Continuous polishing of recombinant antibodies after ...

Continuous polishing of recombinant antibodies aftercontinuous precipitation

Patrícia Sofia Leong Rodrigues

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisor(s): Prof. Alois JungbauerProf. Ana Margarida Nunes da Mata Pires de Azevedo

Examination Committee

Chairperson: Maria Margarida Fonseca Rodrigues DiogoSupervisor: Prof. Ana Margarida Nunes da Mata Pires de Azevedo

Member of the Committee: Prof. Maria Ângela Cabral Garcia Taipa Meneses de Oliveira

October 2020

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Declaration

I declare that this document is an original work of my own authorship and that it fulfills all therequirements of the Code of Conduct and Good Practices of the Universidade de Lisboa.

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Preface

The work presented in this thesis was performed at the Department of Biotechnology (ResearchGroup Jungbauer) of University of Natural Resources and Life Sciences (Vienna, Austria), during theperiod September 2019 and March 2020, under the supervision of Prof. Alois Jungbauer, and within theframe of the Erasmus+ study programme. The thesis was co-supervised at Instituto Superior Técnicoby Prof. Ana Margarida Nunes da Mata Pires de Azevedo.

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Acknowledgements

To Instituto Superior Técnico, I would like to show my gratitude for being my second home thelast 7 years. It has been a long ride, maybe too long of a ride, however every moment contributed tothe person I am today. Even through all the love and hate moments towards this institution, there is nodoubt that everyone that was part of this journey, from professors to colleagues and specially friends,have taught me so much, not only academically but also personally.

A special thanks to my supervisor Professor Ana Azevedo for introducing me to the downstreamprocessing subject and for always teaching so enthusiastically and be available to answer my doubts.

I would like to thank my supervisor Professor Alois Jungbauer for allowing me to do my master’sthesis at the Jungbauer Group. A special thanks to Nico, Daniel and Greg for the mentoring and theknowledge shared during the internship. To Eva and Nico, I also thank them for their time and patiencein teaching me all the laboratory techniques and for sharing their knowledge with me. I would also liketo thank the rest of the working group at BOKU. To my fellow intern colleagues, to the little Portuguesecommunity I was not expecting to find at work and to Edit for their kindness.

I thank Vienna next. The city that was my home for seven amazing months and which hasintroduced to me some of the best people that have entered my life and, hopefully, will continue tobe part of it. To my Vienna family I thank you from the bottom of my heart for making this Erasmusexperience a memorable one. I will never forget any of the weekend trips for which I looked foward to allweek. To Iris, my first friend in Vienna, the best neighbour and city tour guide. To my soulmate Margaridaand my ski partner for life Mariana, thank you for becoming the family I did not know I needed.

To Carla, for the long voice messages venting about our day which were what kept me sane atthe end of some of those days. To Mafs, for being so kind and patient in answering my all doubts, which,ultimately, led to a beautiful friendship. To Carolina, for the random video calls that would make my day.To Cisca and Lena, my girls, to whom without Técnico would not be the same. To Bia, for being alwaysthere, thank you. To everyone who visited me, family (Mãe, Pai, Tio Dido, Tia Verónia and Theo) andfriends (Cisca, Lena, Bia, Carol, Sara, Mafs and Judy), I thank you, because not a month would passwithout home coming to me.

Finally, I want to thank my family. To my grandparents, Vó Lana e Gung Gung, I share myhappiness with you and hopefully, it will reach you. To my grandmother Vó Natália for her love andsupport and for the long phone calls that made me realise how hard it is to be away from family.

And last, but certainly not least, to whom I dedicate this thesis to, to my mom and dad, there isnot enough words to describe how much I thank you for your continuous love and support through goodand bad times. For allowing me to pursue my studies and always believing in me. Without you, this 7-month experience would have not been possible and for all of that and more, I thank you with all my heart.

Thank you.

Danke.

Obrigada.

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Resumo

A substituição de polimento em modo positivo por modo negativo na purificação de anticorposmonoclonais contribui para um processo de manufatura mais contínuo. De modo a inferir como fun-ciona o polimento em modo negativo em diferentes condições de trabalho, o anticorpo recombinanteTrastuzumab (0.29 mg/mL, pI 8.4), obtido após precipitação contínua com ZnCl2, foi purificado usandoduas abordagens, com acetato de sódio como solução tampão: AEX seguida de CEX com não ajuste(N-ADC: pH 3.6, 3.47 mS/cm) e ajuste (ADC: pH 6.0, 4.00 mS/cm) das condições de adsorção doanticorpo.

A AEX em modo negativo demonstrou rendimentos de 82% e 90% para N-ADC e ADC, respeti-vamente. Por sua vez, a CEX não operou completamente em modo negativo, resultando em rendimen-tos globais de 20% e 24% para as experiências AEX-CEX N-ADC e ADC, respetivamente. O produtofinal de Trastuzumab de AEX-CEX ADC apresentou melhores resultados, cujas características foram:DNA < 11×102 ppb, HCP = 830×103 ppm, concentração de anticorpo = 0.08 mg/mL, pureza = 100% eHMWI, LMWI = 0%.

Considerando que o produto final de Trastuzumab da AEX ADC em modo negativo apresentouelevado rendimento (90%) e pureza (> 95%) e, visto que o polimento por CEX não funcionou em modonegativo, este último passo poderia ser dispensável. As experiências que se seguiram, que infelizmentenão puderam ser concluídas, consistiam em verificar se ao utilizar-se uma solução tampão diferente(Tris-HCl) e diferentes pHs (7.0 e 8.0) de trabalho, tal poderia contribuir para um melhor funcionamentoda CEX em modo negativo.

Palavras-chave: Processo de purificação, anticorpo monoclonal, polimento em modo negativo,cromatografia de troca aniónica (AEX), cromatografia de troca catiónica (CEX).

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Abstract

The replacement of bind/elute chromatography by flow-through polishing in mAbs purificationcan contribute towards a more continuous biopharmaceutical downstream process. To infer how flow-through polishing performs under different working conditions, the recombinant antibody Trastuzumab(0.29 mg/mL, pI 8.4), obtained after continuous ZnCl2 precipitation, was purified using two different flow-through polishing trains with sodium acetate buffer: anion exchange chromatography (AEX) followed bycation exchange chromatography (CEX) with non-adjusted (N-ADC: pH 3.6, 3.47 mS/cm) and adjusted(ADC: pH 6.0, 4.00 mS/cm) mAb loading conditions.

The flow-through AEX showed process mAb yields of 82% and 90% for N-ADC and ADC, re-spectively. On the other hand, the CEX did not operate fully in flow-through mode, resulting in overall mAbyields of 20% and 24% for the AEX-CEX N-ADC and ADC trains, respectively. The final Trastuzumabproduct of the AEX-CEX ADC train presented better results and had the following conditions: DNA <11×102 ppb, HCP = 830×103 ppm, mAb titer = 0.08 mg/mL, monomer purity = 100% and HMWI, LMWI= 0%.

Considering that the final Trastuzumab product of the flow-through AEX ADC had a processmAb yield of 90% with high impurity clearance (> 95%) and taking into account the poor performance ofthe flow-through CEX step, it was concluded that the latter could be expendable. Further experiments,which unfortunately were not able to be concluded, were performed using a different buffer, Tris-HCl,and working pH (7.0 and 8.0) in order to infer if different working conditions were more adequate for theflow-through CEX.

Keywords: Downstream processing, monoclonal antibody, flow-through polishing, anion ex-change chromatography (AEX), cation exchange chromatography (CEX).

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Table of contents

Declaration i

Preface iii

Acknowledgments v

Resumo vii

Abstract ix

List of Figures xv

List of Tables xxi

Acronyms 1

1 Introduction 31.1 Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1.1 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.1.2 Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.1.3 Trastuzumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2 Standard mAb manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.3 Continuous manufacturing process vs. batch manufacturing process . . . . . . . . . . . . 71.4 Continuous precipitation with zinc chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.5 Ultrafiltration/Diafiltration (UF/DF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.6 Continuous polishing of mAbs in flow-through mode . . . . . . . . . . . . . . . . . . . . . 11

1.6.1 Process and product-related impurities . . . . . . . . . . . . . . . . . . . . . . . . 121.6.2 Activated carbon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.6.3 Ion exchange chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.6.3.1 Anion exchange chromatography . . . . . . . . . . . . . . . . . . . . . . . 151.6.3.2 Cation exchange chromatography . . . . . . . . . . . . . . . . . . . . . . 15

1.7 Product quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.7.1 Residual DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.7.2 Host cell protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.7.3 Antibody titre quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.7.4 Antibody purity and aggregates (HMWI and LMWI) quantification . . . . . . . . . . 17

1.8 UV signal interpretation in a chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . 181.9 Resin column packing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.10 Aim of thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2 Materials and Methods 232.1 Buffer Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2 Flow-through ion exchange resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.2.1 Anion exchange resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2.2 Cation exchange resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.2.2.1 Eshmuno CP-FT column packing . . . . . . . . . . . . . . . . . . . . . . . 232.3 Flow-through chromatography experiments . . . . . . . . . . . . . . . . . . . . . . . . . . 25

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2.3.1 AEX-CEX (non-adjusted loading conditions) and AEX-CEX (adjusted loading con-ditions) runs with sodium acetate buffer at pH 6.0 . . . . . . . . . . . . . . . . . . . 26

2.3.2 CEX (adjusted loading conditions) "test" runs with Tris-HCl buffer at pH 6.0, 7.0and 8.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.3.2.1 Buffer exchange step to adjust the loading conditions for the CEX "test"

runs at pH 6.0, 7.0 and 8.0 . . . . . . . . . . . . . . . . . . . . . . . . . . 282.3.3 AEX-CEX (adjusted loading conditions) runs with Tris-HCl buffer at pH 7.0 and pH

8.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.3.3.1 AEX-CEX (adjusted loading conditions) run with Tris-HCl buffer at pH 7.0 292.3.3.2 AEX-CEX (adjusted loading conditions) run with Tris-HCl buffer at pH 8.0 30

2.4 Ultrafiltration/Diafiltration (UF/DF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.4.1 Two-step buffer exchange to adjust the loading conditions to pH 6.0 and 4.00

mS/cm conductivity (sodium acetate buffer) . . . . . . . . . . . . . . . . . . . . . . 312.4.2 Two-step buffer exchange to adjust the loading conditions to pH 7.0 and 4.00

mS/cm conductivity (Tris-HCl buffer) . . . . . . . . . . . . . . . . . . . . . . . . . . 312.4.3 Two-step buffer exchange to adjust the loading conditions to pH 8.0 and 4.00

mS/cm conductivity (Tris-HCl buffer) . . . . . . . . . . . . . . . . . . . . . . . . . . 322.5 Analytical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.5.1 HPLC-Protein A affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . 322.5.2 Size exclusion chromatography (SEC) . . . . . . . . . . . . . . . . . . . . . . . . . 332.5.3 Bradford assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.5.4 Residual DNA method quantification . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2.5.4.1 Buffer exchange step to remove the zinc chloride from the sample . . . . 332.5.4.2 Picogreen assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3 Results and Discussion 353.1 Initial experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.2 Flow-through AEX-CEX (N-ADC and ADC) experiments with sodium acetate buffer . . . . 37

3.2.1 Residual DNA content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403.2.2 Total protein, HCP and antibody content . . . . . . . . . . . . . . . . . . . . . . . . 413.2.3 Antibody monomer purity and HMWI and LMWI content . . . . . . . . . . . . . . . 443.2.4 Antibody monomer yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3.3 Flow-through AEX-CEX (ADC) experiments with Tris-HCl buffer . . . . . . . . . . . . . . . 513.3.1 Flow-through AEX-CEX (ADC) train with Tris-HCl buffer at pH 8.0 . . . . . . . . . . 52

3.3.1.1 Residual DNA and total protein content . . . . . . . . . . . . . . . . . . . 543.3.2 Flow-through AEX (ADC) run with Tris-HCl buffer at pH 7.0 . . . . . . . . . . . . . 55

4 Conclusions and Future Perspectives 57

References 61

I Appendix II.1 Appendix I.1 - Materials and Methods of the Initial experiments . . . . . . . . . . . . . . . I

I.1.1 qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II.1.1.1 qPCR standard curve spiked with zinc chloride . . . . . . . . . . . . . . . I

I.1.2 Ultrafiltration/Diafiltration (UF/DF) to adjust the the loading conditions to pH 6.0and 1.98 mS/cm conductivity (sodium acetate buffer) . . . . . . . . . . . . . . . . . I

I.1.3 Flow-through chromatography method . . . . . . . . . . . . . . . . . . . . . . . . . II

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I.2 Appendix I.2 - Results and Discussion of the Initial experiments . . . . . . . . . . . . . . IIII.2.1 Analysis of the precipitated trastuzumab . . . . . . . . . . . . . . . . . . . . . . . . IIII.2.2 Flow-through chromatography experiments . . . . . . . . . . . . . . . . . . . . . . VI

I.2.2.1 Adjustment of the loading conditions . . . . . . . . . . . . . . . . . . . . . VII.2.3 Residual DNA content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII.2.4 Total protein content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X

I.2.4.1 Verification of the Bradford method’s validity . . . . . . . . . . . . . . . . . XII.2.5 Antibody titre determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI

I.3 Appendix I.3 - Results and Discussion of the Flow-through AEX-CEX (N-ADC and ADC)experiments with sodium acetate buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII.3.1 Flow-through AEX-CEX (N-ADC and ADC) trains with sodium acetate buffer . . . XII

I.3.1.1 Residual DNA content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI.3.1.2 Total protein, HCP and antibody content . . . . . . . . . . . . . . . . . . . XVII.3.1.3 Monomer yield, monomer purity, HMWI and LMWI content . . . . . . . . XVIII.3.1.4 Overall results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVIII

I.4 Appendix I.4 - Results and Discussion of the Flow-though AEX-CEX (ADC) experimentswith Tris-HCl buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIXI.4.1 CEX (ADC) "test" runs with Tris-HCl buffer at pH 6.0, 7.0 and 8.0 . . . . . . . . . . XIX

I.4.1.1 Antibody titre determination . . . . . . . . . . . . . . . . . . . . . . . . . . XXII.4.2 Flow-through AEX-CEX (ADC) train with Tris-HCl buffer at pH 8.0 . . . . . . . . . . XXII

I.4.2.1 Residual DNA content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIIII.4.2.2 Total protein content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIV

I.4.3 Flow-through AEX (ADC) run with Tris-HCl buffer at pH 7.0 . . . . . . . . . . . . . XXIV

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List of Figures

1.1 Structural schematic of IgG (Abbreviations: Fab - fragment antigen binding; Fc - fragmentcrystallizable; IgG - immunoglobulin G; Ag - antigen) (Source: Murphy et al., 2016). . . . . 3

1.2 Trastuzumab (Herceptin) mechanism of action to suppress cancer cells growth and prolif-eration (adapted from Source: Genentech). . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.3 Outline of steps involved in a general mAb manufacturing process using mammalian cellculture comprising (1) inoculum expansion, (2) product fermentation, (3) product primaryrecovery, (4) product purification, (5) product formulation (Source: Vázquez-Rey et al.,2010). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.4 Comparison of NFF and TFF. In NFF, the fluid is convected in the direction normal to themembrane under an applied pressure. In TFF, the fluid is pumped tangentially along thesurface of the membrane, where an applied pressure serves to force a portion of the fluidthrough the membrane to the filtrate side (Source: Merck KGaA). . . . . . . . . . . . . . . 10

