Single-Pass VMF of mRNA

Single-Pass, Continuous Concentration of mRNA Using Vibro^® Membrane Filtration

Single-Pass VMF significantly enhances the performance of SPTFF in the downstream concentration and purification of mRNA constructs. It provides more than triple the sustainable flux rates reported for conventional membrane filtration, enabling much higher conversion rates and concentration factors and opening the doors for DSP intensification.

Intensified Downstream mRNA Processing With Vibro^®

Conventionally, single-pass tangential flow filtration of mRNA requires very low flux rates to allow stable operation. VMF, however, uses vibrations to mitigate fouling and can thus handle much higher flux rates, enabling efficient concentration and purification of mRNA in a compact design. This study shows that using < 0.1 m² of membrane area, processing 100 L of in vitro transcription (IVT) crude in 24 h is possible – demonstrating VMF’s huge potential for intensified, continuous processing.

> 3× Sustainable Flux Compared to Conventional TFF

In flux stepping experiments, VMF demonstrated:

• 4 ml/min feed flow: Sustainable flux of 65.1 LMH at a conversion rate of 95% / 20× concentration  for both mRNA constructs
• 5 ml/min feed flow, Fluc (2117 nucleotides): Sustainable flux of 73.7 LMH at a conversion rate of 85% / 6.67× concentration
• 5 ml/min feed flow, Covid-19 (4283 nucleotides)Sustainable flux of 65.1 LMH at a conversion rate of 76% / 4.17× concentration

This corresponds to more > 3× the sustainable flux rates conventional systems achieved under similar conditions and better or comparable conversion rates on less membrane area.

Stable, Long-Term Process Technology

Extended continuous runs (24 h) confirmed VMF’s robustness and high efficiency. At a feed flow of 4 ml/min and flux of 61.7 LMH, intentionally kept slightly below critical flux for long-term operation, VMF demonstrated:

• 90 % conversion / 10× concentration
• Stable operation at low TMP (125 kPa for Fluc / 375 kPa for Covid-19)
• No indication of protein degradation / aggregation

The stable TMP in the last 15 h indicates that much longer operation would have been possible.

Improved Purification

In addition to concentrating the mRNA, ultrafiltration also serves to reduce any impurities (e.g. RNA polymerase) in the IVT crude. VMF removed 63% of all proteins for Covid-19 and 88% for Fluc at a conversion rate of 90%, plus 100% of all residual nucleotides under all conditions, significantly reducing the burden on later chromatography steps.