For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns.
Journal of Chromatography A 1685 (2022) 463638 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Col Liquid Chromatography Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry ES Hecht a,∗ , EC Obiorah a , X Liu b , L Morrison b , H Shion b , M Lauber b,∗ a b Genentech, Inc South San Francisco, CA, USA Waters Corporation, Milford, MA, USA a r t i c l e i n f o Article history: Received 13 August 2022 Revised November 2022 Accepted November 2022 Available online November 2022 Keywords: Size exclusion chromatography (SEC) Microflow Native MS Noncovalent interactions a b s t r a c t For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC) Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns Moreover, the efficient packing of small bore columns is notoriously difficult SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting Here, small bore format online SEC for native MS studies is again investigated but with alternative materials We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 μm organosilica particles as packed into mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST) For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 μL/min) on a × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 μL/min) at the same linear velocity Run times were also shortened by 45% A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte MS LOD values were significantly reduced into the low attomole ranges Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions The ability to detect 0.1 to MDa complexes, with between and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics © 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction The biggest challenges in native mass spectrometry (MS) of proteins include preserving structures, stabilizing non-covalent interactions, and achieving high signal-to-noise detection When successful, native MS provides a wealth of information about the biological properties of intact protein assemblies under nearphysiological conditions [1,2] When used in a biotechnology en∗ Corresponding authors E-mail addresses: hecht.liz@gene.com (E Hecht), matthew_lauber@waters.com (M Lauber) vironment, this information can contribute to early stage understanding of disease states and provide quick insights on the formulation, processing, and purification of therapeutic products [3] Industry adoption of native MS remains limited by the accessibility of instrumentation, the throughput of the experiments, and the reproducibility of the measurements In the field of intact analysis, solutions exist to meet these challenges, with diverse chromatography options ever improving to provide compatibility with time of flight (TOF), Orbitrap, and Fourier transform ion cyclotron resonance (FTICR) MS instrumentation [4] In both native and intact analysis, proteins or large molecule drug targets are typically over-expressed and then isolated from cells for further study Re- https://doi.org/10.1016/j.chroma.2022.463638 0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 verse phase liquid chromatography (RP-LC) [4] or capillary electrophoresis (CE) [5] for intact protein analysis show value in giving reasonable throughput and leading sensitivity by means of oncolumn clean-up and focusing While CE methods have emerged for native mass spectrometry [6], they remain limited by surface interactions that hinder reproducibility, resolution, and sensitivity [7] Ion exchange chromatography and hydrophobic interaction chromatography, when performed with volatile mobile phase components, are compatible with MS and have been used in native applications including the mispairing of bispecific antibodies [8], antibody-drug-conjugate quantification [9], and mass deconvolution of heterogeneous targets [10] However, the high salt and elevated pH conditions required by those techniques can be destabilizing to some protein aggregates or complexes Size exclusion chromatography (SEC) is a compelling and widely used option for these applications, and it separates solutes on the basis of their hydrodynamic radii That said, it is a low sensitivity technique that requires concentrated samples, and, as with any instance of sample handling, it can be difficult to mitigate the disruption of noncovalent interactions [11,12] There are ways to enhance MS sensitivity independent of the protein separation Historically, nano-electrospray (nESI) (static spray) of proteins has achieved the best limits of detection yet does not facilitate rapid screening [13,14] The application of multichannel emitters that are compatible with microflow flow rates bridges the gap between throughput and sensitivity Multichannel emitters most commonly work by splitting a single flow channel into multiple outlets so as to simultaneously spray liquid from more than one tip Each tip within this clustered sprayer has a lower onset voltage and flow rate [15–18] In addition to sensitivity improvements, the reduced onset voltage and an application of sheath gases helps keep proteins