1.5 Schematic of a simple TFF system. A pump is used to generate flow of the feed streamthrough the channel between two membrane surfaces. During each pass of fluid over thesurface of the membrane, the applied pressure forces a portion of the fluid through themembrane and into the filtrate stream. The result is a gradient in the feedstock concen-tration from the bulk conditions at the center of the channel to the more concentratedwall conditions at the membrane surface. There is also a concentration gradient along thelength of the feed channel from the inlet to the outlet (retentate) as progressively morefluid passes to the filtrate side. The flow of feedstock along the length of the membranecauses a pressure drop from the feed to the retentate end of the channel. The flow on thefiltrate side of the membrane is typically low and there is little restriction, so the pressurealong the length of the membrane on the filtrate side is approximately constant (Source:Merck KGaA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.6 Schematic of a flow-through polishing process based on activated carbon, followed byanion exchange and cation exchange chromatography (adapted from Source: Ichihara etal. (2019)). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.7 A schematic analysis of the various mAb process impurities based on their molecularweight and isoelectric point with examples of media types that can be employed for theirselective removal (Source: Gillespie et al., 2015). . . . . . . . . . . . . . . . . . . . . . . . 12

1.8 Ion-Exchange Chromatography (Source: WatersTM). . . . . . . . . . . . . . . . . . . . . . 13

1.9 The net surface charge of a protein is highly pH dependent and will change gradually asthe pH of the environment changes (Source: GE Healthcare). . . . . . . . . . . . . . . . . 14

1.10 Typical high-resolution SEC separation (Source: GE Healthcare). . . . . . . . . . . . . . . 18

1.11 Illustration of a UV curve for acetone in a typical performance test chromatogram fromwhich the HETP and As values are calculated (Source: GE Healthcare). . . . . . . . . . . 20

3.1 Chromatogram of the flow-through AEX run with non-adjusted loading conditions (N-ADC)of the AEX-CEX N-ADC train with sodium acetate buffer. The AEX column was equili-brated with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1mL/min. The AEX column was loaded at the flow rate of 0.2 mL/min with 0.29 mg/mL mAbloading at 225 mL (225 CV). The mAb loading conditions were pH 3.6 and 3.47 mS/cmconductivity. The UV was measured at wavelengths 280 nm and 214 nm. . . . . . . . . . 38

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3.2 Chromatogram of the flow-through CEX run with non-adjusted loading conditions (N-ADC)of the AEX-CEX N-ADC train with sodium acetate buffer. The CEX column was equili-brated with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1mL/min. The CEX column was loaded at the flow rate of 0.2 mL/min with 200 mL (400 CV)of mAb flow-through solution obtained from the AEX N-ADC run. The mAb loading con-ditions were pH 3.6 and 3.51 mS/cm conductivity. The UV was measured at wavelengths280 nm and 214 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.3 Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with sodium acetate buffer. The AEX column was equilibrated with25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min. TheAEX column was loaded at the flow rate of 0.2 mL/min with 0.29 mg/mL mAb loading at225 mL (225 CV). The mAb loading conditions were adjusted to pH 6.0 and 4.00 mS/cmconductivity. The UV was measured at wavelengths 280 nm and 214 nm. . . . . . . . . . 39

3.4 Chromatogram of the flow-through CEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with sodium acetate buffer. The CEX column was equilibrated with25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min.The CEX column was loaded at the flow rate of 0.2 mL/min with 200 mL (400 CV) of mAbflow-through solution obtained from the AEX ADC run. The mAb loading conditions werepH 6.0 and 4.12 mS/cm conductivity. The UV was measured at wavelengths 280 nm and214 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.5 Analysis of the total protein, mAb and HCP content of the samples collected from theAEX-CEX N-ADC (Figure A) (Trastuzumab loading conditions: pH 3.6 and 3.47 mS/cmconductivity, and ZnCl2 is still present) and AEX-CEX ADC (Figure B) (Trastuzumab load-ing conditions adjusted to pH 6.0 and 4.00 mS/cm conductivity, and ZnCl2 has been re-moved). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.6 Trastuzumab product quantity (%) present in the flow-through, wash and elution fractionsof the cation exchange chromatography in comparison to the CEX feed, in both non-adjusted (N-ADC) and adjusted (ADC) mAb loading conditions (analysis of the antibodytitre by HPLC-Protein A affinity chromatography). . . . . . . . . . . . . . . . . . . . . . . . 43

3.7 Size exclusion chromatography analysis of the antibody (monomer) purity (%) and HMWI(%) and LMWI (%) content in the fractions (feed, wash, elution and flow-through) of theAEX-CEX N-ADC (with non-adjusted loading conditions) and AEX-CEX ADC (with ad-justed loading conditions) trains with sodium acetate buffer. Above the 95% dot line isthe target bulk drug specification limit for monomer purity, whereas the HMWI and LMWIcontent should be lower than 1%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.8 Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of thefeed (B), flow-through (C), column wash (D) and elution (E) of the AEX N-ADC (with non-adjusted loading conditions) chromatography run. The monomer peak area is observedbetween 20.96 and 22.04 min. of retention time. . . . . . . . . . . . . . . . . . . . . . . . 45

3.9 Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of thefeed (B), flow-through (C), column wash (D) and elution (E) of the CEX N-ADC (with non-adjusted loading conditions) chromatography run. The monomer peak area is observedbetween 20.96 and 22.04 min. of retention time. . . . . . . . . . . . . . . . . . . . . . . . 45

3.10 Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of thefeed (B), flow-through (C), column wash (D) and elution (E) of the AEX ADC (with adjustedloading conditions) chromatography run. The monomer peak area is observed between20.96 and 22.04 min. of retention time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

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3.11 Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of thefeed (B), flow-through (C), column wash (D) and elution (E) of the CEX ADC (with adjustedloading conditions) chromatography run. The monomer peak area is observed between20.96 and 22.04 min. of retention time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3.12 Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samplesof the feed (B), flow-through (C), column wash (D) and elution (E) of the AEX N-ADC (withnon-adjusted loading conditions) chromatography run. The peak of the retained mAb isobserved between 0.84 and 0.88 min. The mAb titre in each fraction is 0.29 mg/mL (B),0.24 mg/mL (C), 0.08 mg/mL (D) and 0.00 mg/mL (E). . . . . . . . . . . . . . . . . . . . . 48

3.13 Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samplesof the feed (B), flow-through (C), column wash (D) and elution (E) of the CEX N-ADC (withnon-adjusted loading conditions) chromatography run. The peak of the retained mAb isobserved between 0.84 and 0.88 min. The mAb titre in each fraction is 0.24 mg/mL (B),0.07 mg/mL (C), 6.27 mg/mL (D) and 0.38 mg/mL (E). . . . . . . . . . . . . . . . . . . . . 48

3.14 Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samplesof the feed (B), flow-through (C), column wash (D) and elution (E) of the AEX ADC (withadjusted loading conditions) chromatography run. The peak of the retained mAb is ob-served between 0.84 and 0.88 min. The mAb titre in each fraction is 0.29 mg/mL (B), 0.26mg/mL (C), 0.07 mg/mL (D) and 0.00 mg/mL (E). . . . . . . . . . . . . . . . . . . . . . . . 49

3.15 Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samplesof the feed (B), flow-through (C), column wash (D) and elution (E) of the CEX ADC (withadjusted loading conditions) chromatography run. The peak of the retained mAb is ob-served between 0.84 and 0.88 min. The mAb titre in each fraction is 0.26 mg/mL (B), 0.08mg/mL (C), 7.23 mg/mL (D) and 0.14 mg/mL (E). . . . . . . . . . . . . . . . . . . . . . . . 49

3.16 Analysis of the process yield (A) and antibody (monomer) purity (B) of the AEX-CEXN-ADC (with non-adjusted loading conditions) and of the AEX-CEX ADC (with adjustedloading conditions) trains with sodium acetate buffer (based on the AEX feed as startingproduct). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

3.17 Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 8.0. The AEX column was equilibrated with25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The AEXcolumn was loaded at the flow rate of 0.2 mL/min with 0.23 mg/mL mAb loading at 125mL (125 CV). The mAb loading conditions were pH 8.0 and 4.00 mS/cm conductivity. TheUV was measured at wavelengths 280 nm and 214 nm. . . . . . . . . . . . . . . . . . . . 53

3.18 Chromatogram of the flow-through CEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 8.0. The CEX column was equilibrated with25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The CEXcolumn was loaded at the flow rate of 0.2 mL/min with 0.23 mg/mL mAb loading at 100 mL(200 CV). The mAb loading conditions were pH 8.0 and 4.00 mS/cm conductivity. The UVwas measured at wavelengths 280 nm, 254 nm and 214 nm. (Note: * The chromatographyrun had a technical problem and it stopped around the loading at 146 CV. The ÄKTAsystem had to be restarted and the mAb loading was resumed until 220 CV.) . . . . . . . 53

3.19 Analysis of the DNA (Figure A) and total protein (Figure B) content of the Trastuzumabprecipitated material (before buffer exchange) and of the Trastuzumab feed (after bufferexchange) and of the fractions collected in the flow-through, wash and elution from theflow-through AEX run with Tris-HCl buffer at adjusted loading conditions of pH 8.0 and4.00 mS/cm conductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

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3.20 Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 7.0. The AEX column was equilibrated with25 mM Tris-HCl buffer (pH 7.0, 2.29 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The AEXcolumn was loaded at the flow rate of 0.2 mL/min with 0.23 mg/mL mAb loading at 125mL (125 CV). The mAb loading conditions were pH 7.0 and 4.00 mS/cm conductivity. TheUV was measured at wavelengths 280 nm, 260 nm and 214 nm. . . . . . . . . . . . . . . 56

I.1 UV 280 nm HPLC-Protein A affinity chromatogram of the Trastuzumab supernatant solu-tion precipitated with ZnCl2 (mAb feed solution of the AEX). The peak of the retained mAbis observed between 0.84 and 0.88 min. This mAb sample was analyzed the same day(25th Nov 2019) of the ZnCl2 precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . III

I.2 UV 280 nm size exclusion chromatogram (SEC) of the Trastuzumab supernatant solutionprecipitated with ZnCl2, which is the mAb feed solution of the AEX. This mAb sample wasanalyzed the same day (25th Nov 2019) of the ZnCl2 precipitation. . . . . . . . . . . . . . IV

I.3 Analysis of the DNA content of the samples collected from the AEX-CEX trains "1" (TrastuzumAbloading conditions adjusted to pH 6.0 and 17 mS/cm conductivity and ZnCl2 is still present), "2" (Trastuzumab loading conditions adjusted to pH 6.0 and 1.98 mS/cm conductivity andZnCl2 has been removed) and "3" (Trastuzumab loading conditions not adjusted - pH 4.0and conductiviy 2.15 mS - and ZnCl2 is still present), using the Picogreen assay. (The redasterisk (*) means that the DNA content in the collected fractions was bellow the limit ofdetection of the DNA assay (Picogreen assay), therefore the DNA content of these frac-tions cannot be exactly determined and it can be any value bellow the maximum rangeof the respective column graph plotted. The darker coloured columns correspond to thefeed and flow-through fractions and the lighter coloured columns correspond to the washand elution fractions.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII

I.4 UV 280 nm HPLC-Protein A affinity chromatogram of the Trastuzumab supernatant solu-tion precipitated with ZnCl2, which is the mAb feed solution of the AEX. The peak of theretained mAb is observed between 0.84 and 0.88 min. This mAb sample was analyzedtwo months and half (8th Fev 2020) after the ZnCl2 precipitation (25th Nov 2019). Duringthis time, the samples were stored at pH 4.0 and -20°C. . . . . . . . . . . . . . . . . . . . XII

I.5 Zoomed-in chromatogram of the wash (225-235 CV), elution (235-245 CV) and CIP (245-252 CV) steps of the flow-through AEX run with non-adjusted loading conditions (N-ADC)of the AEX-CEX N-ADC train. The AEX column was washed with 25 mM sodium acetatebuffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mM sodium acetatebuffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate.The column was sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min. . . . . . XII

I.6 Zoomed-in chromatogram of the wash (400-410 CV), elution (410-420 CV) and CIP (420-434 CV) steps of the flow-through CEX run with non-adjusted loading conditions (N-ADC)of the AEX-CEX N-ADC train. The CEX column was washed with 25 mM sodium acetatebuffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mM sodium acetatebuffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate.The column was sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2 mL/min. . . . XIII

I.7 Zoomed-in chromatogram of the mAb loading step at 200 mL (400 CV) of the flow-throughCEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with sodiumacetate buffer at pH 6.0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII

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I.8 Zoomed-in chromatogram of the wash (225-235 CV), elution (235-245 CV) and CIP (245-252 CV) steps of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train. The AEX column was washed with 25 mM sodium acetate buffer(pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mM sodium acetate bufferwith 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. Thecolumn was sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min. . . . . . . . XIV

I.9 Zoomed-in chromatogram of the wash (400-410 CV), elution (410-420 CV) and CIP (420-434 CV) steps of the flow-through CEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train. The CEX column was washed with 25 mM sodium acetate buffer(pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mM sodium acetate bufferwith 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. Thecolumn was sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2 mL/min. . . . . . XIV

I.10 Chromatogram of the flow-through CEX test run with adjusted loading conditions (ADC)with Tris-HCl buffer at pH 6.0. The CEX column was equilibrated with 25 mM Tris-HClbuffer (pH 6.0, 2.24 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The CEX column wasloaded at the flow rate of 0.2 mL/min with 2.25 mg/mL mAb loading at 5 mL (10 CV). ThemAb loading conditions were pH 6.0 and 4.00 mS/cm conductivity. The UV was measuredat wavelengths 280 nm and 214 nm. The CEX column was washed (10-20 CV) with 25mM Tris-HCl buffer (pH 6.0, 2.24 mS/cm conductivity, 10 CV) and eluted (20-30 CV) using25 mM Tris-HCl buffer with 1 M NaCl (pH 6.0, 86.2 mS/cm, 10 CV), both steps at 1 mL/minof flow rate. The column was sanitized (30-44 CV) with 0.5 M NaOH for 14 CV at the flowrate of 0.2 mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIX

I.11 Chromatogram of the flow-through CEX test run with adjusted loading conditions (ADC)with Tris-HCl buffer at pH 7.0. The CEX column was equilibrated with 25 mM Tris-HClbuffer (pH 7.0, 2.29 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The CEX column wasloaded at the flow rate of 0.2 mL/min with 2.25 mg/mL mAb loading at 5 mL (10 CV). ThemAb loading conditions were pH 7.0 and 4.00 mS/cm conductivity. The UV was measuredat wavelengths 280 nm and 214 nm. The CEX column was washed (10-20 CV) with 25mM Tris-HCl buffer (pH 7.0, 2.29 mS/cm conductivity, 10 CV) and eluted (20-30 CV) using25 mM Tris-HCl buffer with 1 M NaCl (pH 7.0, 85.7 mS/cm, 10 CV), both steps at 1 mL/minof flow rate. The column was sanitized (30-44 CV) with 0.5 M NaOH for 14 CV at the flowrate of 0.2 mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XX

