Automated Online Buffer Exchange for Native MS and CDMS Analysis of RNA Therapeutics

Casey E. Vantucci, Lauren Schmitt, Carter K. Asef, Suraj Dhungana, and Mason Chilmonczyk
Andson Biotech, Atlanta, GA

Introduction

RNA-based therapeutics represent a rapidly expanding class of biotherapeutics, spanning a diverse range of modalities with distinct physicochemical properties and analytical requirements. These range from short, chemically modified oligonucleotides such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and single guide RNAs (sgRNAs), to substantially larger and structurally complex molecules such as messenger RNAs (mRNAs) and emerging circular and self-amplifying RNA formats. Ensuring the therapeutic potency and safety of these molecules requires accurate characterization, yet each modality presents distinct analytical challenges, from verifying duplex integrity and chemical modifications in smaller species like siRNAs to measuring intact mass and assessing structural heterogeneity in large mRNA constructs.

Native mass spectrometry (MS) has emerged as a powerful analytical tool for RNA therapeutics, offering the ability to measure intact mass and provide structural insights across a wide range of molecular sizes. For larger molecules such as mRNA, charge detection MS (CDMS) extends native MS capabilities into the high molecular weight range, where conventional MS falls short. For both approaches, sample preparation is a critical determinant of spectral quality. RNA molecules are particularly susceptible to cation adduction, and achieving sufficient desalting while preserving structural integrity remains a key challenge. Size exclusion spin columns are commonly used for desalting, but their effectiveness for RNA is limited by tight cation adduct formation, molecular weight cutoff constraints, and RNA’s inherent susceptibility to degradation during handling.

The DynaChip X1™ addresses these limitations through rapid, automated online buffer exchange via tangential flow filtration and microdialysis, efficiently removing salts and interferents while retaining analytes from >10 kDa to 5 MDa+. Designed for direct integration with nano electrospray ionization (nanoESI), the platform reduces workflow time and is gentle enough to preserve RNA structural integrity, enabling high-resolution intact mass characterization from small oligonucleotides to large mRNA constructs.

This application note describes the integration of the DynaChip X1^™ with MS, including CDMS, for the analysis of RNA therapeutics.

Experimental Methods

EPO, OVA, and β-galactosidase (β-gal) mRNA constructs were purchased from TriLink Biotechnologies at 1 mg/mL in sodium citrate buffer (Cat. #L-7209, L-7610, L-7608). GAPDH siRNA (Cat. #AM4605) and AAVS1 sgRNA (Cat. #A35522) were purchased from Thermo Fisher Scientific.
For online buffer exchange, samples were diluted in 200 mM ammonium acetate and then manually loaded into the DynaChip X1^TM sample loop (1.2 µL or 5 µL), injected at a flow rate of 250-500 nL/min (Chemyx Fusion Syringe Pump), and buffer exchanged into 20-200 mM ammonium acetate. The DynaChip^™ outlet was coupled directly to the inlet of the MS. Data were acquired using a Q Exactive Ultra High Mass Range (UHMR) Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with Direct Mass Technology (DMT) mode for CDMS. Samples were ionized with the Nanospray Flex nanoESI ion source using fused silica emitter tips (360 µm OD × 30 µm ID, 15 µm orifice, CoAnn Technologies). Instrument tuning parameters were optimized for each analyte. Data were analyzed using STORIBoard (Proteinaceous) and UniDec v8.1.0 (Michael Marty, University of Texas-Austin).

