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Published in final edited form as: Anal Chem. 2025 Feb 19;97(8):4549–4555. doi: 10.1021/acs.analchem.4c06397

A Robust and Automated Platform for Charge Detection Mass Spectrometry of Megadalton Biotherapeutics

Samuel E Janisse 1, Ryan T Fellers 1,5, Shannon A Raab 2, Michael P Goodwin 3, Kyle P Bowen 3, Zhirui J Lian 2, Kenneth R Durbin 5, Michael W Senko 3, Philip D Compton 4, Jared O Kafader 1, Neil L Kelleher 1
PMCID: PMC12323853  NIHMSID: NIHMS2098420  PMID: 39967556

Abstract

Gene therapies based on adeno-associated viruses are an emerging area with high potential to improve human health. Current quality control techniques to assess contaminates and byproducts from the adeno-associated virus (AAV) production pipelines are lacking in robustness and throughput. To address these limitations, we coupled an automated microfluidic device called SampleStream with orbitrap-based charge detection mass spectrometry (SS-CDMS). We demonstrate that the SS-CDMS workflow performs AAV analysis in under 15 min per sample in a completely autonomous manner. SS-CDMS workflow enables rapid assessment key quality control attributes (CQAs), such as of molecular weight determination and content ratio analysis of AAV formulations with a small sample requirement (<2×109 capsid) without being limited by sample concentration. Additionally, this work shows the potential for the SS-CDMS workflow to be implemented at various stages of the production pipeline through effective sample clean up from more complex AAV matrices such as cell culture media.

Graphical Abstract

graphic file with name nihms-2098420-f0001.jpg

Novel gene therapies, such as adeno-associated virus (AAV)-based biotherapeutics, are a promising avenue to address difficult-to-treat diseases.1 With eight FDA approved AAV-based drugs now on-market, significant new investment in this therapeutic modality forecasts a rapid increase in this number by 2030 with an estimated FDA approval rate of 10–20 per year.2,3,4 Despite forecasted development, analytical techniques, such as analytical ultracentrifugation (AUC) used to characterize these megadalton-sized species in their native form are difficult to implement, lack sensitivity and accuracy, or are low throughput – allowing only a few samples to be processed per day.5–7 The limitations of available technologies hinder widespread standardization of quality control workflows needed for fast composition-bioactivity correlations in AAV research and production pipelines.

Conventional mass spectrometry (MS) is challenged to perform traditional analysis on AAV samples due to their inherent heterogeneity and high molecular weights. Charge detection mass spectrometry (CDMS) largely addresses these limitations.8 CDMS is a powerful technique that simultaneously measures the mass-to-charge ratio (m/z) and charge (z) of individual ion signals. The assignment of charge to single ions enables true mass determination of any species regardless of heterogeneity or megadalton molecular weight values, with several publications denoting its successful application to biotherapeutics including AAV gene therapies.9–12 Commercial Orbitrap-based CDMS workflows have opened this niche MS technique to the broader scientific community.9,13–16 To that end, CDMS is posed to contribute uniquely to the assessment of quality attributes in AAV production by accurately determining the level of genome-containing capsids, which is vital for determining therapeutic efficacy.17 However, current CDMS-based workflows require manual sample pre-cleaning, buffer exchange, and clog-prone sample injection emitters that hinder widespread adoption and formulation assessment for AAVs.

Here, we demonstrate automated sample introduction with Orbitrap-based charge detection mass spectrometry to create a robust AAV analysis workflow composed completely of commercial components (Figure 1). The integrated platform uses a fully self-contained sample processing platform for automated online sample concentration, desalting and buffer exchange, plus injection into CDMS (see Materials and Methods) - providing reproducible results and high sample throughput.

Figure 1.

Figure 1.

