As steadily increasing numbers of mRNA vaccines and therapeutics advance through clinical trials and commercialization, the biopharmaceutical industry is pressed to find ways to make their production increasingly cost-effective. But, as scientists look to model every aspect of mRNA processing, publicly available data on tangential flow filtration (TFF) is noticeably absent.
With this lack of information, “It is challenging to develop mRNA purification processes,” Zoltán Kis, PhD, senior lecturer, University of Sheffield, told GEN. “It is difficult to know the optimal membrane types and process parameters. Moreover, it is not clear what success looks like in terms of impurity removal, losses, and membrane fouling.”
Although those details could be determined experimentally, “mRNA is expensive, therefore these experiments can be costly,” Kis said.
Kis and colleagues at the University of Sheffield, are providing some of that data by studying the ability of TFF to separate high levels of high-purity mRNA from unreacted nucleoside triphosphates in the in vitro transcription reaction mixture. They aimed to maintain mRNA critical quality attributes (CQAs) throughout the separation process and to quantify membrane fouling, thus minimizing a significant product loss.
Transmembrane pressure and membrane fouling were of particular interest in achieving high levels of purified mRNA. Writing in a recent paper, first author Ehsan Nourafkan, PhD, research associate in mRNA vaccine and therapeutics production, and colleagues validated the fouling model and described the adsorption of mRNA on a filtration membrane.
In this experiment, they ran the TFF at a capacity load of ∼19 g/m2, less than 2.5 psi trans-membrane pressure, and feed flux of 300 L/m2/h (LMH), corresponding to a shear rate of 1594 s−1.
Nourafkan, Kis, and colleagues report that a sequential TFF concentration and diafiltration process resulted in no detectable mRNA degradation, complete removal of unreacted nucleoside triphosphates, and a mRNA loss of 30%. They managed to further reduce it to only 3% by washing the TFF membrane using two consecutive wash steps.
“This approach also enables the purification of multiple mRNA drug substance sequences for the treatment of a wide range of different diseases” Kis added.
“For mRNA concentration using the TFF permeate fluxes of less than 40 LMH and less than 1 mg/mL mRNA, concentrations are preferred for achieving stable transmembrane pressures and for minimizing membrane fouling,” Kis said. For diafiltration, higher rates of permeate flux sped TFF filtration. Deviation from the model-predicted transmembrane pressure values could indicate fouling in the filtration process. The most significant factor affecting membrane fouling, they report, is the mRNA concentration in the feed stream.
Using the data gathered in these experiments to populate appropriate models—such as the Hermia model, which describes membrane fouling—allows Kis’ team to accurately predict transmembrane pressure over time. It also allows them to better estimate when the TFF filter should be replaced, they point out, “based on the difference between the membrane’s intrinsic resistance and the resistance observed after membrane washing.”