and noncovalent complexes in their native state [13,14] The degree to which a structure remains native is correlated with charge state, where denaturation leads to higher charge states and lower m/z values [19–21] Nearly all commercial SEC columns have internal diameters (IDs) of 4.6 mm or higher, and are recommended for use with flow rates of 200 μL/min or higher, making microflow compatibility difficult Split-flow methods can be used to access this flow regime, but that comes with higher sample requirements to see significant sensitivity gains [22] There is a paucity of work on microflow SEC applications, especially when compared to the comprehensive literature that can be found for capillary/microflow intact RPLC analyses [23–27] The packing of SEC phases into small column dimensions, compatible with lower flow rates, presents manufacturing challenges brought on by the disproportionate increase of the wall surface area to particle sizes and an increasing likelihood of uneven flow and poor column efficiencies [28] If packed with excessively high flow rates or pressures, bed compression can also limit resolution, when a compressible packing material is used Lastly, any gains in sensitivity from smaller columns must be balanced with refined needs for sample concentration, where the injection volume is ideally < 5% of the column volume [29] Likewise, to access reasonable sensitivity, instrumentation considerations must be made to minimize extra-column dispersion effects, though these issues are bound to be faced in any miniaturized SEC experiment Microflow native MS using polyacrylamide beads packed into 300 μm diameter columns was recently demonstrated [30] Very high sensitivity was achieved, although the columns were not compatible with pressures greater than 400 psi and did not provide separations other than the fractionation of protein samples from salts Two microflow SEC columns are commercially available One, a μm hydrophillic diol-bonded phase is available from Tosoh Biosciences as a × 30 mm column, where the maximum flow rate is 20 μL/min; no LC-MS applications with this hardware dimension were found in literature searches Second, the polyhydroxyethyl A column from PolyLC, Inc is available across a range of IDs and lengths The ideal flow for the 2.1 mm column (various lengths) was 100 μL/min across different studies [31,32], demonstrating significant advantages over larger columns, yet no peer-reviewed literature could be found for a mm ID version of these columns The following work entails the use of 1.7 μm ethylene bridged hybrid (BEH) particles packed into small hardware (1 × 50 mm) dimensions and an investigation of these columns for native MS on noncovalent complexes The effects of hardware dimensions, column hardware surfaces, and particle surface chemistry are assessed over a range of native MS applications and instrumentation We demonstrate that new capabilities can be had with the use of microflow columns constructed from hydrophilic hybrid surface technology (h-HST) column hardware [33] and packed beds made from hydroxy terminated polyethylene oxide silanized BEH (HOPEO BEH) particles [34] These devices make it possible to expand beyond traditional native protein characterization to the study of complexes that otherwise dissociate by high-flow SEC approaches Indeed, the new surface chemistries are seen to minimize chemical forces that are disruptive to complexes, such that is possible to preserve quaternary interactions that have relatively weak dissociation constants ranging from to 40 μM In sum, this work provides compelling evidence for the capabilities of small dimension SEC as an MS-inlet technique that provides sufficient desalting and chromatographic resolution, minimal disruption to native complexes and 99.9% purity 2.2 Column manufacturing SEC packing materials were prepared from organic/inorganic hybrid particles with an empirical formula of SiO2 (O1.5 SiCH2 CH2 SiO1.5 )0.25 [37] One batch of these BEHTM particles was bonded with a hydroxy terminated (HO) polyethylene oxide (PEO) silane [34] This packing material is referred to herein as HO-PEO bonded BEH or HO-PEO BEH The average particle size of this packing material was 1.64 μm in diameter, and the particles were measured to have an average pore diameter of 262 ˚ surface area of 170 m2 /g, surface coverage of 1.15 μmol/m2 and A, pore volume of 1.26 cm3 /g A second BEH packing material was investigated in this study and it was acquired in the form of bulk manufactured diol-bonded BEH particles, just as they are prepared for commercially available columns (ACQUITYTM UPLCTM BEH200 SEC columns, Waters, Corporation, Milford, MA) The selected batch had an average particle diameter of 1.54 μm, average pore ˚ surface area of 225 m2 /g, pore volume of 1.30 diameter of 193 A, cm3 /g and surface coverage of 5.42 μmol/m2 These SEC packing materials were either packed into × 50 mm stainless steel column hardware or × 50 mm column hard2 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 ware that had been treated to have a