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Overview of the SampleStream Charge Detection Mass Spectrometry (SS-CDMS) workflow with key quality control metrics. SampleStream Charge Detection Mass Spectrometry (SS-CDMS) workflow for AAV content analysis. AAV samples are directly injected into the SampleStream flow cell from a temperature controlled Peitler stack held at 6 °C. Samples are concentrated and buffer exchanged on a molecular weight cutoff membrane (100-kDa) followed by direct elution into a heated electrospray ionization (HESI) source coupled to a Q-Exactive UHMR with Direct Mass Technology Mode (DMTM) enabled for single ion charge analysis. Charges of the individual ions are proportional to the slope of the induced current on the orbitrap detection electrodes enabling direct mass analysis. Charge assigned ions are binned and the empty to full AAV capsid ratios are determined through set mass cutoff values.

Experimental Section

Sample Preparation

Empty and CVM-GFP AAV5, AAV8, and AAV9 were purchased from Virovek at a stock concentration of 2×1013 capsids or vector genomes per milliliter (cp/mL or vg/mL). Biopharmaceutical relevant AAV9 was produced by Sf9 cells in-house at Eli Lilly and Company. For ratio experiments, samples were prepared by mixing empty and filled AAVs volumetrically at 0:100, 30:70; 50:50; 70:30; and 100:0 followed by a dilution to 2×1012 cp/mL with 100 mM ammonium acetate. To pre-clean samples prior to SampleStream or nanospray analysis, the samples were buffer exchanged into 100 mM ammonium acetate using a 100-kDa Amicon Ultra centrifugal filter (Merk Millipore) spun at 8,000 × g for 1 min. The concentrated sample was diluted back to 2×1012 cp/mL and the above procedure was repeated five times. For nanospray experiments, borosilicate coted emitters (Thermo Fisher Scientific) were loaded with ~2 μL of buffer exchanged AAV solution (2×1012 cp/mL in 100 mM ammonium acetate) and sprayed with constant backpressure applied by a 10 mL syringe. For the comparison between AAV spiked into 100 mM ammonium acetate and Sf9 cell culture media (Gibco), AAV9 loaded with CMV-GFP was diluted to 5×1011 cp/mL and 20 μL were injected per SampleStream run.

Samples and Pre-Analytical Processing

The design, implementation, and utility of SampleStream has been previously described in detail elsewhere.18,19 In brief, the SampleStream module is a fluidic device that comprises three syringe pumps containing 250 μL syringes, an injection valve, two flow control valves, and a temperature-controlled fluidic channel interfaced directly with a PAL3 robot (CTC Analytics). Each component of the SampleStream system is controlled via a “plugin” that enables seamless control via the PAL3 scripting language. Samples are stored and collected in a temperature-controlled Peltier stack.

For SampleStream operation, data acquisition methods were created and introduced in this work following the following general format: injection, sample push, sample focus, detector priming, and elution into the MS. Samples are collected from the Peltier stack by a 100 μL syringe and injected into the injection port. The injected sample is flowed into the fluidic channel via a sample “push” step from one of the syringe pumps filled with 100 mM ammonium acetate (system liquid) where it is retained on a molecular weight cutoff membrane. The sample in the fluidic channel is further buffer exchanged and concentrated on the 100-kDa MWCO membrane with system liquid by two syringe pumps operating concurrently in opposite flow paths for the “focus” step. The buffer exchanged and concentrated sample is eluted from the channel into a sample loop by “priming” step followed by “elution” into the mass spectrometry at a specified flow rate (μL/min) for analysis. Each step can be optimized for total volume and flowrate to achieve a balance of throughput, sample cleaning, and sensitivity.