I.12 Chromatogram of the flow-through CEX test run with adjusted loading conditions (ADC)with Tris-HCl buffer at pH 8.0. The CEX column was equilibrated with 25 mM Tris-HClbuffer (pH 8.0, 1.87 mS/cm, 10 CV) at a flow-rate of 1 mL/min. The CEX column wasloaded at the flow rate of 0.2 mL/min with 2.25 mg/mL mAb loading at 5 mL (10 CV). ThemAb loading conditions were pH 8.0 and 4.00 mS/cm conductivity. The UV was measuredat wavelengths 280 nm and 214 nm. The CEX column was washed (10-20 CV) with 25mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm conductivity, 10 CV) and eluted (20-30 CV) using25 mM Tris-HCl buffer with 1 M NaCl (pH 8.0, 85.5 mS/cm, 10 CV), both steps at 1 mL/minof flow rate. The column was sanitized (30-44 CV) with 0.5 M NaOH for 14 CV at the flowrate of 0.2 mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XX

I.13 UV 280 nm HPLC-Protein A affinity chromatogram of a trastuzumab thawed solution sam-ple with an antibody titre of 27 mg/mL obtained after Protein A affinity chromatography(capture step) used for the CEX ADC (with adjusted loading conditions) test runs withTris-HCl buffer at pH 6.0, 7.0 and 8.0. The peak of the retained mAb is observed between0.84 and 0.88 min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXI

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I.14 Overlaid UV 280 nm HPLC-Protein A affinity chromatograms of trastuzumab samples ofthe feed, flow-through, column wash and elution of the CEX ADC (with adjusted loadingconditions) chromatography test runs with Tris-HCl buffer at pH 6.0 (A), 7.0 (C), 8.0 (E).The CEX ADC trastuzumab feed solution (2.25 mg/mL) for the test runs at pH 6.0 (B),7.0 (D) and 8.0 (E) originated from a dilution of a trastuzumab thawed solution with anantibody titre of 27 mg/mL. The peak of the retained mAb is observed between 0.84 and0.88 min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXI

I.15 Zoomed-in chromatogram of the mAb loading step at 125 mL (125 CV) of the flow-throughAEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HClbuffer at pH 8.0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXII

I.16 Zoomed-in chromatogram of the wash (125-135 CV), elution (135-145 CV) and CIP (145-152 CV) steps of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 8.0. The AEX column was washed with25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm conductivity, 10 CV) and eluted using 25mM sodium acetate buffer with 1 M NaCl (pH 8.0, 85.5 mS/cm, 10 CV), both steps at 1mL/min of flow rate. The column was sanitized with 1 M NaOH for 7 CV at the flow rate of0.2 mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXII

I.17 Zoomed-in chromatogram of the wash (220-230 CV), elution (230-240 CV) and CIP (240-254 CV) steps of the flow-through CEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 8.0. The CEX column was washed with25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm conductivity, 10 CV) and eluted using 25 mMsodium acetate buffer with 1 M NaCl (pH 8.0, 85.5 mS/cm, 10 CV), both steps at 1 mL/minof flow rate. The column was sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIII

I.18 Zoomed-in chromatogram of the mAb loading step at 125 mL (125 CV) of the flow-throughAEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HClbuffer at pH 7.0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIV

I.19 Zoomed-in chromatogram of the wash (125-135 CV), elution (135-145 CV) and CIP (145-152 CV) steps of the flow-through AEX run with adjusted loading conditions (ADC) of theAEX-CEX ADC train with Tris-HCl buffer at pH 7.0. The AEX column was washed with25 mM Tris-HCl buffer (pH 7.0, 2.29 mS/cm conductivity, 10 CV) and eluted using 25mM sodium acetate buffer with 1 M NaCl (pH 7.0, 85.7 mS/cm, 10 CV), both steps at 1mL/min of flow rate. The column was sanitized with 1 M NaOH for 7 CV at the flow rate of0.2 mL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXV

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List of Tables

1.1 Compression factors for a column at laboratory scale (Source: Merck KGaA). . . . . . . . 19

2.1 To ensure a packed bed volume of 0.50 mL, a settle bed volume of 0.54 mL is neededto pack a stable bed at 2.55 cm bed height (considering 8% compression). Taking intoaccount a 70% slurry concentration, the volume of slurry needed is 0.77 mL. The resin’scompression factor is 1.09 (Equation 1.6), which is within the range found in the literature(Table 1.1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.2 Specifications of the TriconTM 5 mm diameter x 2.5 cm height column at 0.5 mL (GEHealthcare) packed with Eshmuno® CP-FT resin (Merck KGaA) after the performancetest to measure the packed column efficiency. . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.3 AEX flow-through method for the AEX N-ADC and AEX ADC runs. The mAb (0.29 mg/mL)loading conditions for AEX N-ADC were pH 3.6 and 3.47 mS/cm conductivity. The mAb(0.29 mg/mL) loading conditions for the train AEX ADC were adjusted to pH 6.0 and 4.00mS/cm conductivity by buffer exchange using UF/DF. The method flow-rate was 1 mL/min.The UV was measured at wavelengths 280 nm and 214 nm. The AEX column was loadedat the flow rate of 0.2 mL/min with a target of mAb loading at 225 mL (225 CV). . . . . . . 26

2.4 CEX flow-through method for the CEX N-ADC and CEX ADC runs. The mAb loadingconditions for CEX N-ADC were pH 3.6 and 3.51 mS/cm conductivity. The mAb loadingconditions for the train CEX ADC were pH 6.0 and 4.12 mS/cm conductivity. The methodflow-rate was 1 mL/min. The UV was measured at wavelengths 280 nm and 214 nm. TheCEX column was loaded at the flow rate of 0.2 mL/min with a target of mAb loading at200 mL (400 CV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.5 CEX flow-through method "test" run with Tris-HCl at pH 6.0 for adjusted loading conditions(ADC). The mAb (2.25 mg/mL) loading conditions were pH 6.0 and 4.00 mS/cm conduc-tivity. The method flow-rate was 1 mL/min. The UV was measured at wavelengths 280 nmand 214 nm. The CEX column was loaded at the flow rate of 0.2 mL/min with a target ofmAb loading at 5 mL (10 CV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27



2.8 AEX flow-through run with Tris-HCl at pH 7.0 for adjusted loading conditions (ADC). ThemAb (0.23 mg/mL) loading conditions were adjusted to pH 7.0 and 4.00 mS/cm conduc-tivity. The method flow-rate was 1 mL/min. The UV was measured at wavelengths 280nm, 260 nm and 214 nm. The AEX column was loaded at the flow rate of 0.2 mL/min witha target of mAb loading at 125 mL (125 CV). . . . . . . . . . . . . . . . . . . . . . . . . . 29

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2.9 CEX flow-through run with Tris-HCl at pH 7.0 for adjusted loading conditions (ADC). ThemAb loading conditions were pH 7.0 and 4.08 mS/cm conductivity. The method flow-ratewas 1 mL/min. The UV was measured at wavelengths 280 nm, 260 nm and 214 nm. TheCEX column was loaded at the flow rate of 0.2 mL/min with a target of mAb loading at100 mL (200 CV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.10 AEX flow-through run with Tris-HCl at pH 8.0 for adjusted loading conditions (ADC). ThemAb (0.23 mg/mL) loading conditions were adjusted to pH 8.00 and 4.00 mS/cm conduc-tivity. The method flow-rate was 1 mL/min. The UV was measured at wavelengths 280 nmand 214 nm. The AEX column was loaded at the flow rate of 0.2 mL/min with a target ofmAb loading at 125 mL (125 CV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.11 CEX flow-through run with Tris-HCl at pH 8.0 for adjusted loading conditions (ADC). ThemAb loading conditions were pH 8.0 and 3.96 mS/cm conductivity. The method flow-ratewas 1 mL/min. The UV was measured at wavelengths 280 nm, 254 nm and 214 nm. TheCEX column was loaded at the flow rate of 0.2 mL/min with a target of mAb loading at100 mL (200 CV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

I.1 Dilutions for the zinc chloride standard curve. . . . . . . . . . . . . . . . . . . . . . . . . . II.2 AEX flow-through method for the AEX-CEX train "1", "2" and "3" of the initial experiments

with a Trastuzumab titre of 0.34 mg/mL. The mAb loading conditions for AEX "1" wereadjusted to pH 6.0 and 17. mS/cm conductivity with 4 M TrisBase pH 9. The mAb loadingconditions for the train AEX "2" were adjusted to pH 6.0 and 1.98 mS/cm conductivityby buffer exchange using UF/DF. The mAb loading conditions for the train AEX "3" werepH 4.0 and conductivity 2.15 mS/cm. The method flow-rate was 1 mL/min. The UV wasmeasured at wavelengths 280 nm and 214 nm. The AEX column was loaded at the flowrate of 0.2 mL/min with a target of mAb loading at 133 mL (133 CV). . . . . . . . . . . . . II

I.3 CEX flow-through method for the AEX-CEX train "1", "2" and "3" of the initial experimentswith a Trastuzumab titre of 0.34 mg/mL. The mAb loading conditions for CEX "1" were pH6.0 and 17 mS/cm conductivity. The mAb loading conditions for the train CEX "2" were pH6.0 and 1.98 mS/cm conductivity. The mAb loading conditions for the train CEX "3" werepH 4.0 and conductivity 2.15 mS/cm. The method flow-rate was 1 mL/min. The UV wasmeasured at wavelengths 280 nm and 214 nm. The CEX column was loaded at the flowrate of 0.2 mL/min with a target of mAb loading at 80 mL (160 CV). . . . . . . . . . . . . . III

I.4 qPCR DNA quantification using a standard curve spiked with concentrations of ZnCl2ranging from 0.1 mM to 12 mM and a qPCR "normal" DNA standard curve as standardcontrol curve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

I.5 DNA (ng/mL) and total protein (µg/mL) content of the trastuzumab supernatant after ZnCl2precipitation using the Picogreen and qPCR assay, and the Bradford method, respectively.An estimation of the HCP content (Total protein = mAb titer + HCP) of the starting productwas determined considering the mAb titre of 0.34 mg/mL. . . . . . . . . . . . . . . . . . . V

I.6 Results of the DNA content (quantified by the Picogreen assay) of the samples collectedfrom the AEX-CEX train "1" with mAb loading conditions adjusted to pH 6.0 and 17 mS/cmconductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X

I.7 Results of the DNA content (quantified by the Picogreen assay) of the samples collectedfrom the AEX-CEX train "2" with mAb loading conditions adjusted to pH 6.0 and 1.98mS/cm conductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X

I.8 Results of the DNA content (quantified by the Picogreen assay) of the samples collectedfrom the AEX-CEX train "3" with mAb loading conditions of pH 4.0 and 2.15 mS/cm con-ductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X

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I.9 DNA content of the samples collected from the AEX-CEX N-ADC train with non-adjustedmAb loading conditions (pH 3.6 and 3.47 mS/cm conductivity). . . . . . . . . . . . . . . . XV

I.10 DNA content of the samples collected from the AEX-CEX ADC train with adjusted mAbloading conditions (pH 6.0 and 4.00 mS/cm conductivity). . . . . . . . . . . . . . . . . . . XV

I.11 Total protein content, mAb titre and HCP estimation of the samples collected from theAEX-CEX N-ADC train with non-adjusted mAb loading conditions (pH 3.6 and 3.47 mS/cmconductivity). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI

I.12 Total protein content, mAb titre and HCP estimation of the samples collected from theAEX-CEX ADC train with adjusted mAb loading conditions (pH 6.0 and 4.00 mS/cm con-ductivity). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI

I.13 Monomer yield, monomer purity and HMWI and LMWI content of the fractions (feed, flow-through, wash and elution) of the AEX-CEX N-ADC (with non-adjusted loading conditions)train. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII

I.14 Monomer yield, monomer purity and HMWI and LMWI content of the fractions (feed, flow-through, wash and elution) of the AEX-CEX ADC (with adjusted loading conditions) train. XVII

I.15 Overall results of the analytics (DNA, total protein, HCP, mAb yield, mAb purity, HMWIand LMWI) of the AEX-CEX train with non-adjusted loading conditions (N-ADC) at pH 3.6and 3.47 mS/cm). The mAb yield of the AEX N-ADC flow-through was 82%, however theCEX feed has a mab yield of 73% due to the differences in the mAb loading volume. . . . XVIII

I.16 Overall results of the analytics (DNA, total protein, HCP, mAb yield, mAb purity, HMWI andLMWI) of the AEX-CEX train with adjusted loading conditions (ADC) at pH 6.0 and 4.00mS/cm). The mAb yield of the AEX ADC flow-through was 90%, however the CEX feedhas a mab yield of 80% due to the differences in the mAb loading volume. . . . . . . . . . XVIII

I.17 DNA content of the Trastuzumab starting material precipitated (before buffer exchange),Trastuzumab feed (after buffer exchange) and of the samples collected from the flow-through AEX run with Tris-HCl at adjusted loading conditions of pH 8.0 and 4.00 mS/cmconductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIII

I.18 Total protein content of the Trastuzumab starting material precipitated (before buffer ex-change), Trastuzumab feed (after buffer exchange) and of the samples collected from theflow-through AEX run with Tris-HCl at adjusted loading conditions of pH 8.0 and 4.00mS/cm conductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIV

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Acronyms

AC activated carbon

ADC adjusted loading conditions

ADCC antibody-dependent cellular cytotoxicity

AEX Anion Exchange Chromatography

aPES asymmetrical polyethersulfone

BGG bovine gamma globulin

BSA Bovine serum albumin

CEX Cation Exchange Chromatography

CH constant heavy

CHO Chinese hamster ovary

CIP cleaning-in-place

CF compression factor

CV column volume

DBC dynamic binding capacity

DF Diafiltration

DSP downstream processing

dsDNA double-stranded DNA

DNA deoxyribonucleic acid

ELISA Enzyme-linked immunosorbent assay

EMA European Medicines Agency

Fab antigen-binding fragment

Fc fragment crystallizable

FDA Food and Drug Administration

HCl hydrochloric acid

HCP host cell protein

HETP height equivalent to a theoretical plate

HMWI higher molecular weight impurities

HPLC high performance liquid chromatography

IEX Ion Exchange Chromatography

Ig Immunoglobulins

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LMWI lower molecular weight impurities

mAbs monoclonal antibodies

MCE mixed cellulose esters

MWCO molecular weight cut-off

MM molar mass

N-ADC non-adjusted loading conditions

NFF Normal Flow Filtration

NMWL nominal molecular weight limit

LLOQ lower limit of quantification

LOD limit of detection

qPCR quantitive polymerase chain reaction

PBV packed bed volume

pI isoelectric point

SBV settle bed volume

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SEC Size exclusion chromatography

TFF Tangential Flow Filtration

Trp tryptophan

Tyr tyrosine

VH variable heavy

VL variable light

UF Ultrafiltration

USP upstream processing

UV ultraviolet

WHO World Health Organization

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1 Introduction

1.1 Monoclonal Antibodies

Antibodies belong to a large family of glycoproteins, also known as Immunoglobulins (Ig), whichare produced by B lymphocytes in order to stimulate the body’s immune system to identify, neutralizeand destroy compromised cells, malignant proteins, or pathogens, such as foreign bacteria or viruses [1].The Immunoglobulins constitute the humoral branch of the immune system and form approximately 20%of the plasma protein in humans. There are several Ig populations that can be found on the surface oflymphocytes, in exocrine secretions and in extravascular fluids. Antibodies are host proteins producedin response to foreign molecules or other agents in the body and they are able to bind specifically to aparticular foreign substance known as an antigen [2]. This response is a key mechanism used by a hostorganism to protect itself against the action of foreign molecules or organisms. B-lymphocytes carryingspecific receptors recognize and bind the antigenic determinants of the antigen and this stimulates aprocess of division and differentiation, transforming the B-lymphocytes into plasma cells. It is theselymphoid or plasma cells that predominantly synthetize antibodies [3].

