Proven Intelligence Accelerating Next-Generation Therapies.

Executive Summary

High concentrations of empty AAV capsids represent a significant process-related impurity that can increase the total viral protein load administered, potentially elevating immunogenicity risk without contributing to therapeutic efficacy. Effective removal during preclinical vector production is a foundational step for de-risking a therapeutic program. Franklin Biolabs employs established purification methodologies, including iodixanol gradient ultracentrifugation and ion-exchange chromatography, to enrich for genome-containing particles. This focus on purity at the research stage provides a more representative vector for IND-enabling toxicology studies and establishes a scalable purification strategy for future GxP manufacturing.

The Challenge of Process-Related Impurities in AAV Production

In recombinant AAV production using transient transfection of HEK293 cells, the assembly of viral particles is an imperfect process. It results in a heterogeneous mixture of particles, including fully packaged vectors, partially filled vectors, and empty capsids that lack the therapeutic gene cassette. These empty capsids are considered a process-related impurity.

Their presence increases the total quantity of viral protein required to achieve a therapeutic dose of genome-containing particles. This elevated protein load can unnecessarily activate innate and adaptive immune responses, a risk factor that must be minimized long before a candidate enters clinical evaluation.

Purification Strategies for Empty Capsid Removal

A tailored preclinical strategy requires a purification process that effectively separates genome-containing virions from empty capsids. The goal is to enrich the final product for functional, therapeutic particles.

Two primary methodologies are employed for this purpose at research scale:

• Iodixanol Gradient Ultracentrifugation: This density-based separation method has long been a standard for AAV purification. Full capsids, containing the dense DNA genome, exhibit a higher buoyant density than their empty counterparts, allowing for their separation through centrifugation in an iodixanol gradient.

• Ion-Exchange Chromatography: This technique separates molecules based on their surface charge. There are subtle differences in the isoelectric point (pI) between full and empty AAV capsids, which can be exploited for separation using anion-exchange or cation-exchange chromatography. This method is not only effective but also provides a direct path toward a scalable, GxP-compliant downstream process. Analytical methods based on this principle provide precise quantification of full and empty vector particles for lot characterization (PMID: 22428980).

The Immunogenicity Factor: Why Capsid Purity Matters

The rationale for removing empty capsids extends beyond simple product purity. The AAV capsid itself is the primary target for the host immune system. Studies investigating capsid-specific T-cell activation have shown that specific protein motifs can influence dendritic cell uptake and subsequent immune responses (PMID: 16845388). By reducing the total number of capsids administered for a given therapeutic dose, the potential for triggering these immune pathways is lowered.

Achieving a high full-to-empty particle ratio in preclinical vectors ensures that toxicology and efficacy data are generated with a product that is as close as possible to the intended clinical material.

We started collaborating with UPenn Vector core in 2023 and the AAV vector which they manufactured laid a foundation for development of a gene therapy candidate which will enter soon preclinical studies. The key people from UPenn Vector Core joined Franklin Biolabs and our partnership transitioned without interruption from UPenn Vecor Core to Franklin Biolabs Research Vector Division. Franklin Biolabs is an essential collaborator in our AAV-vector based gene therapy candidate development, and we hope to continue the partnership for years to come.
— Biotech Partner

Franklin Biolabs’ Approach to High-Purity Research Vectors

The team at Franklin Biolabs, which includes key personnel from the former Penn Vector Core, has decades of direct experience in optimizing AAV production and purification. Our processes, refined across thousands of vector preparations, are designed to maximize the yield of full capsids while minimizing impurities. This expertise, now operating within a >100,000 sq ft facility, provides a stable and experienced scientific extension for our clients’ programs. Our focus on generating high-purity material at the research stage is a core component of minimizing clinical risk.