Clarification and capture of monoclonal antibodies from ... · Os anticorpos monoclonais (mAbs)...

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

i

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!

ii

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!

iii

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

iv

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

v

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

vi

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

vii

2.5.1. Cell Counting ........................................................................................................................... 41

3. RESULTS AND DISCUSSION ................................................................................ 42

3.1. Characterization of the Protein Media ......................................................................................... 42

3.2. Evaluation of PEG-NaPA ATPS .................................................................................................... 43

3.2.1. Partitioning of mAbs in PEG-NaPA ATPS using LYTAG-ProA ............................................... 43

3.2.2. Integrated Clarification of Hybridoma Cell Cultures ................................................................ 46

3.2.3. Polishing through CEX Chromatography ................................................................................ 47

3.3. Evaluation of PEG-Dextran ATPS ................................................................................................ 49

3.3.1. Partitioning of mAbs in PEG-Dextran ATPS using LYTAG-ProA ............................................ 49

3.3.2. Integrate Clarification of Hybridoma Cell Cultures .................................................................. 52


3.4. Evaluation of PEG-Choline Chloride ATPS ................................................................................. 56

3.4.1. Partitioning of mAbs in PEG-Choline chloride ATPS using LYTAG-ProA............................... 56

3.4.2. Integrated Clarification of Hybridoma Cell Cultures ................................................................ 59


4. CONCLUSIONS AND FUTURE WORK .................................................................. 63

5. BIBLIOGRAPHY ..................................................................................................... 66

6. ANNEXES ................................................................................................................ 70

viii

List of Figures

Figure 1 – (A) Schematic diagram of a conventional IgG molecule. Y-shaped protein composed by two light

chains (orange) and two heavy chains (blue). The heavy chains each consist of three constant sections

(CH1, CH2, and CH3) and one variable section (VH). The light chains each consist of a constant region (CL)

and a variable region (VL). The antigen-binding sites are formed by the juxtaposition of VL and VH domains.

The bottom section of the antibody is the Fc region to which two arms, the Fab regions, are attached.

Disulphide bonds connect heavy and light chains. The variable domains have complementary-determining

regions (CDR) that bind directly to the antigen. (B) A three-dimensional ribbon model of a G-class

immunoglobulin (IgG) molecule. The blue and green sections are the heavy chains, while the orange and

pink sections on the arms of the antibody are the light chains of the molecule. ........................................... 4

Figure 2 – Generation of monoclonal antibodies by hybridoma technology technique (Abbas et al. 1994) 8

Figure 3 – Antibody engineering for humanization. Therapeutic mAbs can be murine (100% murine protein),

chimeric (composed of 67% of human constant domains), humanized (only possess 5-10% of murine

regions) or fully human (100% human proteins) (Carter 2001). .................................................................... 9

Figure 4 – Schematic representation of fed-batch and perfusion culture systems. The fed-batch system is

supplied with a concentrated nutrient solution (no spent culture medium is removed). In perfusion culture

systems fresh nutrient solution is supplied to the vessel at the same rate that spent medium is withdrawn;

cells are, however, returned to the bioreactor. ............................................................................................ 12

Figure 5 – Standard platform downstream process for mAbs. Cell culture supernatant is typically purified

by a capture step with Protein A chromatography. In order to remove all contaminates post Protein A capture

and to obtain purity to regulatory compliance, two additional chromatographic polishing steps are employed.

(Rosa et al. 2010). ....................................................................................................................................... 13

Figure 6 – Schematic protein A HPLC (high pressure liquid chromatography) purification. The Protein A

ligand is immobilized on to the column. Following crude sample loading, the mAb is retained by affinity

binding to Protein A. Washing is employed to remove nonspecific binding. Elution of the mAb is with a low

pH elution buffer. ......................................................................................................................................... 14

Figure 7 – Schematic representation of a phase diagram for ATPS. Bottom phase polymer/salt X (% w/w)

is plotted on the abscissa and top phase polymer Y (% w/w) is plotted on the ordinate. A1, A2, and A3

represent the total compositions of three systems lying on the same tie-line with different volume ratios. The

final composition of the top and bottom phase is represented by nodes T and B, respectively. The ratio of

the segments AB (top phase) and AT (bottom phase) can be approximated graphically by the volume ratio

of the two phases. ....................................................................................................................................... 18

Figure 8 – Representation of the proposed strategy for the predictive development of recovery processes

using ATPS according Benavides and Rito-Palomares (Benavides et al. 2008). ....................................... 21

file:///C:/Users/Isabel/Desktop/Tese_isabel_v4.docx%23_Toc404091824

ix

Figure 9 – Simplified representation of process integration of ATPS and fermentation for intra and extra-

cellular products. The flow diagram represents the extractive fermentation ATPS process in which, the

production and the recovery of the target product can be integrated in one single unit operation. Alternative

to it is represented in the integrated process of cell disruption and ATPS for the recovery of intracellular

products (Rito-Palomares 2004). ................................................................................................................ 23

Figure 10 – Ribbon diagram of the c-terminal domain of the major autolysin, C-LytA, from Streptococcus

pneumonia with β strand assignment .......................................................................................................... 26

Figure 11 – General procedure for purification of C-LytA fused proteins by PEG-phosphate or PEG-dextran

ATPSs. ......................................................................................................................................................... 26

Figure 12 – (A) Photographs taken during purification of GFP-C-LytA in PEG/phosphate by Maestro et al

(Maestro et al. 2008). From left to the right equilibration, wash, elution 1 and elution 2 steps are represented.

(B) Effect of extract concentration on the partition properties of GFP-C-LytA in PEG/phosphate. (A) 0.45

mg/mL; (B) 0.9 mg/mL; (C) 1.9 mg/mL; (D) 4.0 mg/mL. Samples were illuminated with UV light in order to

induce GFP fluorescence (Maestro et al. 2008) .......................................................................................... 27

Figure 13 – Comparing the chemical structure of choline and polyethylene glycol. Both molecules present

a CH2-OH termination, which makes them structural analogues. ............................................................... 28

Figure 14 – Schematic diagram illustrating the behavior of antibodies in presence of LYTAG-ProA in ATPS.

After phase separation, LYTAG-ProA-mAb complexes stay retained in PEG-rich phase, while cell debris

and other proteins go to the PEG poor phase. ............................................................................................ 29

Figure 15 – Purity evaluation of the three types of feed that will be used for ATPS experiments through

SDS-PAGE electrophoresis. M: Protein molecular weight marker; Lane 1: pure IgG; Lane 2: Hybridoma cell

culture; Lane 3: Icosagen supernatant. Position of IgG heavy (H) (50 kDa) and light (L) (25 kDa) chains are

indicated in the right side of the gel. ............................................................................................................ 42

Figure 16 – Effect of LYTAG-ProA on IgG partitioning (Log Kp) (A) and on the recovery yield (B) for each

phase (T: Top phase; B: Bottom phase) in PEG-NaPA systems. System composition: 8% PEG 3350, 6%

NaPA, 10 mM of phosphate buffer pH 7, 500 mM of NaCl, 35% of feedstock and 15% of LYTAG-ProA (only

for systems with ligand, marked as + LYTAG). Results were obtained after quantification of IgG in each

phase, by affinity chromatography. IgG concentration was determined from a calibration curved obtained

using Gammanorn IgG as a standard. The IgG recovery yield in each phase was achieved by the ratio of

the IgG mass in the phase by the IgG mass in the feed extract. ................................................................ 44

