Telma Godinho Barroso Maciel Fernandes Mestre em Engenharia Química e Bioquímica Functional monolithic platforms for antibody purification Dissertação para obtenção do Grau de Doutor em Química Sustentável Orientadores: Prof. Doutora Ana Aguiar-Ricardo Prof. Doutora Ana Cecília Roque January 2014 Functional monolithic platforms for antibody purification “Copyright” Telma Godinho Barroso Faculdade de Ciências e Tecnologia Universidade Nova de Lisboa A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através 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 através de repositórios científicos e de admitir a sua cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que seja dado ao autor e editor. i ii 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 (Márcio 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 (yûûûû 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 tomorrow…you 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 amazing…please hold it), Patrícia Morgado (Thanks for your sympathy and friendship), Ana Silva (For you, the sky is the limit…close your eyes, close your mouth and forget the others, you are “the important” because you have everything to be it), Anita Lourenço (Thank you for your participation in this chapter of my life), Márcia Tavares (“Hard Rock Girl”; It was a pleasure to work with you), Renato Cabral (“Great Engineer”: it was a pleasure to meet you), Vasco Bonifácio (“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: Íris 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 iii work; I will miss our brainstorming about computational modelling), Cláudia Fernandes and Bianca Gonçalves (“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, everything…You 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 José and Isabel Rodrigues for their assistance in bureaucratic work, and to Maria de Palma, Idalina and Conceição 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: Sónia 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, Pêlo, 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, Inês, 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 Fundação para a Ciência 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 Fundação Calouste Gulbenkian. iv 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 scCO drying. A further optimization for opening the 2 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 functionalized natural-based monoliths revealed values of pore size diameter, porosity, and flux between 1-96 µm, 28-88 % and 3-220 (L m-2 h-1). Chitosan/poly(vinyl alcohol)- based monoliths revealed the best binding and elution capacities, 160 mg IgG g-1 support and 97%, respectively, at least over four consecutive cycles. Moreover, tested with crude samples, supports exhibited a good specificity for mAbs, recovering them with 96-98% of purity. KEYWORDS: Biopolymers, monoliths, affinity ligand, plasma technology, supercritical carbon dioxide, green chemistry, antibody purification. v vi RESUMO Este trabalho teve como objectivo o desenvolvimento de suportes monolíticos para a purificação de anticorpos por cromatografia de afinidade. Para tal, utilizou-se uma estratégia sustentável para produzir essas estruturas porosas 3D (monólitos), envolvendo polímeros naturais tais como quitosano, dextrano e agarose. Para melhorar as propriedades mecânicas e biodegradáveis dos monólitos, os polímeros naturais foram misturados fisicamente com polímeros sintéticos. Primeiramente, todos os suportes foram produzidos por liofilização, e numa segunda fase por processos de gelificação, substituição de água por acetona e secagem por scCO . A optimização da abertura da rede porosa foi efectuada recorrendo à capacidade 2 de inchamento dos suportes e subsequente liofilização. Para melhorar a performance dos monólitos, incorporaram-se nanopartículas magnéticas nas redes monolíticas por forma a conferir-lhes a capacidade de resposta magnética e consequente deformação quando sob acção de um campo magnético. Realizaram-se assim eluições assistidas por campo magnétco o que permitiu o aumento do rendimento de recuperação de anticorpo (IgG) ligado (93%) e diminuir o tempo do passo de eluição. A selectividade dos monólitos para o anticorpo foi garantida através da imobilização de dois ligandos sintéticos mimetizando a Proteína A (ligando 22/8 e o novo ligando TPN-BM) na superfície dos suportes. A estratégia de funcionalização do TPN-BM, cuja síntese seguiu os princípios da química verde, foi feita utilizando a tecnologia de plasma. Esta estratégia permitiu reduzir tempo e uso de solventes bem como maximizar todo o processo (em 2 vezes) comparativamente aos procedimentos tradicionais. O mecanismo de ligação/eluição do TPN-BM e IgG foi validado através de estudos de acoplamento molecular e simulações dinâmicas. Em geral, todos os monólitos TPN-BM-funcionalizados exibiram valores de diâmetro de poro, porosidade e fluxo entre 1-96 µm, 28-88 % e 3-220 (L m-2h-1), respectivamente. Os monólitos de quitosano/poli(vinil álcool) revelaram as melhores capacidades de ligação e de eluição, 160 mg IgG g-1 suporte e 97%, respectivamente, pelo menos durante quatro ciclos consecutivos. Adicionalmente, quando testados com extractos brutos, exibiram uma boa especificidade para mAbs, recuperando-os com 96-98% de pureza. . PALAVRAS-CHAVE - Biopolímeros, monólitos, ligandos de afinidade, tecnologia de plasma, dióxido de carbono supercrítico, química verde, purificação de anticorpos. vii viii