There’s no doubt that mass spectrometry has emerged as an essential technique for drug discovery and development. But the denaturing conditions of conventional LC-MS will typically break apart any non-covalent interactions that exist in solution – even though there is often a direct need to understand the very nature of these phenomena. Native mass spectrometry is a facet of MS that can maintain the folded state of the protein and thereby any non-covalent protein-protein and protein-ligand interactions. In drug development this can be particularly useful for assessing:

• Protein aggregation of biotherapeutics that can occur during the manufacturing process or from storage conditions
• Drug-Antibody Ratios (DAR) from covalent and non-covalent payload binding in Antibody-Drug Conjugates (ADCs)
• Protein-drug binding affinities and stoichiometries (fragment-based screening) during small molecule drug development

The space-saving high resolution X500B QTOF system is ideal for these studies because of its high mass range (necessary for native mode analysis), high mass stability, and robust, efficient ionization with ‘soft’ desolvation that preserves the non-covalent interactions. In my recent webinar Native SEC-MS to Study Non-Covalent Protein Interactions, you can see how we apply the technique for determining the identity and relative abundances of protein aggregates (dimers, trimers, etc). Then, for protein-ligand binding, SEC can filter out the non-specific binders from the protein-ligand complexes and determine the relative binding strength of various small molecule inhibitors for fragment-based screening. Additionally, using a fixed protein concentration we can generate ligand titration curves to determine dissociation constants. The stability observed over many sample injections under high salt conditions from the SEC is a testament of the robustness and suitability of the X500B QTOF system for native mode analysis.

Because there was so much interest in this topic, I wanted to take a moment to answer 20 questions that came up during the webinar and afterward. This is truly an exciting area that can greatly extend the capabilities of your laboratory for biotherapeutic and small molecule development!

1. What value do your customers see to implement SEC-MS rather than employing nano-infusion for fragment-based screening? There are several advantages. The first advantage is that the SEC separation provides online desalting, this removes the need for time-consuming and tedious buffer exchange and has the potential to increase assay throughput. The second advantage is that having a chromatographic separation allows for a greater dynamic range response. This is essential when looking for HMW aggregates of protein therapeutics as they are present at very low relative abundances. A third advantage is in the context of fragment-based screening: SEC separates the protein-ligand complex from the excess ligand. This mitigates non-specific protein-ligand formation that can occur during the ionization leading to false positives.

2. What conditions need to be optimized to achieve good protein-ligand data and good protein-protein data? Obviously, buffer conditions need to be optimized to facilitate good protein-ligand or protein-protein binding.  From an instrument standpoint, the main things to optimize are desolvation conditions, you want to have sufficient temperature, ionization voltage, and declustering potential to efficiently ionize the complex and knock off adducts, while still maintaining the non-covalent complexes.

3. Can your native SEC-MS approach for fragment-based screening be employed to analyze a mixture of fragments/ligands? Absolutely.  Although we demonstrated this for a single protein with several individual ligands, there is no reason you could not also do a mixture of proteins and ligands and undertake a competition assay. In fact, that is the next step in the work.

4. How do you know that’s really a dimer spectrum? Couldn’t that just be an artifact of the deconvolution algorithm? There is a really nice feature of SCIEX’s reconstruction software called Hyper Mass that overlays the zero charge reconstructed peaks against the original, multiply-charged spectrum. You can easily see if you are correctly hitting all the charge states in the raw spectrum. If you are not matching enough or matching too many, then those are harmonic artifacts.

5. To achieve the 5 ppm accuracy for 50 injections in an overnight sequence do you have to use a lock mass? No!  Lock mass is NOT necessary. SCIEX TOF ion optics are extremely stable and thus have very stable calibration.

6. How do you make sure that during ionization by ESI the native structure of the protein and its interaction with the ligand are maintained? How efficient is ESI for maintaining the native structure? Can we indirectly determine affinity and avidity based protein-ligand interaction detected by native MS? The two key parameters that enable the complex to be maintained are source temperature and the declustering potential. The optimization of these parameters is more critical for the non-covalent protein-ligand complex which can be a weaker interaction than the protein-protein interaction. However, the process of ESI does still impact the protein-ligand interaction and is particularly true for the weaker binders. For this reason, the best approach is to work with a relative affinity ranking rather than looking to determine the binding affinities. Surface plasmon resonance platforms like Biacore or ForteBio are still the gold standards for binding affinities and kinetics.

