ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis—A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing

Abstract

The genome content of adeno-associated virus (AAV) vectors is critical to the safety and potency of AAV-based gene therapy products. Empty capsids are considered a product-related impurity and a critical quality attribute (CQA) of the drug product, thus requiring characterization throughout the production process to demonstrate they are controlled to acceptable levels in the final drug product. Anion exchange chromatography has been used to achieve separation between empty and full capsids, but requires method development and gradient optimization for different serotypes and formulations. Here, we describe an alternative approach to quantitation that does not rely on achieving separation between empty and full capsids, but instead uses the well-established relationship between absorbance at UV A260/A280 and relation to DNA/protein content, in combination with anion-exchange chromatography to allow one to calculate the relative proportion of empty and full capsids in AAV samples from a single peak. We call this approach ACUVRA: Anion-exchange Chromatography UV-Ratio Analysis, and show the applicability of the method through a case study with recombinant AAV2 (rAAV2) process intermediates and drug substance. Method qualification and GMP validation in a quality control (QC) laboratory results show that ACUVRA is a fit-for-purpose method for process development support and characterization, while also being a QC-friendly option for GMP release testing at all stages of clinical development.

Analytical characterization of full, intermediate, and empty AAV capsids

“…Furthermore, therapeutic AAVs are dosed based on VG titer, meaning that while non-functional empty capsids contribute to the overall viral load they do not contribute to clinical efficacy. A common approach to reduce the overall viral load is the targeted removal of empty capsids from the final product which can be achieved using an optimized AAV manufacturing process [ 11 , 12 ]. Less is currently known about the impact of intermediate capsids on safety and efficacy of AAV products, and their presence in clinical preparations may pose similar risks as described for empty capsids especially if the capsids are identified to be non-functional.…”

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

“…AAV vector production has been widely described as an imperfect process, creating heterogenic vector products with a different range of empty-to-full capsids. Low-quality vector productions with increased empty or defective capsids can limit transduction and lead to even higher adverse immunogenic effects [ 21 , 22 , 23 , 24 ]. The impact of vector quality on dual-AAV vector systems can be even more preeminent, as it can contribute to limiting gene reconstitutions by promoting vector competition and inhibiting co-transduction.…”

A novel method for quantitation of AAV genome integrity using duplex digital PCR

“…Unwanted residual DNA from plasmids or host cells involved in the production process may also be encapsidated [1,3,4]. Current purification processes can efficiently separate empty from full capsids, however analytical methods that rely on separating capsid populations based on density (AUC, Cryo-EM, TEM), mass-to-charge ratios (CDMS), absorbance characteristics (A 260 /A 280 ), or mass (SEC-MALS), vary in their abilities to accurately detect partially filled capsids and cannot differentiate between aberrant versus intended genomes [1][2][3][4][5][6]. It is therefore critical to develop assays to thoroughly characterize rAAV encapsidated content and genome integrity.…”