Improved rna in vitro transcription using dna beads

The RNA in vitro transcription composition of any one of the preceding claims, wherein the IVT buffer comprise a cap analogue, preferably a cap1 analogue.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the IVT buffer comprises an antioxidant at a concentration of less than 5 mM, preferably at 1 mM.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the IVT buffer comprises at least 5 units/ml RNA polymerase, optionally wherein the RNA polymerase is a T7 RNA polymerase.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the composition comprises 10 μg/ml to 100 μg/ml immobilized DNA templates, preferably 50 μg/ml to 70 g/ml immobilized DNA templates.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the composition is essentially free of non-immobilized DNA templates.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the composition comprises 1 mg/ml to 100 mg/ml DNA beads, preferably 20 mg/ml to 40 mg/ml DNA beads.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the bead surface comprises about 0.5 ng DNA/mm2 to about 10 ng DNA/mm2, preferably about 1 ng DNA/mm2 to about 5 ng DNA/mm2.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the DNA beads have a diameter in a range of 1 μm to 10 μm, preferably in a range of 4 μm to 5 μm.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the DNA beads comprise polysterene or a derivative of polysterene.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the DNA beads are magnetic DNA beads, preferably (super) paramagnetic DNA beads.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the DNA beads comprise a biotin-streptavidin immobilized DNA template.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the DNA beads have been produced by site specific immobilization of a biotinylated PCR amplified DNA template on a streptavidin functionalized bead or by site specific immobilization of a biotinylated linearized plasmid DNA template on a streptavidin functionalized bead.

The RNA in vitro transcription composition of any one of the preceding claims, wherein the IVT buffer is configured to reduce or prevent DNA bead agglomeration at RNA concentrations of more than 100 mg/L.

The RNA in vitro transcription composition of any one of the preceding claims, wherein upon incubation of the RNA in vitro transcription composition for at least 1 h, less than 5% of the DNA beads contained in the composition are agglomerated.

Use of an RNA in vitro transcription composition of any one of the preceding claims in a manufacturing process of RNA.

Use of an IVT buffer to reduce or prevent DNA bead agglomeration in an RNA manufacturing process, wherein the IVT buffer comprises less than 2 mM spermidine and Mg2+ to NTP in a molar ratio of not more than 1.4, and, optionally, less than 25 mM Mg2+

Use of a wash buffer comprising a chelating agent to prevent or reduce agglomeration of DNA beads in an RNA manufacturing process, wherein the wash buffer comprises at least one chelating agent preferably at a concentration ranging from 1 mM to 10 mM.