Mass Spectrometry Parameters

SampleStream was connected online to a heated electrospray ionization source (HESI) operating with a +3.8 kV spray voltage and 25 psi sheath gas set to 70 °C. The HESI probe was coupled to a Q Exactive UHMR mass spectrometer. For nanospray experiments, a Nanospray Flex Ion Source (Thermo Fisher Scientific) was used with a spray voltage of +1.2 kV for all measurements. The following mass spectrometer parameters were used for all experiments except for the trapping gas study where the normalized trapping gas setting was altered. Capillary temperature, 320 °C; in-source collisional energy, 0; in-source trapping −10 V for 4 ms. Ion transfer optics and quadrupole settings were set to the default for “high-mass” transfer. Unless otherwise noted, the normalized trapping gas setting was set to 7 (ultra-high vacuum pressure gauge reading: ~3.4×10−10 mbar) with nitrogen as the collision gas. Extended trapping in HCD cell, −10 V. To improve filled AAV transfer out of the HCD cell a 200 V HCD field gradient and 20 ms purge time were used. Single ion measurements were collected with direct mass technology mode (DMTM) enabled. A manual ion injection time was set to 1000 ms and all acquisitions were 1000 ms in length, corresponding to a resolution setting of 100,000 on the UHMR.

Software Processing Parameters

RAW data files were uploaded to STORIboard (Proteinaceous) and filtered using a R2 cutoff of 0.99, duration threshold of 0.42, maximum time of birth of 0.1, minimum time of death of 0.2, signal-to-noise threshold of 3. Ions were frequency corrected according to Goodwin et al.20 The charge of each ion was assigned directly from the calculated slope and was not further processed. The resulting charge assigned ions were extracted from the DMT files and processed in custom python scripts. Ratio calculation proceeded through determining the edge of each distribution through a change-of-slope algorithm applied to a smoothed histogram (kernel density estimation). In cases where the distribution was not sufficiently resolved, manual adjustment of the cutoff was done.

Results and Discussion

Microflow through a Heated Electrospray Ion Source Allows Accurate Assessment of Average AAV Mass and Cargo Loading

As only capsids containing a genome are considered for therapeutic dosages, properly assessing the genome loading in AAVs is vital for treatment efficacy and consistency.12,17 For accurate determination of properly filled AAV, each of the components (empty, filled, extra-filled) needs to be sufficiently resolved, which is usually achieved using small inner diameter (i.d.) emitters. Electrospray through narrow diameter emitters tend to generate smaller droplets that enable better analyte desolvation – translating into narrower mass distributions.21,22 For robust and unattended operation, the SampleStream (SS)-CDMS workflow utilizes a heated electrospray ion source (HESI) with a larger inner diameter (~70 μm) emitter compared to manually-pulled nanospray tips.23 To investigate the influence of a larger diameter emitter and the effectiveness of the flow cell’s 100-kDa molecular weight cutoff (MWCO) membrane on sample cleanup, a volumetrically mixed sample of 50% empty and 50% cytomegalovirus green fluorescent protein (CMV-GFP) genome filled AAV8 (“50E50F”) was prepared with and without prior sample clean-up. There were minimal differences in the full-width, half-maximum (FWHM) of peak intensity of each mass distribution between the sample types (Figure 2A and 2B). Most notably, the FWHM of the mass distribution centered at ~3.80 MDa in the 50E50F AAV8 mixture processed by SampleStream without prior sample cleanup was ~12% (35 kDa) wider than the mass distributions produced by nanospray with offline buffer exchange. These data highlight that SampleStream can produce mass distributions nearly as resolved of those of nanospray without the need for prior buffer exchange – alleviating a time-consuming step and improving robustness to allow for continuous sample processing.24

Figure 2.

Figure 2.

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Nanospray and Heated Electrospray Ionization Comparison. (A) Overlay of KDEs of a 50:50 mixture (V:V) of Empty:Full mixture (“50E50F”) analyzed via nanospray (blue) and SampleStream injection (green and purple). (B) Same as (A) but focusing on the empty component.