hydrophilically modified hybrid organic/inorganic surface In a previous report, this latter type of hardware has been referred to as h-HST hardware, which stands for hydrophilic hybrid surface technology [33] Columns were slurry packed using constant pressure packing conditions and to produce columns with mechanical stability to withstand pressurization to beyond 6,0 0 psi trace From these Gaussian fits, peak width and apex elution time were extracted 2.6 Statistics and curve fitting R was used to fit dilution curves and determine limits of detection Dilution curves used linear or quadratic equations as most appropriate to the data The regression fit of each model was evaluated with the Breusch-Pagan test, and where appropriate, a weighted model was used [38] The limit of detection was calculated as 3.3 x the standard deviation of the intercept/slope The gradient end time was selected as the point at which the A280 spectra returned to baseline after the elution of the last peak (uracil), and the start of the thyroglobulin elution, whose size is above the exclusion limit of the pore size of these SEC particles The theoretical plates (N) reported was calculated as the mean apex protein elution time/standard deviation across the fiveprotein mix The height was calculated as the average plate/the length of the column The peak capacity (P) was calculated as P = + sqrt(N)∗ 0.2 The resolution of the UV peaks was evaluated as the difference in the retention time between two peaks, divided by the original reference retention time 2.3 LC-UV-MS All protein samples were diluted or resuspended in 50 mM ammonium acetate and used within three days of thawing All dilution curve experiments were injected from least to most concentrated, with a minimum of a 10 wash and re-equilibration time between each sample for the small dimension hardware to ensure no carryover Regardless of the concentration, the injection volume was held constant at μL For ConA experiments, all injections were 510 ng (5 picomole based on the tetramer mass), with the exception of split flow experiments, where 153 ng was directly injected at 15 μL/min, or else 1012 ng was injected at 100 μL/min For hexamer experiments, 480 ng was injected onto the column For PLBL2-IgG4 experiments, a 2:1 molar complex, equating to 250 ng of each species, was injected A VanquishTM LC (Thermo Fisher Scientific, Waltham, MA) was operated in isocratic mode with 100% 50 mM ammonium acetate at appropriate micro or high flow rates Regardless of the flow rate, the column outlet was connected to a biocompatible column cooler, a semi-micro bio 2.5 μL flow cell, and then a switching valve via a 100 μm x 750 mm stainless steel line A 0.005" ID peek line was used for connecting a HESI (heated electrospray ionization) source The HESI source was operated at an inlet temperature of 275 °C and an electrospray voltage of kV For low-flow experiments, the sheath gas was set to 15 with no additional heat and for high flow (50 or 300 μL/min) experiments, the sheath gas was set to 20 and a 30 °C auxiliary gas was set to Unless specified, all microflow × 50 mm SEC-MS data was collected with the multichannel emitters A 75 μm x 550 mm nanoViperTM line (Thermo Fisher Scientific, Waltham, MA) was used to connect to the microflow-nanospray Electrospray Ionization (MnESI) source, equipped with 20 μm, nozzle emitters (Newomics, Inc Berkeley, CA) The MnESI source was operated at kV ESI voltage and without the use of sheath gases After ten minutes of isocratic flow, the switching valve changed positions and infused methanol over the multichannel emitters from an external syringe pump (Chemex, Chicopee, MA) at 35 μL/min, while the column was simultaneously washed for 2.5 at 40 μL/min, followed by a pressure re-equilibration to twenty minutes total UV data was collected at 280nm Q ExactiveTM UHMR (Thermo Fisher Scientific, Bremen, DE) mass spectrometer settings were tuned for the sample of interest Results and discussion 3.1 Evaluation of microflow small dimension SEC columns for UV and MS analyses The small dimension SEC columns evaluated in this study were designed to optimize MS sensitivity and speed for the analysis of native proteins and complexes In this work, we have studied samples ranging from a monoclonal antibody (trastuzumab) to a protein test mixture as well as concanavalin A, which forms a concentration-dependent dimer and tetramer Microflow columns yield smaller electrospray droplets that lead to increases in ionization efficiency but it is challenging to achieve efficient separations with them Standard stainless steel (SS) columns or hydrophilic hybrid surface technology columns (h-HST) of 4.6 × 150 mm or × 50 mm dimensions were packed with standard diol bonded BEH or HO-PEO bonded BEH particles Using the MS-compatible mobile phase of 50 mM ammonium acetate, the MS sensitivity of these devices was explored followed by a characterization of their UV capabilities A control experiment was first done to estimate the ionization gains that come with moving from a traditional