7. Are there any differences in instrument setup for the TripleTOF® 6600 LC-MS/MS System vs the X500B QTOF to maintain native conditions vs. denatured conditions? I tried Native MS with WCX separation on the TripleTOF 6600 but I’ve obtained almost only signals from denatured protein. There are differences between the two instruments, however, the instrument parameters and the results that are obtained are fairly consistent between the X500B and TripleTOF 6600.  The instrument itself won’t denature proteins, however using a very high desolvation temperature can cause denaturation and it could be advantageous to optimize the temperature settings. Keep in mind, the buffer compositions for running WCX are often not a buffer system that will preserve the native state of the protein due to the pH gradient.

8. What amount of protein is injected for one analysis? Since we are looking to understand the aggregate species that are present at approximately 1% relative to the monomer we inject as much as possible to ensure we get great signal for the aggregate species. In this case, for the NIST mAb, we injected 20 ug of sample.

9. What is eluting at 1.68 minutes for the NIST mAb aggregate analysis? That peak is the small molecule components that make up the formulation buffer.

10. Do you use different parameters (declustering potential, etc. ) for both HMW and LMW analysis in the run? In the case of looking at the HMW and LMW species of the monoclonal therapeutic, it was not necessary to use different declustering potentials to obtain optimal results for the impurities of different sizes. However, when working with the fragment-based screening approach we are able to create multiple experiments within the same method to ensure we have experimental conditions suited to both the intact protein and for the small molecule within the one run.

11. Which parameters allow soft ESI source conditions to maintain complexes? The two key parameters that enable the complex to be maintained are source temperature and the declustering potential. The optimization of these parameters is more critical for the non-covalent protein-ligand complex which can be a weaker interaction than the protein-protein interaction.

12. What is the highest concentration of ammonium acetate in the mobile phase that one can get away with before it becomes non-volatile? The SEC separation is undertaken with isocratic buffer conditions and we have worked with 10 mM up to 200 mM ammonium acetate. Generally, the higher you go in salt molarity, the lower your signal will be.  We have found we achieve the best results at 50 mM ammonium acetate. This concentration appears to keep the non-covalent complex intact without causing a dramatic loss in the assay sensitivity.

13. Can you please comment on the buffers and columns used for native SEC-MS? Two SEC columns are used in this work. For the therapeutic antibody aggregate work the column employed was a Waters Acquity BEH SEC guard column 200Å, 1.7 µm, 2.1 x 30 mm. For the fragment-based screening work, the column was polyLC polyHYDROXYETHYL A Column 60 Å, 1 mm x 50 mm or 2.1 mm x 50 mm. In both cases, the buffer was 50 mM ammonium acetate. The details of the workflows can be found in these technotes:

• Native Analysis of Intact Monoclonal Antibody Aggregates Using the TripleTOF 6600
• Native Mass Spectrometry using the X500B QTOF System for Fragment-Based Screening

14. What is the flow rate and the buffer for FIA? Flow injection analysis (FIA) was run using a flow rate of 350-μL/min and the buffer employed was 50 mM Ammonium Acetate.

15. Do you deconvolute charge or do you use m/z spectra to determine relative quant of bound and unbound forms? We use the reconstructed data for bound vs unbound determination.

16. What are the pressure parameters in the MS? A higher pressure is often needed in the first pumping stage of many MS instruments to maintain complexes and maximize sensitivities. One key advantage of the SCIEX technology is that the QJet® Ion Guide appears to sufficiently cool and focus the ions so that higher pressures are not required to ensure maximum sensitivities are achieved.

17. Can you analyze polymers with high molecular weight? We have been able to successfully analyze polymers using both the TripleTOF 6600 System and the X500B. However, this is typically undertaken using conventional denaturing buffers.

18. Can the technique be used in any way for the analysis of nano compounds such as nano complexes aggregates? It will largely depend on the molecular weight and charge composition of the nano compound.  There is no reason to think that it will NOT work with nano compounds, but we have not had access to them to test.
19. What are the pH conditions for the protein-protein interactions? The buffer was 50 mM ammonium acetate, the pH was not adjusted.

20. What parameter do you use to acquire the data? The chromatographic and mass spec parameters are outlined for both the aggregate studies and the fragment-based screening workflows in two technotes:

• Native Analysis of Intact Monoclonal Antibody Aggregates Using the TripleTOF 6600
• Native Mass Spectrometry using the X500B QTOF System for Fragment-Based Screening