Correctly measuring the average mass of the AAV components is important for confirmation of genome incorporation. Additionally, accurate average mass measurements provide information on the average capsid virion protein (VPs) ratios, which due to the stochastic assembly process can vary batch to batch as well be optimized for improved transduction efficiency.24–26 In the SS-CDMS workflow, the average mass for each serotype and volumetric mixture is determined through identifying peak apexes (Figure S1A-C). For each serotype (AAV5, AAV8, and AAV9) the average masses, calculated using all ratio experiments, were centered between 3.75 and 3.80 MDa (±<0.02 MDa; n = 12) for empty AAVs (Figure 3A) and between 4.57 and 4.59 MDa (±<0.01 MDa; n = 9) for the genome loaded capsids (Figure 3B). Due to the broad mass distribution of the extra filled capsids arising from the inherent heterogeneity of the capsid, the ability to accurately determine the apex is inhibited at lower ratios of genome filled capsids.26 Nevertheless, the average mass of the doubly-filled capsids was approximately 5.14 to 5.22 MDa (±<0.01 MDa) for the serotypes tested. Depending on the serotype, the observed mass differences between the empty and filled AAV serotypes were 1% to 6% larger than the theoretical 0.782 MDa mass of the CVM-GFP genome. The slightly higher mass than expected has been associated with counterion packing during the capsid assembly process and the data presented here follow very similar trends to previous reports.11

Figure 3.

Figure 3.

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Average mass of CMV-GFP filled and unfilled AAV5, AAV8, and AAV9 determined by SS-CDMS. (A) Boxplot of the average mass of empty AAV5, AAV8, and AAV9. Each boxplot represents every empty distribution detected from each volumetrically mixed sample for a total of 12 measurements per AAV. Black dot represents the mean of the measurements for comparison to the median. (B) Same as (A), but for filled capsids and total number of measurements equal 9, due to the absence of filled in the 100E0F samples.

Using the same method to identify the average mass, the distribution cutoffs were identified and used to calculate the percent ratio of each AAV component in the volumetric mixtures. The observed ratios for each volumetric mixture revealed systematic discrepancies from theoretical ratios for each AAV serotype (Figure 4A–C). For example, for the 70E30F mixture of AAV8, the observed percentage of empty is 81 ± 0.67%. Once establishing that, at sufficient pressures, the use of nitrogen as a background gas does not impart bias in ratio measurements (Figure S2), we turned to potential differences in capsid titer. Comparing the number of charged assigned ions falling within the AAV mass range for each AAV serotype standards (Figure S3) revealed that the discrepancy between theoretical and measured was due to differences in true capsid titer. Correcting for the difference (Figure S4) between capsid titer of the 100%-empty and 100%-filled AAV and the presence of empty capsid in the 100% filled standards resulted in theoretical ratios within just 2% of the observed ratios. The agreement between measured and corrected theoretical ratios illustrates the accuracy of our measurements and lack of bias in AAV ion sampling.

Figure 4.

Figure 4.

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Measured component percentages of AAV5, AAV8, and AAV9 at different volumetric mixtures. (A) Component percentage of AAV5, (B) AAV8, and (C) AAV9. Error bars represent standard deviation (n=3).

Robustness and Reproducibility of The SampleStream-CDMS Workflow For High-throughput AAV Content Ratio Determination

In comparison to orthogonal techniques, the SS-CDMS workflow requires ~100 fold lower sample amount than analytical ultracentrifugation (~1×1011 cp) ~10 fold greater than anion-exchange chromatography (~1–2×1010 cp), and comparable to online-buffer exchange methods (~1×109 cp) mass spectrometry.27,28 As expected, there was strong linearity and reproducibility between the average number of charge-assigned ions and capsid injection amount (Figure S5), and the empty/filled ratios were consistent above an injection amount of 1.25×109 capsids (cp) (Figure 5). Furthermore, The SS-CDMS workflow provided less than 2% sample carryover between analytical injections (Figure S6A) and low sample buildup (Figure S6B) over repeated injections. In contrast to other online buffer exchange methods, dilute AAV samples can be efficiently concentrated in this workflow, with no significant differences in the observed number of charge assigned ions or calculated ratios (Figure S7A and S7B). To highlight the robustness and reproducibility of the automated workflow Figure 6A and 6B show minimal deviation (coefficient of variation of just 3.5%) over 10 repeated cycles of an AAV and blank injections. Further optimization of SS-CDMS workflow revealed a key interdependence between sensitivity and elution flow rate. With a fixed 90 μL elution, there was a 35% decrease in the average number of charge-assigned ions when the flow rate was raised from 10 to 20 μL/min (Figure S8A). Conversely, the component percentages were not influenced by flow rate (Figure S8B). Taken together, the robustness and low carryover of the SS-CDMS workflow enabled 10 samples to be analyzed in under 2.8 hr, and with the tunability of acquisition flow rate the throughput can easily exceed 100 samples per day.