electrospray source (HESI) to multichannel emitters at 15 μL/min (Figure S1) A 3-fold gain in signal-to-noise and a shift to a more native charge state distribution was observed when replacing the HESI source with the multichannel emitter (MnESI) source for the analysis of trastuzumab (Figure S1A) The reduction of 1-2 charges, on average, indicated that the protein remained in a more native state The harshness of the HESI source was further apparent on its effects on the ConA tetramer, which was solely preserved by the MnESI source (Figure S1B) Thus, hereafter, microflow comparisons between small bore columns were made using the multichannel emitters and a HESI-appropriate flow rate of 50 μL/min was used to compare large and small bore columns of the same chemistries A seven-point standard curve of trastuzumab from 1- 400 ng was generated across all columns and analyzed by MS (Fig 1A) With the flow rate controlled at 50 μL/min, the 4.6 × 150 mm column yielded an LOD of 724 attomole, while the × 50 mm SS/BEH Diol column produced an LOD of 253 attomole (Table S1) When operated at 15 mL/min, this same column gave an LOD of 85 attomole, while its h-HST/HO-PEO BEH equivalent produced an LOD with a value of 60 attomole These results suggest that column 2.4 Mass spectrum deconvolution ByosTM software (Protein Metrics Inc, Cupertino, CA) was used for intact mass deconvolution For all intact analysis, the intensity summed across all charge states was used and, if applicable, further summed across glycoforms 2.5 UV Peak fitting MagicPlot software (Magicplot Systems, Saint Petersburg, RUS) was used to peak fit the UV data spectra Gaussian curves were provided with an initial set of parameters defining their approximate elution time, intensity, and width The software then performed a simultaneous multi-fit optimization of these parameters such that the sum of squares was minimized against the A280 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig Standard curves for the MS detection of (A) trastuzumab, (B) IgG, (C) BSA, or (D) myoglobin were generated, with the log2 summed intensity plotted against concentration Confidence intervals (95%) are shown as shaded ribbons miniaturization, low flow rate, and reduced secondary interactions can each contribute to gains in MS sensitivity Standard curves were next built for three of the five proteins injected from a more complex, five-protein test mixture spanning molecular weights from to 600 kDa Thyroglobulin could not be detected by MS due to its large size and heterogeneity, and uracil fell below the mass cutoff of the instrument For BSA, IgG, and myoglobin, the × 50 mm microflow h-HST/HO-PEO BEH column performed the best with LODs of 40, 39, and 14 attomoles, respectively This was a 34%, 58%, and 61% reduction compared to the 4.6 × 150 mm column, respectively (Table S1) All small dimension columns detected proteins over at least three orders of magnitude At low concentrations (less than 100 ng), the signal began to plateau (Fig 1BCD) The Q ExactiveTM UHMR is an ion trapping instrument and all runs maxed out their method injec- tion time (200 ms) (AGC value not reached) It is possible that by increasing the injection time, a larger dynamic range could be achieved at the cost of fewer points across the curve Interestingly for the SS/BEH Diol columns, saturation was observed at high concentrations for trastuzumab, myoglobin, and uracil This suggests that at some point, any potential gains in signal from increased protein loads are mitigated by a corresponding increase in nonspecific binding to non-coated surfaces Columns were also compared based on changes in the signal at the midpoint of the standard curve, rather than at the limit of detection Increases of up to 100-fold gains in absolute signal intensity were observed for the three protein mix analytes compared to approximately 10-fold gains observed for trastuzumab (Fig 1) Similar observations were made upon comparing results from the SS/BEH Diol × 50 and 4.6 × 150 mm columns Thus, it appears that much of the advan4 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig A280 traces for elution of the protein mix are shown in dark blue for the (A) 4.6 × 150 mm column or the (B) SS/BEH Diol (C) h-HST/BEH Diol (D) h-HST/HO-PEO BEH × 50 mm columns The fitted Gaussian peaks that were used for LC peak resolution and capacity calculations are shown as overlaid traces The spectra shown were generated from an injection of protein mix containing uracil, BSA, myoglobin, IgG and thyroglobulin at concentrations 0.02, 1, 0.04, 0.4, and 0.6 μg, respectively Table Separation characteristics of columns, determined by UV, injected with a five protein test mixture As described in Section 2.6, all figures of merit were calculated from Gaussian fits to the LC trace and reported as the average across all proteins For comparison, the theoretical plates calculated from the uracil peak is also provided Dimension (mm) Flow Rate (μL/min) Hardware Particle Theoretical Plates, Uracil Theoretical Plates, 5-Protein Average Avg Height