Overcoming Manufacturing Challenges For Rapid Development Of Genetic Nanomedicines From Discovery To Scale-Up

M. Assadian, A. Armstead, J. Singh, K. Ou, A. Thomas, S.M. Garg, S. Ip, M. Ma, S. Sidhu, T.J. Leaver, A.W. Wild, R.J. Taylor, R. Lockard, E.C. Ramsay
Precision NanoSystems Inc,
Canada

Keywords: manufacturing, rapid development, genetic nanomedicine, robust, in vivo, scale-up, large-scale, disposable

Summary:

Nucleic acid therapies permit access to previously undruggable pathways and allow manipulation of cellular machinery to produce target proteins including antigens for vaccines. To overcome challenges of gene delivery, scientists are optimizing nanoparticles for delivery by fine-tuning their size, composition and surface properties. Although these efforts have yielded substantial results in the laboratory to date, a significant need exists for robust manufacturing technologies to transit these discoveries from lab to clinic. In this context, we present a microfluidic based NanoAssemblrTM platform for production of mL to L of nanoparticles that retains consistent quality, efficacy and safety profiles, at scale, throughout the development process. Factor VII siRNA lipid nanoparticles (LNPs) were prepared on the NanoAssemblrTM Benchtop (Precision NanoSystems, Inc., Vancouver, Canada) and formulation parameters such as concentrations, flow rate ratio and total flow rate were optimized. These optimized process parameters were transferred onto the NanoAssemblrTM Blazeā„¢ and 8X Scale-up System to scale this formulation to 100 mL and 1000 mL respectively. Physico-chemical properties and in vivo activity were measured for particles produced by each instrument to test consistency across the platform. Particle composition was determined by HPLC. Particle size and polydispersity was analyzed using dynamic light scattering and RNA encapsulation efficiency was determined from standard RiboGreen based assay. Finally, in vivo activity was tested by administering LNPs in wild-type mice by i.v. injection and measuring serum Factor VII levels. We obtained LNP particles of about 60 nm (PDI 95% on the NanoAssemblrTM Benchtop. No differences were observed in physicochemical properties of these particles when scaled-up by 10x on Blaze (100 mL) or by 100x on 8X Scale-up System (1000 mL). The particles exhibited consistent lipid composition and N/P ratio within the target specifications. In addition, the particles manufactured across the microfluidic platform showed a similar dose-dependent gene knockdown, achieving > 90% reduction in protein levels at a dose of 1 mg/kg. These studies demonstrated that the NanoAssemblrTM platform provides seamless scale-up and can produce large-scale volumes of lipid nanoparticles with consistent results. The 8X scale-up system can prepare up to 25 L of product under 4.5 hours at 96 mL/min and incorporates a disposable fluid path that eliminates the need for costly and time consuming cleaning validation.