Rapid, Regioconvergent, Solvent-Free Alkene Hydrosilylation with a Cobalt Catalyst.

Alkene hydrosilylation is typically performed with Pt catalysts, but inexpensive base-metal catalysts would be preferred. We report a Co catalyst for anti-Markovnikov alkene hydrosilylation that can be used without added solvent at low temperatures with low loadings, and can be generated in situ from an air-stable precursor that is simple to synthesize from low-cost, commercially available materials. In addition, a mixture of Co catalysts performs a tandem catalytic alkene isomerization/hydrosilylation reaction that converts multiple isomers of hexene to the same terminal product. This regioconvergent reaction uses isomerization as a benefit rather than a hindrance.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up.

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

Novel anion exchange membrane chromatography method for the separation of empty and full adeno-associated virus.

A challenge in the production of recombinant Adeno-Associated Virus (AAV) for gene therapies is the presence of capsids that lack the required gene of interest. The impact of these empty vectors in therapies is not fully understood, however the ability to control the ratio of empty to full particles, which contain the genetic payload, is a necessary step in the purification of these viruses. In this study, a novel anion exchange chromatography elution method for enrichment of full AAV particles is demonstrated. A step gradient with small conductivity increases of around 1 mS cm-1 provides more efficient separation of empty and full AAV serotype 5 across membrane media as compared to conventional linear gradient method. The use of this approach in optimizing a simpler method for manufacturing processes and scalability to a larger chromatographic volume is explored. With this approach, the authors achieved greater than 4-fold enrichment of full capsids, to give a total of ≈50%-60% full capsids, using a 25 mM Bis-Tris Propane pH 9.0 buffer system with NaCl as the eluting salt. Results suggest that this elution method can be implemented into a scalable process and can provide insight into development of elution methods for other AAV serotypes.