Simone Dimartino

3D printed porous media columns with fine control of column packing morphology

In this paper we demonstrate, for the first time, the use of 3D printing (also known as additive manufacturing or rapid prototyping) to create porous media with precisely defined packing morphologies, directly from computer aided design (CAD) models. We used CAD to design perfectly ordered beds with octahedral beads (115 μm apothem) packed in a simple cubic configuration and monoliths with hexagonal channels (150 μm apothem) in parallel and herringbone arrangements. The models were then printed by UV curing of acrylonitrile-butadiene-styrene powder layers. Each porous bed was printed at 1.0, 1.5 and 2.0 mL volumes, within a complete column, including internal flow distributors and threaded 10-32 flow connectors. Close replication of CAD models was achieved. The resultant individual octahedral beads were highly uniform in size, with apothems of 113.6±1.9 μm, while the monolith hexagonal cross-section channels had apothems of 148.2±2.0 μm. Residence time distribution measurements show that the beds largely behaved as expected from their design void volumes. Radial and fractal flow distributor designs were also tested. The former displayed poor flow distribution in parallel and herringbone pore columns, while the fractal distributors provided uniform flow distribution over the entire cross section. The results show that 3D printing is a feasible method for producing precisely controlled porous media. We expect our approach to revolutionize not only fundamental studies of flow in porous media but methods of chromatography column production.

A validated model for the simulation of protein purification through affinity membrane chromatography

A mathematical model is proposed for the description of protein purification through membrane affinity chromatography. The model describes all the three stages of the chromatographic cycle and takes into account convection, axial dispersion and binding reaction kinetics in the porous membrane matrix, while boundary layer mass transfer resistance is shown to be negligible. All the model parameters have a precise physical meaning which enables their evaluation through separate experimental measurements, independent of the chromatographic cycle. Model testing and validation has been performed with experimental chromatographic cycles carried out with pure IgG solutions as well as with complex mixtures containing IgG(1), using new affinity membranes. The comparison between model calculations and experimental data showed good agreement for all stages of the affinity cycle. In particular, for loading and washing steps binding kinetics was found so fast that adsorption equilibrium was sufficient to describe the observed behavior; as a result, the model simulations are entirely predictive for the adsorption and washing phases. On the contrary, in the elution step the reaction rate is comparable to that of the other simultaneous transport phenomena. The model is able to predict the performance of chromatographic purification of IgG from complex mixtures simply on the basis of the parameter values obtained from pure IgG solutions.

Performance of a new protein A affinity membrane for the primary recovery of antibodies.

Recovery of antibodies with Protein A affinity chromatography columns has become the standard for the biotechnology industry. Membrane affinity chromatography has not yet experienced extensive application due to the lower capacity of membrane supports compared to chromatographic beads. In this work, new affinity membranes endowed with an interesting binding capacity for human IgG are studied in view of their application in the capturing step of a monoclonal antibody production process. The membranes have been extensively tested with pure IgG solutions and with a cell culture supernatant containing IgG1. The effects of feed flow rate and IgG concentration on the separation performances have been studied in detail, considering in particular binding capacity, selectivity and recovery. These new high capacity affinity membranes appear good candidates to avoid the throughput limitations and other well‐known drawbacks of traditional bead‐based chromatographic columns.

Dynamic evaluation of polypropylene capillary-channeled fibers as a stationary phase in high-performance liquid chromatography

Polypropylene (PP) capillary-channeled polymer (C-CP) fiber stationary phases are investigated for applications in HPLC. Specifically, the roles that fiber size and shape, linear velocity, interstitial fraction, and column inner diameter play in separation efficiency were evaluated using a uracil and butylparaben mixture eluted under isocratic conditions. Four fiber types, having nominal diameters ranging from 30 to 65 μm, were used in 250 mm × 2.1 mm columns. Optimum flow characteristics, as judged by plate height and resolution, were observed for 40 μm diameter PP C-CP fibers packed at an interstitial fraction of ~0.63, over a broad range of linear velocities (~2 to 37 mm/s). The influence of column inner diameter was studied on 1.5, 2.1, and 4.6 mm columns packed at the optimal interstitial fraction. The best performing column in terms of plate height and resolution was the 2.1 mm inner diameter. C-CP columns were also evaluated for the separation of a protein mixture composed of ribonuclease A, cytochrome c, and transferrin. Results obtained with the biomacromolecules mixture validate the optimal structural and operative conditions determined with the small solutes, laying the groundwork towards biomacromolecule applications, focusing more on the chemical aspects of separations.

Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography

Existing mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behavior are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglobulin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG(1) to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions.

Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification.

