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Projeto de investigação
Precisely Patterned Nanofibers for High Performance Bioseparations
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Computational Modeling of Biopharmaceuticals Purification Systems
Publication . Rodrigues, Jéssica Maria Dias; Barbosa, Arménio; Roque, Ana
Chromatography is widely used in biopharmaceutical purification, particularly for therapeutic antibodies. However, traditional methods, such as using Protein A resin, are costly and present challenges like ligand leakage and antibody denaturation. Additionally, the process requires large amounts of water and generates significant chemical waste, raising environmental concerns. This thesis focuses on the development of affinity ligands to reduce production costs while maintaining efficiency in antibody purification. By leveraging virtual compound libraries, a scaffold was used to generate millions of potential ligands. This approach reduces the number of experimental candidates while ensuring high chemical diversity. The virtual library was screened to identify ligands with strong binding potential to the antibody. The top-performing ligands were further analyzed to gain insights into their molecular interactions. Two ligands showed strong potential for purification. These findings represent a promising advance toward more sustainable and cost-effective antibody purification.
Rational design of a small peptidomimetic ligand to capture a viral spike protein
Publication . Costa, Carlos Filipe Santos; Lychko, Iana; Natal, Carolina Mota; Barbosa, Arménio Jorge Moura; Dias, Ana Margarida Gonçalves Carvalho; Roque, Ana Cecília Afonso; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Elsevier
The availability of purified antigenic proteins is critical to develop agents to prevent, diagnose, or treat infectious diseases. In this context, antigenic proteins are produced by recombinant expression in host cells and further purified, typically by chromatographic methods. Chromatographic steps that allow the one-step capture of the antigenic protein are important to streamline the purification train. Here, we present the design and development of an adsorbent bearing a synthetic affinity ligand to capture the SARS-CoV-2 spike protein, used as a model antigenic protein. A 120-ligand combinatorial library was designed in silico and then synthesised in solid phase, and both were computationally and experimentally screened for binding to the spike protein. One lead ligand was selected for yielding > 95 % binding, and 64–73 % recovery of original strain spike protein, its receptor-binding domain (RBD), and Omicron BA.5 variant spike protein. An enrichment factor of 15 was found when capturing the spike protein from a clarified supernatant sample. Complementary molecular dynamics simulations allowed a better understanding of the interactions between the lead ligand and the spike protein, which mainly consist of hydrophobic interactions, some hydrogen bonds and salt bridges formed with an important ligand carboxyl group. Overall, the methodology is a fast and efficient platform to develop affinity ligands for the purification of antigenic proteins in future pandemics.
Affinity reagents towards SARS-CoV-2
Publication . Costa, Carlos Filipe Santos; Roque, Ana Cecília; Dias, Ana Margarida; Barbosa, Arménio
The interest in biopharmaceuticals has increased over the years, and the recent coronavirus disease 2019 (COVID-2019) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlighted the importance of the continuous improvement of bioprocesses to allow more accessible therapies in large quantities. Biopharmaceuticals are pharmaceutical products of biological origin, like vaccines or monoclonal antibodies (mAb). However, whilst current biopharmaceutical upstream processing can meet their increasing demand, the downstream processing remains largely unoptimised, amounting to the majority of the production costs and suffering from low yields, which further drop with scale-up. Current downstream processing relies heavily on chromatography, the platforms of which are oftentimes expensive and unsustainable, thus representing targets of potential optimisation to improve purification efficiency.
The design of a novel synthetic affinity ligand towards the SARS-CoV-2 spike protein is herein described. This also served as a proof-of-concept for the development a workflow to quickly produce solutions towards COVID-19, and could be easily adapted to future pandemics. A combinatorial library was designed in silico based on existing ligands and screened against spike protein, both experimentally under chromatographic conditions, and computationally, culminating in a lead ligand with high binding and recovery of original strain spike protein, its receptor-binding domain (RBD), and an Omicron variant spike protein, and an enrichment factor of approximately 15.
Finally, a molecular modelling (MD) protocol was optimised using, as a case study, the binding of a mutated Staphylococcus aureus protein A to immunoglobulin G (IgG).
Novel Affinity-Based Technologies for Targeting Distinct Biological Systems
Publication . Almeida, Eduardo Eliziário de; Peixoto, Cristina
O desenvolvimento de estratégias de purificação baseadas em afinidade foi impulsionado pelas vantagens teóricas que esta modalidade oferece para o bioprocessamento de PMTAs (produtos medicinais de terapia avançada), podendo resolver problemas atuais da indústria. A cromatografia de afinidade permitiu que a bioprodução de anticorpos monoclonais (mAbs) atingisse novos patamares, provocando a indústria a desenvolver estas tecnologias para outras biomoléculas, como vetores virais. O foco deste trabalho foi a caracterização/otimização de duas estratégias baseadas em afinidade para a purificação de diferentes biomoléculas, mAbs e vetores lentivirais (LVs). A primeira estratégia, focada em cromatografia, visou o uso de diferentes ligandos de afinidade (Z6Alk-Cys e Petasis-Ugi), imobilizados em diferentes tipos de meios cromatográficos. O ligando com melhor desempenho foi o Z6Alk-Cys imobilizado em esferas de agarose, onde uma recuperação de 83% (rendimento total de 31%) de IgG adsorvido foi obtida. O mesmo ligando foi imobilizado em membranas, porém, nenhuma recuperação foi conseguida. A outra estratégia foi um método não cromatográfico de purificação de LV, baseado em separação de fase líquida através de ultracentrifugação ou filtração de fluxo tangencial (TFF). A separação ocorre devido à presença de um copolipeptídeo termorresponsivo com afinidade para este alvo. Dois lotes desta solução de copolipeptídeo foram avaliados. Uma taxa de recuperação de TU (unidades de transdução) de 100% foi alcançada em escala de bancada e à escala de 200 mL (TFF), um rendimento total de 49,0 ± 25,5% foi obtido. A necessidade de um entendimento mais profundo da termorresponsividade deste copolipeptídeo ficou clara antes de prosseguir com o aumento de escala, tendo esta sido conseguida através de DLS e SEC-HPLC, cujos resultados também estão apresentados. O foco deste trabalho foram tecnologias promissoras baseadas em afinidade e também as adversidades enfrentadas durante o seu desenvolvimento, mostrando que otimização ainda é necessária para seu potencial uso comercial.
Exploring Alternative Culture Media for Recombinant Expression in Bacterial Hosts
Publication . Costa, Renata Miguel Ferreira; Dias, Ana; Roque, Ana
Recombinant protein production is essential in biotechnology, with applications in various fields. However, traditional production methods are costly and environmental unsustainable, relying on highly pure raw materials for bacterial culture media production and consuming significant energy and water during the autoclaving process. This contributes to resources depletion and increase of the environmental footprint, raising concerns about the sustainability of these processes. With the global demand for recombinant protein rising, this dissertation explores sustainable bioprocessing strategies by proving the feasibility of using spent media from eukaryotic cultures to support E. coli growth and protein production.
Reducing sugar analysis showed sufficient carbon sources in spent media to sustain E. coli growth. High throughput screening showed that E. coli BL21(DE) adapted slower but achieved comparable biomass to standard media, while E. coli Rosetta showed superior growth in spent media. Supplementation of the spent enhanced biomass production, surpassing standard media in both cases. Protein expression analysis indicated 2.3 times higher purity in spent media, though absolute yields were lower than standard media. Economic analysis confirmed that reusing spent media significantly reduces protein production costs while maintaining a reasonable yield, supporting its scalability.
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Entidade financiadora
European Commission
Programa de financiamento
H2020
Número da atribuição
899732