Tools to Improve the Performance of ChromatographicApproaches

Chromatography is widely applied industrially. However, some limitations are associated with its common supports, and the impossibility to fully control their structural features is particularly restrictive. Additive manufacturing (AM) is emerging as a fast, highly precise, and reproducible technology for producing chromatographic supports that can improve its performance.

Chromatography is widely applied industrially. However, some limitations are associated with its common supports, and the impossibility to fully control their structural features is particularly restrictive. Additive manufacturing (AM) is emerging as a fast, highly precise, and reproducible technology for producing chromatographic supports that can improve its performance.

Current Trends on the Chromatography Field
Chromatography plays a crucial role in several industries, and the chromatographic market is expected to grow by 6.4% over the next 5 years, reaching US$1670 million in 2024 (https://www.decisiondatabases. com/ip/38033-chromatography-resinmarket-analysis-report).
The most common chromatographic supports are based on microparticulated materials that have a randomly compacted configuration. Monolithic supports are an alternative, but the internal structure of these supports cannot be fully controlled either. Each column has a slightly different internal morphology and structure, which makes its chromatographic performance impossible to predict [1]. This limitation on strictly controlling the morphology and porosity of traditionally manufactured materials can result in low reproducibility, so each column usually needs to be prepared and validated individually (Box 1).
Additionally, since the performance of chromatographic separations and the column efficiency depend on several factors, including the flow of the mobile phase within the column, axial dispersion, and peak widening (resulting in low degrees of purity), recent studies showed that ordered media provides significantly improved chromatographic performance [2].
AM technology can better control the geometry of fabricated pieces, and it is being widely explored for different purposes. For chromatographic support production, it is just starting to be applied: the first uses were reported a few years ago for analytical chemistry [3]. Despite this promise of total customized production, AM technologies are still far from becoming the gold standard for producing chromatographic supports, mainly due to constraints regarding the low resolution, which limits its application in this field [4].
This forum article discusses the potential application of AM developments in the chromatography, pointing out its advantages, limitations, and new trends.

AM Technologies to Produce Chromatographic Structures
AM is starting to be used as a highly accurate and reproducible technology for chromatographic support production [5,6]. The pieces produced using AM are representations of computer-aided design (CAD) models and can be reproduced repeatedly and easily. This strategy allows fine control of the size, shape, position, alignment, and configuration of the (external and internal) structure to create complex constructions that are impossible to produce by conventional methods [7]. Computational fluid dynamics (CFD) can predict several parameters of these structures (e.g., flow dispersion, hydrodynamic constants, molecules diffusion, capacity, and others) with good accuracy.
In chromatography, AM allows the production of a more-defined and uniform convective flow path in opposition to the flow in randomly interconnected pores of a conventional chromatographic support. It should be possible to increase the theoretical plate number and the peak capacity as well as decrease the analytical time needed for the separation of small molecules and proteins [3]. Since the printed pieces are real representations of a design file, it is possible to follow step-by-step instructions to produce a piece with desirable, user-defined characteristics to minimise fouling and clogging. Even if the printed pieces do not totally avoid these limitations, the user will be aware of the feasibility of the chromatographic structure and the developer or producer will be able to predict how many runs/cycles can be performed with the chromatographic support, avoiding the loss of time and money trying geometries that will be not suitable for the desirable application. This could then lead to an increase in the economic viability of the chromatographic process applied in the recovery, purification, and quantification of molecules of interest.
Besides these features, 3D-printed columns are an attractive option for purifying more complex structures, such as viruses, as shown by Moleirinho and colleagues. They demonstrated that 3D-printed chromatographic supports allowed the purification of oncolytic adenoviruses, maintaining their size and shape (which is a common limitation in using conventional purification structures) [8].
So far, several AM techniques, as well as different types of materials, have been applied to produce chromatographic supports. Among the most commonly used AM processes, fused deposition modelling, stereolithography, and selective laser melting have already been applied to producing structures with different geometries and morphologies. Electrospinning has also been applied, mainly in membrane production, but there is some controversy in describing it as an AM technique. This doubt is mainly due to the difficulty of controlling the deposition of the layers, which is now starting to be possible [1].
The materials used in the production of chromatographic supports can highly influence their properties, robustness, and even the separation performance. The material choice is directly related to the intended printing technology because each piece of AM equipment is limited to printing certain materials. Among the materials already used to produce 3Dprinted chromatographic supports are polycaprolactone, poly(methacrylate), and others. Table 1 summarizes the most recent studies on chromatographic support production, listing the printing techniques as well as the main results obtained.
Layer-by-layer methodology can either be used to directly produce chromatographic matrices (as presented in Table 1) or be applied to produce a mould that will originate other chromatographic supports. One US Patent describes a chromatographic structure, whose mould was printed by AM with a very specific geometry (a Schoen gyroid structure). Although only the mould of the structure was produced through 3D printing, the material proved to be quite efficient in separating different biomolecules, compared with a conventional agarose chromatographic column [5]. -the packing of the column cannot be controlled, which means that whenever the matrix is changed in the column, it may be necessary to readjust the conditions used in the chromatographic experiments; -high flows are limited to a few types of supports, where the majority cannot support high pressures, resulting in an increased time for each chromatographic run; -low surface contact, when compared with other technologies on the market.
Membranes are generally used for filtration processes and are simple to use. Additionally, they present increased productivity compared with chromatographic beads. However, there are also some limitations: -low yields and purity degrees; -limitation on temperature control during assays; -fouling and clogging can easily happen.
Monoliths exhibit high porosity and a structure of interconnected pores/channels. However, some limitations have been identified: -fouling and clogging can happen, due to the high pressure that can be achieved inside the monoliths; -impossibility of controlling the temperature at which the chromatographic tests are carried out (they have no recirculating fluid system to maintain the temperature across the chromatographic run); -limited reproducibility due to the difficulty in fully controlling geometry/morphology of the supports, as they are produced by polymerization techniques.

