mountain_topIt’s hard to believe we arrived at Autumn, and in a few more months 2020 is over 😬. But we all still had our objectives for the year, and maybe, like me, you too have recently gone through that usual mid-year review to look at your goals, your planned projects for the year and have been evaluating your past few months.

Have I achieved what I wanted, or could I have done a better job? Did I have the right tools? Maybe if I did not have the right tools, could I still find a way to get what I need, if not for this year’s goals, definitely for next year?

If you are an analytical scientist who works in a BioPharma analytical lab and are always looking for improvements for your analytical methods to achieve the best results possible to help you deliver your objectives, you will likely look for the latest and best tools to do that.

Scientists at NIBRT had a few BioPharma characterization improvement objectives this year, and for that, in collaboration with great application scientists at Thermo Fisher, they looked for and tested a great tool to accomplish what they wanted, and even learned and told us what more you could achieve with the Thermo Scientific™ Orbitrap Exploris™ 240.

They needed a tool for Sensitive profiling of IgG1 monoclonal antibody variants under native conditions. They tested SEC-MS and ion exchange chromatography coupled to MS using the new MS platform.

  • Sensitivity and dynamic range using SEC-MS, as highlighted with trastuzumab, provided exceptionally low limits of detection, enabling native MS to be used for studies where sample is limited, such as at the clone selection stage.
  • Obtaining excellent sensitivity, as well as wide dynamic range, for native MS approaches using standard HPLC flow rates are highly desirable to allow identity testing and monitoring of PTMs even at an early stage of product development.

For their future work, when looking to deliver on their objectives with quick and reliable answers, they found the key benefits using the Orbitrap Exploris 240 are:

  • Operational simplicity for mass spectrometer setup and acquisition under native conditions using standardized tune conditions.
  • Simplified data interpretation from exceptional spectral clarity and confident mass accuracy.
  • Exceptional sensitivity and mass accuracy for intact mass analysis under native conditions allowing confident analysis from low sample loading.

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Figure 1. Sensitivity under native conditions.

  1. A) SEC-MS analysis. BPCs of sample load from 0.05 µg to 50 µg of trastuzumab
  2. B) SEC-MS charge envelopes detected between m/z 5,000 and 7,000 (three scans, acquired with 10 µscans each, were averaged)
  3. C) Normalized intensities plotted to estimate LOD and LOQ.
  4. D) Zoom into the MS spectra showing +26 charge state of the SEC-MS analysis for the different sample amounts of mAb analyzed, demonstrating exceptional spectral quality reproducibility independent of sample loading.

Read more in the Application Brief.

Going further, they tested the Orbitrap Exploris 240 for the Characterization of IgG1 monoclonal antibody (mAb) oxidation variants at intact, subunit and peptide levels

Oxidation is a common post-translational modification (PTM) which must be assessed and monitored because it can impact the stability, safety and efficacy of the final drug product. The NIBRT team performed experiments to investigate the susceptibility of methionine to oxidation by subjecting the IgG1 monoclonal antibody ipilimumab to oxidative stress, then analysed it with LC-MS.

The Orbitrap Exploris 240 mass spectrometer delivers confident tracking of PTMs in mAbs at intact, subunit and peptide level. The latest advances in high-resolution accurate-mass (HRAM) mass spectrometry can be deployed to pinpoint site-specific identification of oxidation and detect with confidence low-level methionine oxidation through diagnostic MS/MS fragmentation at the peptide level.

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Figure 2. Oxidative stress detected at the subunit level.

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  1. A) Zoom of base peak chromatograms (BPC) of separated G0F glycoform of the ipilimumab Fc/2 subunit obtained after IdeS digestion and reduction for the control and both stressed samples (50 and 500 ppm H2O2).
  2. B) Mass spectra of Fc/2 fragments acquired with a resolution setting of 120,000 (at m/z 200) showing near baseline-resolved isotope patterns of the +28 charge state of the Fc/2 G0F subunit, for the control sample plus the singly and doubly oxidized forms detected in the stressed sample (500 ppm H2O2 ).
  3. C) Results obtained upon deconvolution of the entire charge envelope including all Fc/2 subunit glycoforms using Sliding Window Xtract algorithm, obtaining accurate monoisotopic masses.

Read more in the Application Brief here.

And finally, they demonstrated how they can achieve confident peptide mapping and disulfide bond analysis of an IgG2 monoclonal antibody

A comparative analysis of reduced and non-reduced denosumab was used to characterize disulfide linkages. Using BioPharma Finder software, peptides were identified as either free cysteine-containing (reduced sample) or disulfide-linked peptides (non-reduced sample), illustrated as shaded peaks in the chromatogram (Figure 2). Most abundant expected intra- and interchain disulfide bonds were detected and confirmed based on MS/MS spectra with excellent (<3 ppm) precursor and fragment ion mass accuracies.

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Figure 3. Base peak chromatogram (BPC) of a non-reduced digest of denosumab.

Colored shading highlights identified cysteine-containing peptides connected via disulfide bonds. The table lists most abundant expected intra- and interchain disulfide bonds that were identified and confirmed.

“The Orbitrap Exploris 240 mass spectrometer is a reliable and flexible solution for high-performance biopharmaceutical characterization. I am really impressed by the sensitivity achieved for native MS analyses. When the instrument is coupled to the Vanquish Duo UHPLC system, we don’t have to wait during column equilibration. As a result, the mass spectrometer is constantly acquiring valuable data. With Chromeleon CDS software and all of its features, it is fantastic. The system is a must-have instrument for analytical and characterization labs supporting biopharmaceutical development and manufacture.” – Dr. Jonathan Bones, Principal Investigator, NIBRT

Listen to Jonathan Bones’ presentation from the EMEA BioPharma Seminars Week

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The conclusion of NIBRT’s mid-year review would say they evaluated a system that delivered on three BioPharma characterization improvement objectives. If you are also looking for a tool to help you with next year’s characterization challenges, look no further but look deeper into what the Orbitrap Exploris 240 can offer to help you reach your goals in 2020 and in years to come.