Multiple Quality Attribute Characterization of a Ribonucleoprotein Complex by SEC-MALS

Significance of the topic

The structural and compositional quality of ribonucleoprotein (RNP) complexes—particularly CRISPR-associated nucleases complexed with single-guide RNA (sgRNA)—directly determines genome-editing performance, safety, and manufacturability. Analytical methods that quantify oligomeric state, aggregation, and molecular composition under native conditions are therefore essential for development, formulation, and quality control of RNP therapeutics and research reagents.

This application note demonstrates a SEC-MALS workflow that provides absolute molar mass and composition across chromatographic peaks, enabling accurate identification of multiple RNP association states and revealing how sgRNA higher-order structure (HOS) drives complex heterogeneity.

Objectives and study overview

The study aimed to develop and apply a single SEC-MALS assay, augmented by multi-wavelength UV (260/280 nm) and differential refractive index detection and analysed with ASTRA ADC Analysis Module, to:

• Characterize native oligomeric state and purity of CasX apoenzyme and the corresponding sgRNA.
• Quantify the composition and stoichiometry of pre-assembled CasX–sgRNA RNP complexes at each point across the SEC elution profile.
• Detect aggregates and higher-order species that may impair RNP function and to relate RNP heterogeneity to attributes of the individual components.

Methodology

Samples (CasX apoenzyme, sgRNA, and pre-assembled RNP) were provided by Scribe Therapeutics. The key analytical approach combined size-exclusion chromatography under native buffer conditions with multi-angle light scattering for absolute molar mass determination and orthogonal concentration detectors for composition analysis. The mobile phase and chromatographic conditions were selected to preserve native complexes while providing high-resolution separation.

Key method features:

• SEC performed at 25 °C using a 250 Å protein SEC column with 0.5 mL/min flow and phosphate-buffered mobile phase (50 mM sodium phosphate, pH 7.2, 300 mM NaCl).
• Detection: MALS for absolute molar mass, UV absorbance at 260 nm and 280 nm to discriminate nucleic acid vs protein, and differential refractive index (dRI) for concentration.
• Data analysis with ASTRA 8 ADC Analysis Module enabling per-point compositional deconvolution (protein vs nucleic acid contributions) and stoichiometry estimation across each chromatographic peak.

Used instrumentation

• Waters Arc Premier HPLC System with Quaternary Solvent Manager (QSM).
• Waters Arc Premier 2998 UV detector (260 and 280 nm).
• DAWN multi-angle light scattering photometer.
• Optilab differential refractive index detector.
• Waters XBridge Premier Protein SEC Column, 250 Å, 2.5 µm, 7.8 × 300 mm.
• Chromatography software: HPLC CONNECT 4; data analysis: ASTRA 8 with ADC Analysis Module.

Main results and discussion

Free component characterization:

• CasX apoenzyme: predominantly monomeric with measured weight-average molar mass ~108 ± 3 kDa and very low dimer content (~<2% by mass), indicating a monodisperse protein starting material favorable for defined RNP formation.
• sgRNA: strong presence of higher-order structures and aggregates. SEC-MALS identified an sgRNA dimer species that constituted the majority of eluted RNA mass (~~66–70% by mass), plus additional high-molecular-weight (HMW) species and minor impurities. The sgRNA HOS are consistent with self-complementary regions producing duplexed or multimeric RNA assemblies.

RNP complex characterization:

• SEC-MALS with simultaneous UV260/280 and dRI allowed decomposition of each chromatographic feature into protein and nucleic-acid contributions and calculation of per-peak stoichiometries.
• Multiple distinct RNP species were present rather than a single canonical 1:1 complex. The canonical 1 CasX : 1 sgRNA assembly was observed but was a minority species.
• The most abundant complexes reflected non-canonical stoichiometries that correlate with sgRNA HOS in the starting material. Major species included (examples by relative mass fraction): a complex consistent with two CasX bound to an sgRNA dimer (~36% by mass) and a 1 CasX : 2 sgRNA assembly (~29% by mass). A heterogeneous HMW region (predominantly protein by composition, ~80% protein / 20% nucleic acid) suggested CasX bound to aggregated sgRNA or other coeluting species.
• Retention time alone was insufficient to identify species: conformational differences make elution position an unreliable proxy for molar mass, illustrating the necessity of coupling SEC with MALS for accurate mass- and composition-based identification.

Interpretation and implications:

• sgRNA self-association is a major driver of RNP heterogeneity; dimeric or aggregated RNA presents multiple binding sites or altered architectures that permit non-stoichiometric Cas binding despite a single canonical protein binding site.
• Since CasX remains largely monomeric, the observed higher-order RNP stoichiometries are attributed primarily to RNA HOS rather than protein oligomerization.
• Identification of these species is critical because non-canonical assemblies and aggregates can reduce genome-editing potency, alter specificity, and complicate downstream manufacturing and formulation.

Benefits and practical applications of the method

The combined SEC-MALS + UV260/280 + dRI assay provides a single-platform, native-condition characterization that delivers multiple quality attributes relevant to development and QC:

• Absolute molar mass measurement of proteins, RNA, and RNP complexes regardless of conformation.
• Quantitative decomposition of peaks into protein vs nucleic-acid contributions, enabling stoichiometry determination across heterogeneous elution profiles.
• Sensitive detection of aggregates, HOS, and coeluting impurities that affect RNP activity.
• Actionable data to guide sgRNA sequence design, sample handling, formulation development, and complexation protocols to minimize undesirable HOS and maximize functional RNP yield.

Future trends and potential applications

Expected developments and uses that build on this approach include:

• Integration with orthogonal native MS workflows to validate stoichiometries and to provide sequence-resolved mass confirmation while addressing buffer compatibility challenges.
• Design and engineering of sgRNA sequences or chemical modifications to reduce self-association and HOS, informed by SEC-MALS screening early in development.
• Implementation of SEC-MALS as a routine in-process control for RNP manufacturing and for comparability studies during formulation and scale-up.
• Automation and high-throughput adaptations (miniaturized SEC, rapid fraction collection with online MALS) to accelerate screening of sgRNA constructs and complexation parameters.
• Regulatory acceptance of composition-resolved SEC-MALS data as part of analytical control strategies for nucleic-acid–protein therapeutics.

Conclusion

SEC-MALS combined with UV260/280 and dRI detection and analysed with an ADC compositional module provides robust, native-condition measurement of molar mass and composition across chromatographic peaks. Applied to a CasX–sgRNA system, the method revealed extensive sgRNA higher-order structure and multiple non-canonical RNP stoichiometries that correlate with the composition of the RNA starting material. These insights are directly useful for improving RNP formation, maximizing functional material, and defining quality control metrics for CRISPR-based reagents and therapeutics.

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