Performance of various 3D-printed monolith geometries as an alternative to expanded bed adsorption for protein purification.

Here, we compare the performance of various three-dimensional-printed Monolith Adsorption (PMA) columns designed from a triply periodic minimal surface geometry, the Schoen gyroid. The structures examined had designed hydraulic diameters between 203 and 458 µm and voidages of 40%-60%. We compare column efficiency, porosity, static binding capacity and dynamic binding capacity for various load volumes and flow rates. The results show that all structures allowed efficient passage of yeast cells (>97%) over a wide range of interstitial velocities (191 to 1911 cm/h) while maintaining a low pressure drop (<0.1 MPa). The structure with a voidage of 40% and a hydraulic diameter of 203 µm showed the best performance in all aspects evaluated. Bovine serum albumin (BSA) recoveries for all structures (27%-91% when the loaded volume was 180 mL) were significantly affected by hydraulic diameter, mean channel wall thickness, velocity and voidage. Moreover, biomass addition resulted in a decrease in BSA recovery, which became more obvious at high velocities. However, this did not lead to a dramatic reduction in saturated binding capacity, significant changes in axial dispersion, or blockage of channels and could be compensated for by recirculation of the feed, even at high velocity. PMA thus potentially provides an appealing alternative to Expanded Bed Adsorption, retaining the latter's advantages, while eliminating fluidization issues and minimizing both processing time and buffer consumption.

Printed monolith adsorption as an alternative to expanded bed adsorption for purifying M13 bacteriophage.

An ordered 3D printed chromatography stationary phase was used to purify M13 bacteriophage (M13) directly from crude cell culture. This new approach, which offers the same advantages as expanded bed adsorption (EBA) with regard to tolerating solids-laden feed streams but without the corresponding issues associated with fluidized bed stability that affect the latter, can be described as "printed monolith adsorption (PMA)". PMA columns (5, 10 and 15 cm length by 1 cm diameter) were made via a wax templating method from cross-linked cellulose hydrogel and functionalized with a quaternary amine ligand. The recovery of M13 was found to be strongly linked to load flow rate, with the highest recovery 89.7% ± 6% for 1.4 × 1011 pfu/mL of resin occurring at 76 cm/h with a 10 cm column length. A recovery of 87.7% ± 5% for 1.49 × 1011 pfu/mL of media was achieved with a 15 cm column length under conditions comparable to a reported EBA process. The PMA process was completed three times faster than EBA because PMA flow rates can readily be adjusted during operation, with high flow rates and low back pressure, which is unique to the ordered monolithic media geometry used. Equilibration, wash, and cleaning steps were carried out at high flow rates (611 cm/h), minimizing process time and were limited only by the volumetric flow rate capacity of the pumps used, rather than column back pressure (<0.1 MPa at 611 cm/hr). Initial capture of M13 appears to occur on the surface of the monolith solid phase (i.e. the mobile phase channel walls) and subsequently, at a slower rate, within the internal pores of the solid phase media. The difference in binding rate between these two sites is likely caused by slow pore diffusion of the large M13 particles into the pores, with similar slow diffusion out of the pores resulting in tailing of the elution peak. The results indicate that PMA is a promising technology for the efficient purification of viruses directly from crude cell culture.

3D Printing of Gelled and Cross-Linked Cellulose Solutions, an Exploration of Printing Parameters and Gel Behaviour.

In recent years, 3D printing has enabled the fabrication of complex designs, with low-cost customization and an ever-increasing range of materials. Yet, these abilities have also created an enormous challenge in optimizing a large number of process parameters, especially in the 3D printing of swellable, non-toxic, biocompatible and biodegradable materials, so-called bio-ink materials. In this work, a cellulose gel, made out of aqueous solutions of cellulose, sodium hydroxide and urea, was used to demonstrate the formation of a shear thinning bio-ink material necessary for an extrusion-based 3D printing. After analysing the shear thinning behaviour of the cellulose gel by rheometry a Design of Experiments (DoE) was applied to optimize the 3D bioprinter settings for printing the cellulose gel. The optimum print settings were then used to print a human ear shape, without a need for support material. The results clearly indicate that the found settings allow the printing of more complex parts with high-fidelity. This confirms the capability of the applied method to 3D print a newly developed bio-ink material.

Analysis of the Effect of Processing Conditions on Physical Properties of Thermally Set Cellulose Hydrogels.

Cellulose-based hydrogels were prepared by dissolving cellulose in aqueous sodium hydroxide (NaOH)/urea solutions and casting it into complex shapes by the use of sacrificial templates followed by thermal gelation of the solution. Both the gelling temperatures used (40⁻80 °C), as well as the method of heating by either induction in the form of a water bath and hot press or radiation by microwaves could be shown to have a significant effect on the compressive strength and modulus of the prepared hydrogels. Lower gelling temperatures and shorter heating times were found to result in stronger and stiffer gels. Both the effect of physical cross-linking via the introduction of additional non-dissolving cellulosic material, as well as chemical cross-linking by the introduction of epichlorohydrin (ECH), and a combination of both applied during the gelation process could be shown to affect both the mechanical properties and microstructure of the hydrogels. The added cellulose acts as a physical-cross-linking agent strengthening the hydrogen-bond network as well as a reinforcing phase improving the mechanical properties. However, chemical cross-linking of an unreinforced gel leads to unfavourable bonding and cellulose network formation, resulting in drastically increased pore sizes and reduced mechanical properties. In both cases, chemical cross-linking leads to larger internal pores.