After the COVID-19 pandemic, several companies and organisations are actively developing, scaling up and optimising mRNA manufacturing processes to produce their vaccines and therapeutics. Herein, we evaluated tangential flow filtration (TFF) for high-recovery, and high-purity separation of mRNA from unreacted NTPs in the IVT reaction mixture. For the first time, the membrane fouling behaviour caused by mRNA and separation of nucleoside triphosphates (NTPs) was mathematically modelled. The mRNA membrane fouling model is necessary for optimising mRNA separation processes, designing a suitable strategy for membrane clean-up, estimating the end of production life and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ~19 g/m2, <2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach enables the purification of multiple RNA drug substance sequences for the prevention and treatment of a wide range of diseases.