Development of novel purification platform for Fab antibody fragments based on affinity peptides and multimodal ligands
• Ana Margarida Azevedo, Professor @ IST/ULisboa, PT
• Steven Cramer, Professor @ Rensselaer Polytechnic Institute, Troy, NY, USA
• Pankaj Karande, Professor @ Rensselaer Polytechnic Institute, Troy, NY, USA
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.
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.
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
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
Analytical Chemist @ HOVIONE, Sete Casas, 2674 – 506 Loures, Portugal