Microporous membranes are an attractive alternative to circumvent the typical drawbacks associated to bead-based chromatography. In particular, the present work intends to evaluate different affinity membranes for antibody capture, to be used as an alternative to Protein A resins. To this aim, two Mimetic Ligands™ A2P and B14, were coupled onto different epoxide and azide group activated membrane supports using different spacer arms and immobilization chemistries. The spacer chemistries investigated were 1,2-diaminoethane (2LP), 3,6-dioxa-1,8-octanedithiol (DES) and [1,2,3] triazole (TRZ). These new mimetic membrane materials were investigated by static and by dynamic binding capacity studies, using pure polyclonal human immunoglobulin G (IgG) solutions as well as a real cell culture supernatant containing monoclonal IgG(1). The best results were obtained by combining the new B14 ligand with a TRZ-spacer and an improved Epoxy 2 membrane support material. The new B14-TRZ-Epoxy 2 membrane adsorbent provided binding capacities of approximately 3.1mg/mL, besides (i) a good selectivity towards IgG, (ii) high IgG recoveries of above 90%, (iii) a high Pluronic-F68 tolerance and (iv) no B14-ligand leakage under harsh cleaning-in-place conditions (0.6M sodium hydroxide). Furthermore, foreseeable improvements in binding capacity will promote the implementation of membrane adsorbers in antibody manufacturing.

Experimental characterization of the transport phenomena, adsorption, and elution in a protein A affinity monolithic medium

A commercially available convective interaction media (CIM) Protein A monolithic column was fully characterized in view of its application for the affinity capture of IgG in monoclonal antibody production processes. By means of moment analysis, the interstitial porosity and axial dispersion coefficient were determined. The frontal analysis method of characteristic points was employed, for the first time with monolithic media, to determine the dynamic binding capacity. The effects of the flow rate and pH on the total recovery of polyclonal IgG and elution profile were evaluated. A comparison with literature data for Protein A chromatography beads demonstrate the superior bed utilization of monolithic media, which gave better performance at lower residence times.

Experimental and computational analysis of a novel flow channel to assess the adhesion strength of sessile marine organisms

Bioadhesives produced by marine macroalgae represent a potential source of inspiration for the development of water-resistant adhesives. Assessing their adhesion strength, however, remains difficult owing to low volumes of adhesive material produced, low solubility and rapid curing time. These difficulties can be circumvented by testing the adhesion strength of macroalgae propagules attached to a substrate. In this paper, we present a simple, novel flow channel used to test the adhesion strength of the germlings of the fucalean alga Hormosira banksii to four substrates of biomedical relevance (PMMA, agar, gelatin and gelatin + lipid). The adhesion strength of H. banksii germlings was found to increase in a time-dependent manner, with minimal adhesion success after a settlement period of 6 h and maximum adhesion strength achieved 24 h after initial settlement. Adhesion success increased most dramatically between 6 and 12 h settlement time, while no additional increase in adhesion strength was recorded for settlement times over 24 h. No significant difference in adhesion strength to the various substrates was observed. Computational fluid dynamics (CFD) was used to estimate the influence of fluid velocity and germling density on drag force acting on the settled organisms. CFD modelling showed that, on average, the drag force decreased with increasing germling number, suggesting that germlings would benefit from gregarious settlement behaviour. Collectively, our results contribute to a better understanding of the mechanisms allowing benthic marine organisms to thrive in hydrodynamically stressful environments and provide useful insights for further investigations.

Scale-up of affinity membrane modules: comparison between lumped and physical models

Membrane chromatography represents one of the emerging technologies for downstream processing in the biotechnology industry. This process is currently used in polishing steps for antibody manufacturing, while its application is still under development for the capture step. To promote its employment in large‐scale processes, it is crucial to develop a simple, yet reliable, simulation tool able to describe the process performance in a predictive way at all scales. In this work, the physical model for the description of protein purification with affinity membrane chromatography has been used to predict the performance of scaled‐up systems and compared with the lumped model, frequently used for its deceptive simplicity. Two commonly used binding kinetics have been implemented in the models, namely the Langmuir and the bi‐Langmuir equations. The two models describe equally well experimental data obtained in a lab‐scale apparatus, while, on the contrary, important differences are observed in scaled‐up systems even at the early stages of breakthrough, which are particularly relevant in industrial‐scale operations. It is seen that for both kinetics, the physical model is more appropriate and safer to use for scale‐up purposes. Copyright

Microscopic and infrared spectroscopic comparison of the underwater adhesives produced by germlings of the brown seaweed species Durvillaea antarctica and Hormosira banksii.

Adhesives from marine organisms are often the source of inspiration for the development of glues able to create durable bonds in wet environments. In this work, we investigated the adhesive secretions produced by germlings of two large seaweed species from the South Pacific, Durvillaea antarctica, also named ‘the strongest kelp in the word’, and its close relative Hormosira banksii. The comparative analysis was based on optical and scanning electron microscopy imaging as well as Fourier transform infrared (FTIR) spectroscopy and principal component analysis (PCA). For both species, the egg surface presents peripheral vesicles which are released soon after fertilization to discharge a primary adhesive. This is characterized by peaks representative of carbohydrate molecules. A secondary protein-based adhesive is then secreted in the early developmental stages of the germlings. Energy dispersive X-ray, FTIR and PCA indicate that D. antarctica secretions also contain sulfated moieties, and become cross-linked with time, both conferring strong adhesive and cohesive properties. On the other hand, H. banksii secretions are complemented by the putative adhesive phlorotannins, and are characterized by a simple mechanism in which all constituents are released with the same rate and with no apparent cross-linking. It is also noted that the release of adhesive materials appears to be faster and more copious in D. antarctica than in H. banksii. Overall, this study highlights that both quantity and quality of the adhesives matter in explaining the superior attachment ability of D. antarctica.