As parenterally administered drugs, gene therapy products must undergo the same rigorous safety testing required for protein therapeutics. This includes characterizing the stability of the viral vectors, like those derived from adeno-associated virus (AAV), used to deliver the gene of interest.
Subvisible particles in parenteral drug formulations are regulated by the FDA and their monitoring is required for USP compliance. Formation of subvisible aggregates is a key indicator of stability issues with protein drugs as well as for gene therapy vectors. However, assessing vector particle aggregates in the subvisible range is especially challenging where only limited sample is available.
Subvisible Particle Analysis Shouldn’t Require Milliliters of Precious Sample
A combination of dynamic light scattering (DLS) and size exclusion chromatography (SEC) is usually used to monitor particle formation since aggregates vary significantly in size. However, manufacturing complexities impact the availability of sample for formulation or quality testing, which limits the number and type of analytics that can be performed. Standard methods for subvisible particle measurements such as flow microscopy are impractical as they can consume up to 1 mL of sample.
The Aura™ and Horizon® systems use Backgrounded Membrane Imaging (BMI) technology, an automated form of the USP <788> method of Membrane Microscopy that enables particle counting, sizing, and characterization in low volume, high throughput format.
Simple and flexible workflow on the Aura system. As little as 5 µl sample is loaded into wells of a 96-well membrane plate. After vacuum filtration to capture particles on the membrane, the instrument images each well. Total run time is about 1 minute per sample.
Analysis with BMI only requires 5 μL of sample per test,
making detection and characterization of subvisible particles accessible for gene therapy analytics.
Measuring Only in the Sub-Micron Range May Not Give You the Whole Picture
DLS and SEC can miss aggregates in the subvisible range. Large aggregates can be altered or removed in SEC by the column separation mechanism. DLS becomes unreliable when samples become polydisperse or if larger aggregates are present.
mAb samples subjected to mechanical stress show low counts of soluble aggregates as measured by SEC. However, when aggregates in the subvisible range are measured by flow microscopy (FM), the same samples show high aggregate counts. Adapted from Southall, et al. “Particle analysis as a formulation development tool”, Amer. Pharm Rev. (2011).
In other words, subvisible particles can be present even when
smaller aggregates are not detected.
BMI Delivers AAV Aggregate Particle Images, Count, and Size Distribution Information
BMI provides highly resolved particle images, count, and size distribution data. A light scattering feature (SIMI) enables differentiation between protein-containing particles and extrinsic particles such as glass or metal.
Individual AAV aggregate particles > 5 μm, imaged with BMI.
Subvisible Particle Counts Are an Indicator of AAV Stability
Measurement of subvisible particles helps complete the stability picture – which can minimize risk and enable more informed decisions about viral vector formulations and quality assessment. You’ll be able to detect the effects of free-thaw cycles, ionic strength, and different additives to find optimal formulation and storage conditions for your gene therapy product.