Potency is one of the most critical quality attributes for AAV vectors as it is related to the functionality of the product. This presentation will focus on the strategy for the development of the potency assay for SPVN06, a gene-independent treatment for retinitis pigmentosa.

Alignment on upstream and downstream processes and analytical methods from preclinical to GMP manufacturing reduces time to market and de-risks drug development. The use of standardized assays for the characterization of preclinical vectors provides critical insights on potency, identity, and purity, and strengths the value of PoC and IND-enabling studies performed with those vectors.

Viral vectors provide limited or no scope for viral reduction and elimination steps, meaning the control of viral adventitious agents is limited.  In this presentation, the need to consider a wholistic approach to the control of viral agents will be discussed, along with the types of methodology that might be employed, and why.

This paper presents a highly sensitive method, CE-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector, that uses fluorescent labeling to monitor various bioprocess steps and characterize the final product of different viral vectors. Sample purity and capsid protein content of several recombinant Adeno-Associated Viruses produced in different cell systems were analyzed for their viral protein VP1/VP3 ratio. Notably, this method was also successfully implemented for enveloped viruses such as Maraba virus, a chimeric related-virus and lentivirus, showing excellent resolution and sensitivity for measuring purity or vector characterization.

Quantification of vector genome titer by digital PCR is a modern analytical method in gene therapy. Using a single target for titer determination can provide a consistent concentration of the product, but it lacks important information such as data on the full-length transgene. Multiplex dPCR was used to quantify DNA for multiple targets of the vector genome. A quadruplex dPCR method was developed for simultaneous quantification of 4 transgene targets.

The production of viral vectors for gene therapy is focused on pure, safe, and efficacious products. Absence of impurities and presence of full vector genomes play a crucial role. We have addressed the problem of AAV vector genome integrity by long-read sequencing and an advanced dPCR multiplex approach. Both technologies provide useful information on genome integrity and presence of impurities, but dPCR can go a step further providing a quantitative result.

• Vector genome integrity towards becoming a critical quality attribute
• Nanopore long-read sequencing can help in characterization of AAV samples 
•  Advanced multiplex dPCR approaches can help in quantification of genome integrity?

The microtubule-binding protein tau is a key player in Alzheimer’s disease (AD) and frontotemporal dementia. Using Sangamo’s unique zinc finger protein (ZFP) platform it was recently demonstrated that tau down-regulation with AAV ZFP-transcription factors (ZFP-TFs) rescues neuronal damage around amyloid plaques in a mouse model of Alzheimer’s disease. We adopted a matrix approach to determine potency of our AAV ZFP-TF product based on the mechanism of action of our gene therapy product. Design and analytical development of a quantitative PCR-based assay to measure transgene-specific ZFP mRNA for early product characterization and release studies will be presented.

Analytical ultracentrifugation is the gold standard method for the characterization of AAV-based products because it has superior resolving power compared to other biophysical and biochemical methods. In addition, the sedimentation velocity AUC method reports on several attributes at once. Here, we discuss recent developments in AUC methodology which allow reduced sample consumption, higher throughput, and transfer of the method to a quality control environment.

• AUC reports on several attributes at once, therefore it saves time and effort.
• Recent developments enabled a doubling in throughput.
• The method is not restricted to a development environment and can be transferred to a QC setting. 

Distinguishing loaded from empty lipid nanoparticles (LNPs) is challenging, as their overall size and shape may not change proportionally with RNA loading. However, upon loading, their spectral signature and density vary significantly. Using these properties, we developed two analytical ultracentrifuge(AUC) methods that distinguish LNPs. First, density matching AUC, uses varying D2O:H2O ratios to discriminate empty vs loaded LNPs. Second, multi-wavelength AUC, characterizes LNPs based on hydrodynamic and spectral properties.