1. Introduction
In the last decade we have witnessed the booming of additive
manufacturing (AM, also 3D printing), including its related fabrication
technologies, materials, and applications. The biotechnology and
bioprocessing fields have been significantly influenced by AM, with
reports spanning upstream and downstream processing, including sorting
and selection of cell strains (Lin et al., 2016), bioreactors (Saha et
al., 2018), harvesting (Shakeel Syed et al., 2017), filtration (Tan &
Franzreb, 2020), chromatography (Salmean & Dimartino, 2019), and
extraction (H. Wang et al., 2017). One of the most popular AM methods
employed in bioengineering is digital light processing (DLP) where a
three-dimensional model is built, layer upon layer, by selectively
curing a photo-sensible liquid resin. Reasons for the success of DLP in
biotechnology include its relatively low cost, fast speed (litre sized
objects can be printed overnight), and high resolution (generally in the
order of 50 μm).
Historically, AM was primarily in the domain of the automotive,
aerospace, and biomedical industries which favoured materials with
mechanical properties over chemical characteristics and fabricated
non-porous structures where strength and stiffness are key. On the other
hand, bioprocess applications often require porous materials to maximize
the total surface area available for cell adhesion, adsorption, and
allow intraparticle mass transfer. Besides, materials in the
biotechnology industry heavily exploit chemical characteristics such as
electrostatic charge and hydrophobic behaviour to appropriately modulate
their interactions with species as diverse as cells, proteins, and small
metabolites. This requirement contrasts the status quo where the
composition of commercially available AM materials is IP protected,
making it impossible to rationally design material-species interactions
of interest. Proprietary compositions also complicate compliance with
FDA or EMEA requirements, hindering adoption of 3D printing in the
biomanufacturing industry.
Here, we present a novel polymeric formulation for DLP 3D printing which
can be easily tuned to adjust its chemical, porous, and mechanical
properties of printed parts. The formulation consists of a few simple
ingredients, including monomers and crosslinkers to create the polymeric
network (Figure 1a), a UV photoinitiator to trigger the
photopolymerization reaction, a photoabsorber to increase the resolution
of the printed model, and porogenic components. The key feature of the
proposed formulation lays in the use of bifunctional monomers bearing
both a (meth)acrylate functionality for photopolymerization and a
suitable chemical moiety for biomolecular interactions, e.g. charged
groups, alkyl or aryl groups, or reactive groups for successive covalent
immobilization of a desired ligand. By appropriate selection of the
bifunctional monomers, different materials with different surface
derivatizations to suit a range of applications in bioprocess
engineering can be obtained. Furthermore, the nature and relative
concentration of the components making up the overall formulation will
directly impact on the propagation kinetics of the free-radical
polymerization reaction, in turn affecting the morphology of the
resulting polymeric network and its porous microstructure (e.g. surface
area, average pore size, pore size distribution) (Barner-Kowollik et
al., 2014; Buback, 2009). In this work, we manipulated these
characteristics to produce different formulations for DLP printing, and
obtain porous monoliths with different chemical and macroporous
properties for use in chromatography, immobilized enzyme bioreactors,
and biofilm bioreactors.
A Schoen gyroid topology was selected for fabricating the structured
monoliths (Figure 1b,d,f). Gyroids are members of Triply Periodic
Minimal Surfaces (TPMS), highly versatile geometries with maximized
surface area for mass transfer (Femmer et al., 2015) and excellent
load-bearing stiffness. TPMS are described by simple equations and their
properties in terms of size, surface area, hydraulic diameter, bed
porosity, tortuosity, wall thickness, etc. can be tuned by altering the
equation parameters (Schoen, 2012). We first simulated fluid flow and
solute dispersion to validate the suitability of the gyroid topology for
packed beds. The gyroid structure showed 5 fold higher efficiency
(measured in terms of minimum reduced plate height, Figure 1b) and 4
fold higher permeability (6.4 X 10-14m2) compared to traditional random packing of
spherical particles (1.61 X 10-14m2) (Schure et al., 2004). In particular, the
interconnected gyroid lattice ensured appropriate radial intermixing, in
turn reducing axial dispersion and band broadening, while showing
minimal flow resistance with lower pressure drops than random beds.
Use of porogens enabled formation of a highly interconnected porous
network defined by polymeric globules (Figure S1). The final porous
microstructure was principally determined by polymer chemistry, with
acrylate-based formulations producing smaller pores (271 ± 120 nm for
the AETAC and 289 ± 112 nm for the CEA materials, Figure S1,d) than
methacrylate-based formulations (905 ± 410 nm for the MAETAC material).