Avg Peak Capacity × 50 × 50 × 50 4.6 × 150 15 15 15 300 h-HST h-HST SS SS HO-PEO BEH BEH Diol BEH Diol BEH Diol 6281 5052 3566 62939 5709 4848 3457 35100 114 97 69 234 16.1 14.9 12.8 38.5 creased from 35100 on the 4.6 × 150 mm column at 300 μL/min, to approximately 460 (+/- 110 0) on the × 50 mm columns (Table 1) Peak capacity was reduced by up to 63% on the small bore columns However, the addition of h-HST and then additionally HO-PEO BEH particles resulted in small but statistically significant (t-test, p < 0.05) increases in peak capacity and theoretical plates between the small bore devices As noted before, optimization of pre and post column tubing might help in future work to reduce the dispersion of the small volume chromatographic bands that are generated during microflow SEC With the addition of h-HST surfaces and HO-PEO particles, the resolution between peaks of proteins < 10 0,0 0 Da significantly increased, whereas the separation between the larger proteins was less affected (Fig 2) Proteins can exhibit unique types of nonspecific binding depending on their physicochemical properties The small proteins in the text mixture might be subject to pronounced surface interactions as evidenced by their comparatively wider peak widths This effect was further highlighted in a comparison of columns using a single injection of trastuzumab (Figure S2A) Unlike on the 4.6 × 150 mm column, trastuzumab eluted as two peaks on all × 50 mm columns MS analysis confirmed there to be no differences in post translational modifications and the charge state distribution was identical, suggesting no perturbations to structure (Figure S2B) Consequently, when standard curves of trastuzumab were generated on the × 50 mm columns, the main peak showed high nonlinearity compared to the 4.6 × 150 tage conferred to complex mixtures on small bore columns derives from the use of microflow ESI and the related ionization efficiency gains To provide a thorough characterization of these devices, we also compared the separation capabilities for the protein test mixture by online UV detection (Fig 2) It should be noted that the comparisons described used a single LC instrument without any runto-run adaptions The same flow cell and LC capillary lines were used for both small and wide bore columns to model the practical use of an LC in an industry lab, where a user often cannot re-plumb a configuration for a specific application Thus, optimization of the LC system for microflow conditions might be an area of future work that would likely result in improved performance for mm ID SEC analyses Key metrics for column evaluation, including plate heights, peak capacity, and the limits of detection, were determined for the standard five protein test mixture SS/BEH Diol 4.6 × 150 mm columns were run at 300 μL/min and × 50 mm columns at 15 μL/min, which yields comparable linear velocities Certain features were lost entirely in the transition to small columns, including the shoulder observed on the IgG protein, which corresponds to a dimer species (Fig 2) For all mm and 4.6 mm diameter experiments, thyroglobulin eluted at 1.75 and min, respectively This translated to a 41% reduction in elution time A significant loss in observed plate count was expected and observed for the switch to mm ID columns The number of average theoretical plates de5 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig The standard curves for UV detection of (A) trastuzumab (B) myoglobin (C) IgG (D) thyroglobulin (E) BSA or (F) uracil are shown with their confidence intervals Area under the curve was calculated from the Gaussian fits to the raw spectra data for each protein E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig (A) Comparison of the charge summed deconvolved intensity ratio of the tetramer to all peaks for 510 ng ConA on × 50 mm hardware with a 15 μL/min flow rate (B) Values obtained with the h-HST/HO-PEO BEH column from 153 ng of ConA loaded under microflow conditions (15 μL/min) or 1020 ng loaded and run with a 1:6.7 splitflow (153 ng effective MS detection) (C) Comparison of the percent of glucose bound to ConA across the different × 50 mm columns All experiments were performed with three replicates mm columns However, when the sum of the main and secondary peak areas were modeled, the nonlinearity was rescued (Figure S3) The resolution between the first and second trastuzumab peaks increased with the use of h-HST hardware and the HO-PEO BEH particles (Figure S2A) Additional work is needed to understand the behavior of trastuzumab and its split peaks It is possible that system effects, including flow rates, column pressures, and injector processes might also be at play and impacting separation quality For the microflow h-HST/HO-PEO BEH column compared to the 4.6 × 150 mm column at 300 μL/min, the UV LOD for trastuzumab, BSA, IgG, and uracil decreased by 32%, 75%, 89%, and 85% (Table S2) Myoglobin was detected with an approximately equal LOD of picomole, respectively Myoglobin represented the smallest protein eluting from the mix, and