The two sides of the use of supported ionic liquids (SILs) materials

Solid-phase extraction (SPE) is used for purification and removal techniques for target molecules in liquid extracts. In recent years, the introduction of ionic liquids (ILs) in SPE, have emerged as SILs materials and considering their characteristics1 SILs can be excellent adsorbents of a wide range of bioactive compounds, ranging from small organic compounds to complex molecules like antibodies (Abs). Despite the studies that have already been carried out, that prove their role in the extraction and purification of proteins2, there are no indications regarding their performance when applied to Abs. However, it seems to be a viable and sustainable strategy to be used in the downstream processing of Abs. Also, these materials could have the ability to adsorb and remove emerging contaminants that are not being successfully removed from wastewaters by conventional treatment systems. In this work three SILs materials were synthesized and used in IgG purification and in adsorption studies of a pesticide (imidacloprid) from aqueous solutions, being SilPrN(C8)3Cl the more promising SIL for both applications. The development of a new platform such as SILs, showed tremendous potential for the purification of IgG and removal of imidacloprid from water, thus this technique appears to be a viable option for the downstream processing of Abs and to be applied as a filter for the elimination of pesticides from wastewaters.publishe

Supported ionic liquids as customizable materials to purify immunoglobulin G

Over the past few years, antibodies such as immunoglobulin G, IgG, have increased their market share as alternative therapeutics. However, their production at high purity levels is still costly due to the absence of a cost-effective platform for their recovery and purification from the complex biological media in which they are produced. This work describes, for the first time, that materials modified with ionic liquids (ILs) can be designed for the effective capture and purification of antibodies from complex matrices, allowing both the selective adsorption of IgG or the selective adsorption of other proteins present in the media. The best results correspond to IgG with 59 % of yield and 84 % of purity in the aqueous solution, and IgG with 76 % of yield and 100 % of purity on the surface of one SIL due to the selective adsorption of IgG from human serum. The best conditions and materials were then applied to other IgG-containing matrices, namely rabbit serum and Chinese hamster ovary (CHO) cell culture supernatants, proving the robustness of the developed strategy. Furthermore, it is demonstrated that the secondary structure of IgG is preserved during the purification process and that these antibodies remain biologically active. In summary, it is shown that by only changing the IL chemical structure at the material surface it is possible to selectively adsorb IgG or to adsorb other proteins leaving IgG in solution. These findings prove that SILs are customizable materials with future potential to act in the flow-through or bind-andelute modes. Therefore, SILs can be envisioned as potential chromatographic columns capable of substituting the high-cost commercial chromatographic columns based on biological ligands currently used to purify IgG.publishe

Produção e purificação de anticorpos monoclonais humanos anti-interleucina-8

Over the past few years, monoclonal antibodies (mAbs) have gained interest as alternative therapy. In particular, anti-interleukin-8 (anti-IL-8) mAbs have shown high potential for the treatment of inflammatory diseases, since they reduce neutrophil infiltration in the early stage of inflammation. However downstream processing has not evolved at the same rate as upstream, being considered the limiting and most costly step in the production of these biopharmaceuticals. The conventional platform adopted for the purification of IgG, counts on a primary antibody capture step by protein A affinity chromatography. This technique is extremely costly - with two chromatographic purification steps. Nevertheless, the high costs and intrinsic limitations on the use of the ligand itself are other disadvantages of using protein A affinity chromatography. In order to develop an alternative process based on the use of ionic liquids (ILs) as chemical ligands, this dissertation investigated three supported ionic liquid phase materials ([Si][C3C1im]Cl, [Si][N3444]Cl and [Si][N3888]Cl) as alternative matrices for the extraction and/ or purification of antibodies, namely IgG from complex biological matrices (human serum, rabbit serum and Chinese hamster ovary cell culture supernatants). Initially, the operating conditions of the process were optimized, namely the solid: liquid ratio (S:L ratio), pH and contact time (t), in order to maximize the process performance parameters. Best results were obtained using [Si][C3C1im]Cl (S:L ratio= 150 mg.mL-1 ; pH 12; t= 60 min) and [Si][N3888]Cl (S:L ratio= 100 mg.mL-1 ; pH 11; t= 60 min), where it was possible to obtain 59% and 76% IgG yields with 84% and 100% purity levels respectively. Moreover, the IgG purification mechanism was found to be highly dependent on the supported IL structure, being flowthrough-like mode using [Si][C3C1im]Cl and bin-and-elutelike mode with [Si][N3888]Cl. Thus, two new, more efficient and economical strategies for downstream antibody processing have been developed, representing important advances in the biopharmaceutical field.Ao longo dos últimos anos, os anticorpos monoclonais (mAbs) têm vindo a ganhar interesse crescente como terapia alternativa. Em particular, os mAbs anti-interleucina-8 (anti-IL-8) têm apresentado elevado potencial para o tratamento de doenças inflamatórias, uma vez que reduzem a infiltração de neutrófilos no estágio inicial da inflamação. Contudo o processamento a jusante não evoluiu ao mesmo ritmo que o processamento a montante, sendo considerado atualmente como a etapa limitante e dispendiosa na produção destes biofármacos. A plataforma convencional adotada para a purificação de IgG conta com uma etapa primária de captura do anticorpo através da cromatografia de afinidade com a proteína A. Esta técnica é extremamente dispendiosa –,onde apresenta duas etapas cromatográficas de purificação. No entanto, os elevados custos e as limitações intrínsecas ao uso do próprio ligando são outras desvantagens da utilização de cromatografia de afinidade com proteína A. Com vista ao desenvolvimento de um processo alternativo baseado na utilização de líquidos iónicos (LIs) como ligandos químicos, nesta tese de mestrado procedeu-se à investigação de três materiais com líquidos iónicos suportados ([Si][C3C1im]Cl, [Si][N3444]Cl, [Si][N3888]Cl) como matrizes alternativas para a extração e/ou purificação de anticorpos, nomeadamente a IgG de matrizes biológicas complexas (soro humano, soro de coelho e sobrenadantes de culturas celulares de ovário de hamster chinês). Inicialmente, foram otimizadas as condições operacionais do processo, nomeadamente a razão sólido:líquido (razão S:L), p e tempo de contacto (t) de forma a maximizar os parâmetros de performance do processo. Os melhores resultados foram obtidos utilizando [Si][C3C1im]Cl (razão S:L = 150 mg.mL-1 ; pH 12; t = 60 min) e [Si][N3888]Cl (razão S:L = 100 mg.mL-1 ; pH 11; t = 60 min) em que foi possível obter rendimentos de IgG de 59% e 76% com níveis de pureza de 84% e 100%, respetivamente. Mais ainda, verificou-se que o mecanismo de purificação de IgG é altamente dependente da estrutura do LI suportado, sendo “flowthrough-like mode” utilizando [Si][C3C1im]Cl e “bin-and-elute-like mode” com [Si][N3888]Cl. Assim, duas novas estratégias mais eficientes e económicas para o processamento a jusante de anticorpos foram desenvolvidas, representando importantes avanços na área biofarmacêutica.Mestrado em Bioquímic

