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  • Telma Godinho Barroso Maciel Fernandes

    Mestre em Engenharia Qumica e Bioqumica

    Functional monolithic platforms for antibody purification

    Dissertao para obteno do Grau de Doutor em

    Qumica Sustentvel

    Orientadores: Prof. Doutora Ana Aguiar-Ricardo

    Prof. Doutora Ana Ceclia Roque

    January 2014

  • i

    Functional monolithic platforms for antibody purification

    Copyright

    Telma Godinho Barroso

    Faculdade de Cincias e Tecnologia

    Universidade Nova de Lisboa

    A Faculdade de Cincias e Tecnologia e a Universidade Nova de Lisboa tm o direito, perptuo

    e sem limites geogrficos, de arquivar e publicar esta dissertao atravs de exemplares

    impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou

    que venha a ser inventado, e de divulgar atravs de repositrios cientficos e de admitir a sua

    cpia e distribuio com objectivos educacionais ou de investigao, no comerciais, desde

    que seja dado ao autor e editor.

  • ii

  • iii

    ACKNOWLEDGMENTS

    The PhD was more than four years of scientific research combined with my intellectual skills. In

    fact, it was a test to my personal resistance (Mrcio Temtem you were completely right! Thanks

    very much for your advices, and for all friendship and teachings! You were a great professor).

    Fortunately, I believe that I passed it with a feeling of job done. However, PhD was not

    possible without the support and reinforcement of several people that I would like to

    acknowledge.

    First of all, I would like to acknowledge my supervisors Prof. Ana Aguiar-Ricardo and Prof.

    Cecilia Roque that gave me the opportunity to work on this project. I am very grateful for all their

    effort and dedication to me, and to the work that together we were able to develop. Prof. Ana

    Aguiar-Ricardo many thanks to be an exigent and outstanding supervisor, and an especial

    friend when I needed. I will always remember our brainstorming, the phone calls after hours,

    and the happy and frustrating moments together, but the true is: I got here because you never

    gave up of me. Prof. Cecilia Roque, I am very thankful to your patience with me as well as to

    your good vibes that you always transmitted to me (y ooouuu). Without you, the

    biochemical world would be utopic for me! If I have biochemistry in my education, I owe it to

    you.

    I also would like to acknowledge to Polymer Synthesis and Processing Group and to

    Biomolecular Engineering Group. I could find numerous words to say to each one however, it

    will become another thesis. Thus, I reserved some special words for each one of you. Starting

    with Polymer Synthesis and Processing Group: Raquel Viveiros (Yesterday, today and

    tomorrowyou will be forever in my heart, thanks for everything), Vanessa Correia (I will miss

    you, and I will keep great memories of us including brainstorming ), Rita Restani (Gazeada

    Girl: sometimes the life is hard, but at same time is amazingplease hold it), Patrcia

    Morgado (Thanks for your sympathy and friendship), Ana Silva (For you, the sky is the

    limitclose your eyes, close your mouth and forget the others, you are the important because

    you have everything to be it), Anita Loureno (Thank you for your participation in this chapter

    of my life), Mrcia Tavares (Hard Rock Girl; It was a pleasure to work with you), Renato Cabral

    (Great Engineer: it was a pleasure to meet you), Vasco Bonifcio (The Chemist Man: thanks

    for all your patience in try to explain me organic chemistry, it was not easy, but it was possible

    ) and Teresa Casimiro (Great scientist and friend: Thanks to clean my tears and support my

    euphoric moments. You had a fundamental role on this chapter of my life). Now the

    Biomolecular Engineering Group: ris Batalha (Keep Calm Girl: thank you very much for all

    funny and zen moments that only you could give me), Margarida Dias (Special friend and great

    professional: I will miss you a lot), Susana Palma (It was a pleasure to meet you), Henrique

    Carvalho (Alternative Guy: I always remember your good mode), Vijaykumar Dhadge

    (Patents Man: Great Doctor! One day we will be rich!!!), Abid Hussain (Great colleague,

    researcher and English professor: Thanks for everything that you taught me), Ricardo Branco

    (The Modeling Man: I am very grateful for all your effort and dedication for me and for my

  • iv

    work; I will miss our brainstorming about computational modelling), Cludia Fernandes and

    Bianca Gonalves (Recent acquisitions: I adored to meet you). And finally Ana Pina! For you

    all words are not enough. During the last four years you were one of the main pieces of this

    game and when the motivation disappeared, you were the reason that made me to continue. I

    will remember all nights that we spent in the lab, all the balcony moments where we laugh and

    where we cried, all conferences, all days, everythingYou are simply the best.

