumal C Beck A Hernandez Alba O et al Advanced mass spectrometry workflows for accurate quantification of trace level host cell proteins in drug products Benefits of FAIMS separation and gas phase fractionation DIA Proteomics and Systems Biology 2023 23 16 DOI 10 1002 pmic 202300172