Publicação
Fluorinated Ionic Liquids and Nature-inspired Eutectics in Selective Aqueous Biphasic Systems: From Amino Acids to Therapeutic Proteins Purification
| Resumo: | Purification systems for therapeutic proteins are urgently needed in the biopharmaceutical industry, being one of the bottlenecks in downstream procedures to yield proteins with high purity, stability, and activity. Processes that provide selectivity and adaptability while being suitable for a wide range of active biomolecules are required. Protein activity relies on their three-dimensional structure, which is determined by their interactions with the surrounding environment. Ionic liquids (ILs), and more recently eutectics, have emerged as valuable platforms in biological applications for designing task-specific materials by selecting their anions, cations, and hydrogen-bond donors, allowing finetuning of their properties. Herein, FILs and nature-inspired eutectics (formed between choline and carnitine salts and natural short-chain carboxylic acids (SCCA), approved by the EFSA for application as food additives) were used to develop task-specific and more selective aqueous biphasic systems (ABS) for protein purification. The partition systems were validated with increasing biomolecule complexity, from simple amino acids, such as tryptophan (Trp), to conventional proteins, such as lysozyme (Lys) and bovine serum albumin (BSA), and ultimately to high value therapeutic proteins, such as IFN- 2b (interferon). A two-way approach was implemented in developing selective ABS, that are biocompatible with biomolecules, ensuring their activity maintenance. First, the ABS based on nature-inspired eutectics with PPG400 (a widely accepted biocompatible polymer used in many biopharmaceutical applications), that respect the parameters of sustainable chemistry per se, were designed to selectively select the target phase for Trp by eutectic formation (addition of natural SCCA to the ABS based on choline and carnitine salts). Second, the task-specific FIL-based ABS were designed to increase selectivity while still offering a biocompatible medium for proteins. Most biological applications, including protein purification steps, involve aqueous solutions. Insights were attained into taskspecific FILs that limit the impact of water addition on the IL’s hydrogen-bond accepting ability, a key factor in obtaining functionalized materials for protein purification. FILs also enables ABS formation with common protein stabilizers, like sugars. Ultimately, the FIL-based ABS enabled the simultaneous recovery of IFN- 2b and albumin in opposing phases (using BSA as a model; mammalian cell culture supernatants contain IFN- 2b and albumin). Additionally, the interactions between the proteins and ABS-forming solutes were studied to understand the surroundings effect on the structure, activity, and partition behavior of the studied proteins. The designed ABS also enables more efficient production by replacing macroscale batch ABS with flow-through processes (ABS in microfluidic setups). A proof-of-concept was attained for the miniaturization of ILbased ABS for the partition of Trp, assessing microtube batch ABS and microfluidic ABS operation. The outcomes of this thesis demonstrate the feasibility of the proposed ABS to revolutionize the purification systems for therapeutic proteins, with a strong impact on the biopharmaceutical sector. |
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| Autores principais: | Carvalho, Sara |
| Assunto: | Fluorinated Ionic Liquids Choline- and Carnitine-based Eutectics Selective Aqueous Biphasic Systems Tryptophan Lysozyme Albumin |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Purification systems for therapeutic proteins are urgently needed in the biopharmaceutical industry, being one of the bottlenecks in downstream procedures to yield proteins with high purity, stability, and activity. Processes that provide selectivity and adaptability while being suitable for a wide range of active biomolecules are required. Protein activity relies on their three-dimensional structure, which is determined by their interactions with the surrounding environment. Ionic liquids (ILs), and more recently eutectics, have emerged as valuable platforms in biological applications for designing task-specific materials by selecting their anions, cations, and hydrogen-bond donors, allowing finetuning of their properties. Herein, FILs and nature-inspired eutectics (formed between choline and carnitine salts and natural short-chain carboxylic acids (SCCA), approved by the EFSA for application as food additives) were used to develop task-specific and more selective aqueous biphasic systems (ABS) for protein purification. The partition systems were validated with increasing biomolecule complexity, from simple amino acids, such as tryptophan (Trp), to conventional proteins, such as lysozyme (Lys) and bovine serum albumin (BSA), and ultimately to high value therapeutic proteins, such as IFN- 2b (interferon). A two-way approach was implemented in developing selective ABS, that are biocompatible with biomolecules, ensuring their activity maintenance. First, the ABS based on nature-inspired eutectics with PPG400 (a widely accepted biocompatible polymer used in many biopharmaceutical applications), that respect the parameters of sustainable chemistry per se, were designed to selectively select the target phase for Trp by eutectic formation (addition of natural SCCA to the ABS based on choline and carnitine salts). Second, the task-specific FIL-based ABS were designed to increase selectivity while still offering a biocompatible medium for proteins. Most biological applications, including protein purification steps, involve aqueous solutions. Insights were attained into taskspecific FILs that limit the impact of water addition on the IL’s hydrogen-bond accepting ability, a key factor in obtaining functionalized materials for protein purification. FILs also enables ABS formation with common protein stabilizers, like sugars. Ultimately, the FIL-based ABS enabled the simultaneous recovery of IFN- 2b and albumin in opposing phases (using BSA as a model; mammalian cell culture supernatants contain IFN- 2b and albumin). Additionally, the interactions between the proteins and ABS-forming solutes were studied to understand the surroundings effect on the structure, activity, and partition behavior of the studied proteins. The designed ABS also enables more efficient production by replacing macroscale batch ABS with flow-through processes (ABS in microfluidic setups). A proof-of-concept was attained for the miniaturization of ILbased ABS for the partition of Trp, assessing microtube batch ABS and microfluidic ABS operation. The outcomes of this thesis demonstrate the feasibility of the proposed ABS to revolutionize the purification systems for therapeutic proteins, with a strong impact on the biopharmaceutical sector. |
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