Unlocking Method Insights For Novel Engineered AAV Capsids

By Chelsey Mattison, Novartis

Engineered adeno associated virus (AAV) capsids are transforming the possibilities for gene therapy, offering the potential for precise tissue targeting, higher transduction efficiency, and reduced immune response. While these designs represent significant advances, they also introduce a layer of analytical unpredictability. Methods developed for wild type capsids do not always produce consistent or interpretable results with novel variants.

This unpredictability has real-world implications. Developers need confidence that their data accurately reflects the product, regulators require assurance of quality, and patients depend on timely access to effective therapies. Microfluidic capillary electrophoresis platforms, such as labchip, are widely used for assessing capsid integrity, yet structural modifications can generate unexpected migration patterns or signals that may reflect true biological differences or method-specific artifacts.

Recognizing these limitations is critical for supporting innovation in capsid design while maintaining regulatory confidence and ensuring reliable patient access.

Wild-Type Approaches May Not Work For Engineered Serotypes

Analytical platforms like labchip and other capillary electrophoresis methods have long served as key tools for assessing AAV capsid size, purity, and structural integrity. For wild type serotypes, these approaches provide reproducible results that form the foundation of quality control and regulatory documentation.

As engineered capsids become more common, certain variants begin to challenge these established workflows. Structural modifications can generate early migrating peaks, which may initially be concerning, but as reported in this study, are often temperature dependent aggregates rather than degradation. While other engineered capsids could theoretically produce low molecular weight species, these findings emphasize the importance of correctly identifying the nature of these peaks.

To address these challenges, laboratories increasingly rely on variant-specific evaluations and complementary analytical techniques to interpret unexpected signals rather than as general verification of product attributes. Orthogonal methods such as SEC, dynamic light scattering (DLS), and mass spectrometry helped confirm in this study that early peaks represented temperature dependent aggregates. A clear understanding of both the capabilities and limitations of analytical tools allows developers to accurately interpret unexpected results, maintain product quality, and avoid delays in characterization workflows.

Novel Orthogonal Methods Need Refining Before Scaling

Analytical characterization of engineered AAV capsids frequently encounters challenges not observed with wild type serotypes. In recent evaluations, three distinct modified capsid variants were assessed using labchip and CE-UV platforms. One notable observation across these assessments was the emergence of early migrating peaks. Detailed examination indicated that these peaks were not indicative of low molecular weight degradation products but instead represented temperature-dependent aggregate species. As laboratory conditions, particularly temperature, were altered, the relative intensity and migration of these aggregates changed, demonstrating a dynamic response that can confound straightforward interpretation of electropherograms.

Complementary analytical approaches provided critical insight into the nature of these signals. DLS confirmed the presence of higher order species corresponding to early migrating peaks. Mass spectrometry corroborated that no degradation products were present in these fractions, reinforcing the interpretation that there were temperature-sensitive aggregate species arising from the analytical protocol rather than inherent capsid instability. These orthogonal methods collectively strengthened confidence that the observed anomalies reflected recurrent or environmental factors, rather than intrinsic product differences.

Further exploration revealed that formulation conditions influenced aggregate behavior, though inconsistently. For one modified capsid variant, adjustments to dilution buffers resulted in slight attenuation of early migrating peaks, suggesting a potential path for mitigating temperature-dependent aggregation. However, the same adjustments produced minimal or no effect for the other two capsid variants, indicating that mitigation strategies may need to be variant-specific. This variability underscores the importance of cautious interpretation when comparing analytical profiles across multiple engineered capsids and the limitations of applying a single protocol universally.

• These observations highlight practical obstacles faced by developers when characterizing novel capsids. First, analytical signals may be influenced more by method conditions than by product characteristics, leading to potential misclassification of quality attributes.
• Second, buffers, sample preparation, and temperature conditions can unpredictably affect profiles, meaning that method optimization must consider multiple variables in combination.
• Finally, the necessity of employing orthogonal techniques to confirm interpretations introduces added complexity and resource demands but remains essential for ensuring data reliability and regulatory confidence.

Despite these challenges, several actionable lessons emerge. Early recognition that electropherogram anomalies may represent thermally responsive aggregate species can prevent unnecessary concerns regarding product degradation. Employing multiple analytical methods allows for cross-validation and more accurate assignment of observed features. Additionally, a variant-specific mindset when adjusting formulation analytical conditions can improve interpretability without overextending resources on universal adjustments that may have limited impact.

Collectively, these practices enable laboratories to generate reproducible, interpretable data even when working with capsids that diverge significantly from wild type characteristics.

By sharing these insights, the analytical community is better positioned to navigate the complexities of engineered capsid characterization. Understanding the influence of temperature, buffer composition, and method-specific factors on observed signals allows for more confident decision-making, both in development and in communication with regulatory authorities. While engineered AAVs offer transformative potential for gene therapy, careful and nuanced analytical strategies are essential to fully realize their promise without compromising on quality, reproducibility, or interpretability.

Challenges Reveal Tension In Novel Capsid Exploration

While the analytical challenges of engineered AAV capsids are well recognized, several factors complicate efforts to resolve them. One issue stems from the modifications themselves. Structural changes that make these vectors valuable for therapy also generate unexpected results that can be difficult to interpret. Early migrating peaks, for instance, may reflect benign structural shifts, temperature-dependent artifact aggregates, or true product instability. Without a clear attribute, there is a risk of mischaracterizing the nature of these features.

Another limitation is the fragmented nature of current analytical workflows. Microfluidic electrophoresis and CE-UV are useful for size and purity assessment, but they cannot always resolve subtle or temperature-sensitive species. Orthogonal methods like SEC, DLS, and mass spectrometry help fill these gaps, yet they add cost, time, and complexity — factors that are not always sustainable at scale. Even when multiple techniques are applied, the data may remain ambiguous if variant-specific behaviors are not well understood.

These challenges highlight a broader tension: while engineered capsids expand therapeutic potential, their characterization often stretches existing platforms beyond their original design. Without deeper insight into the causes of unusual results, achieving streamlined, broadly applicable methods remains difficult.

Conclusion

Analytical evaluation of engineered AAVs is not just about measuring purity or size, it is about creating confidence in products that will ultimately be used in patients. Meeting this challenge requires both rigor and adaptability – rigor in ensuring data reflect real product attributes and adaptability in refining methods to match the complexity of new designs.

Looking forward, the path to more efficient characterization may lie in understanding why variant-specific behaviors arise in the first place. If unusual peaks and apparent aggregates can be traced back to engineering choices in the capsid, it may become possible to predict, or even prevent, these features. This knowledge could support the development of streamlined high-throughput platforms that capture essential quality attributes without the need for multiple orthogonal assays.

The opportunity  is not only to manage the limitations of current tools but to transform them into a foundation for future workflows. By directly linking capsid design with analytical outcomes, research can move toward faster, more predictive, and more scalable methods. Ultimately, advancing capsid engineering and analytical innovation in parallel will shorten the path from discovery to treatment, ensuring patients benefit more quickly from safe and effective gene therapies.

About The Author:

Chelsey Mattison received her B.S. degree from Worcester State University and started at AbbVie about 10 years ago where she worked on assay support and method development, primarily with DVDs, ADCs, and mAbs, including the FDA-approved drug Skyrizi. She moved to Acceleron, where her group focused on sotatercept, which later became the FDA-approved drug Reblozyl. In her current role at Novartis, her focus is on method development and optimization for AAVs and LVVs.