Qualification of raw materials used in the manufacturing of viral vectors requires the use of risk assessment strategies to categorize the critical components of a manufacturing process. In addition to cell culture supplements, excipients and other formulation components must meet the required quality to ensure consistency in manufacturing, quality, and safety.

Because there are limited options to apply viral reduction or elimination steps in the manufacture of viral vectors, viral control becomes more important. As a consequence, viral testing likely needs to be more comprehensive. Multiple orthogonal methods increase the chances of detection and examples of viral contamination events will be presented.

Potency is one of the most important critical quality attributes for gene therapy products but also the least well understood. This is primarily due to the complex mechanism of action which is inherent to the use of viral vectors for therapeutic gene delivery. Here we describe our approach to developing a potency assay matrix to satisfy the regulatory requirements for a clinical AAV gene editing product.

Part of the success of a gene therapy program is a robust manufacturing process that results in high productivity and highly functional vectors. Developing platform manufacturing processes and platform analytical assays is a CMC strategy to save time and resources. Alcyone will share updates on our efforts to develop a robust platform upstream process as well as a platform in vitro potency assay.

The baculovirus expression vector system (BEVS) to produce gene therapy vectors involves infecting insect cells with a recombinant baculovirus expressing a gene of interest (GOI).  A sufficient amount of virus is needed for infection and expression of the GOI, and hence, an accurate titer would need to be measured.  Presently, we developed a method using droplet digital PCR that offers better precision and accuracy without the reliance on a standard curve.  

To develop a liquid formulation for AAV we looked beyond the standard analytics and assessed colloidal behavior. Examination of the colloidal state using novel analytical technologies for subvisible particles and SLS/DLS complemented existing datasets supporting 2-8°C stability. We use these biophysical analytics to support the liquid formulation development for AAV products. This work confirms a stable AAV liquid formulation, supporting improved logistics and storage conditions for the gene therapy field.

All rAAV products contain varying quantities of non-vector DNA derived from host cells and plasmids used in manufacturing process. The presence of these residual-impurity DNA may impact the safety of gene therapy products. In this study, DNA copy number and length of six residual impurity genes, including HEK293 genomic and plasmid DNA, were evaluated in twenty-one DS/DP lots by dual-color ddPCR assay. Study showed that all residual DNA amounts were low and close on a lot-to-lot basis, indicating a consistent and effective residual DNA removal process. 

Removing and characterizing empty and partial -genome-containing capsids are critical for safety and efficacy of rAAV products. Common characterization tools, such as AUC and TEM, are complicated and labor intensive, and not ideal for routine testing. Here we present different orthogonal methods in characterizing rAAV viral vectors genome. The methods are sensitive in detecting viral vector genome content, and are great additional characterization tools.

Within early-stage development of gene therapeutics, there is a need to perform low volume subvisible particle imaging, counting, sizing and identification to characterize for product stability and impurities. Aura GT is the only tool that rapidly and accurately measures physical stability of viral vector samples and serotypes with just 5µL. Assess for AAV capsid integrity, DNA leakage and their impact on particle formation from early-stage development through drug commercialization.  

Identifying potential critical quality attributes of AAV gene therapy products is challenging due to their inherent complexity and insufficient understanding of the manufacturing process. This presentation will focus on unique properties of different AAV serotypes subjected to forced degradation conditions. For this purpose, various analytical methods and orthogonal approaches were used to evaluate the impact, including chromatography, electrophoresis, LC-MS, molecular biology methods, and effects on potency.

Getting almost any information on AAVs takes too much sample and time. But with Stunner’s AAV Quant assay you can get the titer, empty/full ratio, and aggregation info you need to make your next in-process decision from 2 µL of sample and in less than a minute. When it’s time to pressure test the stability of your capsids, Uncle can take a full look at which capsids and conditions are the most stable by tracking DNA ejection from capsids, and protein unfolding and aggregation.

The quantitation and control of Host Cell Proteins (HCPs) in gene therapy products face significant challenges as manufacturing processes not as well defined as those for traditional biologics, and upstream and downstream processes used by various manufacturers may differ widely. We will discuss a case study of HCP analysis for an adeno-associated virus (AAV)-based gene therapy product, also considerations for choosing specific approaches during different clinical development stages.

Establishing a robust AAV vector genome titering method early in product development is critical.  The vector genome titer method is commonly utilized to make manufacturing, stability, and even clinical dosing decisions. This presentation will discuss the measures taken to develop and optimize a gene-of-interest vector genome titer method using ddPCR.

Lentiviral vectors are utilized in cell and gene therapy programs for both ex vivo and in vivo delivery. Tracking the quantity, size distribution, charge, and protein content of viral particles during production, purification, and formulation development can aid developers in making process decisions. In this presentation, we will discuss the use of DLS, NTA, Flow Virometry, and other methods to characterize lentiviral particles.

Data are presented demonstrating the utility of SV-AUC with case studies covering characterization of AAV vectors and DNA drug substance. For an AAV vector with increased HMW by SEC we show presents as a low molecular weight peak on SV-AUC, with a higher DNA content than the full capsid. Use of pseudo-absorbance and software tools to increase throughput and ultra-low loading concentrations to reduce sample requirements are also discussed.