Altura Size Exclusion 300 Å Allows Significant Gains in Performance at Low Salt Concentrations

Significance of the topic

Size exclusion chromatography (SEC) is the primary analytical technique used to evaluate size-based critical quality attributes of biotherapeutics, including monomer content, oligomer/aggregate formation and conformational changes. Nonspecific adsorption of proteins to column hardware and stationary-phase surfaces—particularly via metal binding to stainless steel components—causes peak tailing, signal loss and reduced resolution. To suppress these interactions laboratories commonly raise mobile-phase ionic strength, but high salt levels can introduce secondary hydrophobic interactions, accelerate instrument wear, and complicate downstream workflows. Reducing metal-induced adsorption while maintaining low-ionic-strength conditions therefore has practical importance for sensitive, robust SEC analyses in pharma and biopharma applications.

Study objectives and overview

This application study compared the performance of the Agilent Altura SEC 300 Å column equipped with Ultra Inert (metal-inert) hardware to an otherwise identical column packed into conventional stainless-steel (SS) housings. The primary aims were to determine whether Ultra Inert column hardware reduces nonspecific metal binding at reduced salt concentrations, improves peak symmetry and sensitivity, and enhances quantification of high-molecular-weight (HMW) aggregates for a panel of proteins, monoclonal antibodies (mAbs) and an antibody–drug conjugate (ADC). Salt was varied systematically (25–500 mM NaCl) in a 50 mM phosphate buffer (pH 7.0) to assess performance across ionic-strength conditions representative of both low- and high-salt SEC practice.

Methodology and experimental details

- Samples: a set of model proteins and biotherapeutics (including lysozyme, α-chymotrypsinogen, myoglobin, ovalbumin) and therapeutic mAbs/ADC (rituximab, infliximab, eculizumab, sacituzumab govitecan) were prepared at 1 mg/mL in 50 mM phosphate buffer, pH 7.0. - Columns: Agilent AdvanceBio/Altura SEC 300 Å, 4.6 × 300 mm, 2.7 µm packing; Altura versions used Ultra Inert coated hardware to block metal interactions while the comparison used stainless-steel housings with the same lot of packing material. - Mobile phase: two eluents (A = 50 mM phosphate pH 7.0; B = same + 500 mM NaCl) were mixed to achieve nominal NaCl concentrations of 25, 50, 100, 250, 400 and 500 mM. - Chromatography: 0.35 mL/min flow, 25 °C, 1 µL injection, UV detection at 220 nm, 20 min runs. - Measurements: peak tailing factors, retention behavior, aggregate area % and resolution between main peak and aggregates were evaluated. Detailed instrumentation is listed in the dedicated instrumentation section below.

Used instrumentation

• HPLC system: Agilent Infinity III Bio configuration components (1260 multiple wavelength detector, multicolumn thermostat, bio multisampler, bio-inert pump).
• Columns: Agilent AdvanceBio SEC 300 Å (standard stainless steel housing) and Agilent Altura SEC 300 Å (Ultra Inert coated housing), both 4.6 × 300 mm, 2.7 µm.
• Software: Agilent OpenLab CDS Acquisition and Analysis v2.8.
• Reagents: HPLC-grade buffer salts and Milli-Q ultrapure water; protein standards and therapeutic samples from commercial suppliers.

Main results and discussion

- Peak symmetry: Ultra Inert Altura hardware substantially reduced peak tailing for proteins and biotherapeutics that have net positive surface charge (pI > 7). At the lowest salt tested (25 mM NaCl), lysozyme and α-chymotrypsinogen showed greater than twofold improvement in tailing factor with Altura compared to stainless steel. For several high-pI mAbs and the ADC, peaks were very low or absent on stainless steel at 25 mM but were detectable with acceptable symmetry on Altura hardware. - Salt dependence: As NaCl concentration increased to very high levels (500 mM), differences between Altura and stainless-steel housings diminished because high ionic strength screens metal–protein interactions. However, raising salt also shifted retention times slightly later and introduced signs of induced hydrophobic interactions and peak fronting in some cases (e.g., α-chymotrypsinogen), underscoring that high salt is not universally beneficial. - Aggregate detection and quantification: Altura columns improved sensitivity and resolution for HMW species. The ADC sacituzumab govitecan showed a larger measured aggregate area when run on Altura hardware (example gain ~1% absolute aggregation) and better resolution between main and aggregate peaks (reported Rs improvement from ~1.33 to ~1.41). Similar resolution gains were observed for rituximab. For proteins with lower pI (e.g., eculizumab, ovalbumin) no meaningful benefit was observed, consistent with lower metal-sensitivity. - Practical interpretation: The data indicate that hardware-related metal binding is a significant contributor to nonspecific adsorption and compromised SEC performance at low ionic strength. Ultra Inert hardware extends the usable salt range toward lower ionic strengths, enabling clearer separation and more accurate aggregate quantitation without relying on very high salt that can mask other interaction modes.

Benefits and practical applications

• Improved peak shape and signal sensitivity for metal-sensitive proteins at reduced ionic strength, enabling more reliable monomer/aggregate quantification.
• Reduced dependence on high-salt mobile phases, lowering risk of salt-induced hydrophobic effects, instrument wear, and incompatibility with downstream techniques.
• Enhanced method robustness and platform transferability across diverse biotherapeutics, reducing sample rework and false negatives for aggregate detection.
• Better discrimination of oligomer species (dimer vs larger multimers) which supports compliance with regulatory requirements for CQA measurement.

Future trends and applications

• Integration of metal-inert SEC hardware with orthogonal detectors (multi-angle light scattering, native mass spectrometry, inline concentration detectors) to improve absolute sizing and composition analysis under low-ionic-strength conditions.
• Broader adoption of inert column housings in regulated QC environments to standardize aggregate measurements and reduce method variability introduced by hardware effects.
• Method development strategies that exploit the extended usable range of ionic strengths to minimize both electrostatic and hydrophobic artifacts for individual proteins, enabling more tailored SEC methods per molecule class.
• Further materials research to reduce other surface interactions (e.g., residual silanols) and to produce inert hardware compatible with harsh cleaning or MS-friendly volatile buffers.

Conclusion

Replacing conventional stainless-steel column hardware with Ultra Inert Altura housings substantially reduces metal-mediated nonspecific adsorption in SEC, particularly for proteins and biotherapeutics with basic surface character. This permits accurate detection and quantification of aggregates at much lower salt concentrations, improving sensitivity, resolution and method flexibility while avoiding some drawbacks of high-ionic-strength mobile phases. Altura Ultra Inert technology therefore supports more robust SEC assays suitable for biopharma method development and QC workflows.

References

1. Kříž Z.; et al. How Ionic Strength Affects the Conformational Behavior of Human and Rat Beta Amyloids – A Computational Study. PLoS ONE. 2013;8(5). doi:10.1371/journal.pone.0062914.
2. Rabe M.; Verdes D.; Seeger S. Understanding Protein Adsorption Phenomena at Solid Surfaces. Advances in Colloid and Interface Science. 2011;162(1–2):87–106. doi:10.1016/j.cis.2010.12.007.
3. International Conference on Harmonisation (ICH). Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products Q6B. 1999.