Size Meets Charge: Chromatography Techniques for AAV Analysis

by | Jan 16, 2025

Size-exclusion chromatography (SEC) and anion-exchange chromatography (AEX) tackle AAV capsid analysis challenges, improving gene therapy production.

Adeno-associated viruses (AAVs) are transforming gene therapy with their potential to treat genetic disorders and advance vaccine development. However, AAV characterization presents significant challenges for analytical chemists tasked with ensuring safety, purity, and efficacy. Measuring critical quality attributes (CQAs)—such as capsid fill levels, aggregate content, and titer—is crucial to upholding biopharmaceutical standards and supporting gene therapy analytics.

This article highlights two key AAV characterization methods: size-exclusion chromatography (SEC) and anion-exchange chromatography (AEX). SEC enables precise separation of AAV aggregates by size, while AEX provides charge-based differentiation of full and empty capsids. Featuring insights from Andrea Krumm, Product Manager at Tosoh Bioscience, and the video series [U]HPLC Analysis of Biopharmaceuticals: Quality Attributes and How to Analyze Them, we explore how these techniques play a vital role in optimizing AAV production and ensuring therapeutic efficacy.

Size-Exclusion Chromatography: Separating AAVs by Size

SEC is a powerful tool for AAV characterization, separating molecules based solely on their size without relying on interactions with the stationary phase. Larger molecules, such as AAV aggregates, bypass the pores in the column resin and elute earlier. Smaller molecules enter the pores, following longer, more intricate pathways before eluting.

SEC’s straightforward mechanism makes it highly effective for analyzing AAV capsids and distinguishing between different capsid populations. Unlike anion-exchange chromatography, SEC avoids chemical or electrostatic interactions with the stationary phase. This lack of interaction ensures consistent and precise AAV aggregate separation and size-based resolution, even for biopharmaceutical products with similar charges or binding properties.

In AAV characterization, SEC can distinguish between:

  • Full capsids: Contain the therapeutic genome and are the desired product.
  • Empty capsids: Lack genetic material, making them slightly smaller and lighter than full capsids, which affects their retention time.
  • Aggregates: Consisting of multiple capsids bound together, aggregates are significantly larger and elute much earlier.

For effective separation of AAV capsids, selecting a column with an appropriate pore size is crucial. Given that AAVs are approximately 25 nanometers in diameter, columns with pore sizes around 100 nanometers are suitable. Tosoh’s TSKgel G5000PWxl column, with its 100-nanometer pore size, is commonly used for such applications.

Advancements in SEC column design further improve AAV characterization. For instance, Thermo Fischer’s SurePac Bio 550 SEC MDI column demonstrates high-resolution separation of AAV capsids and aggregates in under four minutes. Its robust design ensures reproducibility across injection volumes, making it invaluable for both research and industrial applications.

Expanding SEC Capabilities with MALS Detection

Partially filled capsids with incomplete genomes closely resemble fully filled capsids in size and hydrodynamic properties, making them difficult to distinguish using SEC alone. Pairing SEC with multi-angle light scattering (MALS) resolves this issue by precisely measuring molecular weight through light scattering intensity, enhancing SEC’s size-based separations for clearer differentiation.

A practical example comes from the Tosoh Bioscience application note, Sensitive Quantification of AAVs and their Impurities, which highlights the effectiveness of the LenS3 MALS detector combined with the TSKgel GMPWXL column. This setup demonstrated a detection limit as low as 7.0×109 capsids per milliliter, or 2.8×108 capsids in a 40 µL injection. SEC-MALS offers high sensitivity, enabling precise quantification of AAV capsids even at low concentrations—critical for assessing product yield and therapeutic dosing.

Combining SEC-MALS with UV detection unlocks deeper AAV insights. MALS provides molecular weight data, while UV absorbance at 260 and 280 nm distinguishes nucleic acid from protein. This synergy enables precise payload ratio calculations, revealing capsid functionality and payload quality for reliable manufacturing.

Anion-Exchange Chromatography: Differentiating Full and Empty Capsids

While SEC-MALS provides valuable insights, it has limitations. Partially filled and empty capsids often fall within the same molecular weight range as fully filled capsids, making them difficult to distinguish. This challenges the ability of SEC-MALS to always quantify the ratio of full to empty capsids—an essential quality attribute in AAV characterization.

To address this, anion-exchange chromatography offers a complementary solution. Unlike SEC, AEX separates AAV capsids based on their charge, which directly correlates to the presence of nucleic acid payloads. Full capsids, containing negatively charged genomic material, exhibit lower isoelectric points and stronger binding to the positively charged stationary phase of an AEX column. Empty capsids, lacking this payload, are less negatively charged and elute earlier under a salt gradient. This charge-based separation provides the precision needed to quantify and distinguish between full and empty capsids with high accuracy.

AEX has proven particularly effective in a variety of applications, including the use of the TSKgel Q-Stat column with choline chloride as a non-toxic alternative to traditional salt gradients like TMAC. This approach ensures reliable separation while aligning with safety and regulatory standards for industrial manufacturing. Studies have further demonstrated its general applicability across multiple AAV serotypes, making AEX a cornerstone technique for comprehensive AAV characterization.

Conclusion

Blending SEC and AEX provides unmatched precision in AAV characterization, addressing critical challenges such as capsid fill levels, aggregate detection, and purity. These complementary techniques not only ensure safety and efficacy in gene therapy but also highlight the potential for innovation in virus screening and therapeutic design. To explore these techniques in greater detail, watch the Tosoh Bioscience webinar series, available now on Separation Science. Learn how industry-leading experts are tackling AAV characterization challenges with advanced chromatography tools.

Related Content

Advertisment

Advertisment

Advertisement

Advertisement