Figure 5.

Figure 5.

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Limit of ratio quantification. Bar graph illustrating the percentage of empty, filled, and extra filled AAV8 at varying injection amounts of a 50E50F volumetric mixture. Error bars represent standard deviation (n=3).

Figure 6.

Figure 6.

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SS-CDMS provides high robustness and sensitivity at increasing levels of sample complexity. (A) Bar graph representing the number of charge assigned ions per analytical AAV injection. The horizontal dashed line indicates the average number of charge assigned ions (n=10). (B) Histogram (10 spectra per bin) and KDE overlay of a 2×1010 capsid injection followed by a blank injection repeated ten consecutive times. The “B” designation between numerical injection represents a blank injection and the non-acquisition time for sample cleaning.

A nominally 100% genome filled AAV9 was measured to assess the SS-CDMS workflow in characterizing non-standard AAV formulations. The AAV sample was produced via in-house production by Eli Lilly and is representative of those from the biopharmaceutical industry with larger loaded genomes. As previously mentioned, a common challenge of AAV production is co-purified AAVs that contain truncated genomes, unrelated DNA that may or may not also contain the therapeutic genome.12,29 These impurities appear as additional mass distributions in CDMS analysis.11 However, the AAV9 sample contained non-biologically active stuffer DNA that is used to prevent double or “extra” filled AAVs. As a result, the filled distribution is skewed towards lower masses with the most prominent peak centered at ~5.14 MDa (Figure 7A), which contained a single encapsulated genome and benign DNA. Additionally, SS-CDMS analyses revealed an additional distant mass distribution centered at ~3.80 MDa, corresponding to ~6% empty capsids (Figure 7A). Similar to the volumetric mixtures for AAV8 standards, once corrected, the observed ratios for empty- and filled-AAV9 mixtures agree with the theoretical to within ~5% (Figure S9).

Figure 7.

Figure 7.

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(A) KDE of AAV9 with loaded GFP. (B) KDE overlay of a 1×1010 capsid injection (20 μL) of a 0E100F AAV9 standard diluted in 100 mM ammonium acetate with increasing focusing/washing volumes of system solvent (100 mM ammonium acetate). (C) Same as (B), but with cell culture media as the dilutant. (D) Bar graph of the calculated empty, filled, and extra filled AAV9 at increasing focusing/washing volumes. Purple, blue, and green horizontal dashed lines represent the percent empty, filled, and extra filled AAV9 from 100 mM ammonium acetate dilution experiment.

Enrichment of AAV9 from Cell Culture Media

To assess the ability of the SS-CDMS platform to determine the content ratio of AAVs in more complex matrices, known amounts of 100%-filled AAV9 were spiked into Sf9 culture media. Unlike the AAV standard formulations spiked into ammonium acetate, the wash volume during the buffer exchange step needed to be increased from 500 μL to 5000 μL to successfully remove media components (Figure 7B and 7C) The increase in volume corresponds to a 10x increase in sample washing time (0.67 to 6.67 min.). However, once sufficiently washed, the observed empty percentage of AAV9 is within error of that observed from the ammonium-acetate-spiked sample (Figure 7D; blue, green, and purple trace). Despite increases in processing time with higher levels of sample complexity, the demonstrated utility of the SS-CDMS platform to perform content-ratio determination without prior AAV extraction further exemplifies the ability to reduce turnaround time for quality control testing for AAV formulations at any point in the production pipeline. Furthermore, fast quality control checks can help to alleviate the financial and time burden of both process development and production downtime through identifying inefficiencies early in the pipeline.