Monoclonal antibodies as therapeutic agents for inflammatory diseases

Inflammation is a physiological process caused when an agent (chemical, biological or physical) transcends the primary defense barrier of an organism, playing a central role in the fight against those pathogens, setting a series of biological reactions to restore the integrity of such organism. Uncontrolled amplification of these events may lead to undesirable pathological manifestations such as cancer, diabetes, and cardiovascular, neurological, and chronic inflammatory diseases. Monoclonal antibodies (mAbs) were first described in 1975, and since then they have proven to be relevant therapeutic agents in a myriad of diseases. The US Food and Drug Administration (FDA) has already approved more than 90 mAbs for the treatment of several diseases, from which approximately 46% were specifically approved for the treatment of inflammatory diseases, for instance rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis and palmoplantar pustulosis. This chapter aims to provide an overview on the inflammation process and main biochemical mechanisms, a vision on the current state of the art of the mAbs-based biopharmaceuticals market, and describes the mAbs products already approved by regulatory agencies as powerful therapeutic agents for inflammatory diseases, while highlighting the advantages of these biopharmaceuticals and fomenting their widespread use as recurrent therapies.publishe

Use of ionic liquids as cosurfactants in mixed aqueous micellar two-phase systems to improve the simultaneous separation of immunoglobulin G and human serum albumin from expired human plasma

A large fraction of unused clinical transfusion plasma is nowadays discarded. Nevertheless, some proteins, including serum albumin, immunoglobulins, and clot factors, are still viable for use in diagnosis or research. However, lasma is a complex matrix, requiring multiple steps and timeconsuming methods for the isolation of proteins. In this work, aqueous micellar two-phase systems (AMTPS) composed of nonionic surfactants (Triton X-114 or Tergitol 15-S-7) were investigated to simultaneously separate immunoglobulin G (IgG) and human serum albumin (HSA) from human expired plasma. A factorial planning was applied to the nonionic surfactant and plasma concentrations to enhance the systems separation performance. After identifying the best conditions with AMTPS formed by nonionic surfactants, mixed AMTPS formed by Tergitol 15-S-7 and various surface-active ionic liquids (SAILs) acting as cosurfactants were additionally investigated to tailor the proteins partition between the two phases. The mixed AMTPS composed of Tergitol 15-S-7 as the nonionic surfactant and tributyltetradecylphosphonium chloride as the cosurfactant at pH 8.0 improved the simultaneous separation of both proteins to the opposite phases. IgG purification of 1.14- fold in the surfactant-poor phase and HSA purification of 1.36-fold in the surfactant-rich phase were obtained. Method reproducibility was investigated using additional samples of human serum and plasma and serum mixtures. Although improvements in the purification factor of each protein are still a future goal, mixed AMTPS comprising nonionic surfactants and SAILs as cosurfactants may be considered as novel platforms to be used in the simultaneous separation of value-added compounds from complex and natural matrices.publishe