Class 2015
PD/BD/113628/2015
PhD thesis
Development of novel purification platform for Fab antibody fragments based on affinity peptides and multimodal ligands
Supervisors
• Ana Margarida Azevedo, Professor @ IST/ULisboa, PT
• Steven Cramer, Professor @ Rensselaer Polytechnic Institute, Troy, NY, USA
Collaborators
• Pankaj Karande, Professor @ Rensselaer Polytechnic Institute, Troy, NY, USA
PhD Degree complete: Discussion on April 6 2020
THESIS ABSTRACT
Antibody derivates are a class of engineered proteins idealized as alternatives to monoclonal antibodies. The production of Fab fragments, the oldest of this class, in comparison to mAbs is not as efficient, especially the downstream processing. To tackle this limitation, two different approaches were suggested as new solutions to purify Fab fragments. Peptide affinity chromatography and multimodal chromatography were the chosen and evaluated solutions. In this thesis an evolutive approach, based on phage display, was taken to screen and discover peptide affinity ligands capable of binding to Fab fragments. Three different biopanning schemes were designed, with increase complexity and increased focus on the constant regions of Fabs. The outcome were five different peptides with the ability to bind Fab fragments. While a truly universal ligand was not identified, three of those obtained peptides can be considered ligands specific to three or more closely related biologics. The application of one of those peptides as a chromatographic affinity solution was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The potential of multimodal chromatography is as big as its complexity. To rapidly screen chromatographic conditions to purify Fab fragments using multimodal ligands, a highthroughput platform, based on chromatographic microfluidics was developed. An exhaustive study of eleven multimodal resins, targeting Fabs, other proteins and two complex mixtures, at different pH and salt conditions was executed. With this highthroughput approach a sustained selection of the best ligands and chromatographic conditions was taken, and the design of a full multimodal Fab downstream process was made. This work lays the foundation for the discovery new purification solutions for a class of emergent antibody derives. The two different approaches adopted can be considered valuable contributions for a more efficient purification process of Fab fragments and other types of biomolecules.
RESUMO DA TESE
Derivados de anticorpo são uma classe de proteínas idealizadas como alternativas a anticorpos monoclonais. A produção de fragmentos de anticorpo, as proteínas mais velhas desta classe, não é eficiente, especialmente na fase a jusante do processamento. De modo a resolver esta limitação, duas abordagens foram sugeridas como novas alternativas à purificação de Fragmentos de anticorpo. Cromatografia de afinidade usando péptidos e cromatografia multimodal foram as duas soluções escolhidas e avaliadas. Nesta tese, uma abordagem evolutiva, baseada na técnica de phage display, foi usada para rastear e descobrir péptidos de afinidade capazes de ligar a fragmentos de anticorpo. Três diferentes esquemas de selecção de péptidos foram desenhados, onde a complexidade e foco na região constante do fragmento era aumentada. O resultado foram cinco péptidos com a capacidade de ligar a fragmentos de anticorpo. Apesar de nenhum péptido pode ser considerado um ligando universal, três dos cinco podem ser considerados ligandos específicos para três, ou mais, proteínas semelhantes. O uso de um dos péptidos como solução de cromatografia de afinidade foi desenvolvida, tendo a sua eficácia demonstrada através da purificação de fragmentos de anticorpo presente num sobrenadante complexo. O potencial de cromatografia multimodal é tão grande quanto sua complexidade. De modo a fazer um varrimento rápido de diferentes condições de cromatografia, para purificar fragmentos de anticorpo usando ligandos multimodais, uma plataforma microfluídica de alto rendimento foi desenvolvida. Um estudo exaustivo, de onze resinas multimodais, visando fragmentos de anticorpo, outras proteínas e duas misturas complexas, a diferentes pHs e concentrações de sal, foi feito. Com esta abordagem, uma selecção sustentada dos melhores ligandos e condições cromatográficas foi feita, e o desenho de uma processo de purificação totalmente baseado em ligandos multimodais foi concebido. Este trabalho lança bases para a descoberta de novas soluções de purificação para uma classe emergente de derivados de anticorpo. As duas abordagens adoptadas podem ser consideradas contribuições valiosas para um processo de purificação de fragmentos de anticorpo, ou outras biomoléculas, mais eficiente.
PUBLICATIONS
Papers
A. Nascimento, I.F. Pinto, V. Chu, M.R. Aires-Barros, J.P. Conde, A.M. Azevedo, Studies on the purification of antibody fragments, Separation and Purification Technology 195: 388–397, 2018.
A. Nascimento, A. Mullerpatan, P. Karande, A.M. Azevedo, S.M. Cramer, Development of Phage Biopanning Strategies to Identify Affinity Peptide Ligands for Kappa Light Chain Fab Fragments. Biotechnology Progress 35(6):e2884, 2019.
A. Nascimento, M.N. São Pedro, I.F. Pinto, M.R. Aires-Barros, A.M. Azevedo, “Microfluidics as a high-throughput solution for chromatographic process development – The complexity of multimodal chromatography used as a proof of concept”, J. Chromatogr. A 1658: 462618, 2021
Oral Communications
Nascimento A, Mullerpatan A, Azevedo AM, Aires-Barros MR, Karande P, Cramer S, “Phage display-aided identification of affinity peptide ligands against Kappa Fab fragments”, 12th ESBES – European Symposium on Biochemical Engineering Sciences, September 2018, Lisboa, Portugal
A. Nascimento, M.N. São Pedro, I.F. Pinto, A.M. Azevedo. “A Microfluidic high-throughput platform to debottleneck downstream process development”, 5th International conference on High Throughput Process Development – HTDP, November 4-7, 2019 Porto, Portugal
A. Nascimento, M.N. São Pedro, I.F. Pinto, A.M. Azevedo. “A Microfluidic high-throughput platform to debottleneck downstream process development”, Affinity 2019, June 26-28, 2019, Stockolm, Sweden
Poster Communications
A. Nascimento, S.A.S.L. Rosa, M. Mateus, A.M. Azevedo. “Polishing of Monoclonal Antibodies Streams through Convection Flow Devices”, MicroBiotec2015, December 10-12, 2015, Évora, Portugal
A. Nascimento, S.A.S.L. Rosa, M. Mateus, A.M. Azevedo. “Convection Flow Devices for the Polishing of Monoclonal Antibodies”, XVI Latin-American Congress on Chromatography | XVI COLACRO, January, 2016, Lisboa, Portugal
A. Nascimento, A. Mullerpatan, A.M. Azevedo, M.R. Aires-Barros, P. Karande, S.M. Cramer. “Phage display-aided identification of affinity peptide ligands against Kappa Fab fragments”, 255th ACS National Meeting, March 18-22, 2018 New Orleans, Louisiana, USA
A. Nascimento, A. Mullerpatan, A.M. Azevedo, P. Karande, S.M. Cramer. “Development of phage biopanning strategies for the discovery of Fab-binding peptide affinity ligands: an evolutionary approach to universality”, Affinity 2019, June 26-28, 2019, Stockolm, Sweden
DOCTORAL PROGRAM (36 ECS):
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Advanced Topics in Bioengineering and Biosciences (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Animal Cell Technology (6 ECTS)
Advanced Courses: World Talent Campus in Biotechnology Manufacturing, Focus on monitoring and optimization of bioprocesses, Technical University of Denmark, under the supervision of Prof. Krist V. Gernaey and Prof. Franciscus Berg,, August 18-25, 2016, DK
Current position/institution
Analytical Chemist @ HOVIONE, Sete Casas, 2674 – 506 Loures, Portugal