Figure 17 – Qualitative analysis of the purity of both phases from PEG-NaPA ATPS through SDS-PAGE

electroforese. (A) Lane M: Protein molecular weight marker; Lane 1: pure IgG Feedstock; Lane 2 – Top

phase with pure IgG without LYTAG-ProA; Lane 3 – Top phase with Icosagen supernatant without LYTAG-

ProA; Lane 4 – Top phase with pure IgG with LYTAG-ProA; Lane 5 – Top phase with Icosagen supernant

with LYTAG-ProA; Lane 6 – Botttom phase with pure IgG without LYTAG-ProA; Lane 7 – Bottom phase

x

with icosagen supernant without LYTAG-ProA; Lane 8 – Bottom phase with pure IgG with LYTAG-proA;

Lane 9- Bottom phase with Icosagen with LYTAG-ProA. (B) Lane M: Protein molecular weight marker; Lane

1: hybridoma cell culture feedstock; Lane 2 – Top phase with hybridoma cell culture with LYTAG-ProA; Lane

2 – Top phase with hybridoma cell culture without LYTAG-ProA; Lane 4 - Bottom phase with hybridoma cell

culture with LYTAG-ProA; Lane 5 - Bottom phase with hybridoma cell culture without LYTAG-ProA. ...... 45

Figure 18 – Microscopy observation of hybridoma cells in top phase (A), interface (B) and bottom phase

(C) of the PEG-NaPA ATPS. Cells were stained with trypan blue and visualized at 4× magnification and with

25 ms exposure time in an optical microscope. .......................................................................................... 47

Figure 19 – Chromatography runs of the separation of IgG from the ATPS top phases, after loading 1 mL

on two cation exchange columns: Fibers (A) and HiTrap SP FF (B). Adsorption was performed in 0.02 M

sodium acetate at pH 7 and elution was performed in a step gradient with 0.02 M sodium acetate buffer at

pH 7 containing 1 M NaCl............................................................................................................................ 48


phase (T: Top phase; B: Bottom phase) in PEG-Dextran systems. System composition: 6% PEG 3350, 7%

Dextran 500 kDa, 35% of feedstock and 15% of LYTAG-ProA (only for systems with ligand, marked as +

LYTAG). Results were obtained after quantification of IgG in each phase, by affinity chromatography. IgG

concentration was determined from a calibration curved obtained using Gammanorn IgG as a standard. The

IgG recovery yield in each phase was achieved by the ratio of the IgG mass in the phase by the IgG mass

in the feed extract. ....................................................................................................................................... 50

Figure 21 – Qualitative analysis of the purity of both phases from PEG-Dextran ATPS through SDS-PAGE

electroforese. (A) Lane M: Protein molecular weight marker; Lane 1 - pure IgG feedstock; Lane 2 - Top

phase with pure IgG without LYTAG-ProA; Lane 3 – Top phase with pure IgG with LYTAG-ProA; Lane 4 -

Bottom phase with pure IgG without LYTAG-ProA; Lane 5 - Bottom phase with pure IgG with LYTAG-ProA.

(B) Lane M: Protein molecular weight marker; Lane 1 - Top phase with Icosagen supernatant without

LYTAG-ProA; Lane 2 - Top phase with Icosagen supernatant with LYTAG-ProA; Lane 3 – Icosagen

supernatant feedstock; Lane 4 - Bottom phase with Icosagen supernatant without LYTAG-ProA; Lane 5 -

Bottom phase with Icosagen supernatant with LYTAG-ProA. ..................................................................... 51


(C) of the PEG-Dextran ATPS. Cells were stained with trypan blue and visualized at 4× magnification and

with 25 ms exposure time in an optical microscope. ................................................................................... 53


on cation exchange Fibers columns. Adsorption was performed in 0.02 M sodium acetate at pH 7 and the

elution was performed in a step gradient with 0.02 M sodium acetate buffer at pH 7 containing 1 M NaCl

(A); and respective qualitative analysis of the purity of both phases of the ATPS and of flowthrough and

elution pools resulting the chromatography run, through SDS-PAGE electrophorese (B). Lane M: Protein

molecular weight marker in kDa; Lane 1: Hybridoma cell culture feedstock; Lane 2 – Top phase with

xi

hybridoma cell culture without LYTAG-ProA; Lane 3 – Flowthrough pool fraction resulting from a PEG-

Dextran ATPS without LYTAG-ProA; Lane 4 - Eluate pool fraction resulting from a PEG-Dextran ATPS

without LYTAG-ProA; Lane 5 – Top phase with hybridoma cell culture with LYTAG-ProA; Lane 6 -

Flowthrough pool fraction resulting from a PEG-Dextran ATPS without LYTAG-ProA; Lane 7 - Eluate pool

fraction resulting from a PEG-Dextran ATPS with LYTAG-ProA; Lane 8 - Bottom phase with hybridoma cell

culture without LYTAG-ProA; Lane 9 - Bottom phase with hybridoma cell culture with LYTAG-ProA. ...... 54


on cation exchange HiTrap SP FF column. Adsorption was performed in 0.02 M sodium acetate at pH 7 and

the elution was performed in a step gradient with 0.02 M sodium acetate buffer at pH 7 containing 1 M NaCl

(A); and SDS-PAGE analysis of both phases of the ATPS and of flowthrough and eluate fractions collected

during the purification of IgG represented at the left (B). Lane M: Protein molecular weight marker; Lane 1:

Hybridoma cell culture feedstock; Lane 2 – Top phase with hybridoma cell culture without LYTAG-ProA;

Lane 3 – Flowthrough pool fraction resulting from a PEG-Dextran ATPS without LYTAG-ProA; Lane 4 -

Eluate pool fraction resulting from a PEG-Dextran ATPS without LYTAG-ProA; Lane 5 – Top phase with

hybridoma cell culture with LYTAG-ProA; Lane 6 - Flowthrough pool fraction resulting from a PEG-Dextran

ATPS without LYTAG-ProA; Lane 7 - Eluate pool fraction resulting from a PEG-Dextran ATPS with LYTAG-

ProA; Lane 8 - Bottom phase with hybridoma cell culture without LYTAG-ProA; Lane 9 - Bottom phase with

hybridoma cell culture with LYTAG-ProA after diafiltration. ........................................................................ 55


phase (T: Top phase; B: Bottom phase) in PEG-choline chloride systems. System composition: 24% PEG

3350, 7% choline chloride, 5.5% of phosphate buffer at pH 8, 20% of feedstock and 5% of LYTAG-ProA

(only for systems with ligand, marked as + LYTAG). Results obtained after quantification of IgG in each

phase, by affinity chromatography. IgG concentration was determined from a calibration curved obtained

using Gammanorn IgG as a standard. The IgG recovery yield in each phase was achieved by the ratio of

the IgG mass in the phase by the IgG mass in the feed extract. ................................................................ 57

Figure 26 – Qualitative analysis of the purity of both phases from PEG-choline chloride ATPS through SDS-

PAGE electrophorese. Lane M: Protein molecular weight marker; Lane 1 - pure IgG feedstock; Lane 2 - Top

phase with pure IgG without LYTAG-ProA; Lane 3 – Top phase with pure IgG with LYTAG-ProA; Lane 4 -

