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.

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. Graphical abstract.

Innovating a Nonconventional Yeast Platform for Producing Shikimate as the Building Block of High-Value Aromatics.

The shikimate pathway serves an essential role in many organisms. Not only are the three aromatic amino acids synthesized through this pathway, but many secondary metabolites also derive from it. Decades of effort have been invested into engineering Saccharomyces cerevisiae to produce shikimate and its derivatives. In addition to the ability to express cytochrome P450, S. cerevisiae is generally recognized as safe for producing compounds with nutraceutical and pharmaceutical applications. However, the intrinsically complicated regulations involved in central metabolism and the low precursor availability in S. cerevisiae has limited production levels. Here we report the development of a new platform based on Scheffersomyces stipitis, whose superior xylose utilization efficiency makes it particularly suited to produce the shikimate group of compounds. Shikimate was produced at 3.11 g/L, representing the highest level among shikimate pathway products in yeasts. Our work represents a new exploration toward expanding the current collection of microbial factories.