Following the addition of cryo-transmission electron microscopy (cryoTEM) to the updated AAV8 reference standard certificates earlier this year, the U.S. Pharmacopeia (USP) has released a new application note, Strengthening USP AAV Reference Standards with cryoTEM characterization, which presents the cryoTEM data behind its AAV8 Empty and Full Capsid reference standards.
By integrating cryoTEM into the characterization panel, USP further strengthens confidence in the accuracy, reliability, and applicability of its AAV reference standards.
— USP application note, Strengthening USP AAV Reference Standards with cryoTEM characterization
The note details the cryoTEM characterization of USP’s AAV8 (Empty Capsids, Cat. No. 1000301) and AAV8 (Full Capsids, Cat. No. 1000302) reference standards.
In a multi-laboratory study, cryoTEM measured the AAV8 Full Capsid standard at 92% full, 3% partially filled and 5% empty capsids. It measured the Empty Capsid standard at 98% empty and 1% full. These values agree with the five orthogonal methods in the testing panel: AUC, SEC-MALS, CDMS, mass photometry and UV spectroscopy.
As highlighted in the application note, a key advantage of cryoTEM is that it allows for direct visualization of individual capsids, so empty, full and partially filled populations can each be quantified.
Why this matters for gene therapy developers
Empty/full ratio is a critical quality attribute for AAV products, but there is still no single universally used method, which makes method development, validation and bridging studies difficult. With cryoTEM now characterized on USP’s AAV8 standards, developers can qualify and benchmark a cryoTEM method against a pharmacopeial reference, alongside the five other orthogonal techniques. Because cryoTEM gives a direct count of individual particles, it also resolves partially filled capsids, which bulk methods may average out or miss.
CryoTEM for AAV characterization at QuTEM
At QuTEM, cryoTEM is a validated, routine analytical method rather than a research tool. Our automated imaging and analysis workflows classify individual AAV capsids as empty, partially filled or full, and also quantify aggregation in the same sample. We offer GMP-certified cryoTEM services that support everything from early development through QC and release testing.
Learn more about QuTEM’s cryoTEM analysis of AAV capsids.