Bottom phase with pure IgG without LYTAG-ProA; Lane 5 - Bottom phase with pure IgG with LYTAG-ProA

(A). Lane M: Protein molecular weight marker; Lane 1 – Icosagen supernatant feedstock; Lane 2 - Top

phase with Icosagen supernatant without LYTAG-ProA; Lane 3 - Top phase with Icosagen supernatant with

LYTAG-ProA; Lane 4 - Bottom phase with Icosagen supernatant without LYTAG-ProA; Lane 5 - Bottom

phase with Icosagen supernatant with LYTAG-ProA (B) ............................................................................ 58


(C) of the PEG-choline chloride ATPS. Cells were stained with trypan blue and visualized at 4× magnification

and with 25 ms exposure time in an optical microscope. ............................................................................ 59

xii

Figure 28 – Chromatography runs at three different pH for the separation of IgG from the ATPS bottom

phases, after loading 1 mL, on Fibers cation exchange column. Adsorption was performed in 0.02 M sodium

acetate at pH 5/6/7 and the elution was performed in a step gradient with 0.02 M sodium acetate buffer at

pH 5/6/7 containing 1 M NaCl (A). SDS-PAGE analysis of the top and bottom phase from ATPS with

hybridoma cell culture and fractions collected during the purification of IgG represented at the left (B). Lane

M: Protein molecular weight marker; Lane 1: Hybridoma cell culture feedstock; Lane 2 – Top phase with

hybridoma cell culture without LYTAG-ProA; Lane 3 – Bottom phase with hybridoma cell culture without

LYTAG-ProA; Lane 4 - Flowthrough pool fraction resulting from purification at pH 5; Lane 4 - Eluate pool

fraction resulting from purification at pH 5; Lane 6 – Flowthrough pool fraction resulting from purification at

pH 6; Lane 7 - Eluate pool fraction resulting from purification at pH 6; Lane 8 - Flowthrough pool fraction

resulting from purification at pH 7 ; Lane 9 - Eluate pool fraction resulting from purification at pH 7. ........ 60

Figure 29 – Chromatography runs at three different pH for the separation of IgG from the ATPS bottom

phases, after loading 1 mL, on cation exchange HiTrap SPFF column. Adsorption was performed in 0.02 M

sodium acetate at pH 5/6/7 and the elution was performed in a step gradient with 0.02 M sodium acetate

buffer at pH 5/6/7 containing 1 M NaCl (A). SDS-PAGE analysis of the top and bottom phase from ATPS

with Hybridoma cell culture and fractions collected during the purification of IgG represented at the left (B).

Lane M: Protein molecular weight marker; Lane 1: Hybridoma cell culture feedstock; Lane 2 – Top phase

with hybridoma cell culture without LYTAG-ProA; Lane 3 – Bottom phase with hybridoma cell culture without

LYTAG-ProA; Lane 4 - Flowthrough pool fraction resulting from purification at pH 5; Lane 4 - Eluate pool

fraction resulting from purification at pH 5; Lane 6 – Flowthrough pool fraction resulting from purification at

pH 6; Lane 7 - Eluate pool fraction resulting from purification at pH 6; Lane 8 - Flowthrough pool fraction

resulting from purification at pH 7 ; Lane 9 - Eluate pool fraction resulting from purification at pH 7. ........ 62

Figure 30 - Typical calibration curve used for IgG quantification, obtained from IgG stock solutions with

concentrations ranging from 0.2 mg/L to 20 mg/L. ...................................................................................... 70

Figure 31 - Typical calibration curve used for total protein quantification, obtained from BSA standards with

concentrations ranging from 5 mg/L to 400 mg/L. ....................................................................................... 70

xiii

List of Tables

Table 1 – Combined global prescription sales for the top 50 pharmaceutical companies (excluding generic-

drug companies) by molecule type (2009–2014). ......................................................................................... 7

Table 2 – Quantitative purity analysis of each top phase from PEG-NaPA ATPS, for all three feedstocks,

performed after Bradford assays. IgG purity was calculated by the ratio of IgG concentration in each phase,

obtained by affinity chromatography, by the total protein concentration in the same phase. Results are

displayed as mean ± STDV. ........................................................................................................................ 46

Table 3 – Percentage of cells in top phase, interface and bottom phase and concentration of IgG in top and

bottom phase in PEG 3350 Da/ NaPA 8000Da ATPS. Results are displayed as mean ± STDV. .............. 47

Table 4 – Quantitative purity analysis of each phase from PEG-Dextran ATPS for all three feedstocks,

performed after Bradford assays. IgG purity was calculated by the ratio of IgG concentration in each phase,

obtained by affinity chromatography, by the total protein concentration in the same phase. Results are

displayed as mean ± STDV. ........................................................................................................................ 52

Table 5 – Percentage of cells in top, bottom and interface and the concentration of IgG in top and bottom

phase in PEG 3350 Da/ Dextran 500 kDa ATPS. Results are displayed as mean ± STDV. ...................... 53

Table 6 – Comparison of the purity values obtained after purification of IgG directly from top phases of PEG-

Dextran ATPS with HiTrap SP FF and Fibers cationic exchange columns. Values were obtained after total

protein quantification by Bradford method. IgG purity was calculated by the ratio of IgG concentration in

each phase/fraction by the total protein concentration in the same phase/loaded in the column............... 56

Table 7 – Percentage of cells in top, bottom and interface and the concentration of IgG in top and bottom

phase in PEG 3350 Da-choline chloride ATPS. Results are displayed as mean ± STDV. ......................... 59

Table 8 – Summary of the results obtained for PEG-NaPA, PEG-dextran and PEG-choline ATPSs with

Hybridoma cell culture. ................................................................................................................................ 63

Table 9 – Therapeutic monoclonal antibodies approved or in review in the European Union. .................. 71

xiv

List of Abbreviations

ADA Anti-drug antibody

ADCC

AEX

ATPE

Antibody dependent cell-mediated cytotoxicity

Anion exchanger chromatography

Aqueous two phase extraction

ATPS

BSA

Aqueous two-phase system

Bovine serum albumin

CAGR

CDC

CEX

CHO

Compound annual growth rate

Complement-dependent cytotoxicity

Cation exchanger chromatography

Chinese hamster ovary

ChBS Choline binding sites

CDRs Complementarity-determining regions

CV

DHFR

Column volume

Dihydrofolate reductase

DTT

DMEM

DSP

E

Fab

Dithiothreitol

Dulbecco’s modified Eagle medium

Downstream processing

Elution

Antigen-binding fragment

FBS

Fc

Fetal bovine serum

Crystallisable fragment

FDA Food and drug administration

ii

FT

Fv

Flowthrough

Variable fragment

GFP Green fluorescence protein

GS

HEK

Glutamine synthetase

Human embryonic kidney

HGPRT Hypoxanthine-guanine phosphoribosyltransferase

HIC Hydrophobic interaction chromatography

HPLC

IEF

IEX

i.e.

