Increased sensitivity and throughput for native intact mass analysis using a NativePac OBE-1 SEC column and an Orbitrap Ascend Tribrid mass spectrometer

Importance of the topic

The ability to analyze intact proteins under native conditions offers valuable information on glycosylation microheterogeneity and noncovalent interactions of biotherapeutics. Rapid and sensitive workflows support high throughput screening in bioprocess development and quality control.

Study objectives and overview

This study implemented an online buffer exchange SEC column (NativePac OBE-1) coupled to a Vanquish Horizon UHPLC system and Orbitrap Ascend Tribrid mass spectrometer. The aim was to achieve sub-five minute, high sensitivity native mass analysis of monoclonal antibodies using minimal sample consumption and automated reporting.

Methodology and instrumentation

Monoclonal antibody (NISTmAb) samples were prepared at concentrations from 5 to 1000 ng per microliter. The LC separation employed a NativePac OBE-1 SEC column (2.1 × 50 mm, 80 Å, 3 μm) at 65 μL per minute with 50 mM ammonium acetate. A comparison was performed using a MAbPac SEC-1 column at 250 μL per minute. The Orbitrap Ascend Tribrid mass spectrometer with the HMRn+ option collected full MS spectra (m/z 4000–12000) at 15000 resolution. Online buffer exchange removed nonvolatile salts prior to detection.

Instrumentation used

• Vanquish Horizon Flex UHPLC with Binary Pump and Split Sampler
• NativePac OBE-1 SEC column
• MAbPac SEC-1 SEC column for comparison
• Orbitrap Ascend Tribrid mass spectrometer with HMRn+
• BioPharma Finder 5.2 and OptiMSe 1.0 software for automated analysis

Main results and discussion

• Analysis time of 4.5 minutes per sample enabled by the short SEC column and isocratic conditions.
• Detection of NISTmAb down to 5 ng on column with high signal to noise and sub-10 ppm mass accuracy for major glycoforms.
• Compared with conventional SEC at 250 μL per minute, the microflow OBE method improved sensitivity by an order of magnitude.
• Online buffer exchange efficiently separated protein from salts, minimizing instrument contamination and sample preparation.
• Automated deconvolution and reporting with OptiMSe delivered real time results during data acquisition.

Benefits and practical applications

The described workflow offers rapid screening capability for therapeutic protein development, low sample consumption, and high spectral quality. It supports glycoform profiling, batch comparison, and routine QC of biotherapeutics.

Future trends and potential applications

• Expansion to other protein complexes and vaccines under native conditions.
• Integration with machine learning algorithms for automated anomaly detection.
• Further miniaturization and multiplexing to increase throughput.
• Adoption in regulated environments for real time release testing.

Conclusion

The combination of a microflow online buffer exchange SEC column and Orbitrap Ascend Tribrid mass spectrometer achieves fast, sensitive native intact mass analysis of monoclonal antibodies. Automated data processing with OptiMSe enhances throughput and operational simplicity, making this approach well suited for biopharmaceutical development and quality control.

References

1. Thermo Fisher Scientific OptiMSe workflows for fast sample screening.
2. Technical Note 001259: High-throughput native mass spectrometry of large biomolecules, 2022.
3. OptiMSe Workflow brochure, Thermo Fisher Scientific.