Methods for preparing particles and related compositions

Abstract

Methods for preparing particles and related compositions are provided. In some embodiments, the particles include at least one polynucleotide (e.g., mRNA), and in certain embodiments, the particles may include at least one ionizable molecule (e.g., a lipid). A method for preparing a suspension including the particles may comprise one or more filtration steps. In some such embodiments, prior to or during filtration, one or more properties of the particles (e.g., surface charge) and/or one or more properties of the suspension (e.g., pH) may be altered. In some embodiments, altering one or more properties of the particles and/or suspension may improve yield, improve a characteristic of the resulting composition, and/or prevent or reduce certain problems, such as fouling during the filtration process.

1 - 44 . (canceled) 
     
     
         45 . A composition comprising:
 a plurality of particles comprising mRNA comprising greater than or equal to 100 nucleotides and less than or equal to 10,000 nucleotides in length and an ionizable molecule, wherein:   an average cross-sectional dimension of the particles in the composition is less than or equal to about 150 nm,   a coefficient of variation of a cross-sectional dimension of the particles in the composition is less than or equal to about 20%, and   a weight percentage of mRNA in the particles is greater than or equal to about 50% and less than or equal to about 99%, and   wherein the composition is formed by   (ia) changing a pH of a suspension comprising the plurality of particles from a first pH to a second pH, wherein the second pH is greater than the pKa of the ionizable molecule, wherein the pKa of the ionizable molecule is greater than or equal to about 6, or   (ib) changing an average zeta potential of a suspension comprising the plurality of particles, from a first zeta potential to a second zeta potential, wherein the second zeta potential is less than the first zeta potential, and subsequently,   (ii) filtering the suspension.   
     
     
         46 . The composition of  claim 45 , wherein the particles further comprise a molecule capable of reducing particle aggregation. 
     
     
         47 . The composition of  claim 46 , wherein the molecule capable of reducing particle aggregation is a PEG lipid or a PEG-modified lipid. 
     
     
         48 . The composition of  claim 45 , wherein the ionizable molecule comprises a nitrogen. 
     
     
         49 . The composition of  claim 45 , wherein the ionizable molecule is a cationic lipid. 
     
     
         50 . The composition of  claim 49 , wherein the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1.3]-dioxolane, dilinolcyl-methyl-4-dimethylaminobutyrate, and di((Z)-non-2-cn-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate. 
     
     
         51 . The composition of claim  1 , wherein the particle comprises a sterol molecule. 
     
     
         52 . The composition of  claim 51 , wherein the sterol molecule is a cholesterol molecule. 
     
     
         53 . The composition of  claim 45 , wherein the filtered suspension is formed by (ib) and the first zeta potential is greater than or equal to about 0 mV. 
     
     
         54 . The composition of  claim 45 , wherein the filtered suspension is formed by (ib) and the particles have a zeta potential of less than or equal to about −1 mV. 
     
     
         55 . The composition of  claim 45 , wherein the filtered suspension is formed by (ia) and the pH of the suspension prior to the changing step is less than or equal to about the pKa of the ionizable molecule. 
     
     
         56 . The composition of  claim 45 , wherein the filtered suspension is formed by (ia) and the pKa of ionizable molecule is between about 6 and 7. 
     
     
         57 . The composition as in  claim 45 , wherein the pKa of the ionizablc molecule is about 6.2. 
     
     
         58 . The composition of  claim 45 , wherein a coefficient of variation of a cross-sectional dimension of the particles in the filtered suspension is less than or equal to about 15%. 
     
     
         59 . The composition of  claim 45 , wherein the filtering step comprises passing at least a portion of the suspension thorough a porous substrate. 
     
     
         60 . The composition of  claim 45 , wherein the filtering step comprises passing the suspension through a filter having a mean pore size of less than or equal to about 0.2 microns after the tangential flow filtration. 
     
     
         61 . The composition of  claim 45 , wherein the permeate flux throughout the filtering step is less than or equal to about 120 L/m.sup.2h when the transmembrane pressure is between about 1 psi and about 20 psi, or between about 10 psi and about 15 psi. 
     
     
         62 . The composition of  claim 45 , wherein the filtering step comprises tangential flow filtration (TFF).