Ig

High pressure liquid chromatography

Isoelectric focusing

Ion exchange chromatography

id est or in other words

Immunoglobulins

Kp Partition coefficient

mAb Monoclonal antibody

MSX Methionine sulphoximine

MTX Methotrexate

MW

NaPA

Molecular weight

Sodium Poly Acrylate

pAb Polyclonal antibody

PEG Polyethylene glycol

pI

ProA

Isoelectric point

Protein A

SDS-PAGE

SEC

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Size exclusion chromatography

iii

TEMED

TLL

US

WCB

Tetramethylethylenediamine

Tie line length

United States

Working cell bank

1

Background and Aim of Studies

For the last 30 years, monoclonal antibodies (mAbs) have had an ever-growing significance in the

pharmaceutical industry. Drugs based on mAbs are currently one of the leading families of

biopharmaceutical compounds in terms of therapeutic and market potential (Marichal-Gallardo et al. 2012).

Besides their high rate of success, these biopharmaceuticals are also amongst the most expensive drugs

available in the market. The annual cost per patient can reach up to $40 thousand for antibodies that treat

cancer conditions. Given that, in contrast to other biopharmaceutical products, such as several vaccines,

hormones and growth factors, mAbs are typically administered in relatively large doses over long periods

of time in order to achieve the desired level of efficacy, the treatment costs shoot even further (Rosa et al.

2010).

The demand to efficiently supply the biopharmaceutical market with mAbs, led to the need for production

processes that rapidly produce large quantities of pharmaceutical monoclonal antibodies at moderate costs

and in a consistent and reproducible manner (Sommerfeld et al. 2005).

The greatest capacity constraints in current manufacturing platforms of monoclonal antibodies, are no longer

found in the upstream production processes, where cell culture productivity has dramatically increased over

the past decade, but in the downstream purification (DSP) area (Bernardo 2014); (Azevedo, Rosa, Ferreira,

Pisco, et al. 2009). DSP has been considered responsible for the major cost factor with 50-80% of total

production costs Challenges in the purification of bioproducts include developing robust purification

processes that allow integration of the upstream and downstream processing, in order to develop efficient,

sustainable and cost-effective processes. Currently, the established platform for the purification of mAbs

usually includes three chromatographic steps, in which the mAb is firstly adsorbed to an affinity resin, almost

invariably a protein A (ProA) affinity column, followed by two further chromatography steps, which will allow

the removal of the remaining host cell proteins, DNA, leached proA and aggregates, as well as provinding

an adequate level of overall viral clearance (Rosa et al. 2010). ProA chromatography takes advantage of

the highly specific interaction between the Fc region of mAbs and immobilized ProA, which is a cell wall

component of Staphylococcus aureus, rendering purities greater than 98% in a single step (Li et al. 2007).

However, ProA affinity chromatography does suffer from several limitations, being the high cost of the resin

the worst enemy – which can be up to 10 times as expensive as conventional chromatographic supports

(Gottschalk 2008).

In an attempt to overcome the limitations posed by ProA affinity chromatography, various non-

chromatographic alternatives purification protocols have generated a long-standing interest. Aqueous two-

phase systems (ATPSs) are an example of a valuable option. It can combine a high biocompatibility and

selectivity with an easy and reliable scale up and capability of continuous operation. Moreover, it can

2

overcome some of the technical drawbacks currently encountered using the established purification

platform, such as high cost, batch operation, low productivities, scale-related packing problems, diffusional

limitations and low chemical and proteolytic stability. Furthermore, ATPSs allows process integration and

can be used in an early stage of the bioproducts purification platform to integrate clarification and capture

of bioproducts from non-clarified cell culture medium.

The main goal of this project was to design an innovative downstream process based on an affinity ATPS

step for the purification of mAbs from a complex medium, comprising cell separation and antibody selective

extraction, envisaging process integration and intensification.

Different aqueous two-phase systems non-functionalized or functionalized with an affinity ligand (LYTAG-

ProA) for the specific capture of mAbs from animal complex media were screened. The efficacy of the

LYTAG ligand was evaluated and the optimal conditions for extraction established. The partitioning of mAbs,

protein impurities and cells was also evaluated.

3

1. Introduction

1.1. Monoclonal Antibodies (mAbs)

Antibodies (Abs), also named as immunoglobulins (Ig), are circulating glycoproteins produced in vertebrates

in response to exposure to foreign structures known as antigens. They are incredibly diverse and specific

in their ability to recognize foreign molecular structures, being the primary mediators of the immune

response. Abs together with major histocompatibility complex (MHC) molecules and T cell antigen receptors

are the three classes of molecules of the immune system that bind to antigens (at a specific epitope). Of

these three, antibodies are the ones that recognize the widest range of antigens, have the greatest ability

to discriminate between different antigens and bind to them with the greatest strength (Abbas et al. 1994).

B lymphocytes are the only cells responsible for antibodies synthesis. Antibodies can exist either on the

surface of B lymphocytes – membrane-bound antibodies – functioning as receptors for antigen, or can

reside in the circulation, tissues and mucosal sites – secreted antibodies – neutralizing toxins, preventing

the entry and spread of pathogens and eliminating microbes (Abbas et al. 1994). Because most antigens

are highly complex, they present numerous epitopes that are recognized by a large number of lymphocytes.

Each lymphocyte is activated to proliferate and differentiate into plasma cells, and the resulting antibody

response is named polyclonal (pAb). In contrast, monoclonal antibodies are antibodies produced by a single

B lymphocyte clone (Lipman et al. 2005).

1.1.1. Antibody Structure and Functional Features

All antibody molecules share the same basic structural characteristics but display remarkable variability in

the regions that bind the antigens. Each molecule has a symmetric core structure composed of two identical

light chains and two identical heavy chains that are covalently linked by disulfide bonds. The light chain has

a molecular weight (MW) of around 25 kDa and the heavy chain around 50 kDa, being the total molecular

weight of the molecule 150 kDa. Both light and heavy chains consist of amino-terminal variable (V) regions

responsible for the antigen recognition and a carboxyl-terminal constant (C) region responsible for mediating

effector functions (Abbas et al. 1994). Light chains consist of one V domain (VL) and a single constant

domain (CL), whereas heavy chains include one V domain (VH) and three constant domains (CH1, CH2,

and CH3) (See Figure 1 A).

The binding to the antigen is made in the antigen-binding fragment (Fab) by the complementary-determining

regions (CDRs). These CDRs are composed by different amino acid sequences according the type of

antigen they will bind, and that is why they are also called hypervariable regions (Abbas et al. 1994). In the

4

constant regions of the heavy chains there is a region called the Fc (Fragment crystallizable) region which

is common to all antibodies of the same class.

There are five types of constant heavy chains, named µ, γ, α, δ and ε that define the five classes of

immunoglobulins, namely, IgM, IgG, IgA, IgD and IgE, respectively. The difference between these classes

include the amino acid sequence of the heavy chain constant domains, their immune response and their

interaction mode with Fc receptors (Abbas et al. 1994). From a biotechnology perspective, IgG is the most

important class of antibodies (Kim et al. 2005), since they are the most abundant immunoglobulins in the

blood (representing 75% of the antibodies) and the dominant format of therapeutic antibodies.

The two arms (Fab) of the antibody molecule containing the antigen-binding domains and the stem (Fc) are

connected by a region rich in proline, threonine and serine, known as the hinge (Abbas et al. 1994). This

region imparts lateral and rotational movement to the antigen-binding domains, providing the antibody the

ability to interact with a variety of antigen presentations. The hinge, which contains the principal disulfide

linkages between the heavy chains, is susceptible to proteolysis by different proteases, including papain or

pepsin enzymes. Papain will cleave the antibody above the disulfide bridge, generating two monovalent Fab

fragments and a single Fc fragment. In contrast, pepsin cleaves the antibody below the disulfide bridge,

generating a single bivalent Fab fragment containing both antigen binding domains, and a partially digested

Fc region (See Figure 1) (Lipman et al. 2005).