The Y-shape structure of antibodies was discovered by Rodney Porter and Gerald M Edelman,who were awarded a Nobel Prize for Medicine and Physiology for this finding in 1972. The typical IgGmolecule consists of four polypeptide chains: two heavy (50 kDa each) and two light chains (25 kDaeach) organized in a "Y"-shaped format. Each of these chains contains one variable region and mul-tiple constant regions, which are linked by noncovalent bonds and disulfide bridges (shown in red inFigure 1.1), resulting in a molecule with a molecular weight of approximately 150 kDa. These Y-shapedmolecules have two main functional regions: the antigen-binding fragment (Fab) and the fragment crys-tallizable (Fc) region. The Fab portion is composed of variable heavy (VH) and variable light (VL) regions,and the Fc portion, which is important for immune signaling and effector functions, is composed of theconstant heavy (CH) regions (CH1, CH2, and CH3) [2,4].

Figure 1.1: Structural schematic of IgG (Abbreviations: Fab - fragment antigen binding; Fc - fragment crystallizable;IgG - immunoglobulin G; Ag - antigen) (Source: Murphy et al., 2016).

In mammals, immunoglobulins are divided into five major classes according to their heavychains: IgG (γ), IgA (α), IgM (µ), IgD (δ) and IgE (ε). Differences in the amino acid sequence in theconstant region (Fc) of the heavy chain allow these Ig to function in different types of immune responsesand at particular stages of the immune response [3]. In addition to the major Ig classes, several Ig sub-

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classes exist in all members of a particular animal species, which differ based on the heavy chain typeof each Ig class. In humans there are four subclasses of IgG: IgG1, IgG2, IgG3 and IgG4, and twosubclasses of IgA: IgA1 and IgA2 (numbered in order of decreasing concentration in serum) [5].

The hybridoma technique, introduced by Georges Köhler and César Milstein in 1975, revolu-tionized antibody research and paved the way to clinical advances due to the discovery of how celllines could be made in order that they produce an antibody of know specificity [6]. Monoclonal antibod-ies (mAbs) are highly specific antibodies produced from hybridoma cells that are created by isolatingplasma cell precursors, which are then fused with immortal cells. The hybridoma cells can be single-cellcloned and expanded as individual clones that secret only one antibody type, a monoclonal antibody,that has a high specificity which is a significant advantage for therapeutical applications. Productionof monoclonal antibodies (mAbs) using the hybridoma technology has been successful for the pro-duction of mouse monoclonal antibodies, however this has meant that therapeutical applications havealways been associated with the risk of immunogenic reactions (with the exception of human antibodieswhich are nonimmunogenic to humans). Nowadays, mAbs are often generated by isolating or transform-ing antibody-producing cells taken directly from immunized animals or patients, and transplanting theantibody-encoding genes of these cells into suitable producer cell lines, rather than using the hybridomatechnology [6]. For antibodies to be most effective when used as therapeutic agents, they should havea long serum half-life, low immunogenicity, a high affinity for the antigen and be able to neutralize theactivity of the antigen. These are all features that can be enhanced by genetic manipulation using re-combinant antibodies in order to overcome the problem of the high immunogenicity of mouse mAbs [3].

1.1.1 Applications

Even though monoclonal antibodies are routinely used in biochemistry, molecular and cell bi-ology, and medical research, the most beneficial application is their use as therapeutic drugs for thepredominant treatment of human diseases, especially in the areas of oncology, immunology and hema-tology. The Food and Drug Administration (FDA) has approved several mAbs for clinical use againstcancer-related diseases (lymphoma, myeloma, melanoma, glioblastoma, neuroblastoma, sarcoma, col-orectal, lung, breast, ovarian, head and neck), auto-immuno disorders (rheumatoid arthritis and Crohn’sdisease), asthma, transplant rejection and cardiovascular and infectious diseases [7,8]. Oncology is themedical specialty where mAb treatments are most used in. Monoclonal antibodies are used in antitumortherapy where mAbs are directed to their tumoral targets (antigen) without affecting healthy tissues orwith minimal effects on them [8].

Human, humanized, chimeric and murine antibodies respectively account for 51%, 34.7%, 12.5%and 2.8% of all mAbs in clinical use, with human and humanized mAbs (a total of 85.7%) being thedominant modalities in the field of therapeutic antibodies [7]. Humanization of mouse mAbs has beenimplemented on a large scale, due to the availability, low cost and quick production time for mousemAbs [7]. The use of humanized antibodies has helped greatly to improve tolerance of mAb therapeu-tics. Furthermore, intricate control over antibody sequences has opened the door to engineering mAbsfor a wide range of possible applications in medicine. One of the most well-known humanized antibodiesis Trastuzumab (Herceptin), which is the monoclonal antibody used in this study (Section 1.1.3).

1.1.2 Market

The first monoclonal antibody was commercialized and approved by the FDA in 1986, and eversince then, therapeutic monoclonal antibodies have grown to become the dominant product class within

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the biopharmaceutical market [2]. Monoclonal antibodies represent around 36% of the total biophar-maceutical market, with growth sale per year of in between 7.2% and 18.3% [9], making mAbs thesingle largest class of biological drugs nowadays and the fastest-growing sector of the pharmaceuticalindustry [10]. In 2018, the global therapeutic monoclonal antibody market was valued at approximatelyUS$115.2 billion and a revenue of $150 billion was expected by the end of 2019 and $300 billion by2025. This explosive growth of the market of therapeutic drugs is directly related to the approval of newdrugs for treating various human diseases, including many cancers, autoimmune, metabolic and infec-tious diseases. As of December 2019, a total of 79 therapeutic mAbs have been approved by the FDAand are currently on the market, including 30 mAbs for the treatment of cancer [7].

The global mAb market is currently dominated by seven companies: Genentech (Roche Group)(30.8%), Abbvie (20.0%), Johnson & Johnson (13.6%), Bristol-Myers Squibb (6.5%), Merck Sharp &Dohme (5.6%), Novartis (5.5%), Amgen (4.9%), with other companies comprising the remaining 13% [7].

With the increasing and aging of the world’s population, there is a further expansion of the globalmarket of biopharmaceuticals, which, in consequence, fuels the growth in monoclonal antibody productsales. The high demand of monoclonal antibody therapeutic doses results in a crucial need to obtainhigh amounts of pure mAbs, which in spite of their effectiveness and safety for human administration,the access to this type of therapy has been hampered by high manufacturing costs, making it imperativeto develop effective and economical methods to purify antibodies [2]. In the past few years, the down-stream processing of mAbs has been the critical step in the overall production due to its high costs ofmanufacturing related with high-titers, representing 80% of the total production costs. The downstreamprocessing development has not evolved at the same pace as of the upstream processing improvements,which, consequently, has created bottlenecks that need to be overcome by new innovative manufacturingin order to reduce costs and increase productivity without sacrificing process robustness [2,11].

1.1.3 Trastuzumab

Trastuzumab is an anti-HER2 humanized IgG1 monoclonal antibody produced in CHO cell cul-tures. Trastuzumab (also know for its brand name Herceptin® from Genentech (Roche Group)) was firstdeveloped by Genentech in the 1990s and had its first regulatory approval for treatment of human epi-dermal growth factor receptor (HER)-2+ metastatic breast cancer in 1998 by the FDA, and in 2000 bythe European Medicines Agency (EMA) [2,7,12]. It was later approved, in 2010, for treatment of gastricand gastroesophageal junction adenocarcinoma [12]. In 2018, trastuzumab was the fourth most soldmonoclonal antibody drug and the most sold drug for breast cancer treatment with an annual revenue of$7 billion, with sales hovering around that amount over the last few years [7].

The anti-HER2 isotype family, which was generated by recombinant DNA technology, featuresthe variable region of trastuzumab. Trastuzumab targets the HER2 receptor that is found on the cellmembrane of epithelial cells (normal cells and HER2+ tumor cells). HER2 has an important role in nor-mal cell growth and differentiation, however, in certain types of cancers, particularly breast cancer, HER2is over-expressed and causes uncontrollable cell proliferation. Binding of trastuzumab to HER2 recep-tors blocks intracellular signaling pathways by attenuating the signal transduction downstream, whichmay promote cell death through different mechanisms including antibody-dependent cellular cytotoxic-ity (ADCC) and phagocytosis. Activation of natural killer cells expressing the Fc gamma receptor, whichcan be bound by the Fc domain of Trastuzumab, is mainly what leads to ADCC [13,14]. The mechanismof action of Trastuzumab is illustrated in Figure 1.2.

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Figure 1.2: Trastuzumab (Herceptin) mechanism of action (mechanism sequence: A → B → C → D) to suppresscancer cells growth and proliferation (adapted from Source: Genentech).

1.2 Standard mAb manufacturing process

Recombinant mAbs are produced from cell culture/fermentation of genetically modified eukary-otic host cells - Chinese hamster ovary (CHO) cells [15]. A standard monoclonal antibody manufacturingprocedure using mammalian cell culture is illustrated in Figure 1.3. The first step of the upstream pro-cessing (USP) is the inoculum expansion where cells are thawed and cultured in small flasks and aftera period of approximately two weeks, the cells are grown in increasingly larger volumes to provide aseed culture for the fermentation tanks. Once the cells are inoculated into the production bioreactor,they are grown under controlled conditions to an optimal density for maximum productivity. At the end ofthe production stage, the harvest step separates the cells from the cell culture supernatant to recoverthe product in the liquid phase. This is typically done by either centrifugation and/or microfiltration [8].

The bioreactor harvest is followed by at least three unit operations, including primary capture,secondary purification, and final polishing. The purification process in the downstream processing (DSP)of mAbs usually involves capture with a Protein A-based chromatography media, which results in a highdegree of purity and recovery in a single step [16,17]. In most cases, since mAbs elute at low pH values(between pH 3.0 and 4.0), a virus innactivation step can follow at a low pH [8]. The purification and pol-ishing steps generally incorporate Cation Exchange Chromatography (CEX) and Anion Exchange Chro-matography (AEX), which can be performed in either bind/elute (binding of the mAb) or flow-throughmode (non-binding of the mAb). Nevertheless, hydrophobic interaction chromatography, mixed modechromatography or hydroxyapatite chromatographies may also be polishing steps that can be used [16].In the first polishing step, product-related impurities such as aggregates, fragments, as well as host cellprotein (HCP) are, often, removed by step or linear gradient elution using cation-exchange resins in bindand elute mode. A second polishing step is usually performed using anion-exchange chromatographyin flow-through mode with pH adjusted, such that only residual impurities, such as deoxyribonucleicacid (DNA), bind to the resin and the desired product is not adsorbed [18]. These purification steps pro-

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vide additional viral, residual host cell protein (HCP) and DNA clearance, and they remove aggregates,unwanted product variant species and other minor contaminants [16].

Furthermore, to guarantee product safety, potential viral contaminants can be removed by fil-tration using nanofilters. Once product impurities have been removed and conditioning buffer has beenadded, the product is filled into bags, bottles or stainless steel tanks for subsequent storage until filledinto vials [8].

Figure 1.3: Outline of steps involved in a general mAb manufacturing process using mammalian cell culture com-prising (1) inoculum expansion, (2) product fermentation, (3) product primary recovery, (4) product purification, (5)product formulation (Source: Vázquez-Rey et al., 2010).

1.3 Continuous manufacturing process vs. batch manufacturing process

Batch manufacturing of monoclonal antibody purification processes has been the trusted ap-proach for biopharmaceutical manufacturing [18]. Batch manufacturing involves manufacturing pharma-ceutical products in multiple steps, where all materials are charged before the start of processing anddischarged at the end of processing, which results in production stopping before moving on to the nextstep, resulting in hold times that can vary depending on the next stage of the process [19, 20]. Thesehold times are one of the downsides of batch manufacturing, since they increase the risk of materialdegradation and, overall, the length of the manufacturing process, which makes scaling-up the produc-tion a challenge, considering it would also result in the scale-up of the equipment, which can take time,money and additional space [19]. For example, in a typical batch downstream process, the product pool

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between unit operations is collected in product pool tanks, adjusted for pH and/or conductivity, and thenprocessed through the next unit operation [11]. The main benefit of batch manufacturing is the fact thatit is well established and pharmaceutical companies have approval from regulators for their productsbased on them being produced using batch manufacturing techniques [20].

In recent years, there has been a growing interest in continuous biopharmaceutical processingdue to the advantages of small footprint, increased productivity, consistent product quality, high processflexibility and robustness, facility cost-effectiveness, and reduced capital and operating costs [20]. Con-tinuous manufacturing is characterized as simultaneously charging and discharging of material from aprocess at a uniform flow rate. Broader definitions of continuous manufacturing allow the feed and outputof chromatographic processes to occur periodically or in packages, which are significantly smaller thanthe packages of a batch chromatography process that would be used for the same purpose. The entireprocess takes place in one facility, start to finish, without hold times [18].

Several groups have demonstrated that upstream, as well as downstream, processing cansuccessfully be performed continuously, even considering hybrid processes with batch upstream andcontinuous-based downstream or vice-versa. For instances, even when the upstream process is notcontinuous, adopting a more productive and/or continuous downstream process can be of significantadvantage [21]. A 40% reduction in the cost of goods of an end-to-end continuous mAbs manufacturingprocess compared with a conventional batch manufacturing process, for a 10-year scenario, has beenreported [22,23].

Furthermore, FDA has shown support for continuous processing for pharmaceutical manufac-turing [19] and regulatory definitions are clear that there is no reason to avoid continuous manufacturingfrom a regulatory standpoint [24]. The large-scale demand of mAbs production over the last severalyears (as seen in Section 1.1.2), has triggered a necessity for an increase in manufacturing capacity,which consequently, has led also to more cost-effective, efficient and flexible process purification solu-tions to be found by mAb manufacturers [16]. Furthermore, significant upstream advances have beenleading to high titer cell culture processes that create bottlenecks on the downstream process, such asthe cell culture supernatant containing a more elevated number of impurities (e.g. agreggates, HCPs,residual host cell DNA) that need to be separated from the target molecule [17]. Therefore, a greaterdemand as been put on downstream processing to address the high titers of mAbs in harvested cellculture fluids resulting from the improved productivity in the upstream. The FDA believes that there areseveral advantages of a continuous mode of operation that would solve those problems, which includeintegrated processes with fewer steps, decreased buffer consumption, smaller equipment and facilities,and on-line monitoring and control, which would increase product quality assurance in real-time andultimately lead to better defined, characterized and safer therapeutic drugs [18].