This is consistent with the higher reactivity of acrylates over
methacrylate groups (Barner-Kowollik et al., 2014), with acrylates
generating a higher number of polymerization nuclei, in turn leading to
smaller globules and smaller pores than methacrylates. Fine tuning of
the porous characteristics of the 3D printed matrices can be achieved by
adjusting the overall composition as its nature and concentration
directly affects the kinetic and thermodynamic properties of the
mixture. This ultimately enables modulation of diffusional mass transfer
within the 3D printed scaffold and the surface area available for
adsorption and reaction.
We first demonstrate use of the proposed formulation for chromatography
applications. In particular, strong anion and weak cation exchange
monoliths were fabricated using resins with bifunctional acrylates
bearing positive quaternary amine (AETAC monomer) and negative carboxyl
groups (CEA monomer) respectively (Figure 1a). Ion-exchangers with
varying ligand densities were obtained by altering the concentration of
the functional monomers in the formulation, enabling adjustment and
optimization of the adsorption characteristics towards the target
solute. The ion exchangers were initially 3D printed as hollow cylinders
(Figure 1c-e), and adsorption of pure Bovine Serum Albumin (BSA) and
lysozyme (LYS) was measured in batch conditions on the AETAC and CEA
materials, 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. 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 tuned to enable bind
and elute operation of the ion-exchangers.
The potential to fabricate immobilized enzyme bioreactors for
biotransformations was tested by covalently immobilizing trypsin onto
the free carboxylic groups of the CEA material. In particular, 3D
printed supports were exposed to trypsin solutions having different
enzyme concentrations, leading to materials with progressively
increasing immobilized trypsin, up to a maximum density of 7.5 ± 0.04 mg
of trypsin per g of support, corresponding to 66% utilization of the
theoretical carboxyl groups. Enzymatic activity was tested in batch
experiments by introducing N-α-benzoyl-L-arginine ethyl ester
hydrochloride (BAEE) to 3D printed hollow cylinders, and monitoring the
formation of N-α-benzoyl-D,L-arginine (BA) as the product of the enzyme
catalyzed reaction. Results indicated that the immobilized trypsin
retained its hydrolytic activity (Figure 3a). Dynamic experiments were
carried out using 3D printed gyroids to demonstrate the operation of the
bioreactor in steady-state, continuous mode. Five flow rates in the
range of 0.5 – 8 mL/min were tested and product formation was verified
for all of them. As the flow rate increased, the concentration of
product in the effluent stream descreased due to a combination of mass
transport phenomena and actual reaction kinetics (Figure 3b). Yet,
productivity, expressed in terms of product formed per unit time, showed
a proportional growth with increased flow rates due to higher
throughputs. This is consistent with other case studies for continuous
packed bed reactors bearing immobilized enzymes (Halim et al., 2009).
The bioreactor concept was extended to whole-cell biocatalysis. We
selected the biodesulfurization reaction catalyzed by immobilizedRhodococcus opacus as a case study for an emerging
eco-friendly alternative to traditional desulfurization techniques
employed in oil refineries (Mohebali & Ball, 2016). R. opacushas negatively charged cell membrane rich in mycolic acid. Accordingly,
the support material was engineered with positively charged and
hydrophilic bifunctional monomers displaying quaternary amine (MAETAC)
and hydroxyl groups (HEMA, Figure 1a) to enhance cell immobilization. A
methacrylate functionality was chosen to enable autoclaving of the
supports for repeated use. Immobilization experiments with standard
culture medium revealed that R. opacus bacteria formed stable and
healthy biofilms on the 3D printed supports (Figure 4a). In particular,
a biofilm covering the external surface of the supports was obtained,
with rod-like morphology typical of filamentous aggregates of matureRhodococcus cells (approximately 4.0 and 0.5 microns in length
and width, respectively). Since the pore size of the matrix is between
0.4 and 1.0 microns, the biofilm could penetrate into the porous
architecture, aiding cell adhesion and biofilm stability. The
gyroid-based bioreactor was tested for the biodesulfurization of
benzothiophene (BT) in perfusion mode. Results reveal conversion of BT
into phenolic end products (Figure 4b,c), proving the biodesulfurization
potential of the 3D printed bacterial biofilm bioreactor. In addition,
production of phenolic compounds increases over time, indicating
adaptation of the biofilm to the reacting conditions and confirming the
biocompatibility of the support material with a thriving bacterial
population.
Taken together, the versatile material formulation here presented could
be used to create complex three-dimensional matrices for chromatography
and bioreactors. The chemical, physical, mechanical and microporous
properties of the supports can be easily tuned by changing the
composition of the DLP resin formulation. For example, a combination of
bifunctional monomers bearing alkyl chains and polar groups could be
employed to tune the hydrophobic/hydrophilic as well as charged state of
the matrices. We expect this versatility, coupled with cost-effective
and rapid DLP 3D printing (all models were 3D printed in a few hours),
will truly enable fabrication of complex three dimensional architectures
to suit a range of diverse experimental requirements for a multitude of
applications in bioprocess engineering.