Figure 1 – (A) Schematic diagram of a conventional IgG molecule. Y-shaped protein composed by two light chains

(orange) and two heavy chains (blue). The heavy chains each consist of three constant sections (CH1, CH2, and CH3)

and one variable section (VH). The light chains each consist of a constant region (CL) and a variable region (VL). The

antigen-binding sites are formed by the juxtaposition of VL and VH domains. The bottom section of the antibody is the

Fc region to which two arms, the Fab regions, are attached. Disulphide bonds connect heavy and light chains. The

variable domains have complementary-determining regions (CDR) that bind directly to the antigen. (B) A three-

dimensional ribbon model of a G-class immunoglobulin (IgG) molecule. The blue and green sections are the heavy

chains, while the orange and pink sections on the arms of the antibody are the light chains of the molecule.

In order to inhibit or neutralize the infectivity of microbes as well as the potential injurious effects of infection

antibodies can operate through various mechanisms. When the Fab part of an antibody binds to the antigen

5

it blocks its interaction with a ligand. Signaling occurs when the binding of the antibody to a receptor delivers

an agonist signal. These functions can be independent of the Fc part of the molecule, although interactions

of the Fc portion with other molecules can enhance these mechanisms (Hansel et al. 2010). Antibody

dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) are two

ways of action. In ADCC responses, antibodies bind to antigens on target cells and the antibody Fc domain

engage Fc receptors on the surface of effector cells, such as macrophages and natural killer cells. These

cells in turn trigger phagocytosis or lysis of the targeted cell. In CDC responses, antibodies kill the targeted

cells by triggering the complement cascade at the cell surface

1.1.2. Polyclonal Versus Monoclonal Antibodies: Biotechnological Value

The decision regarding whether to use polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs)

depends on several factors, being the most important their intended use, since each type has its own

advantages and disadvantages.

PAbs can be generated much more rapidly, with less expense, and with less technical skill than is required

to produce MAbs. Further, since they are heterogeneous and recognize a variety of epitopes at the surface

of a certain antigen, they can be very helpful in amplifying signals from a target protein with low expression

level, as the target protein will bind in more than one antibody molecule on the multiple epitopes. Another

advantage of pAbs is that they are more tolerant to changes occurring on a single or small number of

epitopes (e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation) than mAbs.

PAbs are also more stable over a broad pH and salt concentration, whereas mAbs can be highly susceptible

to small changes in both.

Regarding mAbs, homogeneity and consistency are their principal advantages. The monospecificity

provided by these molecules are useful in evaluating changes in molecular conformation, protein-protein

interactions and phosphorylation/glycosylation states, and in identifying single members of protein families.

It also allows for the potential of structural analysis (e.g., x-ray crystallography or gene sequencing) to be

determined for the antibody on a molecular level. However, the monospecificity of mAbs may also be

considered a limitation since small changes in the structure of an epitope can markedly affect the function

of a mAb. For that reason, mAbs should be generated to the state of the antigen to which it will eventually

need to bind (Lipman et al. 2005).

Another key advantage of mAbs is that once the desired hybridoma (see below Section 1.1.4.1 Hybridoma

Technology) has been generated, mAbs can be generated as a constant and renewable resource. PAbs

generated to the same antigen using multiple animals will differ among immunized animals, and their avidity

may change as they are harvested over time. Also, the quantity of pAbs obtained is limited by the size of

the animal and its lifespan.

6

PAbs frequently have better specificity than mAbs because they are produced by a large number of B cell

clones each generating antibodies to a specific epitope, and polyclonal sera are a composite of antibodies

with unique specificities. However, the concentration and purity levels of specific antibody are higher in

mAbs. The concentration of specific antibody in polyclonal sera is typically 50 to 200 µg/mL, and the range

of total Ig concentration in sera is between 5 and 20 mg/mL.

Nevertheless, many of the disadvantages of mAbs can be overcome by pooling and using multiple mAbs of

desired specificities. The pooled product is consistent over time and available in limitless quantity. However,

it is frequently difficult, too expensive, and too time consuming to identify multiple mAbs of desired specificity

(Lipman et al. 2005).

1.1.3. Market Considerations

As medicine progresses into a new era of personalized therapy, the use of monoclonal antibodies to treat a

wide range of diseases lies at the heart of this new forefront. For instance, in the treatment of cancer,

researchers are focused on studying and identifying molecular targets on cancer cells in order to generate

personalized treatments that are based upon an individual’s molecular profile. An interesting case is the one

seen by the binding of the Fc-gamma receptor III, which stimulates ADCC, to the Fc region of Rituxan®

(rituximab) [anti-CD20] and Herceptin® (trastuzumab) [anti-HER2/neu] in xenografted mice. These shown

to be one of the mechanisms by which they may mediate their antitumor effects on non-Hodgkin’s lymphoma

and breast cancer, respectively (Lipman et al. 2005).

Since the licensing of the first monoclonal antibody for clinical use 30 years ago, the monoclonal antibody

industry has expanded exponentially and is now valued at billions of dollars (Liu 2014).

In 2009-2012, the market size of mAb agents grew at a CAGR (compound annual growth rate) of 13%, far

higher than the overall growth rate of biopharmaceuticals in the same period. Between 2009 and 2014 (See

Table 1) mAbs alone will generate an additional $23 billion, thus enticing a growing number of companies

to expand in this field with the hope of ensuring long-term growth. In fact, it is expected that 36 of the top 50

pharmaceutical companies (excluding generics companies) have a presence in the mAb therapeutic protein,

or vaccines sector by this year of 2014. Thanks to robust market demand, approval of new products and

new indications as well as launch of monoclonal antibody generic drugs, the global mAb market size will

ascend by more than 12% in 2013-2017, hitting in 2017 $141 billion (ResearchandMarkets 2013). By July

2014, a total of 36 therapeutic mAbs were approved by the US Food and Drug Administration (FDA) and 7

were still under review (see Table 9 - Annexes) (Food and Drug Administration [Online] n.d.).

Propelled by the optimistic market prospect, advancement of monoclonal antibody technologies and the

upcoming patent expiration of several key monoclonal antibody agents (infliximab (Remicade from Johnson

& Johnson), trastuzumab (Herceptin from Roche), and adalimumab (Humira from Abbott) are three

7

examples), the research and industrialization of monoclonal antibody agents has become a global

investment highlight, wherein the future market competition will be evident (ResearchandMarkets 2013).

In fact, the development of clear, regulatory approval pathways for biosimilars in emerging markets is

creating large, additional opportunities for biosimilar mAbs. Compared to the relatively small protein-based

drugs for which biosimilars have been approved by European Medicines Agency, such as epoetins,

filgrastims, growth hormones, and follitropin alfa, mAbs are much larger and more complex molecules that

can be difficult to fully characterize and thus demonstrate biosimilarity (Challener 2014). This complexity

requires more complex manufacturing processes and, consequently, new competitive downstream

processes. So the discovering of alternative purification processes might be the way of biosimilar mAbs to

get a great approval in the market.

Table 1 – Combined global prescription sales for the top 50 pharmaceutical companies (excluding generic-drug

companies) by molecule type (2009–2014).