In terms of purification of mAbs towards an end-to-end continuous manufacturing, chromatog-raphy seems to still remain the primary downstream processing method, since it consistently delivershigh-purity products [25]. Due to high costs of protein A chromatography as a capture step, the substi-tution or improvement is still a challenge [22], however, a variety of technologies have been investigatedin recent years to replace chromatography, including precipitation [26] in continuous mode, as it willbe seen in Section 1.4. In continuous polishing of mAbs, different but complementary chromatographiccolumns can be typically combined to improve overall process performance and avoid unnecessaryproduct storage, while still being able to achieve typical purity and yield values similar to those obtainedin a batch manufacturing polishing process [11,18].

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1.4 Continuous precipitation with zinc chloride

The capture of recombinant antibodies from cell culture broth is the first critical step of down-stream processing with the main goal of isolating the product from most of the impurities and to concen-trate the product as much as possible, while avoiding the product dilution. Continuous precipitation is anew unit operation that has been described as an economically viable option for the continuous captureof antibodies from clarified culture supernatants [27]. Precipitation has shown to be a good alternativecapture step for a transition from a batch to a continuous operation due to its robustness, high yield,purity and recovery rates [28]. Literature studies have shown that it is possible to develop a continu-ous antibody capture process based on a precipitation method with ZnCl2 that results in an antibodyprecipitate with yield and purity similar to those achieved with protein A affinity chromatography [27,28].Burgstaller et al. reported that the combination of ZnCl2 and PEG resulted in a high yield (> 90%) captureof recombinant antibodies.

Immunoglobulins have a higher precipitation rate than other proteins due to the higher numberof surface-exposed histidines that favor the protein cross-linking. Furthermore, metal cations, such asZn2+, have the tendency to form metal-protein complexes by binding to the amino acid side chains.When it comes to immunoglobulins, Zn2+ has shown to form relatively stable complexes by bindingthose surface-exposed histidines and cysteines residues, possibly through exposed imidazole and thiolgroups, respectively. The Zn2+ bridges to the immunoglobulins, cross-linking the monomers and creatingprotein clusters, which reduces the protein solubility and leads to precipitation [28].

In the study performed by Dutra et al., they were able to precipitate most of Trastuzumab (pI 8.4)with 12 mM of ZnCl2 added to the clarified culture supernatant with an optimal pH for the precipitationwith this recombinant antibody of pH 7. To ensure good removal of impurities, a wash step with 50 mMTris-HCl pH 7 buffer prior to resolubilization with 100 mM sodium acetate pH 5 was added. They wereable to recover most of the Trastuzumab after precipitation, wash and resolubilization with yields above90% and a 7.6-fold removal of impurities. The continuous precipitation was integrated with a tangentialflow filtration (TFF) for the continuous separation and washing of the precipitate with the optimal precip-itation conditions of 12 mM ZnCl2 and pH 7. The overall process had a yield of approximately 73% andthe relative monomer purity was around 85%, which was very comparable with previous studies.

Dutra et al. was able to demonstrate that the crosslinking nature of divalent cations, namelyZnCl2, without the use of PEG as precipitation or co-precipitation agent, allowed to keep a low viscosityof the supernatant as well as low resolubilization dilution factors, which are quite beneficial character-istics for a transition from a traditional precipitation process to a precipitation in a continuous mode.Additionally, this ZnCl2 precipitation-based method is able to reduce the process overall cost and envi-ronmental footprint.

1.5 Ultrafiltration/Diafiltration (UF/DF)

A typical biopharmaceutical process uses a Ultrafiltration (UF)/Diafiltration (DF) step precedinga chromatographic column step, or in between chromatographic column steps, in order to prepare theproduct for the following chromatography stage, since, usually, all columns are operating under differentpH or molarity conditions. UF/DF concentrates and resuspends the product in the correct buffer beforeit is placed into the column, which results in a faster and more efficient process [29].

Filtration is a pressure driven separation process that uses membranes to separate componentsin a liquid solution or suspension based on their size and charge differences. The solution is in contact

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with the membrane under an applied pressure that forces the solvents’ or buffer’ salts and smallermolecules to migrate through the membrane, while the membrane retains the larger molecules, such asproteins [29]. Filtration can be broken down into two different operational modes: Normal Flow Filtration(NFF) and Tangential Flow Filtration (TFF). The difference in fluid flow between these two modes isillustrated in Figure 1.4. In both operational modes, particulates and macromolecules that are too largeto pass through the membrane pores are retained on the upstream side. However, in TFF, the retainedcomponents do not build up at the surface of the membrane, instead, they are swept along by thetangential flow, which makes it an ideal process for finer sized-based separations [30].

Figure 1.4: Comparison of NFF and TFF. In NFF, the fluid is convected in the direction normal to the membraneunder an applied pressure. In TFF, the fluid is pumped tangentially along the surface of the membrane, where anapplied pressure serves to force a portion of the fluid through the membrane to the filtrate side (Source: MerckKGaA).

Ultrafiltration (UF) is one of the most widely used forms of TFF and is used to separate proteinsfrom buffer components for buffer exchange, desalting, or concentration. Depending on the protein to beretained, a membrane’s molecular weight cut-off (MWCO) can be in the range of 1 kDa to 1000 kDa.MWCO is a method of characterization used in filtration to describe pore size distribution and retentioncapabilities of membranes. It is defined as the minimum molecular weight (in Daltons) of a solute thatis 90% retained by the membrane. When choosing the appropriate molecular weight cut-off for specificapplications, many factors must be considered including sample concentration, composition, molecularshape, and operating conditions such as temperature, pressure, and cross-flow velocity. However, there

Figure 1.5: Schematic of a simple TFF system. A pump is used to generate flow of the feed stream through thechannel between two membrane surfaces. During each pass of fluid over the surface of the membrane, the appliedpressure forces a portion of the fluid through the membrane and into the filtrate stream. The result is a gradientin the feedstock concentration from the bulk conditions at the center of the channel to the more concentrated wallconditions at the membrane surface. There is also a concentration gradient along the length of the feed channelfrom the inlet to the outlet (retentate) as progressively more fluid passes to the filtrate side. The flow of feedstockalong the length of the membrane causes a pressure drop from the feed to the retentate end of the channel. Theflow on the filtrate side of the membrane is typically low and there is little restriction, so the pressure along the lengthof the membrane on the filtrate side is approximately constant (Source: Merck KGaA).

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is no set industry standard for MWCO determination, therefore, according to the literature, it is advisableto select a molecular weight cut-off that is at least 2 times smaller than the molecular weight of the solutethat is being retained [31]. Dialfiltration (DF) is a TFF process that can be performed in combinationwith UF, where the buffer is introduced into the recycle tank while the filtrate is removed from the unitoperation. In processes where the product is in the retentate, DF washes components out of the productpool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species.When the product is in the filtrate, DF washes it through the membrane into a collection vessel. Aschematic of a simple TFF system is shown in Figure 1.5.

1.6 Continuous polishing of mAbs in flow-through mode

The purification of antibodies using chromatography involves the separation of antibodies presentin a complex fluid mixture, known as the mobile phase, by passing them through a fixed-bed of a solute-interacting material, known as the stationary phase, and allowing the antibodies to bind or pass throughdepending on whether "bind and elute" or "flow-through" chromatography methods are employed [4,25].The nature of the polishing steps is determined by the nature of the product and the impurities present,with ion exchange chromatography being usually the method applied [2]. Typically, Ion Exchange Chro-matography (IEX) is used in bind and elute mode, where the target molecule (mAb) binds to the column.However it can also be used in flow-through mode where impurities bind to the column, while the mAbpasses through, which will be the case in this study.

Several studies to replace this conventional bind and elute chromatography polishing processfor a flow-through chromatography process have been reported [11,15], and since, either ion exchangechromatography step can be performed in bind and elute or flow-through mode, depending upon thephysicochemical properties of the target protein and impurities, the flow-through concept seems the log-ical thought considering the advantages. Increase in productivity for feedstocks in which the impuritiesare more abundant than the product, cost reduction of manufacturing through a reduction of media vol-umes and reduction of buffer consumption are some of the advantages of this replacement [15, 16, 32].The flow-through polishing has previously been mentioned in the literature where the reduction of in-termediate product hold tanks has been a success using a two flow-through purification/polishing chro-matography steps in a single operation [21], as well as the use of this polishing mode without any bufferadjustments needed [16] and both studies enabling the possibility of a pool-less concept. Furthermore,it has been shown that a process in flow-through mode based on activated carbon, followed by an-ion exchange and cation exchange chromatography (Figure 1.6) is very effective in removing residualimpurities after the capture step [11,16].

Figure 1.6: Schematic of a flow-through polishing process based on activated carbon, followed by anion exchangeand cation exchange chromatography (adapted from Source: Ichihara et al. (2019)).

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1.6.1 Process and product-related impurities

The polishing of a mAb reduces the impurities present in the post-capture step to levels that areclinically acceptable. Therefore, when developing a downstream polishing process using ion exchangeflow-through chromatography, the choice of the media is very important since the media will be in chargeof removing a wide variety of impurities that persist after the capture step. These impurities can be clas-sified as process and product related impurities. Product-related impurities include molecular variantsof the product such as aggregates, oxidized forms or unwanted glycoforms. Process-related impuritiesderive from the manufacturing process, which can include HCPs, residual DNA, components in cell cul-ture broth (e.g., medium components, antibiotics), and components from downstream processing steps(e.g., column leachates) [33,34].

By analysing the different types of impurities considering the molecular weight and the isoelectricpoint, it is possible to identify which flow-through adsorption media is the most appropriate to use [15].Acidic isoelectric point (pI) is considered to be bellow pI 7.0 and basic pI is considered to be abovepI 8.0 [35, 36]. Figure 1.7 illustrates that there are three broad categories of impurities where differ-ences in physicochemical properties can be used to enable their extraction from the mAb supernatantsolution obtained from the capture step. The first category is composed of lower molecular weight im-purities (LMWI) (bellow of approximately 150 kDa, taking into account the molecular weight of a typicalIgG of around 150 kDa [2,3]), including low molecular weight HCP and DNA, mAb fragments, and resid-ual small molecules (e.g., cell culture components). The selective removal of many of these impuritiescan be achieved using activated carbon (AC) [11] (see Section 1.6.2). The second category is com-posed of negatively charged impurities including acidic HCP, DNA and viruses, which are removed byanion exchange chromatography (AEX) (see Section 1.6.3.1). The third category is composed of highermolecular weight impurities (HMWI) (above of approximately 150 kDa, taking into account the molecu-lar weight of a typical IgG of around 150 kDa [2, 3]), including product-related impurities, such as mAbaggregates that are particularly difficult to remove since they share many of the same characteristics asthe mAb product. CEX is responsible for the removal of these larger basic species [11,15].

Figure 1.7: A schematic analysis of the various mAb process impurities based on their molecular weight andisoelectric point with examples of media types that can be employed for their selective removal (Source: Gillespie etal., 2015).

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1.6.2 Activated carbon

Activated carbon (AC) is a low-cost, porous material with a higher surface area than chromatog-raphy resins that adsorbs molecules through non-covalent interactions [37]. It has been used in waterpurification and in the food beverage industry for the nonselective removal of lower concentrations ofproteins [38].

The use of AC in the purification of mAbs has been applied before where it exhibited effectiveclearance of lower molecular weight impurities (LMWI), including HCP and DNA [11, 37, 38]. AC hasusually been used as the first in a sequence of polishment steps (Figure 1.6) because it is the leastexpensive step and it reduces the concentration of impurities that would otherwise be capacity limiting tothe more expensive AEX and CEX media [15]. Ichihara et al. (2019) demonstrated that the addition of ACresulted in a much better clearance of HCP and DNA and delivered a higher product yield, concludingthat activated carbon intensified the AEX step.

1.6.3 Ion exchange chromatography

IEX is a liquid chromatography technique that separates proteins based on differences in theirnet surface charge, generating high-resolution separations with high sample loading capacity. Ions areseparated by their interaction with oppositely charged ion exchange groups immobilized on an insolublesupport (stationay phase). An IEX medium comprises a matrix of spherical particles substituted withionic groups that are negatively (cation exchanger) or positively charged (anion exchanger) (see Figure1.8). The matrix is usually porous to give a high internal surface area and the medium is packed intoa column to form a packed bed. The bed is then equilibrated with buffer which fills the pores of thematrix and the space in between the particles [39]. The mobile phase in an IEX is aqueous because theformation of ions is favored in aqueous solutions and buffers are usually adjusted to a particular pH [40].

Figure 1.8: Ion-Exchange Chromatography (Source: WatersTM).

IEX relies on the pI value of the protein to achieve separation, which is the pH at which a proteinhas no net charge. The net surface of proteins depends on protonatable groups (amino acid side chainson the protein that can be acidic or basic) and it will vary according to the surrounding pH. IEX is usuallyconducted at pH between 4 and 9, since most proteins do not tolerate extremes of pH [40]. A proteinthat has no net charge at a pH equivalent to its pI will not interact with a charged resin. However, at apH above its pI, a protein will bind to a positively charged resin (anion exchanger). At a pH below its pI,a protein will bind to a negatively charged resin (cation exchanger) (Figure 1.9) [39].

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Figure 1.9: The net surface charge of a protein is highly pH dependent and will change gradually as the pH of theenvironment changes (Source: GE Healthcare).

In an IEX in flow-through mode, the antibody is loaded onto the column in a buffer that maintainsa steady pH and contains a relatively low level of salt such as NaCl (conductivity < 5 mS/cm), ensuringthat the antibody flows through the column. Some salt is beneficial to keep the protein soluble butshould be low enough so that it does not interfere with the ionic interaction between protein and matrix.In this thesis, impurities are eluted isocratically from the column by increasing the ionic strength (saltconcentration) of the elution buffer [4,39]. This increase in salt concentration will make salt ions, competewith the target impurities for the charged ligands of the matrix. Isocratic elution is traditionally associatedwith a dilution of the separated products, where the adsorption equilibrium is essential linear, meaningthat each component travels through the column independently of other species [25]. Since the IEX isin flow-through mode, the eluted components are impurities, therefore there is no need to recover themseparately according to their affinity for the mobile phase.

IEX in flow-through mode is composed of the following main steps [39]:

1) Equilibration: the stationary phase is equilibrated to the desired starting conditions. Whenequilibrium is reached, all stationary phase charged groups are bound with exchangeable counterions,such as sodium (Na+) or chloride (Cl-). The pH and ionic strength of the start buffer are selected toensure that, when sample is loaded, the antibody flows through and the impurities bind to the medium.

2) Sample application and wash: the goal in this step is to bind the impurities to the column,while antibody passes through and wash out all unbound material. The sample buffer should have thesame pH and ionic strength as the start buffer in order to bind all charged target impurities. Oppositelycharged biomolecules bind to ionic groups of the IEX medium, becoming concentrated on the column.While uncharged proteins, or those with the same charge as the ionic group (antibody), pass through thecolumn at the same speed as the flow of buffer, eluting during or just after sample application, dependingon the total volume of sample loaded. When all the sample has been loaded, the column is washed withstarting buffer so that all non-binding proteins and impurities have passed through the column.