Trends in Biotechnology
Despite this promising potential and results, AM manufacturing does not per se have the ability to structurally adapt the printed filaments/beads to a target sample. Some of the most important parameters in this context are the pore dimension, the volume of the piece produced, the production time, and the ability to print mono-or multimaterial pieces. Some existing companies, such as Nanoscribe, have printers that can create porosities close to what is required for chromatographic applications. However, only some details on the printed structure could achieve nanometer resolution, with micrometre or millimetre scales being more common [9]. Additionally, the maximum printed piece can only reach 100 × 100 × 8 mm 3 and the process takes a long time (~20 h). This printing technology cannot satisfy industrial demands, where the bed volume could reach several litres. Therefore, the maximum printable piece volume possible so Cellulose

Purification of viral particles
Oncolytic adenoviruses were successfully purified with a recovery yield of 69 ± 6%, while maintaining their size and shape. Additionally, lentiviral vectors had a recovered yield of 57%. [8] SLM Use high power-density laser to melt and fuse metallic powders. The pieces are built by selectively melting and fusing powders within and between layers. It enables the production of pieces impossible to be produced through other processes.
-Slow production process and extremely expensive when compared with other subtractive processes -Produces a lot of waste -Usually needs post-processing processes Effect of column geometry on the separation of several proteins and enzymes 3D serpentine column provided a 58% reduction in the analysis time and 74% increase in the peak capacity for the isocratic separations of the small molecules and proteins, when compared with a 3D spiral column. [14] Titanium Protein separation The monoliths were successfully used to separate a mixture of four intact. Further chromatographic characterisation showed a permeability (Kf) of ∼4 × 10 -15 m 2 and a total porosity of 60%. far would not be enough to achieve the desired surface area, and the printing process is too time consuming, which would create problems when scaling up this technology to meet the needs of industry.
In this regard, it is crucial to invest in the development of new printing systems or work on combining existing printing methods. For example, combining electrospinning technology with other AM techniques could help to decrease the dimensions of the pores. Indeed, this combination was already used in the field of tissue engineering [10], where it achieved decreased pore dimension and increased contact surface area for enhanced cell adhesion. Although a different application, what is intended hereincreasing the surface area and decreasing the pore volumeis similar and should be achieved by this proposed technology. Additionally, this technology will become more valuable if it can fully control the fibre design instead of only the fibre alignment [11]. This adaptation or combination of printing mechanisms still needs some improvement, especially when industrial applications are envisioned. However, it will be a critical step in the functional and effective applications of AM in the chromatographic world.
Another key factor for using AM in the largescale production of chromatographic supports could be the possibility to directly immobilise specific ligands onto the support surface during the printing process. This would be an important advance, mainly because the target molecules could be specifically captured, representing a huge gain in the final product recovery and purity. Indeed, the purity of the samples is a key point in the pharmaceutical sector, mainly because impurities present in the final formulations could lead to serious problems in patients' health [12].

Concluding Remarks and Future Perspectives
AM technology is a promising future trend in the chromatography field mainly in predicting the structural configuration expected from a specific geometry of chromatographic support, and in achieving a total reproducibility of the produced structures. Additionally, this production approach will make it possible to develop supports with an increased superficial area of contact and large pores, not only allowing a higher number of binding sites to the desired molecule but also promoting a better flow of molecules within the support. In this regard, innovation in the technologies used to produce supports is critical. AM has the capacity to respond to this challenge and will lead to the production of organized chromatographic supports, with fully controlled geometry that can adapt to the target compound. Considering the translational application and transfer potential of this technology, the successful application of AM to chromatographic support production may result in progress in various industrial sectors, but with especially great impact on the biomedical field in diagnosis, prognosis, and disease therapy.