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- PolyThymine cryogels as a novel approach to the purification of mRNA encoding anti- ADAM17 antibodiesPublication . Rodrigues, Bruno Daniel Lopes; Tomaz, Cândida Ascensão Teixeira; Sousa, Fani Pereira deCurrently, the metalloprotease "A disintegrin and metalloproteinase 17" (ADAM17) has been gaining prominence due to its important action in signalling pathways where the soluble interleukin receptor - 6 (sIL-6R), the epidermal growth factor receptor (EGF-R) and the tumour necrosis factor alfa receptor (TNF-aR) participate, establishing ADAM17 as an attractive target in cancer therapy and infectious diseases. Thus, due to the high specificity of antibodies to recognize a particular epitope, it was possible to develop an anti-ADAM17 antibody, named MEDI3622, which has been shown to have the ability to specifically block the activity of ADAM17. Furthermore, the messenger Ribonucleic Acid (mRNA) has been showing great potential and applicability in several areas, namely in passive immunization, by the in vivo expression of antibodies, making mRNA a highly attractive method. Thus, in the present work, an mRNA sequence was prepared with the coding sequence of MEDI3622 for expression of this anti-ADAM17 antibody. Therefore, given the great therapeutic potential of mRNA, the high demand from the biopharmaceutical industry and the need to obtain it in a stable and pure form, it is important to optimize the production and purification processes of this biomolecule. The cryogels have emerged as a new alternative to conventional particulate chromatographic media for the purification of high molecular weight molecules, and their potential has already been demonstrated in the purification of the supercoiled isoform of plasmidic deoxyribonucleic acid (pDNA). Cryogels also have a supermacroporous structure that allows for facilitated diffusion, high flow rates, and high capacity. Currently, the strategy of choice for mRNA purification is affinity chromatography, using nucleotides as ligands, whose adenine and thymine base pairing specificity promotes highly selective and specific interactions. Thus, the aim of this work was to synthesize thymine-functionalized cryogels to purify the mRNA sequence produced by in vitro transcription, that encodes the anti-ADAM17 antibody MEDI3622. To this end, the ligand 1-(2-propenyl) thymine (ProT) was synthesized and characterized by nuclear magnetic resonance (NMR), the cryogels were subsequently prepared based on the monomer 2-hydroxyethyl methacrylate (HEMA), by copolymerization of the monomers HEMA and ProT, with different mass ratios of ProT and subjected to cryogenic treatment. The cryogels were characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDXA), and rehydration capacity tests. Subsequently, to evaluate the behaviour of poly-thymine cryogels, purification tests were first performed under different conditions, varying the ionic strength, the type of salt (sodium chloride (NaCl) or ammonium sulphate ((NH4)2SO4)) and the pH in the mobile phase, taking advantage of the affinity between ProT ligands and adenines present in low molecular weight RNA (LMW RNA) used as a model sample, aiming to identify the best conditions under which binding and elution occurred. In the second phase, having already optimized the binding and elution conditions, the ability of cryogels to purify the mRNA was tested. In this phase, the NaCl concentration in the elution buffers was varied in order to achieve the separation between mRNA and pDNA used for its production. As the main results, the synthesis of the ligand 1-(2-propenyl) thymine was successfully achieved. Furthermore, in this task, it was also possible to isolate the unreacted thymine and a by-product of the reaction in which di-alkylation of thymine occurred (1,3-bis(2- propenyl) thymine). The fact that it was possible to isolate these two compounds makes the present work fit in with the principles of circular economy, reducing the costs of the synthesis of the ligand by reusing the isolated thymine, while eliminating the waste of by-products by the possibility of using di-alkylated thymine as a cross-linking agent instead of N, N'-Methylenebisacrylamide. However, traces of a second by-product (3-(2- propenyl) thymine) were found, and it was not possible to fully isolate the ligand of interest due to its structural similarity to this by-product, thus the isolation step needs to be optimized in the future. Regarding the characterization of the cryogels, the functionalized ones did not show significant changes in rehydration capacity compared to the non-functionalized one. Differences in morphology were visualized, which may denote the presence of the ligand in the polymeric matrix of the cryogels. Also, they may have resulted from the conditions used in the synthesis process of cryogels. Furthermore, through the EDXA, it was possible to confirm that there was functionalization of the cryogels, however, this method is not the most indicated for this type of confirmation, other methods, such as elemental analysis and solid-state NMR, can be used in the future to corroborate the results at the functionalization level. The results of the chromatographic assays demonstrate that the ProT functionalized cryogels were able to bind and elute mRNA and pDNA. Moreover, they showed potential to separate pDNA and mRNA, demonstrating a higher affinity for mRNA. The control cryogel (with no ProT ligand incorporated) also demonstrated the ability to bind and elute both mRNA and pDNA, however, it showed no aptitude to separate these two biomolecules. Thus, ProT functionalized cryogels appear to be a novel approach to overcoming challenges in mRNA purification.