had the greatest peak overlap with other proteins in the × 50 mm ID columns (Fig 2), potentially accounting for there being no change in LOD Thyroglobulin was detected with reduced sensitivity on microflow columns Thyroglobulin is largely excluded from the intraparticle pores of the applied BEH packing material With a compressed elution time in microflow columns, this could cause a decrease in sensitivity, and this issue would likely be solved through use of larger pore size particles The microflow columns showed high reproducibility, with minimal retention time shift, sensitivity loss, and column degradation over 150 injections Full robustness testing was beyond the scope of this study, where the focus ultimately was to provide a base level characterization of the column behavior and demonstrate their utility for MS experiments Some aspects of the robustness of the microflow SEC device can be predicted from the performance of the applied BEH particle and its history of use in analytical scale applications To this end, it can be noted that batch-to-batch reproducibility for the HO-PEO BEH packing material has been previously reported [34] Particles corresponding to different manufacturing batches were studied in 4.6 mm ID column hardware and used to separate NIST mAb reference material 8671 with a phosphate buffered saline mobile phase Elution times, area %, USP resolution and USP tailing values were compared for the monomer main peak as well as high molecular weight species RSD% values were all less than or equal to 7% Column lifetime was also previously investigated for a 4.6 × 300 mm packing of 1.7 μm Fig (A) The ∼900 kDa intact RGY antibody hexamer (black) elutes from 2.22.5 and undergoes gas-phase dissociation into monomer, dimer, trimer, and tetramer units (B) The RGY hexamer’s in-solution monomer (red) independently elutes at 2.7 particles No change was observed in elution times and area% values after the course of 10 0 repeat injections E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig A 443 kDa hexamer complex of protein trimers (18 proteins total) was observed by the × 50 mm h-HST/HO-PEO BEH SEC microflow column Extracted ion chromatograms of the (A) hexamer, (B) tetramer, (C) dimer, and (D) monomeric species are shown, where the “monomeric unit” is considered to be the protein trimer (E) The average spectra showing the in solution dimer, tetramer, and hexamer from 2.3-2.7 (F) The average spectra from 3-3.4 showed the tailing of the hexamer, a gas-phase generated dimer, and the monomeric species The microflow SEC devices described in this work showed improved LODs for both MS and UV detection Overall, the × 50 mm columns offered a more sensitive platform compared to the large bore columns, compounding the benefits of reduced surface interactions and improved ionization efficiency The small bore columns had peak capacities approaching 20 (Table 1), differentiating the column from alternative desalting columns, such as those packed with compressible large particle size materials [30,39,40] The × 50 mm SEC columns constructed with h-HST hardware and HO-PEO BEH particles offered a highly sensitive column for MS analysis with fast LC run times that enabled attomole level protein detection [42] Sensitivity to pH makes it potentially more susceptible to gasphase dissociation due to the acidic nature of electrospray ConA binds glucose and PNM with dissociation constants of 5.7 [43] and 40.9 μM [44], respectively While ConA has been extensively studied, it is well documented that it can present problems when analyzed by chromatography During purification of glycoproteins where ConA crosslinked to sepharose is used in an affinity column, leaching of ConA is a historic and persisting problem [45] Likewise, while ConA is well studied in the field of mass spectrometry, to our knowledge, all analyses of the native tetramer by MS has been done via direct infusion [46,47] As discussed earlier and shown in Figure S1B, the ConA tetramer could not be observed from the high flow setup, even with up to μg injections, due to the harshness of the electrospray source Across the microflow columns, differences in ConA tetramer detection were observed The ConA tetramer was quantified from the SS/BEH Diol, h-HST/BEH Diol, and h-HST/HO-PEO BEH microflow columns at 0.5%, 3.7%, and 7% of the total protein signal, respectively (Fig 4A) The combination of hydrophilically optimized particles and column surfaces maximized the amount of multimer detected, suggesting that secondary surface interactions could be responsible for complex dissociation To further investigate effects that can influence tetramer recovery, split flow and higher linear velocity experiments were performed The same h-HST/HO-PEO BEH column was evaluated for a 153 ng, μL injection at 15 μL/min and a 1020 ng, μL injection at 100 μL/min For the latter scenario, post column flow was split at a 1:6.7 ratio to ensure equal protein concentrations were elec- 3.2 Noncovalent complex stability as a function of particle chemistry and hardware The