Micropatterned charge heterogeneities via vapor deposition of aminosilanes.

Aminosilanes are routinely employed for charge reversal or to create coupling layers on oxide surfaces. We present a chemical vapor deposition method to pattern mica surfaces with regions of high-quality aminosilane (3-aminopropyltriethoxysilane, APTES) monolayers. The approach relies on the vapor deposition of an aminosilane through a patterned array of through-holes in a PDMS (poly(dimethylsiloxane)) membrane that acts as a mask. In aqueous solutions the surfaces have regular patterns of charge heterogeneities with minimal topographical variations over large areas. This versatile dry lift-off deposition method alleviates issues with multilayer formation and can be used to create charge patterns on curved surfaces. We identify the necessary steps to achieve high quality monolayers and charge reversal of the underlying mica surface: (1) hexane extraction to remove unreacted PDMS oligomers from the membrane that would otherwise deposit on and contaminate the substrate, (2) oxygen plasma treatment of the top of the membrane surfaces to generate a barrier layer that blocks APTES transport through the PDMS, and (3) low of the vapor pressure of APTES during deposition to minimize APTES condensation at the mica-membrane-vapor contact lines and to prevent multilayer formation. Under these conditions, AFM imaging shows that the monolayers have a height of 0.9 ± 0.2 nm with an increase in height up to 3 nm at the mica-membrane-vapor contact lines. Fluorescence imaging demonstrates pattern fidelity on both flat and curved surfaces, for feature sizes that vary between 6.5 and 40 µm. We verify charge reversal by measuring the double layer forces between a homogeneous (unpatterned) APTES monolayers and a mica surface in aqueous solution, and we characterize the surface potential of APTES monolayers by measuring the double-layer forces between identical APTES surfaces. We obtain a surface potential of +110 ± 6 mV at pH 4.0.

Separating Empty and Full Recombinant Adeno-Associated Virus Particles Using Isocratic Anion Exchange Chromatography.

The development of recombinant adeno-associated virus (rAAV) gene therapies is becoming an increasing priority in the biotherapeutic landscape. One of the challenges associated with the production of rAAV is the formation of empty AAV particles that do not contain a therapeutic gene. The concerns about the impact of empty particles on clinical safety and rAAV-mediated gene expression have necessitated the development of purification processes to remove these species. The development of a robust and scalable purification process to separate empty and full AAV particles at large scale remains a challenge. In this study, a novel anion exchange chromatography process based on isocratic wash and elution steps to enrich full rAAV2 particles is presented. An operating design space is identified to ensure the robustness of the process. The isocratic chromatography provides several advantages over the traditional shallow linear gradient elution, including lower buffer consumption, smaller intermediate pool volumes, and more robust manufacturing.

Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development.

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability with both the transmission electron microscopy and analytical ultracentrifugation methods used in empty-to-full capsid ratio quantification, while providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes/mL.