Demonstration of protein capture and separation using three‐dimensional printed anion exchange monoliths fabricated in one‐step

Abstract

Three-dimensional printing applications in separation science are currently limited by the lack of materials compatible with chromatographic operations and three-dimensional printing technologies. In this work, we propose a new material for Digital Light Processing printing to fabricate functional ion exchange monoliths in a single step. Through copolymerization of the bifunctional monomer [2-(acryloyloxy)ethyl] trimethylammonium chloride, monolithic structures with quaternary amine ligands were fabricated. The novel formulation was optimized in terms of protein binding and recovery, microporous structure, and its swelling susceptibility by increasing its cross-link density and employing cyclohexanol and dodecanol as pore forming agents. In static conditions, the material demonstrated a maximum binding capacity of 104.2 ± 10.6 mg/mL for bovine serum albumin, in line with commercially available materials. Its anion exchange behavior was validated by separating bovine serum albumin and myoglobin on a monolithic bed with Schoen gyroid morphology. The same column geometry was tested for the purification of C-phycocyanin from clarified as well as cell-laden Arthrospira platensis feedstocks. This represents the first demonstration of one-step printed stationary phases to capture proteins directly from solid-laden feedstocks. We believe that the material presented here represents a significant improvement towards implementation of three-dimensional printed chromatography media in the field of separation science.

Flexible material formulations for 3D printing of ordered porous beds with applications in bioprocess engineering

“…Also, approximately 90% of the proteins adsorbed were recovered during elution for both materials, demonstrating that the strength of the electrostatic interactions can be appropriately adjusted to enable bind and elute operation of the ion exchangers. The broad elution peaks confirm mass transfer limitations from the bulk of the 3D printed media to the liquid phase during elution (Simon et al 2020), an open challenge requiring improvements in the resolution achievable with current 3D printers. Appropriate process development also needs to be carried out to determine the optimal operating conditions to maximize the overall yield of the separation step.…”

Flexible Material Formulations for 3D Printing of Porous Beds with Applications in Bioprocess Engineering

“…Maximum binding capacities of 104.2 ± 10.6 mg of BSA per mL of AETAC-based support, and 108.1 ± 25.9 mg of LYS per mL of CEA-based material were recorded (Figure 2a and 2b), about 5 fold higher than for commercial monoliths (Hahn et al, 2002) and chromatographic membranes (Boi et al, 2020) and in line or above standard chromatographic resins (Staby et al, 2005). Testing in dynamic conditions was carried out using Schoen gyroid columns (Figure 1f-h) by loading BSA and myoglobin (MYO) onto the AETAC material (Figure 2c, Simon et al, 2020) and BSA and LYS on the CEA material (Figure 2d). The chromatograms reveal elution patterns in line with the electrostatic interactions established at the buffer's pH, thus con rming the availability of the surface quaternary amine and carboxyl groups to establish appropriate electrostatic interactions with the protein models.…”

Flexible material formulations for 3D printing of porous beds with applications in bioprocess engineering

“…Figure 2. Adsorption isotherms of (a) bovine serum albumin (BSA) on anion exchangers (based on AETAC monomer, adapted from Simon et al, 2020), and (b) lysozyme (LYS) on cation exchangers (based on CEA monomer). Ligand densities of 0 (control), 0.57, 1.14, 1.73, 2.33 mmol/mL and of 0 (control), 0.77, 1.56, 2.36, 3.23 mmol/mL were tested for the anion and cation exchanger, respectively.…”

“…Maximum binding capacities of 104.2 ± 10.6 mg of BSA per mL of AETAC-based support, and 108.1 ± 25.9 mg of LYS per mL of CEA-based material were recorded (Figure 2a and 2b), about 5 fold higher than for commercial monoliths (Hahn et al, 2002) and chromatographic membranes (Boi et al, 2020) and in line or above standard chromatographic resins (Staby et al, 2005). Testing in dynamic conditions was carried out using Schoen gyroid columns ( Figure 1f-h) by loading BSA and myoglobin (MYO) onto the AETAC material ( Figure 2c, Simon et al, 2020) and BSA and LYS on the CEA material ( Figure 2d). The chromatograms reveal elution pattern in line with the electrostatic interactions established at the buffer's pH, thus confirming the availability of the surface quaternary amine and carboxyl groups to establish appropriate electrostatic interactions with the protein models.…”