omniDAWN Detector: Bridging the Best of Both Worlds

Significance of the topic

The combination of ultra-performance liquid chromatography (UPLC) separations with multi-angle light scattering (MALS) detection addresses a persistent analytical challenge: preserving the high resolution and speed of UPLC while obtaining absolute molar mass and size information that is typically measured with larger-volume HPLC-compatible MALS detectors. This capability is critical for biopharmaceutical development and quality control where sample is limited and minor species (aggregates, fragments, conjugates, viral particles, lipid nanoparticles) must be accurately characterized.

Study objectives and overview

This application note evaluates the omniDAWN MALS Photometer configured downstream of an ACQUITY Premier UPLC system and ACQUITY Premier Protein SEC columns. The primary goals were to demonstrate that omniDAWN preserves MALS accuracy and dynamic range in a UPLC workflow, to quantify performance (sensitivity, reproducibility, ability to resolve and quantify minor species), and to compare UPLC-omniDAWN results to conventional HPLC-DAWN MALS measurements.

Methodology

The study used bovine serum albumin (BSA) as a model analyte prepared at 5 mg/mL, 0.10 mg/mL and 0.01 mg/mL and filtered to 0.02 µm. Injections ranged from 100 ng to 10 µg with five replicates per condition. UPLC separations employed ACQUITY Premier Protein SEC Columns (250 Å, 1.7 µm, 4.6 x 300 mm) with phosphate buffered saline (PBS) mobile phase. For comparison, conventional HPLC and UHPLC column formats (XBridge Premier Protein SEC 250 Å, 2.5 µm, 7.8 x 300 mm; WTC-030S5 300 Å, 5 µm, 7.8 x 300 mm) were used with a DAWN MALS and Optilab dRI detector. Data acquisition and analysis were performed using HPLC CONNECT and ASTRA Software. Analysis utilized dn/dc = 0.185 mL/g and a UV extinction coefficient of 0.667 mL·mg⁻1·cm⁻1.

Used Instrumentation

• ACQUITY Premier UPLC System with Binary Solvent Manager and Flow-Through Needle Sample Manager
• ACQUITY Premier Protein SEC Column (250 Å, 1.7 µm, 4.6 x 300 mm)
• omniDAWN MALS Photometer (18-angle MALS)
• microOptilab differential refractive index (dRI) Detector
• ACQUITY Premier PDA eλ UV Detector (280 nm)
• Comparison instruments: DAWN MALS Photometer and Optilab dRI Detector used with HPLC columns
• Software: HPLC CONNECT for control and ASTRA for data analysis

Key results and discussion

• Run time and peak broadening: UPLC runs with the omniDAWN configuration completed injections in under 10 minutes versus approximately 20 minutes or more for standard HPLC. Reduced dispersion in UPLC resulted in less dilution of eluting species.
• Detection of minor species: Improved concentration and narrower peaks in the UPLC configuration facilitated detection and accurate quantitation of low-abundance species (e.g., trimers, higher-order aggregates and fragments) that were not reliably quantified with HPLC-DAWN due to lower signal-to-noise.
• Molar mass accuracy and precision: Monomer weight-average molar mass (Mw) across tested conditions was 67.4 ± 0.80 kDa with relative standard deviation <2% for each injection mass, demonstrating repeatability comparable to HPLC-MALS while offering higher sensitivity to minor peaks.
• Dynamic range and sizing capability: The omniDAWN’s 18-angle geometry provides a broad dynamic range sufficient for robust size and molar mass determinations up to ~10^9 g·mol⁻1 when combined with UV and RI detectors and ASTRA software calculations.
• Practical comparison: When chromatograms are scaled to relative column volume, both omniDAWN-UPLC and DAWN-HPLC quantified monomer and dimer peaks equivalently; however, omniDAWN-UPLC uniquely enabled accurate quantitation of certain high molecular weight species and resolved fragment peaks with measurable molar mass (example: a fragment at ~48.3 ± 0.7 kDa).

Benefits and practical applications

• Retains full MALS analytical capabilities (absolute molar mass, radius, conjugation analysis, branching) while leveraging UPLC speed, solvent/sample economy, and resolution.
• Improves detection and quantitation of low-abundance and labile species important in biopharma: aggregates (including AAV aggregates), conjugates (e.g., protein–polysaccharide), lipid nanoparticles, and branched polymers.
• Enables faster throughput and reduced sample consumption without sacrificing data quality or reproducibility—useful for method development, QC screening, and limited-sample workflows.
• Smooth integration into existing UPLC methods and ASTRA-based analytical workflows simplifies technology adoption.

Future trends and potential applications

• Broader adoption of UPLC-compatible MALS in regulated biopharma workflows for lot release and stability testing as method validation strategies mature.
• Extension to characterizing more complex modalities: viral vectors (AAV), lipid nanoparticles for mRNA delivery, antibody–drug conjugates, and heterogeneous conjugates where minor species are critical quality attributes.
• Integration with orthogonal detectors and techniques (multi-wavelength UV, mass spectrometry orthogonal interfaces, and advanced RI/viscometry) for richer physicochemical profiles.
• Automation, miniaturization, and higher-throughput SEC-MALS platforms to accelerate screening during formulation and bioprocess development.

Conclusion

The omniDAWN MALS Photometer, when paired with ACQUITY Premier UPLC and short-particle SEC columns, successfully bridges UPLC performance and classical MALS analytical capabilities. It provides accurate and reproducible molar mass and sizing while increasing speed, sensitivity to minor species, and reducing sample dilution. This configuration broadens the practical applicability of SEC-MALS to modern high-throughput and sample-limited biophysical characterization tasks.

References

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2. Martini C.; et al. Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate. mBio 2024, 15, e02952-23.
3. Hsieh V. H.; Wyatt P. J. Measuring proteins with greater speed and resolution while reducing sample size. Scientific Reports 2017, 7, 10030.