Proven Intelligence in Vector Process Development.

Executive Summary

Improving the ratio of full (genome-containing) to empty adeno-associated virus (AAV) capsids is a primary objective in process development for viral vectors. High concentrations of empty capsids can increase the total viral particle dose required for efficacy, potentially elevating immunogenicity risk without contributing to therapeutic effect. This asset details process development strategies for enriching full AAV capsids, ensuring a more potent and consistent product profile for IND-enabling toxicology studies and clinical manufacturing.

Optimizing Upstream Parameters for Capsid Packaging

The foundation for a high-quality AAV product is established during the upstream production phase. The stoichiometry of the triple-plasmid transfection system is a key variable. Adjusting the relative amounts of the plasmid containing the gene of interest (GOI), the Rep/Cap plasmid, and the helper plasmid directly influences the efficiency of genome packaging into nascent AAV capsids.

Our process development teams systematically evaluate these ratios for specific AAV serotypes (e.g., AAV8, AAV9, and engineered capsids) and production systems. This data-driven approach moves beyond theoretical starting points to define an optimized plasmid ratio that favors the assembly of full particles, forming the basis of strategic process blueprints that accelerate technology transfer.

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 our primary scientific and manufacturing collaborator for our AAV-vector based gene therapy candidate development, and we hope to continue the partnership for years to come.

Downstream Purification: Separating Full from Empty

While upstream optimization is preventative, downstream purification provides a direct mechanism for enriching full capsids. Standard purification processes may co-purify full and empty particles, but advanced methods can effectively separate them.

We leverage specialized analytical ultracentrifugation (AUC) and anion-exchange chromatography (AEX) methods to achieve this separation. By refining buffer conditions, pH, and salt gradients, we can exploit the subtle biophysical differences between full and empty capsids to preferentially elute and collect the therapeutically active, genome-containing particles. This level of process control is documented to support Investigational Medicinal Product Dossier (IMPD) submissions for Advanced Therapy Medicinal Product (ATMP) programs targeting EU approvals.

The consequences of administering a product with a high percentage of empty capsids extend to in vivo performance. Unnecessary viral protein load can heighten the risk of innate and adaptive immune activation. Managing in vivo biological responses is a complex challenge that requires a multi-faceted approach, from preclinical model selection to clinical trial design (PMID: 35211641). Mitigating this risk begins with robust CMC and process development that yields a highly pure and potent vector.

Featured: Franklin Biolabs Vector Production Facilities

Our process development and manufacturing operations are housed within a >100,000 sq ft facility designed for advanced therapy programs. This infrastructure supports the 18-24 month timelines our teams target to get candidates to IND.

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For a comprehensive look at our capabilities, view the full facility tour [here]).