Lyophilization of RNA

A method for lyophilizing a mRNA, wherein the method comprises the following steps:

• a) providing a liquid having a glass transition temperature comprising at least one mRNA and at least one lyoprotectant, wherein the glass transition temperature of the liquid is in a range from −15° C. to −50° C.;
• b) introducing the liquid provided into a freeze drying chamber of a freeze dryer;
• c) cooling the liquid to a freezing temperature, wherein the cooling is performed at a defined cooling rate in a range from 0.1° C./min to 2° C./min;
• d) freezing the liquid having the glass transition temperature at the freezing temperature in order to obtain a frozen liquid having the glass transition temperature, wherein the freezing temperature is in a range from 0.5° C. to 25° C. below the glass transition temperature of the liquid provided in step a);
• e) reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure;
• f) drying the frozen liquid obtained in step d) in order to obtain a lyophilized composition comprising the at least one single-stranded RNA and at least one lyoprotectant, wherein drying comprises heating the frozen liquid obtained in step d) to a drying temperature;
• and
• g) equilibrating the pressure in the freeze drying chamber to atmospheric pressure and removing the lyophilized composition obtained in step f) from the freeze drying chamber to provide a lyophilized mRNA composition,
• wherein the at least one mRNA comprises i) a 5′ cap;
• ii) at least one coding region;
• iii) a poly(A) sequence of about 50 to about 100 adenine nucleotides and wherein the at least one mRNA has a length of from 200 to 15,000 nucleotides.

A method for lyophilizing a mRNA composition, wherein the method comprises the following steps:

• a) providing a liquid having a glass transition temperature comprising at least one mRNA and at least one lyoprotectant, wherein said at least one mRNA is in complex with at least one cationic or polycationic compound to form a nanoparticle, wherein the glass transition temperature of the liquid is in a range from −15° C. to −50° C.;
• b) introducing the liquid provided into a freeze drying chamber of a freeze dryer;
• c) cooling the liquid to a freezing temperature, wherein the cooling is performed at a defined cooling rate in a range from 0.1° C./min to 2° C./min;
• d) freezing the liquid having the glass transition temperature at the freezing temperature in order to obtain a frozen liquid having the glass transition temperature, wherein the freezing temperature is in a range from 0.5° C. to 25° C. below the glass transition temperature of the liquid provided in step a);
• e) reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure;
• f) drying the frozen liquid obtained in step d) in order to obtain a lyophilized composition comprising the at least one single-stranded RNA and at least one lyoprotectant, wherein drying comprises heating the frozen liquid obtained in step d) to a drying temperature;
• and
• g) equilibrating the pressure in the freeze drying chamber to atmospheric pressure and removing the lyophilized composition obtained in step f) from the freeze drying chamber to provide a lyophilized mRNA composition,
• wherein the at least one mRNA comprises i) a 5′ cap;
• ii) at least one coding region;
• iii) a poly(A) sequence of about 50 to about 100 adenine nucleotides and wherein the at least one mRNA has a length of from 200 to 15,000 nucleotides.