AdvanceBio SEC: Overcoming Challenges in Modern Biotherapeutic Aggregate Analysis

Size Exclusion Chromatography (SEC) remains a fundamental technique for characterising biotherapeutic products, offering essential insights into aggregation and size variants – key factors that impact product safety, efficacy, and regulatory compliance. However, conventional SEC methods are often limited by inadequate resolution, nonspecific interactions, and extended run times, which can limit their effectiveness in both research and quality control settings.

As the biotherapeutic landscape expands – from peptides and monoclonal antibodies (mAbs) to more complex modalities such as adeno-associated viruses (AAVs), virus-like particles (VLPs), and nucleic acid-based therapeutics – SEC must adapt to address increasingly diverse analytical challenges. Precise aggregate quantification is critical at every stage of development, from establishing critical quality attributes (CQAs) during early process development to verifying acceptable aggregate levels in final product release.

Agilent’s AdvanceBio SEC columns were developed to meet these evolving requirements, delivering high-resolution, reproducible separations across a wide range of molecular sizes and structural complexities.

Optimising Pore Size for SEC Separations

SEC separates analytes based on their size in solution, more specifically their hydrodynamic radius. Molecules penetrate the porous stationary phase to different extents depending on their size. Smaller analytes spend more time inside the pores and elute later, while larger analytes are excluded from the pores and elute earlier (Figure 1).

Figure 1: Schematic of pore interaction as a function of analyte size.

Selecting an appropriate pore size is crucial for achieving reliable resolution. While SEC pore sizes are typically matched to protein molecular weights based on globular proteins, this relationship breaks down when analysing newer generation biotherapeutics. Selecting the

correct pore size is therefore crucial for achieving accurate and high-resolution separations. AdvanceBio SEC columns are available in a broad range of pore sizes – from 120 Å to 1000 Å – enabling analysts to tailor separations to specific analyte types (Figure 2).

Figure 2: Recommended pore size selection based on molecular weight range of proteins [1]

For conventional biomolecules like peptides and proteins, where the size-to-weight relationship is relatively predictable, pore selection can follow standard molecular weight guidelines:

Small therapeutic proteins and peptides:

AdvanceBio SEC 130 Å, 2.7 μm: Routine aggregate analysis (100 Da–120 kDa)
AdvanceBio SEC 120 Å, 1.9 μm: High-resolution, rapid profiling (1–80 kDa)

Monoclonal antibodies and ADCs:

AdvanceBio SEC 300 Å, 2.7 μm: General aggregate screening (1 kDa–1.25 MDa)
AdvanceBio SEC 200 Å, 1.9 μm: mAb fragment analysis (2–700 kDa)

The AdvanceBio SEC 120 Å 1.9 µm, 4.6 x 300 mm column delivers superior resolution (Rs = 4.03) for the analysis of high molecular weight (HMW) species in insulin samples, enabling accurate quantification (Figure 3). Improved resolution is also evident in the insulin monomer peak, where lower molecular weight fragments are more clearly separated compared to columns from other vendors.

Figure 3: SEC analysis of stressed insulin with aggregates and low molecular weight fragments. Full method conditions in application note 5994-1566EN. [2]

For next-generation biologics, the correlation between molecular weight and pore size is often non-linear, as the hydrodynamic radius can differ significantly from what is predicted based on molecular weight alone. For example, adeno-associated viruses (AAVs), despite their high molecular weight, have compact, tightly folded structures with a hydrodynamic diameter of approximately 25 nm. Consequently, columns with smaller pore sizes than expected may provide superior resolution.

In contrast, virus-like particles (VLPs), which are self-assembling protein structures typically ranging from 20 to 150 nm in diameter, require columns with larger pore sizes to accommodate their extended size and maintain effective separation. As shown in Figure 4 the AdvanceBio range demonstrates superior resolution for these ultra-large biotherapeutics.

AAVs and VLPs:

AdvanceBio SEC 500 Å, 2.7 μm: Suitable for AAVs (5 kDa–5 MDa or Rh 3–25 nm)
AdvanceBio SEC 1000 Å, 2.7 μm: Ideal for larger VLPs (15 kDa–10 MDa or Rh 5–40 nm)

Figure 4: AdvanceBio SEC columns show higher resolution compared to other vendor columns for protein-based next-generation therapeutics [1]

Oligonucleotides and mRNA molecules often exhibit extended conformations, significantly increasing their hydrodynamic radius beyond what is expected from linear molecular weight estimates. Larger pore columns improve resolution for these structures (Figure 6).

Oligonucleotides:

AdvanceBio SEC 500 Å or 1000 Å: Selection depends on sequence length and secondary structure (Figure 5)

Figure 5: Estimated resolving ranges for DNA and RNA molecules across different column pore sizes [3]

Figure 6: 50 bp DNA ladder ranging from 50 bp to 3,000 bp, showing excellent resolution particularly for larger oligonucleotides [1]

For biomolecules exceeding the resolving range of the 1000 Å columns, Agilent Bio SEC-5 with 2000 Å pores is available, offering a suitable option for ultra-large biotherapeutics up to 10 MDa or Rh ~75 nm.

Minimising Nonspecific Interactions

A key challenge in SEC is the occurrence of nonspecific interactions between analytes and the stationary phase. This is particularly problematic for hydrophobic or charge-sensitive species such as ADCs, where secondary interactions can distort peak shapes, reduce recovery, and compromise quantitation accuracy.

To mitigate these effects, AdvanceBio SEC columns feature a proprietary hydrophilic polymer coating that creates an inert, low-interaction surface This allows for accurate analysis without the need for high salt concentrations or organic modifiers, preserving sample integrity. The result is improved accuracy and peak symmetry, even with challenging analytes such as ADCs (Figure 7).

Figure 7: Excellent peak shape for ADC samples (top) using AdvanceBio SEC which are more hydrophobic than the related mAb (bottom) [1]

The coating is mechanically robust, maintaining consistent performance over extended use. As shown in Figure 8, the column delivers stable results even after 1,000 injections, demonstrating long operational lifetime. This durable surface chemistry also enhances batch-to-batch consistency and method robustness, enabling reliable performance across different laboratories and instrument platforms. Such reproducibility is critical for validated methods, regulatory submissions, and successful method transfer.

Figure 8: Column stability data for AdvanceBio SEC 500 Å and 1000 Å formats. The red line indicates a 10% drop in efficiency [4]

Reducing Analysis Time Without Sacrificing Resolution

SEC is inherently slower than many chromatographic techniques, often requiring lengthy run times to achieve baseline resolution. Traditional columns with 300 mm length are widely used but longer run times can be a bottleneck in busy labs or early-stage screening.

AdvanceBio SEC columns address this issue by using optimised optimized small particles with high pore volume, enabling the use of shorter 150 mm formats without compromising resolution. This significantly reduces analysis time – often by 50% – without compromising data quality. This efficiency is particularly beneficial in high-throughput settings, enabling faster decision-making, higher instrument utilisation, and reduced overall cost per analysis.

Figure 9: Faster elution using a shorter column format to increase throughput [1]

Conclusion: Advancing SEC Performance to Meet the Complex Demands of Modern Biotherapeutics

Agilent AdvanceBio SEC columns offer a technically robust solution for the evolving challenges of size-based analysis in biotherapeutic development. With a wide range of pore sizes, inert surface chemistry, and the ability to shorten analysis time without compromising data quality, these columns are well-suited for both traditional and next-generation therapeutics.

Whether analysing proteins, viral particles, or complex nucleic acid structures, researchers can rely on AdvanceBio SEC for reproducible, high-resolution separations in both research and regulated workflows.