1.1.4. Upstream Processing of mAbs

The clinical and commercial success of mAbs has led to the need for very large-scale production in

mammalian cell culture. This has resulted in rapid expansion of global manufacturing capacity, an increase

in size of reactors and a greatly increased effort to improve process efficiency with concomitant

manufacturing cost reduction (Birch et al. 2006). For this purpose, genetic engineering and cell engineering

had to ally themselves in order to develop new media and reactors that lead to the optimization of

mammalian cell culture conditions, at higher scale.

Hybridoma technology was the first to make possible the production of large quantities of mAbs from murine

origin, but rapidly new very efficient expression systems were developed in order to allow the full exploitation

of antibody potential, both in terms of efficiency and cost-effectiveness.

Sales ($billion)

Molecule type 2009 2010 2011 2012 2013 2014 Difference in sales

between 2009 and 2014

Small molecules 411 414 415 405 394 394 -4%

Therapeutic proteins

65 68 70 72 74 76 17%

Monoclonal antibodies

38 43 48 53 58 62 63%

Vaccines 21 22 24 25 27 28 33%

8

1.1.4.1. Hybridoma Technology

MAbs were first recognized in sera of patients with multiple myeloma in which clonal expansion of malignant

plasma cells produced high levels of an identical antibody resulting in a monoclonal expansion of malignant

plasma cells produce high levels of an identical antibody resulting in a monoclonal gammopathy. The

discovery of monoclonal antibodies produced by

these tumors led to the idea that it may be possible

to produce similar mAbs of any desired specificity by

immortalizing individual antibody-secreting cells

from an animal immunized with a known antigen. In

1975, Georges Kohler and Cesar Milstein (Nobel

Prize in Physiology or Medicine in 1984) developed

a technique named hybridoma technology that relies

on fusing B cells from an immunized animal

(typically a mouse) with a myeloma cell line and

growing the cells under conditions in which the

unfused normal and tumor cells cannot survive. In

this procedure, spleen cells from a mouse that has

been immunized with a known antigen or mixture of

antigens are fused with an enzyme-deficient partner

myeloma cell line.

The myeloma partner used is one that does not

secrete its own Igs. These hybrid cells are then

placed in a selection medium that permits the

survival of only immortalized hybrids; these hybrid

cells are then grown as single cell clones and tested

for the secretion of the antibody of interest. The

selection medium includes hypoxanthine,

aminopterin, and thymidine and is therefore called

HAT medium. There are two pathways of purine

synthesis in most cells, a de novo pathway that

needs tetrahydrofolate and a salvage pathway that

uses the enzyme hypoxanthine-guanine

phosphoribosyltransferase (HGPRT). Myeloma cells that lack HGPRT are used as fusion partners, and they

normally survive using de novo purine synthesis. In the presence of aminopterin, tetrahydrofolate is not

made, resulting in a defect in de novo purine synthesis and also a specific defect in pyrimidine biosynthesis,

namely, in generating thymidine monophosphate (TMP) from deoxyuridine monophosphate (dUMP). Hybrid

cells receive HGPRT from the splenocytes and have the capacity for uncontrolled proliferation from the

Figure 2 – Generation of monoclonal antibodies by

hybridoma technology technique (Abbas et al. 1994)

9

myeloma partner; if they are given hypoxanthine and thymidine, these cells can make DNA in the absence

of tetrahydrofolate. As a result, only hybrid cells survive in HAT medium.

Each hybridoma produces only one Ig and the antibodies secreted by many hybridoma clones are screened

for binding to the antigen of interest, and this single clone with the desired specificity is then selected and

further expanded. The products of these individual clones are monoclonal antibodies and are specific for a

single epitope on the antigen or antigen mixture used to identify antibody secreting clones (Abbas et al.

1994).

In the late 1980s, murine mAbs started their clinical development, however, the first generations, as

therapeutic agents, suffered a number of drawbacks. Murine mAbs are often associated with allergic

reactions, and the induction of anti-drug antibodies (ADAs). They also exhibit a relatively short serum half-

life, when comparing to human IgG, insufficient activation of human effector functions and development of

human anti-mouse-antibody (HAMA) responses in patients, especially when repeated administrations were

necessary (Buss et al. 2012). Finally, murine mAbs are relatively poor recruiters of effector function, which

can be critical for their efficacy, especially in oncology indications (Stern et al. 2005).

Hence, chimeric mouse–human antibodies were developed. This was enabled by grafting the entire antigen-

specific variable domain of a mouse Ab onto the constant domains of a human Ab using genetic engineering

techniques, resulting in molecules that are approximately 65% human. These chimeric mAbs exhibit an

extended half-life in man and show reduced immunogenicity, but nevertheless, the propensity of chimeric

mAbs to induce ADAs was still considerable. To further improve mAb properties, humanized mAbs were

developed by grafting just the murine hypervariable regions onto a human Ab framework, resulting in

molecules that are approximately 95% human. Whilst humanized mAbs appeared to overcome the inherent

immunogenic problems of murine and chimeric mAbs, humanization does have limitations and can be a

laborious process (Buss et al. 2012). (See Figure 3).

Figure 3 – Antibody engineering for humanization. Therapeutic mAbs can be murine (100% murine protein), chimeric

(composed of 67% of human constant domains), humanized (only possess 5-10% of murine regions) or fully human

(100% human proteins) (Carter 2001).

10

1.1.4.2. Recombinant DNA Technology

The development of very efficient expression systems is essential to the full exploitation of antibody

potential, both in terms of efficiency and cost-effectiveness. The expression of functional, correctly folded

antibodies or antibody fragments and its scale up to commercial levels is a major goal in therapeutic

antibodies development (Laffly et al. 2005).

Since antibody therapies may require large doses over a long period of time, manufacturing capacity

becomes an issue because the drug substance must be produced in large quantities with cost and time

efficiency to meet clinical requirements. In response to the strong demand, companies have built large scale

manufacturing plants containing multiple 10,000 L or larger cell culture bioreactors (Li et al. 2010).

Therapeutic antibodies are mainly produced in mammalian cell expression systems due to their ability to

produce large amounts of mAbs with a consistent quality and to adapt well to culture in large-scale

suspension bioreactors. Another reason, and probably the most important, for the dominance of mammalian

cells is their capability to perform the required protein folding, assembly and post-translational modifications,

such as glycosylation, so that the produced mAb would be chemically similar to human forms for increased

product efficacy and safety (Ho et al. 2013). However, Escherichia coli remains the production system of

choice for antibody fragments used in therapeutic applications, due to be the easiest, quickest and

economical method for protein expression. E. coli production can offer rapid means to progress from

antibody selection to good manufacturing practice (GMP) production of antibodies, due to the ease and

speed of making productive cell lines compared to eukaryotic cell lines. Besides, high production levels of

antibody fragments are usually attainable when E. coli is used as the production organism (Andersen et al.

2004). The only reason for not using E. coli for mAbs production is their inability to make complex post-

translational modifications.

Cell culture process development starts with cell line generation and selection, followed by process and

media optimization in small scale systems, including 96-well plates, shaker flasks, and bench-scale

bioreactors, for high throughput screening purposes. Once conditions are defined, the process is often

transferred to a pilot scale to test scalability and produce material for preclinical toxicology studies, and then

larger scale manufacturing for production of clinical material under current good manufacturing practices

(cGMP) regulations. Once the development of a commercial cell culture process for production of a

biological product is completed at the laboratory and pilot scales, the commercialization process begins with

process characterization, scale-up, technology transfer, and validation of the manufacturing process (Li et

al. 2010).