detection of noncovalent complexes is a particular challenge for native SEC-MS, where the column can cause complexes to dissociate The stability of a complex can be affected by pressure, nonspecific interactions, shear, buffer, and pH Protein complexes of interest are also often found at low relative abundances Accordingly, we studied the effects of the microflow columns for several well characterized protein-protein and protein-small molecule systems ConA is a tetrameric lectin with the capacity to bind up to four glucose and mannose type sugars [41] Formation of the ConA tetramer is reversible, with the tetrameric form stabilized at neutral to high pH, and the dimer favored under acidic conditions E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Fig The PLBL2-IgG4 complex as observed by (A) the h-HST/HO-PEO BEH SEC microflow device (black) or (B) static spray (red) The peaks corresponding to the complex are shown with stars trosprayed into the mass spectrometer for both conditions There was a statistically significant loss of tetramer (29%) observed when the column flow rate was increased to 100 μL/min (Fig 3B) ConA forms in a concentration dependent fashion An increased concentration would theoretically increase the relative percent starting tetramer in solution So the observed reduction could in fact be an underestimation of the amount loss Between the 100 μL/min and 15 μL/min flow rates, the pressure increased from 41 to 237 bar, and the elution time decreased from 3.7 to 0.6 As the split-flow and microflow experiments were performed under laminar flow conditions, the only changes to shear forces would be in direct correlation to the change in flow rate Therefore, it is possible for the equilibrium of the complex to have been affected by the high flow and >200 bar pressure conditions Additional experiments with controlled flow restriction might help better elucidate the operational boundaries to consider for these types of microflow SEC-MS experiments and application of SEC to weakly bound complexes Small molecule binding was next studied Each ConA protomer has the ability to bind small ligands Glucose was detected bound to the tetrameric form of ConA by microflow-SEC-MS only when the h-HST column hardware was employed (Fig 3C) Interestingly, there was no difference in the ratio of tetramers with glucose bound between the HO-PEO BEH and BEH Diol particles In all cases, a high concentration of ConA (∼5 picomol) was applied and evidence of column overload can be seen in the form of peak tailing Nevertheless, a comparison of the MS1 total ion chromatogram (TIC) between control, PNM, and Glu binding experiments showed clear differences, with new peaks corresponding to multiple binding events detected (Figure S4) In future work, it might be of interest to assess the limits of detection for protein-ligand complexes across a range of binding affinities 3.3 Application of small ID hardware to characterize therapeutic complexes For therapeutic complexes, reproducibility, sensitivity, and specificity gains must be balanced with the speed of analysis We sought to benchmark the utility of the consistently best performing column, the × 50 mm h-HST/HO-PEO BEH column, across protein therapeutic applications Antibody hexamer structures, for example, routinely need characterization to qualify higher order structure features, including relative quantification of subunit to intact species, clipped species and glycoforms Yet, due to sensitivity issues exacerbated by the challenge to efficiently transmit high m/z ions, native MS is generally performed with direct infusion and static spray tips even when an SEC-UV or SEC-MALS method has already been established [35,48-50] For the first time, we have detected a 900 kDa RGY antibody hexamer species from online native LC-MS This allows the accurate quantification of the hexamer to monomer ratio and to look at monomer glycoform enrichment within the hexamer As shown in Figure S5, the hexamer species is chromatographically resolved from the antibody monomer The most abundant free monomer Ab species was 529.1 Da less in mass than the hexamer-dissociated monomer (Fig 5) This mass difference corresponds to a HexNAcHex2 residue, confirming prior work that showed higher-mass glycans are enriched in the hexamer complex [35] This online SEC approach extends to hexamers formed from different noncovalent protein subunits In Fig 6, the elution profiles of a three-protein complex (74 kDa) are shown This protomer structure assembles into a larger hexamer complex to form a 18 protein ternary structure of ∼443 kDa There was a 0.3 difference between the hexameric protein and monomer elution times, enabling relative quantification and the potential to screen across batches of drug product (Fig 6ABCD) In Fig 6F, the trimer pro9 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 463638 Metrics, Inc Q ExactiveTM is a trademark of Thermo Fisher Scientific tomer spectra is clearly observed Comparing the spectra of 6E and 6F, two unique m/z distributions of the dimer are observed, which