Clarification and capture of monoclonal antibodies from complex media using aqueous two-phase ...

93 Pages·2014·2.04 MB·English

Clarification and capture of monoclonal antibodies from
complex media using aqueous two-phase systems

Isabel Cardoso Alves de Campos Pinto

Thesis to obtain the Master of Science Degree in
Biotechnology

Supervisors: Doctor Ana Margarida Nunes da Mata Pires de Azevedo
Professor Maria Raquel Múrias dos Santos Aires Barros

Examination Committee
Chairperson: Professor Luís Joaquim Pina da Fonseca
Supervisors: Doctor Ana Margarida Nunes da Mata Pires de Azevedo
Member of the committee: Doctor Ana Gabriela Gonçalves Neves Gomes

December 2014

The real voyage of discovery consists not in seeking new landscapes, but in seeing with new eyes.
Marcel Proust, 1913

Acknowledgments

This thesis could never have been made without the help of my supervisors Doctor Ana Azevedo and
Professor Raquel Aires Barros. I would like to thank them, first, for giving me the opportunity to integrate
the Bioseparation Engineering Laboratory (BEL) and, second, for all your dedication, guidance, availability
and (very precious) knowledge given during all this work. A special thanks to Dr. Ana Azevedo for the
encouragement and comprehension. I know you gave your best and I hope I have been a good pupil.
I would like to thank the European Committee for supporting and funding the European Project INTENSO,
in which my work is included. And also to the direct partners that were involved in my work, namely IcoSagen
and Biomedal.
To Sara Rosa for all your work spent in providing me the cell cultures and time spent in teaching me several
techniques.
To all my lab colleagues Dragana Barros, Edith Espitia Saloma, António Grilo, Inês Pinto, Sara Rosa,
Raquel Santos, Maria João Jacinto and Sandra Bernardo. Not only for sharing with me your knowledge and
ideas but also for your fellowship. To Inês, in particular, I want to thank you for your sincere friendship, which
I pretend to keep.
To my master colleagues who have embarked on this adventure of biotechnology with me. Specially to
Diana Marques, Rita Fernandes, Ana Faria, Liliana Brito, Cátia Jorge, Fábio Gonçalves and Carlos
Rodrigues. I also have to thank to Pedro Pereira, who despite not being neither my master's colleague nor
lab partner, often brightened my day. You all gave me extraordinary moments, which I certainly will never
forget.
To my always and forever friends Inês, Gonçalo, Brito, Kiko and Mafalda. You helped me in so many ways
(even though you were not aware you were doing it). I will always need you to cheer up my life.
To João, the most amazing person I’ve ever met, in every single way, I thank you for always believing in me
and in my work. I truly thank you for everything!
Finally to my family. My mum and dad in particular, thank you for your patience and unconditional love, you
mean everything to me. Thank you to my big sister, my almost second mum. Thank you for having giving
me the most perfect nieces in the world that so much rejoice my life! Thank you to my grandfathers for the
wisdom and inspiration. I will never be thankful enough for what you all gave to me!
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This thesis is dedicated to every individual person here referred. You have taught me so many things in so
different ways. I feel I grew up a million times not only as a scientist but as an individual human being. It
was a great year and a wonderful experience. Thank you all!

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Resumo
Os anticorpos monoclonais (mAbs) constituem uma das classes de produtos biofarmacêuticos mais
importantes da indústria farmacêutica. A sua elevada procura tornou necessária a criação de processos de
fabrico capazes de produzir rapidamente elevadas quantidades de mAbs, a preços competitivos e de um
modo consistente e reprodutível. Atualmente, as principais limitações das plataformas de fabrico
encontram-se nos processos de purificação, onde o principal desafio consiste no desenvolvimento de um
processo robusto capaz de fazer a integração da produção e da purificação. Os sistemas de duas fases
aquosas (ATPS) são uma alternativa válida às plataformas estabelecidas devido ao seu “scale-up” fácil, à
sua capacidade de operação em contínuo e ao seu elevado rendimento. Além disso permite integrar os
processos de clarificação, concentração e purificação numa única etapa.
Neste trabalho foi desenvolvido um processo de purificação, com base nos ATPS, capaz de purificar mAbs
a partir de um meio de cultura complexo, abrangendo a separação de células e a extração seletiva de
anticorpos. Posteriormente, foi também realizado um passo de polimento por cromatografia de troca
catiónica com o objetivo de aumentar a pureza do anticorpo. Foram realizados estudos de partição de
mAbs a partir de sobrenadantes de células CHO utilizando diferentes tipos de ATPS, nomeadamente, PEG-
NaPA, PEG-dextrano e PEG-cloreto de colina. O efeito da presença do ligando LYTAG-ProA na partição
dos anticorpos foi ainda avaliado. Os sistemas PEG-NaPA apresentaram elevados rendimentos a nível da
extracção de mAbs (sem recurso ao uso do ligando LYTAG-ProA) e mostraram-se ideais no passo de
clarificação de células.