Conclusion

In conclusion, we present an automated platform for rapid analysis of AAV cargo. The SS-CDMS workflow enables ratio calculations accurate to <5%, with better than 5% reproducibility. The platform functions at a rate of up to 100 samples per day and alleviates the need for tedious sample clean up. The source configuration provides robustness with sample processing in under 15 minutes, creating the most reliable, scalable and integrated AAV analysis platform to link with bioactivity assessments using commercially available components. We envision widespread adoption of SS-CDMS platform to rapidly monitor the purification process for deleterious components as well as near real-time assessment for optimal AAV production kinetics in post plasmid-transfected cell line-based production systems. With a powerful platform for academic and biopharmaceutical labs alike, the development of groundbreaking AAV therapeutics enabling the treatment of diseases once thought to be undruggable will be accelerated.

Supplementary Material

SM

Supporting Information file contains KDE plots of AAV5, AAV8, and AAV9 at varying volumetric ratios; bar graphs of calculated ratios and charge assigned ions at varying trapping gas pressure, elution flow rates, injection volumes; bar graphs illustrating line plot illustrating the differences in capsid titer of the AAV standards; method and example for correcting; scatter plot showing linearity of injection amount (cp) and charge assigned ions; bar graphs showing carryover between AAV injections; KDE of non-standard AAV.

Acknowledgements

This work was supported by National Institutes of Health P41 GM108569 (N.L.K.), the NIH Office of Director award S10 OD025194 (P.D.C.), and the National Institutes of Health P30 CA060553 (awarded to the Robert H. Lurie Comprehensive Center).

Footnotes

M.P.B., M.P.G., and M.W.S. are employees of Thermo Fisher Scientific. S.A.R. and Z.J.L., are employees of Eli Lilly and Company. P.D.C. and N.L.K. are stakeholders in Integrated Protein Technologies. J.O.K. and N.L.K. serve as consultants to Thermo Fisher Scientific. R.T.F., and K.R.D., are employees of Proteinaceous and N.L.K. is a stakeholder. S.E.J. declares no competing interests.

Software and Data Availability

STORIboard is freeware that can be downloaded from https://www.proteinaceous.net to process RAW files and view processed DMT files. The unprocessed (RAW) and processed (DMT) files have been posted on the MassIVE repository under the identifier MSV000096253.