provides distinction between the in-solution and gas phase generated species Protein-antibody complexes may also be examined with microflow SEC-MS Lipase-antibody complexes are historically difficult to analyze due to the extensive glycan heterogeneity of the lipase and the micromolar dissociation constants of the affinity interactions [51,52] Interrogating these complexes is critical to refining downstream process parameters for a new drug, because host cell lipases can bypass purification steps and be carried through to therapeutic products [53] To design effective purification strategies, the nature of these interactions must be characterized We recently published work showing that certain complexes can be detected by direct infusion static spray, ion mobility, and microscale thermophoresis (MST), but these approaches are not amenable to use as high throughput screening techniques [36] In the case of MST, there can also be issues with labeling artifacts Thus, an SEC method has long been desired As shown in Fig 7, a PLBL2-IgG4 complex was detected at ∼5% the level observed by static spray However, SEC enabled the analysis of protein:Ab complexes at a 2:1 ratio, rather than a 10:1 lipase: Ab ratio Microflow SEC seems to have minimized ion suppression problems encountered with direct infusion This advantage opens up the possibility of generating concentration dose curves for complex formation, which would not be achievable by static spray The utility of the small dimension columns seems therefore to lie in its sensitivity gains, its preservation of native states, and its subunit-level separations Data availability The authors not have permission to share data Acknowledgements The authors would like to thank Yeliz Sarisozen and Nicole Lawrence for providing SEC packing materials, Mathew DeLano for helping to procure different types of column hardware, and Steven Byrd for the preparation of packed columns For assistance in obtaining protein samples, we thank Bingchuan Wei and Shrenik Mehta We thank Wayne Fairbrother for scholarly conversations Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2022.463638 References [1] E Boeri Erba, C Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, Protein Sci 24 (8) (2015) 1176–1192, doi:10.1002/pro.2661 [2] A.C Leney, 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where screening of native protein-ligand binding must be performed in an automated and high throughput manner SEC-MS is traditionally a slow technique (at least ten minutes long) but it offers online separations, reproducibility, and a potentially universal, broadly applicable platform Here, fast elution times of less than minute and no more than 3.5 minutes are achieved to better meet throughput demands Moreover, 10-fold and higher increases in signal are reported Limits of detection were driven into the high attomole and lower ranges by employing the microflow devices Microflow SEC as shown here also affords minor, but reproducible, separations between subunits As such it is possible to detect multiple types of quaternary structures Coupled to multichannel emitters, we have also demonstrated an improvement in maintaining the native state of protein complexes such that it has become possible to detect micromolar affinity complexes Clearly, there is a penalty to miniaturizing SEC without simultaneously making wholesale changes to the LC flowpath The extra column tubing and dispersion effects are of significant influence to the apparent performance of the mm ID column Nevertheless, a device that is capable of achieving half the effective peak capacity of an optimized 4.6 mm ID SEC column represents a step forward in downsizing size exclusion chromatography and creating increasingly powerful hyphenated analytical approaches Declaration of Competing Interest The authors declare the following competing financial interest(s): Several of the authors are employed by Waters Corporation, the manufacturer of the prototype columns used for this work and several are employed by Genentech, Inc., which develops and markets drugs for profit BEHTM , ACQUITYTM , and UPLCTM are trademarks of Waters Technologies Corporation VanquishTM is a trademark of Dionex Softron GmbH ByosTM is a trademark of Protein 10 E Hecht, E Obiorah, X Liu et al Journal of Chromatography A 1685 (2022) 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biopharmaceuticals, Biotechnol Prog 34 (4) (2018) 828–837, doi:10.1002/btpr.2640 ... antibodies and heavily glycosylated macromolecular immune complexes by size- exclusion chromatography multi-angle light scattering, native charge detection mass spectrometry, and mass photometry,... Valliere-Douglass, Native size- exclusion chromatography- mass spectrometry: suitability for antibody-drug conjugate drug -to- antibody ratio quantitation across a range of chemotypes and drugloading... weights from to 600 kDa Thyroglobulin could not be detected by MS due to its large size and heterogeneity, and uracil fell below the mass cutoff of the instrument For BSA, IgG, and myoglobin,