3) Elution: the target impurities that bound to the column are gradually released from the resinby a change in the buffer composition. The bound molecules are usually eluted by increasing the ionicstrength (salt concentration) of the buffer or, occasionally, by shifting the pH. In this study, the elution wasisocratic, where the pH was mantained, however the salt concentration was increased with 1M NaCl. Asionic strength increases, the salt ions (Na+, Cl-), depending on the type of ion exchanger, compete

Page 14

with the bound components for charges on the surface of the medium and one or more of the boundspecies begin to elute and move down the column. The target molecules with the lowest net charge atthe selected pH will be the first ones eluted from the column as ionic strength increases. Similarly, themolecules with the highest charge at a certain pH will be most strongly retained and will be eluted last.The higher the net charge of the target molecule, the higher the ionic strength that is needed for elution.

4) Sanitization: to guarantee the removal of all biological contaminants still bound to the column,a cleaning-in-place (CIP) is performed to ensure that the full capacity of the stationary phase is availablefor the next run. All columns should be sanitized on a regular basis [41].

5) Re-equilibration: the column is re-equilibrated in start buffer for immediate reuse or transferredinto a storage solution.

1.6.3.1 Anion exchange chromatography

The use of AEX in flow-through mode has been more common than in CEX. The operationalpH is below the protein’s pI, ensuring that the mAb is positively charged and, therefore, easily washedaway from the AEX resin, while negatively charged impurities remain attached [2]. Flow-through anionexchange chromatography around neutral pH and at low conductivity is often used as a polishing stepfor acidic impurity removal of acidic HCP, DNA and many viruses, which bind to the resin due to theirnegative charge, while the antibodies will not bind but pass through the column into the flow-throughfraction [16,42]. The target impurities that bound to the column will be gradually released from the resinas the ionic strength increases. The Cl- salt ions compete with the bound acidic impurities for chargeson the surface of the medium, resulting in the elution of the bound species down the column.

The anionic exchange resin used in this study is Capto Q, which is a high capacity and stronganionic exchange medium with -N+(CH3)3 charged groups. Capto Q resin couples a quaternary ammo-nium (Q) strong anion exchanger to a highly cross-linked agarose matrix, which provides particle rigiditywithout compromising the pore size. In addition, dextran surface extenders coats the agarose matrix.This combination allows for a fast mass transfer, resulting in high dynamic binding capacities of Capto Qat high flow rates, which also makes the resin suitable for high volume process scale applications [43,44].

1.6.3.2 Cation exchange chromatography

In a traditional mAb polishing process (see Section 1.2), CEX is used in bind and elute mode,in which the protein of interest is first retained (operational pH bellow the protein’s pI), by binding to thecharged residues on the matrix, and later eluted using a pH or a salt gradient, separating the protein ofinterest from other bound proteins and impurities [39].

Operating CEX chromatography in the flow-through mode under solution conditions (low con-ductivity and operational pH below the protein’s pI) that strongly bind both the monomer and aggregatecan be categorized as frontal separation [16], where the least retained mAb monomer (antibody) is ob-tained from a fast breakthrough followed by the breakthrough of the more retained species (aggregates).Cation exchangers are used for the removal of larger basic species including product related aggregatesand fragments. Several studies have been reported where mAb aggregrate clearance was obtained us-ing flow-through mode on CEX resins [15,16], however, the weaker binding of impurities and the narrowoperating window of pH and conductivity has been one of the disadvantages reported of this mode.Therefore, it is of great importance the choice of a specific resin that is compatible with the flow-throughmode of operation.

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The cationic exchange resin used in this study is Eshmuno® CP-FT, which is a strong cationexchanger with sulfoisobutyl as a functional group. The resin’s beads are composed of a hydrophilicpolyvinil ether polymer that enables high flow rates resulting in shorter process times. Eshmuno® CP-FTis specifically developed for the flow-through removal of mAb aggregates under strong binding conditions(pH 4.0-5.5, 3.00-7.00 mS/cm) that favor frontal chromatography. Under these conditions, both the mAbmonomer product (less retained species) and the mAb aggregates (more retained species) will initiallybind to the Eshmuno® CP-FT resin. The least strongly adsorbed species (monomer product), will breakthrough much earlier than the mAb aggregates in a series of fronts [45]. A large amount of feed can beprocessed before the impurities start to break through from the column [25].

1.7 Product quality

Protein aggregates, HCP, DNA and other contaminants must be removed to acceptable levelsvia appropriate purification steps (mentioned in Section 1.6) in order to ensure product safety, otherwise,if a therapeutic protein is not stabilized adequately, it will lose partially or totally its therapeutic propertiesor even cause immunogenetic reactions which could potentially endanger the patient’s health [8].

To ensure the product quality of a therapeutic drug, a series of analytical techniques need to beperformed after the polishing experiments to guarantee the medicine’s safety according to organizationssuch as EMA, FDA and World Health Organization (WHO).

1.7.1 Residual DNA quantification

Host cell DNA is a common process-related impurity derived from the CHO cells used in themanufacturing of mAbs. Residual DNA poses a concern related with the immunogenicity and safety(oncogenicity, infectivity, and immunomodulatory effects) of the patient’s health, therefore it is of extremeimportance to remove this impurity to levels that guarantee a medicine’s safety approval. According withWHO and EMA, the residual DNA content should be lower than 10 pg DNA/mg IgG (10 ppb), accordingto the requirement of protein biopharmaceuticals [11,25].

Among the methods of detecting residual DNA, quantitive polymerase chain reaction (qPCR)is considered to be the most practical for residual DNA quantification due to its sensitivity, accuracy,precision and time-saving [46]. There are commercially available qPCR kits for quantitatively detectingthe residual DNA of CHO cells [47]. A traditional method usually used is the Picogreen assay, in whicha Picogreen® dye binds to double-stranded DNA (dsDNA) and generates fluorescence, whereas un-bound dye emits little fluorescence, as it will be described in Section 2.5.4.2 [46]. The advantage of thePicogreen method is its ease of use, low cost and its utility for samples with high levels of DNA. On theother hand, Picogreen is an assay with poor sensitivity and it is prone to interference from other com-ponents (including high recombinant protein and zinc chloride, for example). The maximum acceptableconcentration for ZnCl2 is 5 mM and for IgG proteins is 0.1%, with a signal change of 8% decrease and19% increase, if the concentration values are above those limits [48].

1.7.2 Host cell protein quantification

HCPs are endogenous proteins derived from host cells, constituting a major group of process-related impurities. The clearance of HCPs is a challenging issue due their abundance and heteroge-neous nature in the harvest product pool, which is dependent, not only, upon the type of host cell usedbut also on the cell culture and harvest conditions. Due to their non-human nature, HCPs can have a

Page 16

potential impact on the patient’s health, by eliciting an immune response. In addition to safety concerns,the presence of HCPs is also known to have an impact on product quality, by triggering aggregation orfragmentation of the therapeutic protein. As HCPs cause both safety and efficacy issues, they must beremoved as completely as possible since they compromise product stability and safety even at traceconcentrations [42,49,50]. The FDA requires that the levels of HCP in the final product must be reducedto lower than 100 ng HCP/mg IgG (100 ppm) [42].

Enzyme-linked immunosorbent assay (ELISA) is a common method used to quantify the HCPcontent of a given sample, however it lacks coverage of the complete spectrum of HCPs and is morelikely to miss weak or nonimmunogenic species. Furthermore, ELISA is an expensive and complexprocedure, specially when many samples need to be analyzed. Therefore, using this analytical methodfor HCP routine tracking during a bioprocess can be regarded as not economically feasible [51]. Onthe other hand, the Bradford assay, as it will be described in Section 2.5.3, is a simple and inexpensiveassay method based on the the binding of protein molecules to a dye reagent under acidic conditionswhich results in a colour change from brown to blue. The Bradford method quantifies the total proteincontent, which can be used to estimate the HCP content in culture supernatants. The total protein is thesum of product (measured using a product-specific titer assay (Section 1.7.3)) and HCPs. Subtractionof the product concentration from the total protein provides an estimate of the non-product protein, HCP,in mg/mL [52].

1.7.3 Antibody titre quantification

During mAb manufacturing, CHO cell supernatant samples must be screened in order to deter-mine their mAb titers for accurate determination of monoclonal antibody concentration. HPLC-Protein Aaffinity chromatography is a simple, but highly effective method used to determine mAb concentration.By absorbing the IgG molecule onto a Protein A affinity chromatography column, the remaining impu-rities and by-products can be removed. Elution of the purified monoclonal antibody and quantificationby comparing the peak area to a calibration curve allows rapid measurement of the protein concentra-tion [53,54].

1.7.4 Antibody purity and aggregates (HMWI and LMWI) quantification

Size exclusion chromatography (SEC) is a high resolution analytical method that separatesmolecules based on size (molecular weight) without fractionation (Figure 1.10), as they pass througha SEC resin packed in a column [55]. SEC has been the predominant technique for determination of pu-rity content and analysis of biotherapeutic protein aggregation (high molecular weight impurities (HMWI)and low molecular weight impurities (LMWI)). HMWI can include dimers, trimers, tetramers, etc., formedof monomers that can be either covalently or non-covalently linked. Often, these large protein impuritiesconsist of misfolded monomers in which surfaces of the monomer are exposed that typically would notbe in the monomeric form. They are believed to primarily impact safety, but also can impact efficacy [56].LMWI include clipped species and half molecules for compounds that are intended to be dimeric, suchas monoclonal antibodies and bispecific antibodies that must also be controlled during downstreamprocessing.

These aggregate molecules can impact both safety, due to the potential to cause immunogenic-ity responses, and efficacy, as the result of missing structural features, such as complementary deter-mining regions, which play a role in determining the antigen-binding specificity of antibodies and T-cellreceptors [56]. An additional degradation for monoclonal antibodies that can be observed by SEC is a

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Figure 1.10: Typical high-resolution SEC separation (Source: GE Healthcare).

non-enzymatic peptide bond hydrolysis in the hinge region of these proteins, resulting in antibodies thatare missing one of both Fab arms [54,57]. These high and low molecular weight fragments are inducedby various stress conditions and formed during the process of drug development. According to typicalspecifications values, aggregates should be removed to values bellow 1% [43] and the monomer purityshould be above 95% [11].

1.8 UV signal interpretation in a chromatogram

In a chromatography run it is important to choose which ultraviolet (UV) wavelengths are goingto be monitored over time to gather more information on the respective absorbing species. The UV signalwas monitored at 214 nm, 254 or 260 nm and 280 nm wavelengths in the chromatography runs of thisstudy.

The concentration of a protein in solution is often quantified by UV absorbance due to absorptionby the aromatic amino acids tyrosine (Tyr), tryptophan (Trp) and phenylalanine and the disulfide bridges[58]. The adsorption maxima for proteins is typically around 280 nm, because of the strong absorbanceof the aromatic amino acid tryptophan. The specific absorbance of proteins varies with the relativecontent of the aromatic amino acids Trp and Tyr and, to a lesser extent, of the disulfide bridges [59].However, the measurement of protein at 280 nm is not strictly quantitative for all protein since proteinswith few Trp, Tyr and phenylalanine amino acids, may not absorb as strongly as expected [60].

UV absorbance at 214 nm is much more sensitive for proteins and peptides than 280 nm, dueto the adsorption maxima for peptide bond being around the 214 nm wavelength, considering the amidebond. This wavelength can be used to detect a peptide deficient in tryptophan, tyrosine and phenylala-nine or all the proteolytic digestion products of a protein which otherwise will not be detectable with280 nm as some fragments may be deficient in the aromatic amino acids, as mentioned before [59].Additionally, this wavelength allows use of buffers that might be problematic at 206 nm (backgroundabsorbance) [60].

Nucleic acids have an absorbance maximum at 254 nm or 260 nm [60], which can interferesubstantially with protein determinations at 280 nm. Thus, when nucleic acids are simultaneous presentin solution, corrections must be made in order to determine protein concentration from absorbancevalues at 280 nm. The purity of DNA is estimated by the ratio of absorbance at 260 nm and 280 nm, andfor pure double-stranded DNA, this ratio is between 1.8 and 2.0. If the ratio is lower, it may indicate thepresence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. In contrast

Page 18

to proteins, the absorbance of nucleic acids is quite sensitive to pH, and decreases at lower pH values[25,58].

1.9 Resin column packing

In the case that a resin has to be individually packed into a column, it is important to perform anaccurate determination of the slurry volume and slurry concentration in order to achieve good packingresults. An error can result in inaccurate packed bed compression, giving rise to high or low operatingpressures and possibly poor height equivalent to a theoretical plate (HETP) and asymmetry (As) values,which will affect directly the efficiency of the packed column [41].

The packed bed volume (PBV) can be determined by the following formula (Equation 1.1):

PBV = π × (column diameter2 )2 × bed heigh (1.1)

In order to calculate the settle bed volume (SBV) required at a given percent compression for atarget packed column bed volume, it is necessary to know the recommended compression factor (CF)(Equation 1.2) or the recommended compression percent (Equation 1.3) of the column (Table 1.1).

Table 1.1: Compression factors for a column at laboratory scale (Source: Merck KGaA).

Column Size Recommended Compression Factor (CF) Recommended Compression Percent

Lab Scale 1.06 to 1.09 6 to 8%

SBV = PBV × CF (1.2)

or

SBV = PBV

(100% − % compression) (1.3)

Equation 1.4 calculates the slurry volume required for a target bed height:

slurry volume = SBV

slurry concentration(1.4)

Where:

slurry concentration = gravity settled volume of resin

total slurry volume(1.5)

The compression factor (CF) can be calculated using Equation 1.6:

CF = 100%100 − % compression

(1.6)

The quality of the packed bed of a column is checked by performing a column performancetest. Tests should be made directly after packing and at regular intervals during the working life ofthe column and also when separation performance is seen to deteriorate. The parameters to describe

Page 19

column efficiency are the HETP and As, which will depend on the specific test conditions (concentrationand volume, flow rate and system tubing/pipework) [41]. The column performance test calculates HETPand As from the integrated UV curve (Figure 1.11) and these values are stored as data in the ÄKTApure’s software Unicorn 7.1 (GE Healthcare Life Sciences).

The HETP (Equation 1.7) is the ratio between the column’s bed height (L) (cm) and the numberof theoretical plates (N ). The number of theoretical plates (N ) can be determined with Equation 1.8,where Wh is the peak width at half height and VR is the retention volume (Wh and VR are in the sameunits).

HETP = L

N(1.7)

N = 5.54 × ( VR

Wh)2 (1.8)

It is important to note that the calculated plate number will vary according to the test conditionsand it should only be used as a reference value. Furthermore, test conditions should be kept constantso that the results are comparable. Changes of solute, solvent, eluent, sample volume, flow velocity,temperature, etc. will influence the results [61].

The peak asymmetry As factor can be determined using Equation 1.9, which represents theration between the 2nd half peak width at 10% of peak height (b) and the 1st half peak width at 10% ofpeak height (a) (see Figure 1.11).

As = b

a(1.9)

The As factor should be as close to 1 as possible, nevertheless, values between 0.7 and 1.6 areusually acceptable [41,61]. Furthermore, the peak should be symmetrical. A change in the shape of thepeak is usually the first indication of bed deterioration due to excessive use.

Figure 1.11: Illustration of a UV curve for acetone in a typical performance test chromatogram from which the HETPand As values are calculated (Source: GE Healthcare).

