Process intensification for lentiviral vector manufacturing using tangential flow depth filtration.

For gene therapies to become more accessible and affordable treatment options, process intensification is one possible strategy to increase the number of doses generated per batch of viral vector. Process intensification for lentiviral vector manufacturing can be achieved by enabling perfusion in the production bioreactor when applied in tandem with a stable producer cell line, allowing for significant expansion of cells and production of lentiviral vectors without the need for transfer plasmids. Tangential flow depth filtration was used to achieve an intensified lentiviral vector production by enabling perfusion to expand cell density and allow for continuous separation of lentiviral vectors from producer cells. Hollow-fiber depth filters made of polypropylene with 2- to 4-µm channels demonstrated high filter capacity, extended functional life, and efficient separation of lentiviral vectors from producer cells and debris when used for this intensified process. We anticipate that process intensification with tangential flow depth filtration at 200-L scale from a suspension culture can produce on the order of magnitude of 10,000 doses per batch of lentiviral vectors required for CAR T or TCR cell and gene therapy that would require approximately 2 × 109 transducing units per dose.

Microfluidic droplet liquid reactors for active pharmaceutical ingredient crystallization by diffusion controlled solvent extraction.

A novel method for crystallization utilizing solvent/antisolvent extraction in microfluidic droplet liquid reactors has been developed for rapid and low-cost screening of crystal polymorphism (i.e. molecular crystallographic arrangement or internal structure) and habit (i.e. crystallographic shape or external structure). The method involves a ternary solvent system consisting of a dispersed phase of two miscible fluids, one in which the active pharmaceutical ingredient (API) is soluble (solvent) and one in which the API is insoluble (antisolvent). The solvent/antisolvent dispersed phase is immiscible with a third continuous phase. Crystallization of an API, GSK1, was controlled within droplets by altering the rate of solvent extraction from droplets into the continuous phase, thereby decreasing API solubility. Crystal size, habit, and population per droplet were directly impacted by the solvent's rate of extraction. Single crystals were grown in individual droplets by slow extraction of solvent into the surrounding continuous phase, which occurs when crystal growth gradually reduces API concentration such that it is maintained within the metastable zone throughout extraction. Rapid extraction of solvent from droplets results in API concentration significantly exceeding its metastable limit, producing a greater number of crystal nuclei compared to slow extraction conditions. When holding constant solubilized API mass per droplet, crystal sizes were larger for slow extraction rates (l = 96.3, w = 16.6 µm) compared to fast extraction rates (l = 48.8, w = 9.5 µm) as a result of crystal growth occurring on fewer crystal nuclei per droplet. Crystal habit can be controlled by adjusting the solvent extraction rate and consequently the saturation, where minimal saturation resulted in a rhombohedral habit and comparatively higher saturation resulted in an acicular habit with a higher aspect ratio. Antisolvents were tested using two hydrophobic APIs demonstrating the method's capability for rapidly identifying favorable crystal morphologies for downstream manufacturability using miniscule amounts of API.