Agilent Altura SEC Columns: Ultra Inert Size Exclusion Chromatography for Biomolecule Analysis

Size exclusion chromatography (SEC) plays an important role in biopharmaceutical analysis, from monitoring protein aggregation and degradation to characterising peptides, monoclonal antibodies and increasingly complex therapeutic modalities. However, interactions between biomolecules and metal surfaces within the LC flow path can introduce unwanted adsorption, resulting in peak tailing, reduced recovery and loss of sensitivity.

Agilent Altura SEC columns have been developed to address these challenges by combining proven SEC stationary phase technology with Agilent Ultra Inert column hardware. Covering pore sizes from 130 Å to 1000 Å, the range supports size-based separations across a broad spectrum of biomolecules, from therapeutic peptides through to viral vectors and large oligonucleotides.

What is Altura Ultra Inert Technology?

Altura SEC columns use the same SEC media as Agilent AdvanceBio SEC columns, based on high-porosity silica particles with hydrophilic bonding chemistry designed to provide stable size-based separations while minimising unwanted secondary interactions. The key difference is the addition of Ultra Inert column hardware.

An advanced coating is applied to the column hardware to block active metal sites and create a more inert sample flow path, while retaining the strength and pressure tolerance associated with conventional stainless-steel columns. For metal-sensitive biomolecules, reducing these interactions can improve sample recovery, peak symmetry and sensitivity and help produce more consistent chromatographic results. Altura SEC columns can also replace an equivalent particle- and pore-size AdvanceBio SEC column where improved inertness is required.

Altura SEC Pore Sizes

The Altura SEC range includes five pore sizes, with four columns available in 2.7 µm particles and one high-efficiency 1.9 µm option. This range provides flexibility to select the most appropriate pore and particle size for different biomolecule types and SEC performance requirements.

130 Å, 2.7 µm – designed for peptides and smaller therapeutic proteins.
200 Å, 1.9 µm – a high-resolution, high-throughput option for monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs).
300 Å, 2.7 µm – suited to mAbs, ADCs and other proteins.
500 Å, 2.7 µm – designed for larger biotherapeutics including adeno-associated viruses (AAVs), large proteins and oligonucleotides.
1000 Å, 2.7 µm – provides the larger pore structure required for virus-like particles (VLPs), oligonucleotides and other large biotherapeutic molecules.

How Altura Ultra Inert Hardware Improves SEC Performance

Example 1: Protein and Antibody Aggregate Analysis

Protein aggregation is an important critical quality attribute in biopharmaceutical analysis, and SEC is widely used to quantify high- and low-molecular-weight species. However, proteins can interact non-specifically with exposed metal surfaces in conventional stainless-steel column hardware, particularly under lower ionic-strength conditions. These interactions can lead to peak tailing, reduced recovery and loss of sensitivity, potentially making low-level aggregates and fragments more difficult to detect accurately.

To assess the effect of the hardware itself, Agilent packed the same lot of SEC material into conventional stainless-steel and Altura Ultra Inert hardware and compared the performance of proteins, monoclonal antibodies and an antibody-drug conjugate across NaCl concentrations from 25 to 500 mM. As shown in Figure 1, the greatest improvements were seen at lower salt concentrations, where metal-analyte interactions were more pronounced. For proteins with a pI above 7, the Altura hardware provided at least a two-fold improvement in peak symmetry at the lowest salt conditions tested.

Figure 1: Comparison of peak tailing of proteins, mAbs, and ADCs at varying NaCl concentrations [1]

The impact of the Ultra Inert hardware at low salt concentrations is illustrated more clearly in the representative chromatograms in Figure 2. At 25 mM NaCl, globular proteins such as lysozyme and α-chymotrypsinogen showed substantial peak tailing with stainless-steel hardware, which was markedly reduced with Altura. The effect was even more significant for larger biotherapeutics: rituximab and the ADC sacituzumab govitecan produced extremely low or no detectable signal with stainless steel, while peaks remained observable with the Ultra Inert column. These results demonstrate how reducing metal-related adsorption can improve peak symmetry and analyte recovery, particularly for metal-sensitive biomolecules.

Figure 2: Representative chromatograms comparing Ultra Inert hardware against stainless steel at low salt concentrations (50 mM phosphate buffer + 25 mM NaCl) [1]

At higher salt concentrations, increased ionic strength helped suppress metal binding and the performance of the two hardware types became more comparable. However, higher-salt mobile phases can also increase hydrophobic interactions and place greater demands on the LC system, particularly with non-bio-inert instrumentation. By reducing reliance on elevated salt concentrations to control secondary interactions, Altura Ultra Inert hardware provides greater flexibility during SEC method development and supports robust analysis across a wider range of mobile phase conditions.

Example 2: GLP-1 and Therapeutic Peptide Analysis

GLP-1 analogues can be challenging to analyse by SEC because both their aggregation behaviour and chromatographic performance are strongly influenced by method conditions. During evaluation, liraglutide, semaglutide and exenatide all showed pH-dependent changes in elution behaviour, while the fatty-acid modifications present in liraglutide and semaglutide can further increase hydrophobicity and contribute to peak-shape changes. Alongside these effects, non-specific interactions with residual silanol groups and exposed metal surfaces in conventional stainless-steel hardware can cause additional peak tailing and reduced recovery.

To isolate the effect of the column hardware, Agilent packed the same SEC stationary phase into stainless-steel and Altura Ultra Inert columns and compared performance at pH 2, 7.4 and 8.5 with 50% acetonitrile. As shown in Figure 3, the Altura hardware improved peak symmetry under most conditions tested, with the greatest differences seen at the more extreme pH values where adsorption to stainless-steel surfaces was more pronounced. Peak tailing was reduced by more than 60% for liraglutide, around 60% for exenatide and around 30% for semaglutide under most conditions.

Figure 3: Comparison of Stainless-steel hardware compared to Ultra Inert hardware on the peak symmetry of GLP-1 analogues under different pH conditions at 50% acetonitrile [2]

Representative chromatograms in Figure 4 show the improvement in peak shape for liraglutide and semaglutide at pH 8.5 and exenatide at pH 2. For semaglutide, the peak width at half height remained comparable between the two hardware types, indicating that the main improvement was associated with reduced tailing rather than a change in column efficiency. This is important because excessive tailing can compromise the detection and resolution of fragment impurities and aggregate species. By reducing metal-related secondary interactions, Altura Ultra Inert hardware provides greater flexibility to explore pH during SEC method development for GLP-1 analogues.

Figure 4: Representative chromatograms of the gain in performance with the Alutra SEC 130 A (blue) versus stainless steel (green) for Liraglutide (A), Semaglutide (B) and Exenatide (C) under pH 8.5 (A, B) and pH 2 (C) with 50% acetonitrile. [2]

Conclusion: Expanding SEC Method Development with Ultra Inert Hardware

Agilent Altura SEC columns build on established AdvanceBio SEC stationary phase technology with the addition of Ultra Inert column hardware designed to minimise unwanted metal-analyte interactions. By improving the inertness of the chromatographic flow path, the columns can support better peak shape, recovery and sensitivity, particularly for biomolecules that are prone to nonspecific adsorption.

With five pore sizes spanning therapeutic peptides, proteins and antibodies through to viral vectors, VLPs and large oligonucleotides, the Altura SEC range provides a flexible platform for both established and emerging biopharmaceutical SEC applications. The ability to explore a wider method development space while reducing unwanted surface interactions makes Altura particularly relevant as biomolecules and analytical workflows continue to become more complex.