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Rational design of affinity ligands for bioseparation
Publication . Matos, Manuel J. B.; Pina, Ana S.; Roque, Ana Cecília A.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Elsevier Science B.V., Amsterdam.
Affinity adsorbents have been the cornerstone in protein purification. The selective nature of the molecular recognition interactions established between an affinity ligands and its target provide the basis for efficient capture and isolation of proteins. The plethora of affinity adsorbents available in the market reflects the importance of affinity chromatography in the bioseparation industry. Ligand discovery relies on the implementation of rational design techniques, which provides the foundation for the engineering of novel affinity ligands. The main goal for the design of affinity ligands is to discover or improve functionality, such as increased stability or selectivity. However, the methodologies must adapt to the current needs, namely to the number and diversity of biologicals being developed, and the availability of new tools for big data analysis and artificial intelligence. In this review, we offer an overview on the development of affinity ligands for bioseparation, including the evolution of rational design techniques, dating back to the years of early discovery up to the current and future trends in the field.
Anything but Conventional Chromatography Approaches in Bioseparation
Publication . Roque, Ana Cecília Afonso; Pina, Ana Sofia; Azevedo, Ana Margarida; Aires-Barros, Raquel; Jungbauer, Alois; Di Profio, Gianluca; Heng, Jerry Y. Y.; Haigh, Jonathan; Ottens, Marcel; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Wiley
While packed bed chromatography, known as conventional chromatography, has been serving the biopharmaceutical industry for decades as the bioseparation method of choice, alternative approaches are likely to take an increasing leading role in the next few years. The high number of new biological drugs under development, and the need to make biopharmaceuticals widely accessible, has been driving the academia and industry in the quest of anything but conventional chromatography approaches. In this perspective paper, these alternative approaches are discussed in view of current and future challenges in the downstream processing field.
A purification platform for antibodies and derived fragments using a de novo designed affinity adsorbent
Publication . Matos, Manuel J. B.; Trovão, Filipa; Gonçalves, João; Rothbauer, Ulrich; Freire, Mara G.; Barbosa, Arménio M. J. B.; Pina, Ana Sofia; Roque, Ana Cecília Afonso; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Elsevier
Antibody technologies are the most representative success-case in the biopharmaceutical industry. Widely available purification technologies fail in providing a dedicated universal purification platform that can accommodate antibodies structural diversity, namely antibodies from non-human sources, as chicken IgY, and antigen-binding fragments. In this work, we took inspiration from natural and engineered antibody-binding ligands, to rationally design affinity adsorbents able to capture full-length antibodies and fragments. The one-pot Petasis and Ugi combinatorial reactions were sequentially employed to rapidly generate a library of putative solid-phase adsorbents. The best performing adsorbent yielded a single-step recovery, under mild conditions, of human and chicken whole antibodies, antigen-binding fragments and engineered single-domain antibodies from different complex feedstocks. Due to its simple preparation, the lead antibody adsorbent finds broad applicability as a universal purification platform to increase the availability of antibody technologies in research and development.
Natural Multimerization Rules the Performance of Affinity-Based Physical Hydrogels for Stem Cell Encapsulation and Differentiation
Publication . Fernandes, Cláudia S. M.; Rodrigues, André L.; Alves, Vitor D.; Fernandes, Tiago G.; Pina, Ana Sofia; Roque, Ana Cecília A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; ACS - American Chemical Society
Tissue engineering and stem cell research greatly benefit from cell encapsulation within hydrogels as it promotes cell expansion and differentiation. Affinity-triggered hydrogels, an appealing solution for mild cell encapsulation, rely on selective interactions between the ligand and target and also on the multivalent presentation of these two components. Although these hydrogels represent a versatile option to generate dynamic, tunable, and highly functional materials, the design of hydrogel properties based on affinity and multivalency remains challenging and unstudied. Here, the avidin-biotin affinity pair, with the highest reported affinity constant, is used to address this challenge. It is demonstrated that the binding between the affinity hydrogel components is influenced by the multivalent display selected. In addition, the natural multivalency of the interaction must be obeyed to yield robust multicomponent synthetic protein hydrogels. The hydrogel's resistance to erosion depends on the right stoichiometric match between the hydrogel components. The developed affinity-triggered hydrogels are biocompatible and support encapsulation of induced pluripotent stem cells and their successful differentiation into a neural cell line. This principle can be generalized to other affinity pairs using multimeric proteins, yielding biomaterials with controlled performance.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BPD/97585/2013