The cell line development process starts from transfection of a mammalian cell line with plasmid vectors

carrying the light chain (LC), the heavy chain (HC), and a selection marker genes. The plasmid vector comes

in various designs, optimized for mAb production. Several cell types can be used. The most common

mammalian hosts include chinese hamster ovary (CHO) cells, murine lymphoid cells (NS0,Sp2/0-Ag14),

11

baby hamster kidney (BHK) cells, human embryonic kidney (HEK-293) cells and human embryonic

retinoblast derived (PER.C6) cells. Within these, CHO cells are the most widely used cell line since have

attractive process performance attributes such as rapid growth, high expression, and the ability to be

adapted for growth in chemically-defined media (Kelley 2009). CHO cells have also a proven track record

of producing safe, biocompatible and bioactive mAbs, enabling products from these cells to gain regulatory

approval more easily (Ho et al. 2013).

The selection of the right expression system is determined by its capability of producing high concentrations

of product, their ability to consistently produce the antibody with the desired characteristics (glycosylation

pattern), their speed to reach a high yielding cell line, and also their ability to grow in suspension. Highly

productive cell lines result from using a host cell line that has the desired characteristics, an appropriate

expression system, and a good transfection and selection protocol (Birch et al. 2006).

In order to efficiently select for stable transfected cells, selection marker genes that confer resistance to

certain antibiotics or growth advantage in a nutrient-deficient condition are used. The most frequently used

selectable marker genes are the ones based on dihydrofolate reductase (DHFR) and on glutamine

synthetase (GS). DHFR is involved in the reduction of dihydrofolate to tetrahydrofolate, which is in turn

needed for nucleic acid metabolism. First, selection is conducted in a media devoid of hypoxanthine and

thymidine, so that only cells that have incorporated the DHFR gene are able to survive. Amplification can

be further carried out adding a folic acid analogue, methotrexate (MTX), which inhibits DHFR activity. In

order to survive, cells will need to amplify the DHFR gene copy. The mAb genes located on the same

transfected vector or in nearby sites are also amplified, increasing the gene copies and, thus, expression

levels after several stepwise increase in MTX levels.

The GS selection marker catalyzes the formation of glutamine from glutamate and ammonia, allowing the

successfully transfected cells to survive in media lacking in glutamine. The use of this system with

mammalian cell with endogenous levels of GS requires the use of methionine sulphoximine (MSX), a GS

inhibitor. Similar to using MTX with DHFR, using MSX with GS forces cells to co-amplify the GS gene and

the product gene (Ho et al. 2013). The GS system has a time advantage over the DHFR system during

development, and requires fewer copies of the recombinant gene per cell, allowing a faster selection of

high-producing cell lines.

After the selection of transfectants and amplification, single clones are chosen for scale-up and

characterization of product quality and long-term expression (Ho et al. 2013).

1.1.4.3. Large Scale Production

The need for massive production of mAbs led to dramatic increase in capacity in the industry and an

increase in the scale of reactors used for production.

12

A typical cell culture process starts with thawing a frozen vial of a working cell bank (WCB), followed by

expanding the cell population through a series of seed trains in different culture vessels. The culture is then

transferred to a production bioreactor where the cells continue to grow and the expressed product is

excreted into the culture broth (Li et al. 2007).

For large-scale manufacture of mAbs two of the most popular process modes of culture system used are

fed-batch and continuous perfusion culture (see Figure 4).

Figure 4 – Schematic representation of fed-batch and perfusion culture systems. The fed-batch system is supplied with

a concentrated nutrient solution (no spent culture medium is removed). In perfusion culture systems fresh nutrient

solution is supplied to the vessel at the same rate that spent medium is withdrawn; cells are, however, returned to the

bioreactor.

In fed-batch culture, small volumes (less than 10% of the reactor volume) of key nutrients are fed to the

culture during the fermentation process to maintain a certain level of nutrients, and the culture is harvested

at the end of the batch cycle. Optimization of feeding strategies has been a major factor contributing to

improvements in growth and productivity in recent years.

In perfusion culture systems, fresh medium is added continuously to the reactor and spent medium,

containing product, is continuously removed. In these systems, cells are retained in the reactor and a variety

of retention devices have been described which may be internal or external to the reactor. This system

involves more complexity in the process and is more time consuming, although it has a throughput of

antibody 10 times higher than can be achieved in a batch or fed-batch system (Birch et al. 2006).

Although stainless steel bioreactors are still the major choice for large-scale production, disposable

bioreactor systems have become available. For example, the Wave Bioreactor system, which uses a plastic

disposable bag, is commonly used during seed culture expansion. These types of disposable bioreactor

systems can benefit the manufacturing process by eliminating the clean-in-place (CIP) and steam-in-place

13

(SIP) operations and by reducing the expensive capital investment for stainless steel bioreactors (Li et al.

2007).

1.1.5. Downstream Processing of mAbs

The efficient recovery and purification of biopharmaceuticals has been referred as a critical part of the

production process (Rosa et al. 2010). The primary considerations during downstream process

development are the purity and the speed of process development. Other key considerations include overall

yield and process throughput, and in addition, the process must meet several manufacturability criteria

including robustness, reliability and scalability (Shukla et al. 2007).

The explosion in the number of mAbs entering clinical trials has created the need for employing a rather

templated approach to process development. The purification process of mAbs needs to reliably and

predictably produce a product suitable for use in humans, while impurities such as host cell proteins, DNA,

adventitious and endogenous viruses, endotoxins, aggregates and other species must be removed while

an acceptable yield is maintained (Liu et al. 2010). Therefore it became necessary to have a generic process

that could be employed for all mAbs candidates, reducing the time and resources needed for process

development.

The established platform for the purification of mAbs (shown in Figure 5) usually includes three

chromatographic steps, in which the mAb is firstly adsorbed to an affinity resin, almost invariably a proA

affinity column – since proA ligand has high affinity for the Fc area of the mAb, enabling the mAbs capture

from the cell culture fluid – followed by two further chromatography steps, which will allow the removal of

the host cell proteins, DNA, any leached proA and aggregates as well as will provide an adequate level of

overall viral clearance (Rosa et al. 2010).

Figure 5 – Standard platform downstream process for mAbs. Cell culture supernatant is typically purified by a capture

step with Protein A chromatography. In order to remove all contaminates post Protein A capture and to obtain purity to

regulatory compliance, two additional chromatographic polishing steps are employed. (Rosa et al. 2010).

The first step in the recovery of an antibody from a mammalian cell culture is harvest. Since mAbs are

typically produced using high density mammalian cell culture, the removal of cells and cell debris from

Upstream Initial recovery Affinity chromatography

Viral inactivation

Polishing steps Viral filtration UF/DF

Purified

mAb

14

culture broth to yield a clarified, filtered fluid suitable for chromatography is required. In typical processes,

the accepted range for solids concentration in a culture broth from mammalian cell culture is usually 40-

50%, and by the end of the harvesting process solids concentration is expected to be negligible, although

turbidity may remain. This step is generally accomplished through the use of centrifugation, depth filtration

and sterile filtration, although other approaches may be applied. Harvesting operations, in terms of capital

cost and energy consumption, can account for up to 25% of the cost of the entire downstream process

(Marichal-Gallardo et al. 2012).