    Sincerely, thank you all for everything. This thesis has pieces of you thus, it is also yours.

    To Professor Manuel Nunes da Ponte I would like to express my sincere acknowledgments for

    all teachings in green chemistry which formatted me to be a sustainable engineer. A special

    thanks also to professor Ana Maria Rego for all XPS discussions and friendship.

    A special thanks to Maria Jos and Isabel Rodrigues for their assistance in bureaucratic work,

    and to Maria de Palma, Idalina and Conceio for providing me clean laboratory material to

    develop me research. For all, a distinctive thank also for your love and kindness. I want also to

    acknowledge the Analytical Services Laboratory of REQUIMTE for the characterization of

    materials, and to the Animal Cell Technology Unit of ITQB-UNL/IBET (Dr. Paula M Alves and

    Dr. Ana Teixeira) for providing the cell culture bulks of antibodies.

    To all my family, I am very grateful for all your support and patience. Due to all of you (father,

    sister, nephew (Ti), cousins, uncles, aunts, brothers-in-law, Manuela e Armando) I am the

    person that was able to get here. I am sorry for the moments that I missed with you (especially

    with my nephew, Tiago) but this work was hard. However, believe in me, you were always in my

    heart. Specially, I would like to acknowledge two persons: Snia Barroso that more than a

    sister, she was and is the mother that I never had. I love you! The second person is my

    husband Nuno Fernandes that was the light that appeared in my life, and make me believe that

    I was able to do everything that I wanted. Thank you for your love, friendship, patience and

    encouraging words. Without you my life will be uncoloured. Love you! To all my friends, that are

    a lot, but you know who I am referring, many thanks for always being there for me! Beetocada

    (all members and staff) thanks very much for all sportive and relax weekends and moments.

    Nephews (Joana, Porco, Li, Central, Plo, Caixinha, and all others) for you a special thanks

    for all happy moments that you gave me, and that I will always keep in my heart. I love you all in

    the same way. For the ones that life turned difficult their presence (Bru, Ins, Meguy and

    Tropa), a special kiss. You were also my driving force to continue. You are great friends.

    Lastly, I would like to thank the financial support from Fundao para a Cincia e Tecnologia,

    Portugal, through contracts PEst-C/EQB/LA0006/2011, MIT-Pt/BS-CTRM/0051/2008,

    PTDC/EBB-BIO/102163/2008, PTDC/EBBBIO/ 098961/2008, PTDC/EBB-BIO/118317/2010

    and doctoral grant SFRH/ BD/62475/2009, and Fundao Calouste Gulbenkian.

  • v

    ABSTRACT

    This work aimed at the development of monolithic chromatographic platforms for antibody

    purification. A sustainable strategy, comprising the use of natural polymers such as chitosan,

    agarose and dextran, was employed to create 3D porous structures. In order to improve the

    mechanical properties and biodegradability of monoliths, natural polymers were physically

    blended with synthetic ones. All supports were, in a first stage, produced by freeze-drying

    methods while in a second attempt were prepared by an integrated approach involving gelation

    process, water-acetone substitution and scCO2 drying. A further optimization for opening the

    porous network was evaluated involving swelling and freeze-drying procedures. To optimize the

    efficacy of monoliths, magnetic nanoparticles were embedded in monoliths structure to confer

    them a magnetic responsive behaviour. This additional feature improved antibody recovery

    when performing a magnetically-assisted elution (93% recovery of bound IgG) complemented to

    less time processing. The selectivity of monoliths for antibody, IgG, was guaranteed by the

    immobilization of ligand 22/8 (artificial Protein A) and a new triazine-based ligand (TPN-BM)

    onto their surfaces. The functionalization strategy of TPN-BM, which synthesis followed the

    principles of green chemistry, was induced by plasma technology. This alternative strategy

    allowed the reduction of time and solvents consumption while maximizing the functionalization

    yield of supports (2-fold, comparing to the traditional procedures). Moreover, the binding/elution

    mechanism between TPN-BM and IgG at a molecular level was validated through molecular

    docking studies and dynamic simulations.

    Overall, TPN-BM func