Compositions and methods for the manufacture of lipid nanoparticles

Abstract

The invention relates to methods, processes and apparatuses for the manufacture of lipid nanoparticles having a therapeutic payload.

1 . A method of preparing a formulation comprising lipid nanoparticles comprising an RNAi agent payload comprising;
 (a) mixing a first solution with a second solution in a mixing connector, wherein
 (i) said first solution comprises an ethanolic solution comprising one or more lipids and having a total lipid concentration of approximately 30 mg/mL, and 
 (ii) said second solution comprises citrate buffered aqueous solution comprising one or more RNAi agents and having an RNAi agent concentration of approximately 1 mg/mL and a pH of between 3 and 6, and 
   (b) diluting the mixture produced in (a) in a vessel containing a buffer solution thereby producing a formulation comprising lipid nanoparticles comprising an RNAi agent payload;   
       wherein the linear flow rate of said first solution into the mixing connector is approximately 606,267 cm/h and the linear flow rate of said second solution into the mixing connector is approximately 1,818,801 cm/h and the volume ratio of said first solution to said second solution is approximately 1:3. 
     
     
         2 . The method of  claim 1  wherein the buffer solution of (b) is phosphate buffered saline (PBS). 
     
     
         3 . The method of  claim 2 , wherein the vessel contains sufficient PBS to dilute the mixture resulting from step (a) by a factor of between 4 and 10 fold. 
     
     
         4 . The method of  claim 3 , wherein the factor is 5 fold and the final concentration of ethanol in the formulation is equal to or less than 5%. 
     
     
         5 . The method of  claim 1 , further comprising;
 (c) ultrafiltration of said formulation comprising;
 (i) concentration of said formulation such that the lipid nanoparticle concentration is increased by a factor of between 1 and 10 fold, 
 (ii) diafiltration of the concentrated formulation of (i) using at least 10 volume exchanges with buffer solution, wherein the ethanol concentration is reduced to less than 1%, and 
 (iii) concentration of the filtered formulation of (ii) to produce an RNAi agent concentration of between 2.5 and 3 mg/mL. 
   
     
     
         6 . The method of  claim 5 , further comprising;
 (iv) adjusting the RNAi agent concentration of the formulation of (iii) to a concentration of 2 mg/mL by the addition of PBS.   
     
     
         7 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the total lipid to RNAi agent w/w ratio is between 10:1 and 14:1 based on the total lipid concentration of said first solution and the RNAi agent concentration of said second solution as determined prior to mixing using HPLC. 
     
     
         11 . A system for the manufacture of a formulation comprising lipid nanoparticles comprising an RNAi agent payload comprising;
 (a) a first reservoir providing a first solution, wherein said first solution is an ethanolic solution comprising one or more lipids,   (b) a second reservoir providing a second solution, wherein said second solution is a buffered aqueous solution comprising one or more RNAi agents,   (c) a first pump, operably connected to said first reservoir and configured to regulate the flow of said first solution at a linear flow rate of between 303,133 and 909,400 cm/h,   (d) a second pump, operably connected to said second reservoir and configured to regulate the flow of said second solution at a linear flow rate of between 1,515,667 and 2,121,934 cm/h,   (e) a mixing connector comprising at least a first inlet, a second inlet and an outlet, wherein said first inlet receives flow from said first pump and said second inlet receives flow from said second pump,   (f) at least one heat exchanger operably connecting each of said first and said second pumps to said inlets of the mixing connector, respectively, and   (g) a vessel for receiving effluent from the outlet of said mixing connector.   
     
     
         12 . The system of  claim 11 , further comprising an ultrafiltration system configured to receive effluent from said vessel, said effluent comprising the lipid nanoparticle formulation. 
     
     
         13 . The system of  claim 12 , wherein the ultrafiltration system comprises a rotary lobe pump or a diaphragm pump which allows for lipid nanoparticle formulation retentate circulation and permeate transport across a membrane filter. 
     
     
         14 . (canceled) 
     
     
         15 . The system of  claim 13 , wherein the filter is a polyethersulphone membrane. 
     
     
         16 . The system of  claim 13 , wherein the transmembrane pressure across the membrane filter is between 5 and 15 psi. 
     
     
         17 . The system of  claim 13 , wherein the permeate flow rate is between 50 and 400, 60 and 300 or 100 and 200 liter/m 2 /h. 
     
     
         18 . The system of  claim 11 , further comprising;
 (g) at least one filter or filtration device operably engaged in front of each of said first and said second reservoirs.   
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The system of  claim 11 , wherein the planar angle between said first inlet and said second inlet is between 5 and 180 degrees. 
     
     
         22 . The system of  claim 21 , wherein the planar angle between said first and said second inlet is 120 degrees. 
     
     
         23 . The system of  claim 11 , wherein the mixing connector comprises 3, 4, 5, 6, 7 or 8 inlets, each configured to receive flow from either said first pump or said second pump. 
     
     
         24 . The system of  claim 23 , wherein the inlets of the mixing connector are positioned equidistant from one another about a central axis. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . A lipid nanoparticle formulation comprising an RNAi agent payload prepared by the process of  claim 1 . 
     
     
         28 . The method of  claim 1 , wherein the RNAi agent is selected from the group consisting of siRNA, dsRNA, miRNA, and nucleotide sequences encoding the same.