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1.10 Aim of thesis

Ever since the approval and commercialization of the first monoclonal antibody in 1986, ther-apeutic monoclonal antibodies have grown to become the most dominant biological drug within thebiopharmaceutical market [2]. The high demand of these therapeutic drugs have pressured mAb man-ufacturers to find new innovative manufacturing processes to reduce costs and increase productivitywithout sacrificing process robustness. In recent years, there has been significant improvement in theupstream processing of mAbs, however, the downstream processing has not evolved at the same pace,which has created bottlenecks in the production of therapeutic mAbs. A transition from batch to contin-uous mAb process manufacturing and adoption of flow-through chromatography in the polishing stepsin replacement of bind and elute mode chromatography have been some of the solutions proposed tointensify the downstream processing of mAbs [11,16].

In 2019, Ichihara et al. [11] reported a polishing approach for therapeutic monoclonal antibodypurification (after Protein A affinity chromatography as a capture step) with three fully connected flow-through polishing columns, without in-line pH/conductivity adjustment between each column. The prin-ciple of the connected flow-through reported in the article was composed of in-series activated carbon(AC) for the removal of lower molecular weight impurities (LMWI) including HCP and DNA; flow-throughanion exchange chromatography (AEX) for the removal of negatively charge impurities including acidicHCP, DNA and viruses; and flow-through cation exchange chromatography (CEX) for the removal ofhigher molecular weight impurities (HMWI) including product related aggregates.

This thesis study on "Continuous polishing of recombinant antibodies after continuous precip-itation" was based on the flow-through chromatography polishing method described by Ichihara et al.,however instead of having a previous capture step using Protein A affinity chromatography, the mAb tobe purified in this thesis was Trastuzumab (pI 8.4) and it was obtained after continuous precipitation withzinc chloride (ZnCl2).

The aim of this thesis was to compare two flow-through chromatography polishing trains with dif-ferent loading conditions, after continuous ZnCl2 precipitation. In the first train of experiments (AEX-CEXwith non-adjusted loading conditions (N-ADC)), the mAb was filtered and loaded onto the AEX columnwithout adjustment of the loading conditions (no pH or conductivity adjustment), followed by loading ontothe CEX column. In the second train of experiments (AEX-CEX with adjusted loading conditions (ADC)),the loading conditions (pH and conductivity) were adjusted by performing a two-step buffer exchangeusing UF/DF, before the filtered mAb was loaded onto the AEX column, followed by loading onto theCEX column. Samples were collected before and after each polishing step and analyzed to determineyield, purity, HMWI, LMWI, DNA and HCP content, using analytical methods such as high performanceliquid chromatography (HPLC)-Protein A affinity chromatography, size exclusion chromatography (SEC),Bradford assay and Picogreen assay.

A new set of experiments with adjustment of the mAb loading conditions had been planned inorder to further study how a different starting buffer solution, as well as varying its pH, would work incomparison with the first experiments of the train AEX-CEX ADC. Furthermore, an activated carbon (AC)polishing step, previous to the AEX step, was going to be introduced in order to see how its addition tothe mAb puritication process would influence the removal of HCP and DNA by the AEX, since accordingwith the findings reported by Ichihara et al., the use of AC intensified the AEX. However, due to COVID-19, the laboratory at the Department of Biotechnology of the University of Natural Resources and LifeSciences (Vienna, Austria) closed and I had to return to Portugal, therefore the AC experiment was neverstarted and the AEX-CEX ADC experiments with a different starting buffer and pH were not concluded.

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2 Materials and Methods

2.1 Buffer Preparation

All buffering chemical components were from Merck KGaA (Darmstadt, Germany), unless statedotherwise. Sodium acetate (CH3COONa, molar mass (MM) = 82.03 g/mol), sodium chloride (NaCl,MM = 58.44 g/mol), tris(hydroxymethyl)aminomethane (NH2C(CH2OH)3, MM = 121.14 g/mol), sodiumhydroxide pellets (NaOH, MM = 40.00 g/mol) and 96% (v/v) ethanol, were used to prepare buffers ofspecific pH and concentrations.

For the buffer preparation, all chemical components were weighed in a Mettler Toledo balanceand stirred in a IKA Werke magnetic stirrer. The pH was adjusted using a SevenEasy pH meter, fromMettler Toledo, by adding solutions of 25% hydrochloric acid (HCl) prepared from a 1 M HCl stock so-lution, or by adding 10 M NaOH, to decrease or increase the pH, respectively. In the case of the pHadjustment of the sodium acetate buffers, it was added a 20% acetic acid solution prepared from an100% acetic acid stock solution (glacial acetic acid supplied by Acros Organics (Belgium)). To adjust thebuffers conductivity, a 2 M NaCl solution was added to increase the conductivity and a FiveGo conduc-tivity meter (Mettler Toledo) with a conductivity range of 0.1 to 199.9 µS/cm was used to measure it. AfterpH and conductivity adjustment, all buffers were filtered using a 0.22 µm mixed cellulose esters (mixedcellulose esters (MCE)) membrane filter supplied by Merck Millipore (Darmstadt, Germany), followed bydegassing of the buffer for 15 minutes in an Elmasonic S100 unit ultrasonic bath.

2.2 Flow-through ion exchange resins

2.2.1 Anion exchange resin

The AEX column was a 1 mL pre-packed HiTrap® column with Capto QTM resin from GE Health-care (Buckinghamshire, UK) with a pre-column pressure limit of 0.50 MPa and a delta-column pressurelimit of 0.30 MPa. Capto Q is a strong anionic exchange medium with -N+(CH3)3 charged groups. CaptoQ resin couples a quaternary ammonium (Q) strong anion exchanger to a highly cross-linked agarosematrix. In addition, dextran surface extenders coats the agarose matrix [44].

2.2.2 Cation exchange resin

The CEX Eshmuno® CP-FT (Merck KGaA) resin was individually packed into a 0.5 mL Tricon 5x 2.5 column (described in Section 2.2.2.1) with a pre-column pressure limit of 0.29 MPa and a delta-column pressure limit of 0.07 MPa. Eshmuno® CP-FT is a strong cation exchanger with sulfoisobutyl as afunctional group and the resin’s beads are composed of a hydrophilic polyvinil ether polymer. Eshmuno®

CP-FT is specifically developed for the flow-through removal of mAb aggregates under strong bindingconditions (pH 4.0-5.5, 3.00-7.00 mS/cm) that favor frontal chromatography [41,45].

2.2.2.1 Eshmuno CP-FT column packing

In order to perform the cation exchange chromatography runs, the CEX resin had to be indi-vidually packed into an appropriate column housing. The Eshmuno® CP-FT resin supplied by MerckKGaA (Darmstadt, Germany), was packed into a TriconTM 5 mm x 2.5 cm column housing (0.5 mL),provided by GE Healthcare (Buckinghamshire, UK) using a 150 mM NaCl packing buffer and a flow

Page 23

rate of 1 mL/min. The resin slurry was prepared according to the Eshmuno® CP-FT ChromatographyResin User Guide [41] and it was stored in 20% ethanol solution containing 150 mM NaCl at room tem-perature (between 5°C to 30°C), until further usage. The chromatography run for the column packingprocedure was performed on an ÄKTATM pure supplied by GE Healthcare Life Sciences (Sweden) andthe operating software used for the system control and data analysis was the Unicorn 7.1, also sup-plied by GE Healthcare Life Sciences (Sweden). The packing procedure followed is described in theEshmuno® CP-FT Chromatography Resin User Guide [41] and a TricornTM 5 medium filter kit, suppliedby GE Healthcare, was used.

The bed height of the resin Eshmuno® CP-FT was calculated to ensure that the PBV of thecolumn was 0.5 mL, according with the Equation 1.1. The bed height value calculated of 2.55 cm was,then, measured and marked on the column housing, in order to have a reference when packing the resinslurry into the column and, also, to avoid the reducing of the bed height due to the top filter compres-sion. Taking into account the 8 % percent compression of the resin [41] (see Section 1.9), a settle bedvolume (Equation 1.3) of 0.54 mL was determined, considering a PBV of 0.50 mL. Therefore, 0.54 mLof Eshmuno® CP-FT resin was needed to pack a stable bed at 2.55 cm bed height in order to have aPBV of 0.50 mL. The resin was supplied in a storage solution (20 % ethanol solution containing 150 mMNaCl, 70 % slurry concentration), therefore, the volume of slurry needed was 0.77 mL (Equation 1.4) toguarantee a good packing result. Table 2.1 summarizes the calculations made for an accurate packedbed compression in order to achieve good packing results.

Table 2.1: To ensure a packed bed volume of 0.50 mL, a settle bed volume of 0.54 mL is needed to pack a stablebed at 2.55 cm bed height (considering 8% compression). Taking into account a 70% slurry concentration, thevolume of slurry needed is 0.77 mL. The resin’s compression factor is 1.09 (Equation 1.6), which is within the rangefound in the literature (Table 1.1).

PBV (mL) Bed heigh (cm) SBV (mL) Slurry volume (mL) CF

0.50 2.55 0.54 0.77 1.09

In order to evaluate the quality of the packed column, the column was run at a flow rate of 1mL/min for 10 CV with 25 mM sodium acetate (pH 6, conductivity 1.98 mS/cm) starting buffer (equilibra-tion buffer of the flow-through chromatography experiences [11]). The packing quality was determinedby injecting 0.1 mL of 1% acetone with a syringe through the loop and measuring the asymmetry fac-tor of the corresponding peak, which is represented in Table 2.2. The asymmetry factor is in the rangesuggested by the resin’s user guide (between 0.7 and 1.6) and the number of theoretical plates is ap-proximately 4000/m (which was calculated from the HETP value, by the Unicorn 7.1 software), as it isrecommended at laboratory scale [41]. The column was then stored in 20% ethanol at room temperature.

Table 2.2: Specifications of the TriconTM 5 mm diameter x 2.5 cm height column at 0.5 mL (GE Healthcare) packedwith Eshmuno® CP-FT resin (Merck KGaA) after the performance test to measure the packed column efficiency.

Bed height

(cm)

Volume

(mL)

Asymmetry

factor

Plate height

(HETP) (cm)

Plates per meter

(N)

Max column

pressure (MPa)

Delta column

pressure (MPa)

2.45 0.48 1.07 0.02636 3793.3 0.29 0.07

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2.3 Flow-through chromatography experiments

The mAb to be purified was Trastuzumab (pI 8.4), a humanized IgG1 monoclonal antibody,produced in CHO cells in a perfusion system bioreactor. The mAb solution to be purified in the continuouspolishing steps was obtained after continuous ZnCl2 precipitation of the cell culture broth. The continuousZnCl2 precipitation method, used as a capture step of the recombinant antibody in this study, can befound in the the article Dutra et al. [28]. A concentration of 12 mM of ZnCl2 was used for the precipitationconditions. The mAb supernatant solution was stored in the cold room at 2-8°C, prior to the polishingchromatography runs.

A SevenMultiTM dual meter for protein solutions (Mettler Toledo) was used to measure the pHand conductivity of all mAb supernatant solutions. Before loading onto the chromatography columns, themAb supernatant solution was filtered with a 0.2 µm asymmetrical polyethersulfone (aPES) membranein a 250 mL Nalgene rapid-flow filter unit (Thermo Fisher Scientific).

All chromatography runs were performed on an ÄKTATM pure supplied by GE Healthcare LifeSciences (Sweden) and the operating software used for the system control and data analysis was theUnicorn 7.1, also supplied by GE Healthcare Life Sciences (Sweden).

The method for all of the ion exchange chromatography runs used in this study was the samefor either anion exchange or cation exchange chromatography, unless stated otherwise. The methodwas defined in the UnicornTM 7 software in the ÄKTATM pure. The mobile phases (equilibration, washingand elution buffer and sanitization solution) used on the AEX and the CEX runs varied depending onthe conducted experiments (see Sections 2.3.1, 2.3.2 and 2.3.3). The method based unit used was incolumn volume (CV). The UV wavelengths measured were 280 nm (UV1) and 214 nm (UV2). The flowrate of the equilibration, column wash and elution steps of all polishing experiences was of 1 mL/minand the flow was controlled during the whole run in order to avoid overpressure. After equilibration of thecolumns, the mAb sample was loaded onto the column at a flow rate of 0.2 mL/min in all the chromatog-raphy runs. The residence times of columns were: AEX = 5.0 min. and CEX = 2.5 min. (Residence time =Total resin volume/Flow rate). The loading conditions of the mAb supernatant solution loaded dependedon the experiments (see Sections 2.3.1, 2.3.2, 2.3.3.1 and 2.3.3.2). The antibody solution was injecteddirectly onto the column using a sample pump as the inlet and a fixed sample volume. The flow-throughpool was collected using a fraction collector with a fixed volume fractionation of 15 mL in 15 mL conicalcentrifuge Falcon tubes. The column was washed with equilibration buffer and the column wash wascollected using a fixed outlet valve (outlet 2) for further analysis. The elution step was performed isocrat-ically and the fractions from the elution step were collected using a fixed outlet valve (outlet 3) for furtheranalysis. Following, the elution step, a column CIP was performed in order to wash the column in a lineargradient at a 0.2 mL/min flow rate (35 min. of incubation time). 1 M NaOH was used for the sanitizationof the AEX column and 0.5 M NaOH for the sanitization of the CEX column. The system in by-passmode, as well as the waste outlet and outlet 1 were also washed with 1 M NaOH or 0.5 M NaOH at a 20mL/min flow rate. In case the column was not reused the following day, the column was washed with 2CV of ultrapure water, followed by 2 CV of 20% ethanol and stored at 4°C to 30°C. Finally, to ensure theremoval of all residues of antibody or buffer in the system, the system is washed with ultrapure waterat a flow rate of 20 mL/min, followed by washing with 20% ethanol at the same flow rate. At the end ofeach run, every inlet and outlet tube used was pumped washed with ultrapure water and followed by20% ethanol. In the case of the sample inlet, outlet 1, 2 and 3 (which are all tubes where the antibodysolution was in contact with), they were all pumped washed with 1 M NaOH or 0.5 M NaOH, followed byultrapure water and 20% ethanol.

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2.3.1 AEX-CEX (non-adjusted loading conditions) and AEX-CEX (adjusted loading conditions)runs with sodium acetate buffer at pH 6.0

The mAb supernatant obtained from the continuous precipitation with ZnCl2 had a titre of 0.29mg/mL, which was determined by HPLC-Protein A affinity chromatography (see method in Section2.5.1), and a pH value of 3.6 and conductivity of 3.47 mS/cm, before adjustment of the loading con-ditions. Two flow-through chromatography trains were tested: AEX-CEX with non-adjusted loading con-ditions (N-ADC) and AEX-CEX with adjusted loading conditions (ADC). MAb solution volumes of 225mL (225 CV) and 200 mL (400 CV) were injected, respectively, onto the AEX and the CEX columns (CV= Feed volume/Total resin volume) of both trains (N-ADC and ADC) studied.

In the AEX-CEX N-ADC, the mAb supernatant solution (precipitated material) was filtered anddirectly loaded onto the AEX column with no adjustment of the loading conditions. The mAb solutionwas loaded onto the column with pH 3.6 and a conductivity of 3.47 mS/cm.

In the AEX-CEX ADC, the conditions of the mAb supernatant solution (precipitated material)were adjusted prior to the loading onto the AEX column. With the objective of removing the zinc chloridefrom the precipitation step and to adjust the loading conditions according to the flow-through chromatog-raphy method reported by Ichihara et al., a two-step buffer exchange by UF/DF was performed (Section2.4.1). The loading conditions of the mAb solution were adjusted to pH 6.0 and 4.00 mS/cm conductivity.Before, loading onto the AEX column, the mAb supernatant solution was filtered.

