3D printed platform stationary phases for downstream processing: development of porous platform inks, characterization of 3D printed chromatography columns and testing for industrial bioprocessing

Abstract

Liquid chromatography is the central purification technique in analytical, preparative and industrial scale applications thanks to its simplicity and high selectivity. In traditional chromatography, a glass or steel column is packed with functionalised porous beads that can selectively retain the therapeutic target (e.g. an antibody) while eluting contaminants. Currently, porous beads are the material that dominates the market of chromatography stationary phases. While they are still indispensable in biomanufacturing, they are slowly becoming a problem due to their limitations in productivity and adaptability to new classes of therapeutics. Innovative solutions such as 3D printed ordered stationary phases can make a real impact on downstream processes by offering geometrical control and unprecedented flexibility. However, the availability of 3D printable materials for chromatography applications has been limited, thereby hindering their use in biomanufacturing.

The work presented in this thesis aimed to fabricate 3D printed chromatography columns with sub-micrometre scale geometrical features and bespoke flow channels within the sub-millimetre range. The initial objective involved the formulation of novel inks with two key components. First, pore forming agents responsible for generating inherent porosity, ensuring higher surface area. Second, glycidyl methacrylate, a functional monomer suitable for the immobilisation of chromatography ligands. The formulated inks were 3D printed leveraging the advantages of digital light processing (DLP) 3D printing and polymerisation induced phase separation, enabling the fabrication of large polymer structures with hierarchical porosity. Fine tuning of the material composition led to the fast fabrication (100 mL/h) of high resolution (in the micrometer range) inherently porous structures with epoxy chemistry. A range of different ionisable groups was immobilised to the 3D printed structure walls by a change in pH in aqueous solutions, which was found to be sufficient to allow good conversions. The modified ion exchanger adsorbents were tested for the purification of model proteins and their capture from industrial feedstocks with dynamic binding capacities up to 16 mg/mL.

The platform developed ink was used to fabricate 3D printed ordered porous columns with affinity and cation exchange ligands for application in monoclonal antibody (mAb) manufacturing. The 3D printed protein A column enabled flow through of harvested cell culture fluid at 4 g/L mAb titre expressed in the Apollo™ X expression system. Binding capacities remained unchanged over the range of residence times tested (6-2 min), and were in line with the first generation of protein A resins. Multiple cycles ensured HCP clearance and co-eluted protein A in agreement with regulatory expectations. In addition, a similar column with cation exchange functionality was tested for application in the polishing step showing recovery exceeding 80%. While recognizing the potential of this technology, efforts to scale up revealed remaining challenges in relation to the fabrication of larger columns. The experimental results obtained in this work laid the foundations to compare the economic viability of using 3D printed integrated clarification and capture columns to replace current state of the art of mAb downstream processing. Our models predicts that integration of harvest and capture can alleviate the cost of mAb manufacturing through a significant reduction in materials and labour costs. In addition, potential optimisation strategies should focus on enhancing the capacity of 3D printed columns which could lead to significant reduction in production costs.