References

  • (1).Dunbar CE; High KA; Joung JK; Kohn DB; Ozawa K; Sadelain M Gene Therapy Comes of Age. Science 2018, 359 (6372), eaan4672. 10.1126/science.aan4672. [DOI] [PubMed] [Google Scholar]
  • (2).Wang J-H; Gessler DJ; Zhan W; Gallagher TL; Gao G Adeno-Associated Virus as a Delivery Vector for Gene Therapy of Human Diseases. Sig Transduct Target Ther 2024, 9 (1), 78. 10.1038/s41392-024-01780-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (3).Li C; Samulski RJ Engineering Adeno-Associated Virus Vectors for Gene Therapy. Nat Rev Genet 2020, 21 (4), 255–272. 10.1038/s41576-019-0205-4. [DOI] [PubMed] [Google Scholar]
  • (4).Kuzmin DA; Shutova MV; Johnston NR; Smith OP; Fedorin VV; Kukushkin YS; Van Der Loo JCM; Johnstone EC The Clinical Landscape for AAV Gene Therapies. Nat Rev Drug Discov 2021, 20 (3), 173–174. 10.1038/d41573-021-00017-7. [DOI] [PubMed] [Google Scholar]
  • (5).Gimpel AL; Katsikis G; Sha S; Maloney AJ; Hong MS; Nguyen TNT; Wolfrum J; Springs SL; Sinskey AJ; Manalis SR; Barone PW; Braatz RD Analytical Methods for Process and Product Characterization of Recombinant Adeno-Associated Virus-Based Gene Therapies. Molecular Therapy - Methods & Clinical Development 2021, 20, 740–754. 10.1016/j.omtm.2021.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (6).Schnödt M; Büning H Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities. Human Gene Therapy Methods 2017, 28 (3), 101–108. 10.1089/hgtb.2016.188. [DOI] [PubMed] [Google Scholar]
  • (7).Wang C; Mulagapati SHR; Chen Z; Du J; Zhao X; Xi G; Chen L; Linke T; Gao C; Schmelzer AE; Liu D Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2. Molecular Therapy - Methods & Clinical Development 2019, 15, 257–263. 10.1016/j.omtm.2019.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (8).Keifer DZ; Pierson EE; Jarrold MF Charge Detection Mass Spectrometry: Weighing Heavier Things. Analyst 2017, 142 (10), 1654–1671. 10.1039/C7AN00277G. [DOI] [PubMed] [Google Scholar]
  • (9).Barnes LF; Draper BE; Jarrold MF Analysis of Thermally Driven Structural Changes, Genome Release, Disassembly, and Aggregation of Recombinant AAV by CDMS. Molecular Therapy - Methods & Clinical Development 2022, 27, 327–336. 10.1016/j.omtm.2022.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (10).Wörner TP; Snijder J; Friese O; Powers T; Heck AJR Assessment of Genome Packaging in AAVs Using Orbitrap-Based Charge-Detection Mass Spectrometry. Molecular Therapy - Methods & Clinical Development 2022, 24, 40–47. 10.1016/j.omtm.2021.11.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (11).Barnes LF; Draper BE; Chen Y-T; Powers TW; Jarrold MF Quantitative Analysis of Genome Packaging in Recombinant AAV Vectors by Charge Detection Mass Spectrometry. Molecular Therapy - Methods & Clinical Development 2021, 23, 87–97. 10.1016/j.omtm.2021.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (12).McColl-Carboni A; Dollive S; Laughlin S; Lushi R; MacArthur M; Zhou S; Gagnon J; Smith CA; Burnham B; Horton R; Lata D; Uga B; Natu K; Michel E; Slater C; DaSilva E; Bruccoleri R; Kelly T; McGivney JB Analytical Characterization of Full, Intermediate, and Empty AAV Capsids. Gene Ther 2024, 31 (5–6), 285–294. 10.1038/s41434-024-00444-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (13).Kafader JO; Melani RD; Durbin KR; Ikwuagwu B; Early BP; Fellers RT; Beu SC; Zabrouskov V; Makarov AA; Maze JT; Shinholt DL; Yip PF; Tullman-Ercek D; Senko MW; Compton PD; Kelleher NL Multiplexed Mass Spectrometry of Individual Ions Improves Measurement of Proteoforms and Their Complexes. Nat Methods 2020, 17 (4), 391–394. 10.1038/s41592-020-0764-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (14).McGee JP; Melani RD; Yip PF; Senko MW; Compton PD; Kafader JO; Kelleher NL Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry. Anal. Chem 2021, 93 (5), 2723–2727. 10.1021/acs.analchem.0c03282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (15).Du C; Cleary SP; Kostelic MM; Jones BJ; Kafader JO; Wysocki VH Combining Surface-Induced Dissociation and Charge Detection Mass Spectrometry to Reveal the Native Topology of Heterogeneous Protein Complexes. Anal. Chem 2023, 95 (37), 13889–13896. 10.1021/acs.analchem.3c02185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (16).Kostelic MM; Zak CK; Liu Y; Chen VS; Wu Z; Sivinski J; Chapman E; Marty MT UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data. Anal. Chem 2021, 93 (44), 14722–14729. 