Evaluating Volume Effects in SEC for Large Biomolecules on the Alliance™ iS Bio System With GTxResolve™ SEC 2000 Å Columns

Summary: Evaluating Volume Effects in SEC for Large Biomolecules on the Alliance iS Bio System with GTxResolve 2000 Å Columns

Significance of the topic

The expansion of nucleic-acid modalities alongside traditional biotherapeutics increases demand for robust analytical workflows that can characterize both proteins and large DNA vectors using size-exclusion chromatography (SEC). Understanding how method parameters such as column geometry, mobile-phase ionic strength, and injection volume affect apparent size distributions is essential to avoid masking structurally relevant features (for example, plasmid topology-dependent peak shoulders) or introducing hardware-related artifacts. The work summarized here investigates practical method choices that enable reliable characterization of high-molecular-weight biomolecules and informs transfer into routine or QC laboratories.

Objectives and overview of the study

• Compare SEC behavior of large proteins and nucleic acids on a single platform and column format (GTxResolve 2000 Å, 3 µm, 7.8 × 300 mm) implemented on the Alliance iS Bio HPLC System.
• Assess the influence of mobile-phase ionic strength (1X DPBS versus 1X DPBS + 1.0 M NaCl) on protein and plasmid peak shapes.
• Use enzymatic relaxation (topoisomerase I) to determine whether observed plasmid chromatographic shoulders originate from sample topology versus chromatographic artifact.
• Evaluate linearity and usable injection-volume headroom for both proteins and DNA mixtures to determine practical sensitivity gains without loss of interpretability.

Methodology

• Samples analyzed: thyroglobulin (2 mg/mL), a BEH450 multi-protein SEC standard mix, a DNA Vector Mixture Standard (pBR322 dsDNA, ΦX174 ssDNA, uracil void marker), and topoisomerase I–treated DNA mixture.
• Column and conditions: GTxResolve 2000 Å SEC Column, MaxPeak Premier (3 µm, 7.8 × 300 mm); mobile phases were 1X DPBS (without Ca/Mg) and DPBS spiked to 1.0 M NaCl; isocratic runs at 0.60 mL/min, 35 °C column temperature, 31 min run time.
• Injection volumes: standard 50 µL for most experiments; injection-volume studies ranged up to 100 µL for thyroglobulin and up to 100 µL for the DNA mixture to test linearity.
• Topoisomerase treatment: DNA standard incubated with Topoisomerase I (NEB) in manufacturer buffer at 37 °C for 15 min, then heat-inactivated; injected without further cleanup.
• Detection/data: PDA UV detection at 280 nm for proteins and 260 nm for DNA; data acquired and processed in Empower 3 with consistent integration settings for comparability.

Instrumentation used

• Alliance iS Bio HPLC System with integrated PDA detector and built-in 300 mm column holder; inert flow path and MaxPeak High Performance Surfaces (HPS) to minimize nonspecific adsorption.
• Alliance iS HPLC Photodiode Array Detector with analytical 8.4 µL flow cell (10 mm pathlength), 20 Hz sampling, 1 nm digital resolution.
• Empower CDS (version 3.10.x) for acquisition and processing.
• GTxResolve 2000 Å SEC Column, MaxPeak Premier Column, 3 µm, 7.8 × 300 mm.

Main results and discussion

• Protein performance and robustness
  • Triplicate injections of the BEH450 protein standard in 1X DPBS showed strong repeatability across a wide molecular-weight range, demonstrating stable retention and peak shape suitable for routine SEC profiling.
  • Thyroglobulin injection-volume studies (25–100 µL) preserved oligomer, dimer, and monomer peak structure while showing linear increase in UV peak area, indicating the 7.8 × 300 mm format supports higher injection volumes as a sensitivity lever without degrading interpretability.
  • Adjusting ionic strength to 1.0 M NaCl produced negligible change in thyroglobulin SEC profile (less than ~4% difference in integrated area), indicating protein SEC readouts were largely insensitive to the salt increase under these conditions.
• Plasmid/nucleic-acid behavior and method sensitivity
  • In contrast to proteins, the DNA vector mixture displayed pronounced sensitivity to mobile-phase ionic strength. In 1X DPBS, pBR322 and ΦX174 peaks exhibited clear shoulder features and additional late-eluting signals; these shoulders were substantially suppressed when NaCl was increased to 1.0 M, producing simpler-looking chromatograms.
  • Topoisomerase I treatment in 1X DPBS produced a similar directional effect to high-salt: shoulder areas decreased significantly (reported decreases ~41–52% in highlighted regions), supporting the interpretation that those shoulders arise from plasmid conformational/topological heterogeneity (e.g., supercoiling states) rather than purely column artifact.
  • The combination of salt-sensitivity and enzymatic modification indicates plasmid SEC requires analyte-specific interpretive frameworks: method choices can either reveal or obscure structurally informative subpopulations.
• Injection-volume headroom for DNA
  • DNA injection-volume series (12.5–100 µL) demonstrated proportional and linear UV responses across the tested range for major DNA regions and late-eluting low-MW signals, expanding usable sample mass from ~3 µg to 25 µg per DNA component while maintaining chromatographic interpretability.
  • The wider-bore, longer column format (7.8 × 300 mm) provided greater separation space and reduced influence of system dispersion relative to shorter, narrower formats—resulting in higher resolution of late-eluting species and improved ability to observe load-sensitive subpopulations.

Benefits and practical applications

• Single-platform workflow: The Alliance iS Bio System supports direct implementation of the 7.8 × 300 mm SEC format without auxiliary column management hardware, facilitating method adoption and potential QC transfer.
• Low adsorption and inert flow path reduced nonspecific interactions and hardware-derived artifacts, improving confidence that observed features are sample-derived.
• Increased injection-volume capability provides a pragmatic sensitivity improvement for both proteins and large nucleic acids without requiring changes to fundamental SEC conditions.
• Orthogonal experiments (ionic-strength modulation and enzymatic relaxation) enable mechanistic assignment of plasmid peak components, assisting in accurate characterization of plasmid topology and subpopulations critical in gene-therapy and vector development workflows.

Future trends and applications

• Method standardization across modalities: As DNA- and RNA-based therapeutics proliferate, there will be growing need for standardized SEC protocols that deliberately preserve or collapse topology-dependent information depending on regulatory or QC objectives.
• Orthogonal characterization integration: Combining SEC with online multi-angle light scattering (MALS), native MS, or fraction collection followed by topology-specific assays may improve assignment of SEC-resolved subpopulations.
• Column and surface chemistry advances: Continued optimization of low-adsorption chemistries and stationary phases tailored for very large biomolecules will further reduce method artifacts and extend dynamic range.
• Automation and QC deployment: Systems like the Alliance iS Bio that support larger bore, longer columns in an integrated package will simplify method transfer into regulated environments where repeatability and robustness are essential.

Conclusion

The study demonstrates that a 7.8 × 300 mm GTxResolve 2000 Å SEC Column on the Alliance iS Bio System provides high-resolution, repeatable SEC separations for both proteins and large nucleic acids while allowing larger injection volumes with linear UV response. Proteins showed robust behavior across ionic-strength variations, whereas plasmid peak structure proved method-sensitive and responsive to enzymatic topology modification. These findings emphasize the need for analyte-specific method choices when interpreting SEC data for nucleic-acid modalities and support the practical utility of wider-bore, longer SEC formats for expanded analytical windows and improved resolution of late-eluting species.

References

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