Palavras-chave: Sistemas de duas fases aquosas; anticorpos monoclonais; purificação; clarificação

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Abstract

Monoclonal antibodies (mAbs) are within the most important biopharmaceutical products of the
pharmaceutical industry. Their great demand, led to the need to create production processes that rapidly
produce large quantities of pharmaceutical mAbs at moderate costs and in a consistent and reproducible
manner. Major limitations in current manufacturing platforms are no longer found upstream but in the
downstream processing. Challenges in purification include developing robust processes with integration of
the upstream and downstream, allowing efficient, sustainable and cost-effective processes. Aqueous-two
phase systems (ATPS) shown to be a valuable alternative to the established platforms due to its easy
scalability, capacity of continuous operation and high capacity. Besides that, clarification, concentration and
purification can be achieved in just one step, using a biocompatible environment.
In this work, the design of a downstream process was developed based on ATPS for the purification of
mAbs from a complex medium, comprising cell separation and antibody selective extraction, envisaging
process integration and intensification. Subsequently, it was also performed a polishing step of cation
exchange chromatography, in order to increase the purity of the antibody. Partition studies of mAbs from
CHO cell supernatants were investigated using different types of ATPS, namely PEG-NaPA, PEG-dextran
and PEG-choline chloride. The effect of the ligand LYTAG-ProA was also evaluated in the partition of the
antibodies. PEG-NaPA systems showed high yields of extraction of mAbs (without the use of ligand-LYTAG
ProA) and shown to be an optimal system for the clarification of cells, with 100% of elimination of cells from

the IgG-rich phase.

Keywords: Aqueous two-phase systems; monoclonal antibodies; downstream processing, cell

clarification

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Index
ACKNOWLEDGMENTS.................................................................................................... I
RESUMO ......................................................................................................................... III
ABSTRACT .................................................................................................................... IV
INDEX ............................................................................................................................. V
LIST OF FIGURES ....................................................................................................... VIII
LIST OF TABLES ........................................................................................................ XIII
LIST OF ABBREVIATIONS ......................................................................................... XIV
BACKGROUND AND AIM OF STUDIES......................................................................... 1
1. INTRODUCTION ....................................................................................................... 3
1.1. Monoclonal Antibodies (mAbs)...................................................................................................... 3
1.1.1. Antibody Structure and Functional Features ............................................................................. 3
1.1.2. Polyclonal Versus Monoclonal Antibodies: Biotechnological Value .......................................... 5
1.1.3. Market Considerations ............................................................................................................... 6
1.1.4. Upstream Processing of mAbs .................................................................................................. 7
1.1.4.1. Hybridoma Technology .......................................................................................................... 8
1.1.4.2. Recombinant DNA Technology ........................................................................................... 10
1.1.4.3. Large Scale Production ....................................................................................................... 11
1.1.5. Downstream Processing of mAbs ........................................................................................... 13
1.1.5.1. Downstream processing of mAbs: Alternative processes ................................................... 15
1.2. Aqueous Two-Phase System ....................................................................................................... 17
1.2.1. Principles ................................................................................................................................. 17
1.2.2. Two Phase Formation Phenomena ......................................................................................... 19
1.2.3. Factors Influencing Partitioning ............................................................................................... 19
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1.2.4. Development of an Aqueous Two-Phase Extraction Process ................................................. 20
1.2.4.1. Physicochemical Characterization of the Feedstock ........................................................... 21
1.2.4.2. ATPS Type Selection .......................................................................................................... 21
1.2.4.3. System Parameters Selection ............................................................................................. 22
1.3. Process Integration Using ATPSs ............................................................................................... 22
1.3.1. Process Integration Using ATPSs: Some Studies ................................................................... 23
1.4. Affinity Partition in ATPSs ............................................................................................................ 24
1.4.1. Affinity Partition Driven by LYTAG ........................................................................................... 25
1.4.2. Preliminary Studies in Partitioning of GFP-LYTAG ................................................................. 27
1.4.3. Potential of LYTAG-Protein A in the Recovery of mAbs ......................................................... 28
1.5. Aqueous Two-Phase Systems vs ProA Chromatography ........................................................ 29
1.5.1. Integrating ATPS Extraction with Ion Exchange Chromatography (IEX) ................................ 31
1.6. ATPSs: A Tool for the Purification of mAbs (some studies) .................................................... 31
2. MATERIALS AND METHODS ................................................................................ 33
2.1. Chemicals ....................................................................................................................................... 33
2.2. Biologicals...................................................................................................................................... 33
2.2.1. CHO cell supernatant – Icosagen............................................................................................ 33
2.2.2. Cell Culture (Hybridoma cells) ................................................................................................. 34
2.2.3. LYTAG-Protein A ..................................................................................................................... 35
2.3. Preparative Methods ..................................................................................................................... 35
2.3.1. Aqueous Two-Phase Systems (ATPS).................................................................................... 35
2.3.2. Aqueous two-phase extraction (ATPE) ................................................................................... 36
2.3.3. Capture of IgG – Cation Exchange Chromatography.............................................................. 37
2.3.4. Diafiltration ............................................................................................................................... 37
2.4. Analytical Methods ........................................................................................................................ 38
2.4.1. IgG quantification – HPLC ....................................................................................................... 38
2.4.2. Total protein quantification – Bradford assay .......................................................................... 38
2.4.3. Purity evaluation – Protein gel electrophoresis ....................................................................... 39
2.4.4. Cell Counting ........................................................................................................................... 39
2.5. Extraction performance parameters ............................................................................................ 40
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