After successful harvesting, protein A affinity chromatography is the first step of choice for most industrial

processes. ProA is a naturally occurring polypeptide found anchored in the wall of Staphylococcus aureus.

The MW of the intact native molecule is 54 kDa but typically recombinant ProA used for IgG purification

(produced as a secreted extracellular protein in E. coli) is engineered to have the cell wall domain deleted.

Therefore, its MW is reduced to ~42 kDa. The main industrial manufacturers of ProA resins are General

Electric® (GE) and Millipore® (Marichal-Gallardo et al. 2012).

The natural high affinity of ProA for the Fc region of IgG-type antibodies forms the basis for the purification

of IgG, IgG fragments and subclasses. The IgG-ProA binding mechanism primarily consists of hydrophobic

interactions related to specific hydrogen bonds that are established as a function of pH. At alkaline pH,

histidyl residues on the binding site of IgG–Protein remain uncharged. This contributes to bonding involving

hydrophobic interactions. At low pH, these histidyl residues become charged and mutually repellant, thereby

providing a means for easy detachment of the IgG from ProA.

This chromatography procedure typically involves passage of clarified cell culture supernatant over the

column at pH 6–8, under which conditions the antibodies bind and unwanted components such as host cell

proteins and cell culture media components and putative viruses flow through the column. An optional

intermediate wash step may be carried out to remove non-specifically bound impurities from the column,

followed by the final elution of the product with a low pH (2.5–4) elution buffer which causes the protonation

of histidine residues on the mAb and ProA, which are next to each other in the binding area. This leads to

charge-charge repulsion and elution. The column is then regenerated for further use (see Figure 6).

Figure 6 – Schematic representation of a protein A affinity chromatography purification. The Protein A ligand is

immobilized on to the column. Following crude sample loading, the mAb is retained by affinity binding to Protein A.

Washing is employed to remove nonspecific binding. Elution of the mAb is with a low pH elution buffer.

Column loading Affinity Binding Washing Elution and regeneration

15

ProA chromatography is typically used as the first step in an antibody purification process due its high

selectivity, high flow rate, cost effective binding capacity and its ability for extensive removal of process-

related impurities such as host cell proteins, DNA, cell culture media components and endogenous and

adventitious virus particles. In the end, this step yields a relatively pure product, more stable due to the

elimination of proteases and other media components that could cause degradation.

Polishing steps are then performed for a final removal of trace contaminants from the solution in order to

achieve acceptable concentrations of these and to obtain the final solution required, according to the

particular formulation to be used.

The nature of the polishing steps is determined by the nature of the product and the impurities present, but

usually one or two additional chromatography polishing steps are applied. Most mAb purification processes

will include at least one ion exchange chromatography step, for reducing high molecular weight aggregate,

charge-variants, residual DNA and host cell protein, leached ProA and viral particles. The use of anion

exchange chromatography (AEX) is more common than cation exchange chromatography (CEX) as this

resin is often used in flowthrough mode (in which the product does not bind to the column whereas impurity

species are retained) (Marichal-Gallardo et al. 2012).

Since mammalian cells used in the manufacture of mAbs can produce endogenous retroviruses and are

occasionally infected with adventitious viruses during the upstream processing, a virus clearance step is

always required, prior to the final product formulation. In fact, virus removal is a key control step in antibody

downstream processing. If present, endogenous viruses can proliferate in the upstream process during cell

multiplication and in addition, cells within process streams may be infected by external viral contamination

(Marichal-Gallardo et al. 2012). Due to safety requirements, mammalian cell-derived products may contain

less than one virus particle per million doses, which translates to approximately 12-18 log10 clearance for

endogenous retrovirus and 6 log10 clearance for adventitious virus (Liu et al. 2010). Virus filters are typically

operated at constant pressure and, depending on the membrane, volumetric loads can be in the range 200-

400 L/m2 before flow decay is noticeable (Marichal-Gallardo et al. 2012). This viral removal is normally

accomplished by a filtration operation using a membrane with a pore size adequate to the type of the virus.

Finally, an ultrafiltration can be used for protein concentration and buffer exchange (Liu et al. 2010).

1.1.5.1. Downstream processing of mAbs: Alternative processes

Downstream processing would never have developed as an individual sector of the bioprocessing industry

without chromatography, whose inherent simplicity and selectivity has made it the key enabling technology

in all bioseparation processes (Gottschalk 2008). However, chromatography has been the major cost center

mainly due to media cost and relatively long cycle times (Rosa et al. 2010), so lower cost alternatives have

been sought. Two viable options can be considered in the DSP of mAbs: the replacement of ProA affinity

16

chromatography by other chromatographic processes, or elimination at all of chromatography by using non-

chromatographic methods. Consequently, several alternatives to the established process have generated

interest either to replace column chromatography or to decrease the number of chromatography steps by

reducing the load of impurities in the feed stream (Gottschalk 2008). Among the non-affinity chromatography

options, the use of cation exchange resins as a capture step has been shown to be a viable and promising

choice, since they allow the removal of host cell proteins to levels comparable to the traditional ProA

process, are relatively inexpensive, and can provide dynamic binding capacities as high as >100 g/L (Liu et

al. 2010). Additionally, new generations of mixed mode ligands appear as versatile alternatives to standard

purification platforms, exploiting multiple types of interactions and gaining their position as powerful tools for

monoclonal antibodies purification (Follman et al. 2004). Flocculation, precipitation, crystallization, high

gradient magnetic fishing, membrane processing and liquid–liquid extraction are non-chromatographic

techniques that can also be used for the purpose (Rosa et al. 2010).

Flocculation and precipitation can be used in combination with conventional cell separation techniques such

as centrifugation and microfiltration in order to enhance the removal of residual particulates and soluble

impurities that might increase the burden on downstream polishing steps.

Crystallization is another inexpensive technology that has been recognized for many years as a powerful

technology because of its ability to simultaneously concentrate, purify and stabilize the target product.

Nevertheless, it has limited application in antibody purification not only due to the inherent complexity of the

process, but also as a result of the low yields and difficulties with process control.

Membrane chromatography is also emerging as an attractive alternative to traditional column

chromatography that is already used in many bioprocesses. However, it has some limitations that need to

be overcome before routine successful process-scale production, such as distorted or poor inlet flow

distribution, non-identical membrane pore size distribution, uneven membrane thickness and lower binding

capacity. Membrane chromatography behaves similarly to packed chromatography columns, but in the

format of conventional filtration modules, which usually has multiple layers containing functional ligands

attached to the internal pore surface throughout the membrane structure (Gottschalk 2008).

High gradient magnetic separation technology is also a promising approach that has been adapted from the

chemical and mineral processing industries. Advantages of magnetic columns according to conventional

affinity chromatography include the efficient fluid-solid mass transfer properties, low pressure drop, good

fluid-solid contact, elimination of clogging and continuous countercurrent operation (Denizli 2002). Higher

adsorption values of immunoglobulin G from human plasma of up to 320 mg/g and a purity of 87% were

observed in previous studies (Denizli 2002).

Conventional liquid

Date post:	30-Jan-2021
Category:	Documents
Upload:	others
View:	3 times
Download:	0 times

Clarification and capture of monoclonal antibodies from ... · Os anticorpos monoclonais (mAbs)...

Documents