BioAccord™ LC-MS System Enhancements for Improved Native MS Analysis

This application brief demonstrates the significance of recent enhancements to the BioAccord LC-MS System for biopharmaceutical analysis workflows requiring native mass spectrometry (MS) analysis. The extension of the mass detection window on the BioAccord System's ACQUITY™ RDa™ Detector, now reaching up to m/z 9000 in positive ion mode, enables more effective MS analysis of larger protein complexes and protein conjugates, broadening the system’s applicability for characterization, attribute monitoring, and quality control for these emerging biopharmaceutical modalities.

Benefits

• Extended mass detection up to m/z 9000 in positive ion mode widens the acquisition mass range sufficiently for analysis of larger protein species such as monoclonal antibody (mAb) aggregation, noncovalent protein complexes, and native MS analysis of antibody conjugates with higher drug-to-antibody ratio (DAR) values.
  
  

The BioAccord LC-MS System (Figure 1), originally launched in 2019, is a high-resolution compact benchtop time-of-flight mass spectrometry (TOF MS) system engineered for robust biopharmaceutical analysis. It has demonstrated capability for the analyses of intact mAbs and proteins,^1–2 peptide mapping,^3–4 antibody-drug conjugates (ADCs),⁵ released glycans,^6–7 oligonucleotides,^8–9 and emerging biotherapeutic modalities such as mRNA^10–11 and viral vectors.¹² Key features such as automated instrument setup, workflow-based analyses, and the compliance-ready waters_connect™ Informatics Platform facilitate rapid adoption by analysts— regardless of their MS experience levels— across discovery, development, manufacturing, and QC/release organizations.

The BioAccord System incorporates a source design that is optimized for gentler ionization, making it particularly effective for native MS applications, where the preservation of the protein tertiary structure is critical. Examples of native MS analysis that are becoming more routine for biopharmaceutical analysis include: the structural assessment of non-covalent protein complexes or conjugates and for pairing with nondenaturing chromatographic techniques such as ion exchange chromatography (IEX)^13–14 and size exclusion chromatography (SEC).⁵

Notably, raw combined mass spectra differ significantly between denatured and native MS conditions (Figure 2).  For example, under native conditions (Figure 2B), the MS signal for the National Institute of Standards and Technology (NIST) mAb Reference Material 8671 appears with fewer charge states at higher m/z values (corresponding to lower charge states) vs. the typical denaturing LC-MS pattern (Figure 2A). This difference reflects the reduced chargeable surface area of folded proteins compared to their denatured counterparts. The original BioAccord System supported a positive ion m/z range of up to 7000, which proved sufficient for most mAb analysis, denaturing or native. However, as biopharmaceutical development continues to push the boundaries of innovation with more complex molecules and conjugates, the BioAccord System has been enhanced to offer an extended mass range now up to m/z 9000, providing greater flexibility for the analysis of larger or more complex protein species. 

For this study, native SEC-MS was carried out on an ACQUITY UPLC™ Protein BEH™ SEC Column, 200 Å, 1.7 µm, 2.1 x 150 mm (p/n: 186008471) maintained at 30 °C using a 10-minute isocratic flow of 50 mM ammonium acetate, pH 6.8. The ACQUITY RDa Detector was set to collect MS data in electrospray ionization (ESI) positive mode, comparing two mass ranges: 1) High Mass Range (HMR): m/z 400-7000 and 2) the new Extended Mass Range (EMR): m/z 400-9000. Calibration of both modes can be accomplished with the existing standard BioAccord calibration solution, also useful for lower m/z mode system calibrations.

The protein samples of interest in this case were yeast alcohol dehydrogenase (ADH), a known multimeric protein, and a stressed infliximab sample which contains a small percentage of high molecular weight (HMW) species detectable by SEC-MS.

yADH is a cytosolic protein that naturally self-associates into a tetrameric complex.¹⁵ Using the standard HMR (m/z 400–7000) acquisition mode, the ACQUITY RDa Detector readily identified ADH tetramer (~148 kDa) and dimer species (~ 74 kDa) (Figure 3A). Applying the EMR (m/z 400-9000) mode, the additional octamer¹⁶ (~ 296 kDa) was observed (Figure 3B), demonstrating the capability for analysis of larger non-covalent protein complexes and aggregates. 

In an additional set of experiments, a sample of infliximab was stressed by freeze/thaw cycles in the attempt to induce aggregation. A small percentage of HMW species was detected in the UV280 chromatogram (Figure 4A, highlighted in red). This HMW species was also observed by mass detection on the BioAccord System (Figure 4B inlay, red spectrum) and compared with the spectrum from the monomer peak (Figure 4A inlay, black spectrum). The observed HMW (dimer) species appears in the m/z 6500-8500 range, enabled by the EMR mode of the ACQUITY RDa Detector, and has a mass consistent with the expected mass for the dimer of infliximab (Figure 4B, bottom panel). This dimer species would not have been detectable under the original HMR settings.

The extension of the mass detection window on the BioAccord System to m/z 9000 in positive mode allows for effective analysis of higher molecular weight species or those shifted to higher m/z by conjugation or LC inlet conditions. This study showed successful detection of ADH protein higher-level multimers and a mAb dimer species resolved via native SEC-MS analysis. The EMR mode of the BioAccord System directly addresses growing industry demands for characterizing larger protein complexes, protein conjugates with increasingly complex payloads (e.g. ADCs or antibody-oligonucleotide conjugates), and for the study of aggregation prone therapeutics.

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