Proven Intelligence Accelerating Next-Generation Therapies.

Executive Summary

Adeno-associated virus (AAV) vector aggregation during downstream processing presents a significant obstacle to achieving the purity, potency, and safety profiles required for clinical progression. Aggregates can trigger unwanted immune responses and reduce therapeutic efficacy, jeopardizing IND and Investigational Medicinal Product Dossier (IMPD) submissions. Effective mitigation requires a tailored process development strategy focused on optimizing buffer conditions, chromatography methods, and formulation excipients specific to the vector serotype and payload. This approach moves beyond a platform methodology to directly address the biophysical drivers of aggregation, ensuring a stable and monomeric final product.

The Challenge of Vector Integrity in Downstream Purification

High-purity AAV vector preparations are a prerequisite for successful gene therapy programs. The presence of aggregates, even at low levels, can compromise data from IND-enabling toxicology studies and create significant delays. The biophysical properties of AAV capsids are highly dependent on their specific serotype, a principle observed both in transduction patterns and manufacturing behavior (PMID: 18714307). A purification process optimized for AAV8 may not be suitable for AAV9 or a novel engineered capsid, necessitating a data-driven approach to process development.

The objective is to deliver a clinical candidate with a clean profile and persistent transgene expression, which requires meticulous control over the final product’s key quality attributes (PMID: 34258325).

Learn more about initiating successful AAV vector programs by viewing the full webinar at: https://franklinbio.com/resource/vector-ready-where-aav-projects-begin-and-how-they-succeed/

Root Causes and Mitigation Strategies

Controlling aggregation requires a deep understanding of the vector’s behavior throughout the downstream purification train. The transition from cell lysate to a highly purified bulk substance exposes the vector to a range of conditions that can induce instability.

Key areas for process optimization include:

• Buffer & Excipient Screening: Systematic screening of pH, salt concentration (e.g., NaCl, KCl), and stabilizing excipients (e.g., surfactants, sugars, amino acids) is performed to identify conditions that maximize the colloidal stability of the target serotype.

• Chromatography Resin Selection: Affinity, ion-exchange, and multimodal chromatography steps must be optimized. The choice of resin, gradient slope, and flow rate can either prevent or promote aggregation.

• Tangential Flow Filtration (TFF): Parameters for concentration and diafiltration steps, such as transmembrane pressure and shear rates, are carefully controlled to minimize stress on the capsid structure.

“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 integral to our AAV-vector based gene therapy candidate development and we hope to continue the partnership for years to come.”
— Biotech Partner

Analytical Characterization for GxP-Compliant Manufacturing

Robust analytical methods are required for monitoring and controlling aggregation. A phase-appropriate approach ensures that assays are qualified and validated to meet GxP requirements as a program advances toward clinical manufacturing. Our >100,000 sq ft facility is equipped to develop and execute these complex assays, supporting programs on an 18-24 month timeline to IND. This analytical framework provides the data package necessary for regulatory submissions, building on a history that includes a 100% successful IND rate for programs initiated since 2019, prior to the Franklin Biolabs brand launch in 2024.