The downstream processing and purification of glycans has been challenging for biopharmaceutical manufacturers because of the intricate mixtures of sugars, nucleosides, and buffer molecules that are present after glycan synthesis. A continuous, single-pass double-membrane nanofiltration module developed by researchers at Germany’s Karlsruhe Institute of Technology (KIT) may solve that challenge.
Designed to purify enzymatically-synthesized glycan products, this nanofiltration module
“nearly doubled the saccharide recovery rate,” according to a recent paper. The product recovery rate was high, too, and product purities exceeded—in some cases—99%. Those results are comparable to running several sequential diafiltration cycles.
A team led by first author Ulrich Thiele, a doctoral student at KIT, and senior researcher Katharina Bleher, PhD, group leader, analytical chemistry at KIT, designed a 3D-printed nanofiltration membrane that featured a molecular weight cut-off between 300 and 500 Da. With that range, it separated the disaccharide lactose from the nucleoside uridine and, they suggested, may also separate larger oligosaccharides.
While examining the effect of the transversal diafiltration medium stream on product yield within the module, Bleher’s team reported that strong convection forces increased product accumulation on the membrane surface and the diffusion effect through the membrane, which ultimately decreased yields. To counter this, they developed a static mixer. This fluid-guiding element “increased dispersion, disrupting the concentration polarization layer on the membrane and thereby reducing diffusive effects through the membrane and improving product yields,” they noted. Buffer choice also affected product purity and yield.
“Generally…the module’s performance improves with the molecular weight of the…saccharide,” they reported. To their surprise, as they noted, “The charge of the HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer at pH 7.5 significantly hinders the separation effect between saccharides and nucleotides in the single-pass nanofiltration/diafiltration operation model…which was not observed in the conventional single pass tangential flow filtration experiments.”
Key scale-up considerations
When scaling up this filtration method, there is a real risk that high operating pressures will damage the membranes. Distributing the pressure more evenly mitigates that risk. To do that, the scientists used modules with large middle compartments and large membrane surface areas.
To reduce costs and downtime, they advised selecting inexpensive, easy-to-replace flat-sheet membranes rather than the more expensive cassette systems often used in ultrafiltration.
The 3D-printed modules they developed support a scale-out strategy in which filtration units run in parallel. “This modular setup not only lowers production and maintenance costs, but also reduces downtime,” they noted.
Next steps in this research may include optimizing the static mixer and fine-tuning operating parameters and membrane types to further increase yields, as well as investigating the influence of the buffer concentration on purification to support the separation of even larger glycans.