10.1021/acs.analchem.1c03181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (17).Gao K; Li M; Zhong L; Su Q; Li J; Li S; He R; Zhang Y; Hendricks G; Wang J; Gao G Empty Virions in AAV8 Vector Preparations Reduce Transduction Efficiency and May Cause Total Viral Particle Dose-Limiting Side Effects. Molecular Therapy - Methods & Clinical Development 2014, 1, 9. 10.1038/mtm.2013.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (18).Park HM; Winton VJ; Drader JJ; Manalili Wheeler S; Lazar GA; Kelleher NL; Liu Y; Tran JC; Compton PD Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry. Analytical Chemistry 2020, 92 (2), 2186–2193. 10.1021/acs.analchem.9b04826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (19).Santos Seckler H. dos; Park H-M; Lloyd-Jones CM; Melani RD; Camarillo JM; Wilkins JT; Compton PD; Kelleher NL New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry. J. Am. Soc. Mass Spectrom 2021, 32 (7), 1659–1670. 10.1021/jasms.1c00026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (20).Goodwin MP; Grinfeld D; Yip P; Bowen KP; Kafader JO; Kelleher NL; Senko MW Improved Signal Processing for Mass Shifting Ions in Charge Detection Mass Spectrometry. J. Am. Soc. Mass Spectrom 2024, 35 (4), 658–662. 10.1021/jasms.3c00435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (21).Schmidt A; Karas M; Dülcks T Effect of Different Solution Flow Rates on Analyte Ion Signals in Nano-ESI MS, or: When Does ESI Turn into Nano-ESI? J. Am. Soc. Mass Spectrom 2003, 14 (5), 492–500. 10.1016/S1044-0305(03)00128-4. [DOI] [PubMed] [Google Scholar]
  • (22).Wilm M Principles of Electrospray Ionization. Molecular & Cellular Proteomics 2011, 10 (7), M111.009407. 10.1074/mcp.M111.009407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (23).Harvey SR; Gadkari VV; Ruotolo BT; Russell DH; Wysocki VH; Zhou M Expanding Native Mass Spectrometry to the Masses. J. Am. Soc. Mass Spectrom 2024, 35 (3), 646–652. 10.1021/jasms.3c00352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (24).Ebberink EHTM; Ruisinger A; Nuebel M; Thomann M; Heck AJR Assessing Production Variability in Empty and Filled Adeno-Associated Viruses by Single Molecule Mass Analyses. Molecular Therapy - Methods & Clinical Development 2022, 27, 491–501. 10.1016/j.omtm.2022.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (25).Wang Q; Wu Z; Zhang J; Firrman J; Wei H; Zhuang Z; Liu L; Miao L; Hu Y; Li D; Diao Y; Xiao W A Robust System for Production of Superabundant VP1 Recombinant AAV Vectors. Molecular Therapy - Methods & Clinical Development 2017, 7, 146–156. 10.1016/j.omtm.2017.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (26).Wörner TP; Bennett A; Habka S; Snijder J; Friese O; Powers T; Agbandje-McKenna M; Heck AJR Adeno-Associated Virus Capsid Assembly Is Divergent and Stochastic. Nat Commun 2021, 12 (1), 1642. 10.1038/s41467-021-21935-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (27).Guapo F; Füssl F; Strasser L; Bones J Mass Spectrometry Friendly pH-Gradient Anion Exchange Chromatography for the Separation of Full and Empty Adeno-Associated Virus (AAV) Capsids. Anal. Methods 2023, 15 (43), 5788–5792. 10.1039/D3AY01560B. [DOI] [PubMed] [Google Scholar]
  • (28).Cotham VC; Wang S; Li N An Online Native Mass Spectrometry Approach for Fast, Sensitive, and Quantitative Assessment of Adeno-Associated Virus Capsid Content Ratios. J. Am. Soc. Mass Spectrom 2024, 35 (7), 1567–1575. 10.1021/jasms.4c00151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (29).Zhang J; Guo P; Yu X; Frabutt DA; Lam AK; Mulcrone PL; Chrzanowski M; Firrman J; Pouchnik D; Sang N; Diao Y; Herzog RW; Xiao W Subgenomic Particles in rAAV Vectors Result from DNA Lesion/Break and Non-Homologous End Joining of Vector Genomes. Molecular Therapy - Nucleic Acids 2022, 29, 852–861. 10.1016/j.omtn.2022.08.027. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SM
NIHMS2098420-supplement-SM.docx (566.1KB, docx)

Data Availability Statement

STORIboard is freeware that can be downloaded from https://www.proteinaceous.net to process RAW files and view processed DMT files. The unprocessed (RAW) and processed (DMT) files have been posted on the MassIVE repository under the identifier MSV000096253.

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