Results & Discussion

To evaluate the DynaChip X1^™ for online buffer exchange and native MS analysis of RNA therapeutics, a panel of molecules spanning a broad size range was analyzed, from a 20-nucleotide siRNA duplex to a 3,421-nucleotide mRNA construct. Online buffer exchange produced high-quality native mass spectra for both small RNA species. The GAPDH siRNA (20 nt) and AAVS1 sgRNA (100 nt) each yielded well-resolved charge states with narrow peak widths. Deconvolution resulted in experimental masses of 13.28 kDa and 32.32 kDa, respectively, in close agreement with the expected theoretical masses of ~13.2 kDa and ~33 kDa (Figure 1A, B). For larger constructs, resolved charge-state envelopes were obtained for EPO (859 nt), OVA (1,438 nt), and β-gal (3,421 nt) mRNAs (Figure 1C), confirming effective salt removal even for larger RNA molecules with higher charge density. While the charge-state envelopes were resolved, the overlapping signals and broad distributions characteristic of these large mRNA constructs made conventional deconvolution impractical. CDMS using DMT mode was therefore employed to enable accurate mass determination of these species.

Figure 1: Native MS of RNAs. Raw mass spectra (left) and deconvoluted masses (right) of (A) GAPDH siRNA (20 nt) and (B) AAVS1 sgRNA (100 nt). (C) Raw mass spectra of EPO (859 nt), OVA (1438 nt), and β-gal (3421 nt) mRNAs, showing resolved charge-state envelopes across a broad size range.

CDMS analysis extended mass characterization across the full mRNA panel. Deconvoluted spectra revealed distinct monomer peaks at 283.1 kDa for EPO mRNA, 468.9 kDa for OVA mRNA, and 1,193.5 kDa for β-gal mRNA, in good agreement with the theoretical masses of 282.9, 474, and 1105.75 kDa, respectively (Figure 2). In addition to the primary monomer species, populations corresponding to truncations, extensions, and dimers were observed. These data provide critical insight into sample heterogeneity, including the presence of incomplete transcripts and higher-order aggregates, which are important quality attributes for mRNA therapeutics.

Figure 2: CDMS of mRNAs. Deconvoluted masses of (A) EPO mRNA (859 nt), (B) OVA mRNA (1438 nt) and β-gal mRNA (3421 nt).

The DynaChip X1™ offers flexibility in controlling elution time for CDMS by adjusting flow rate and sample loop volume. Because CDMS relies on the accumulation of individual ion measurements to build statistically robust mass distributions, longer acquisition windows can substantially improve spectral quality and mass precision. As shown in Table 1 and Figure 3, extending the elution from approximately 5 minutes (Run 1) to 25 minutes (Run 2) increased charge-assigned ions more than threefold (12,410 to 38,236) and improved observed monomer mass accuracy from +0.21% to +0.06% of the theoretical mass.

Table 1: Using the DynaChip X1TM to Vary Elution Time for CDMS of EPO mRNA

Figure 3: Effect of Elution Time on CDMS of EPO mRNA. Deconvoluted mass for EPO mRNA for both (A) shorter (~5 min, Run 1) and (B) longer (~25 min, Run 2) elution times. Peaks corresponding to truncations, monomers, extensions, and dimers are indicated.

The longer elution also provided better resolution of subpopulations, including more clearly defined truncation and dimer species (Figure 3B). These results illustrate how the DynaChip X1^™ can be tuned to balance throughput and data quality, offering rapid screening for routine analysis and a higher-sensitivity mode for detailed characterization.

Conclusions

This application note demonstrates that the DynaChip X1^™ provides effective automated online buffer exchange for native MS and CDMS analysis of RNA therapeutics across a broad size range, from a 20-nucleotide siRNA to a 3,421-nucleotide mRNA. The platform efficiently removed cation adducts to yield well-resolved charge-state envelopes and accurate deconvoluted masses for all analytes tested. For CDMS, tunable elution parameters enabled optimization of ion statistics and mass accuracy, facilitating detailed characterization of sample heterogeneity including truncations, extensions, and dimers. By providing automated inline desalting with direct coupling to nanoESI, the DynaChip X1^™ simplifies sample preparation, reduces handling-related degradation, and supports high-quality mass spectrometric analysis of diverse RNA therapeutic modalities.

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