The methods for the ÄKTATM pure and the mobile phases used on the AEX-CEX trains for non-adjusted (N-ADC) and adjusted (ADC) loading conditions for the anion exchange chromatography (AEX)and the cation exchange chromatography (CEX) are represented in Tables 2.3 and 2.4, respectively.

Table 2.3: AEX flow-through method for the AEX N-ADC and AEX ADC runs. The mAb (0.29 mg/mL) loadingconditions for AEX N-ADC were pH 3.6 and 3.47 mS/cm conductivity. The mAb (0.29 mg/mL) loading conditionsfor the train AEX ADC were adjusted to pH 6.0 and 4.00 mS/cm conductivity by buffer exchange using UF/DF. Themethod flow-rate was 1 mL/min. The UV was measured at wavelengths 280 nm and 214 nm. The AEX column wasloaded at the flow rate of 0.2 mL/min with a target of mAb loading at 225 mL (225 CV).

Method block Buffer solution Duration (CV)

Equilibration 25 mM sodium acetate, pH 6.0 conductivity 1.98 mS/cm 15

Sample loading 225 mL mAb supernatant (pI 8.4), (loading conditions in the caption) 225

Wash 25 mM sodium acetate, pH 6.0 conductivity 1.98 mS/cm 10

Elution 25 mM sodium acetate, 1 M NaCl, pH 6.0 conductivity 85.1 mS/cm 10

CIP 1 M NaOH 7

Re-equilibration 25 mM sodium acetate, pH 6.0 conductivity 1.98 mS/cm 5

CIP ultrapure water 2

CIP 20% ethanol 2

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Table 2.4: CEX flow-through method for the CEX N-ADC and CEX ADC runs. The mAb loading conditions for CEXN-ADC were pH 3.6 and 3.51 mS/cm conductivity. The mAb loading conditions for the train CEX ADC were pH 6.0and 4.12 mS/cm conductivity. The method flow-rate was 1 mL/min. The UV was measured at wavelengths 280 nmand 214 nm. The CEX column was loaded at the flow rate of 0.2 mL/min with a target of mAb loading at 200 mL(400 CV).






CIP 0.5 M NaOH 14



CIP 20% ethanol 2

2.3.2 CEX (adjusted loading conditions) "test" runs with Tris-HCl buffer at pH 6.0, 7.0 and 8.0

A trastuzumab thawed solution with an antibody titre of 27 mg/mL (pH 12.4, conductivity 4.40mS/cm) obtained after Protein A affinity chromatography (capture step) was used to perform cationexchange chromatography (CEX) "test" runs (with adjustment of the loading conditions) with Tris-HClbuffer at pH 6.0, 7.0 and 8.0. The trastuzumab solution was diluted to a titre of 2.25 mg/mL.

Each mAb solution was previously adjusted, by a buffer exchange step (see Section 2.3.2.1)to the respective loading condition and filtered, before loading onto to the CEX column. The antibodysolution was injected directly onto the column using a sample pump as the inlet and a fixed samplevolume of 5 mL (10 CV) was injected. The flow-through was collected using a fraction collector with afixed volume fractionation of 5 mL in 15 mL conical centrifuge Falcon tubes.

The methods for the ÄKTATM pure and the mobile phases used on the CEX "test" runs foradjusted (ADC) loading conditions with Tris-HCl buffer at pH 6.0, 7.0 and 8.0, are represented in Tables2.5, 2.6 and 2.7, respectively.

Table 2.5: CEX flow-through method "test" run with Tris-HCl at pH 6.0 for adjusted loading conditions (ADC). ThemAb (2.25 mg/mL) loading conditions were pH 6.0 and 4.00 mS/cm conductivity. The method flow-rate was 1mL/min. The UV was measured at wavelengths 280 nm and 214 nm. The CEX column was loaded at the flow rateof 0.2 mL/min with a target of mAb loading at 5 mL (10 CV).


Equilibration 25 mM Tris-HCl, pH 6.0 conductivity 2.24 mS/cm 10

Sample loading 5 mL mAb supernatant (pI 8.4), pH 6.0 conductivity 4.00 mS/cm 10

Wash 25 mM Tris-HCl pH 6.0 conductivity 2.24 mS/cm 10

Elution 25 mM Tris-HCl, 1 M NaCl, pH 6.0 conductivity 86.2 mS/cm 10

CIP 0.5 M NaOH 14

Re-equilibration 25 mM Tris-HCl, pH 6.0 conductivity 2.24 mS/cm 5


CIP 20% ethanol 2

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Wash 25 mM Tris-HCl, pH 7.0 conductivity 2.29 mS/cm 10


CIP 0.5 M NaOH 14



CIP 20% ethanol 2







CIP 0.5 M NaOH 14



CIP 20% ethanol 2

2.3.2.1 Buffer exchange step to adjust the loading conditions for the CEX "test" runs at pH 6.0,7.0 and 8.0

Three CEX "test" runs using Tris-HCl buffer were performed at mAb loading conditions of pH6.0 and 4.00 mS/cm conductivity, pH 7.0 and 4.00 mS/cm conductivity, and pH 8.0 and 4.00 mS/cmconductivity. The buffer used for the buffer exchange was 25 mM Tris-HCl, conductivity 4.00 mS/cm andpH 6.0, 7.0 or 8.0, depending on the respective CEX test run.

Briefly, 200 µl of mAb solution was pipetted into Amicon® Ultra 0.5 mL centrifugal filters (MerckKGaA) with a molecular weight cut-off of 3 kDa (Merck KGaA). The centrifugal filters were placed in1.5 ml reaction tubes and centrifuged at 14,000 rcf for 5 min, at 4°C (Eppendorf centrifuge 5415R;Eppendorf). Then, 300 µl of 25 mM Tris-HCl buffer at the respective pH (6.0, 7.0 or 8.0, depending onthe chromatography run) and conductivity 4.00 mS/cm (equilibration buffer), was added to the sample.An Heidolph REAX 2000 vortex was used and, the sample was centrifuged again. This buffer exchangeprocedure was repeated five times.

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2.3.3 AEX-CEX (adjusted loading conditions) runs with Tris-HCl buffer at pH 7.0 and pH 8.0

The new mAb supernatant obtained from the continuous precipitation with ZnCl2 had a titre of0.23 mg/mL, pH of 9.8 and conductivity of 7.02 mS/cm. Two flow-through chromatography trains, usingTris-HCl buffer, with adjustment of the loading conditions were tested: AEX-CEX at pH 7.0 (Section2.3.3.1) and AEX-CEX at pH 8.0 (Section 2.3.3.2). The loading conditions of the mAb supernatantsolution (precipitated material) were adjusted prior to the loading onto the AEX column. A two-stepbuffer exchange using UF/DF was performed with the objective of removing the zinc chloride from theprecipitation step and to adjust the mAb loading conditions. Before, loading onto the AEX column, themAb supernatant solution was filtered. MAb solution volumes of 125 mL (125 CV) and 100 mL (200 CV)were injected, respectively, onto the AEX and the CEX columns.

2.3.3.1 AEX-CEX (adjusted loading conditions) run with Tris-HCl buffer at pH 7.0

The two-step buffer exchange method by UF/DF to remove the zinc chloride and adjust the mAbloading conditions to pH 7.0 and 4.00 mS/cm conductivity using Tris-HCl buffer is explained in Section2.4.2.

The methods for the ÄKTATM pure and the mobile phases used on the AEX-CEX train withadjustment of the loading conditions at pH 7.0 for the anion exchange chromatography (AEX) and thecation exchange chromatography (CEX) are represented in Tables 2.8 and 2.9, respectively.

Table 2.8: AEX flow-through run with Tris-HCl at pH 7.0 for adjusted loading conditions (ADC). The mAb (0.23mg/mL) loading conditions were adjusted to pH 7.0 and 4.00 mS/cm conductivity. The method flow-rate was 1mL/min. The UV was measured at wavelengths 280 nm, 260 nm and 214 nm. The AEX column was loaded at theflow rate of 0.2 mL/min with a target of mAb loading at 125 mL (125 CV).






CIP 1 M NaOH 7



CIP 20% ethanol 2

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Table 2.9: CEX flow-through run with Tris-HCl at pH 7.0 for adjusted loading conditions (ADC). The mAb loadingconditions were pH 7.0 and 4.08 mS/cm conductivity. The method flow-rate was 1 mL/min. The UV was measuredat wavelengths 280 nm, 260 nm and 214 nm. The CEX column was loaded at the flow rate of 0.2 mL/min with atarget of mAb loading at 100 mL (200 CV).






CIP 0.5 M NaOH 14



CIP 20% ethanol 2

2.3.3.2 AEX-CEX (adjusted loading conditions) run with Tris-HCl buffer at pH 8.0

The two-step buffer exchange method by UF/DF to remove the zinc chloride and adjust the mAbloading conditions to pH 8.0 and 4.00 mS/cm conductivity using Tris-HCl buffer is explained in Section2.4.3.

The methods for the ÄKTATM pure and the mobile phases used on the AEX-CEX train withadjustment of the loading conditions at pH 8.0 for the anion exchange chromatography (AEX) and thecation exchange chromatography (CEX) are represented in Tables 2.10 and 2.11, respectively.

Table 2.10: AEX flow-through run with Tris-HCl at pH 8.0 for adjusted loading conditions (ADC). The mAb (0.23mg/mL) loading conditions were adjusted to pH 8.00 and 4.00 mS/cm conductivity. The method flow-rate was 1mL/min. The UV was measured at wavelengths 280 nm and 214 nm. The AEX column was loaded at the flow rateof 0.2 mL/min with a target of mAb loading at 125 mL (125 CV).






CIP 1 M NaOH 7



CIP 20% ethanol 2

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Table 2.11: CEX flow-through run with Tris-HCl at pH 8.0 for adjusted loading conditions (ADC). The mAb loadingconditions were pH 8.0 and 3.96 mS/cm conductivity. The method flow-rate was 1 mL/min. The UV was measuredat wavelengths 280 nm, 254 nm and 214 nm. The CEX column was loaded at the flow rate of 0.2 mL/min with atarget of mAb loading at 100 mL (200 CV).






CIP 0.5 M NaOH 14



CIP 20% ethanol 2

2.4 Ultrafiltration/Diafiltration (UF/DF)

The ultrafiltration cassettes used in this study were a Biomax® Pellicon® 2 "mini" filter mem-brane with a molecular weight cut-off of 8 kDa and a Biomax® Pellicon® XL membrane with a molecularweight cut-off of 5 kDa, both from EDM Millipore Corporation (USA). Both membranes were composedof polyethersulfone (PES). The filtration areas of the Biomax® 8 kDa and 5 kDa membranes were 0.10m2 and 50 cm2, respectively. Each membrane, when used, was placed with the two die-cut silicon gas-kets, in the holder which was tightened with a Quatrolit 2179-60 torque wrench from Elora (10-60 N.m;9-45 Lb.ft) by setting the torque wrench settings to 20.3-22.6 N.m prior to use. The UF/DF runs wereperformed on an ÄKTA flux system (GE Healthcare).

2.4.1 Two-step buffer exchange to adjust the loading conditions to pH 6.0 and 4.00 mS/cm con-ductivity (sodium acetate buffer)

The feed tank was filled with 250 mL of mAb supernatant solution (titre 0.28 mg/mL, pH 3.6, con-ductivity 3.47 mS/cm) and the retentate was continuously stirred (30-60 rpm) and recirculated throughthe Biomax® 8 kDa membrane using a peristaltic pump at a maximum transfer flow rate of 50 mL/min.The run was operated at a maximum operating pressure (feed to permeate) of 2.0 bar. The mAb super-natant solution was concentrated for 5 volumes with 25 mM sodium acetate buffer pH 4.0 and conduc-tivity 2.02 mS/cm (to guarantee the zinc chloride removal), and then diafiltered for 5 volumes with 25mM sodium acetate buffer pH 6.0 and conductivity 4.00 mS/cm (adjustment of the loading conditions) ata constant volume by continuously feeding diafiltration buffer into the retentate vessel. The system andmembrane were washed with 0.1 M NaOH, by recirculating the solution for 30 minutes. The system wasstored in 20% ethanol and the membrane was stored in 0.1M NaOH.

2.4.2 Two-step buffer exchange to adjust the loading conditions to pH 7.0 and 4.00 mS/cm con-ductivity (Tris-HCl buffer)

The feed tank was filled with 140 mL of mAb supernatant solution (titre 0.23 mg/mL, pH 9.8, con-ductivity 7.02 mS/cm) and the retentate was continuously stirred (30-60 rpm) and recirculated throughthe Biomax® 5 kDa membrane using a peristaltic pump at a maximum transfer flow rate of 50 mL/min.

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The run was operated at a maximum operating pressure (feed to permeate) of 2.0 bar. The mAb su-pernatant solution was concentrated for 5 volumes with 25 mM Tris-HCl buffer, pH 9.0 and 2.01 mS/cmconductivity (to guarantee the zinc chloride removal), and then diafiltered for 5 volumes with 25 mMTris-HCl buffer pH 7.0 and conductivity 4.00 mS/cm (adjustment of the loading conditions) at a constantvolume by continuously feeding diafiltration buffer into the retentate vessel. The system and membranewere washed with 0.1 M NaOH, by recirculating the solution for 30 minutes. The system was stored in20% ethanol and the membrane was stored in 0.1M NaOH.

2.4.3 Two-step buffer exchange to adjust the loading conditions to pH 8.0 and 4.00 mS/cm con-ductivity (Tris-HCl buffer)

The feed tank was filled with 140 mL of mAb supernatant solution (titre 0.23 mg/mL, pH 9.8, con-ductivity 7.02 mS/cm) and the retentate was continuously stirred (30-60 rpm) and recirculated throughthe Biomax® 5 kDa membrane using a peristaltic pump at a maximum transfer flow rate of 50 mL/min.The run was operated at a maximum operating pressure (feed to permeate) of 2.0 bar. The mAb su-pernatant solution was concentrated for 5 volumes with 25 mM Tris-HCl buffer pH 9.0 and 2.01 mS/cmconductivity (to guarantee the zinc chloride removal), and then diafiltered for 5 volumes with 25 mMTris-HCl buffer pH 8.0 and conductivity 4.00 mS/cm (adjustment of the loading conditions) at a constantvolume by continuously feeding diafiltration buffer into the retentate vessel. The system and membranewere washed with 0.1 M NaOH, by recirculating the solution for 30 minutes. The system was stored in20% ethanol and the membrane was stored in 0.1M NaOH.

2.5 Analytical techniques

All samples were collected before and after every polishing step in order to be analyzed to de-termine yield, purity, HMWI, LMWI, DNA and HCP. Analytical methods such as HPLC-Protein A affinitychromatography, SEC, Bradford assay and Picogreen assay were used in this study as it will be describenext.

2.5.1 HPLC-Protein A affinity chromatography

HPLC-Protein A affinity chromatography was used to determine the antibody concentration. ADionex UltiMate 3000 HPLC system equipped with a diode array detector (Thermo Fisher Scientific)was used. Mobile phase A was 50 mM phosphate buffer, pH 7.0. Mobile phase B was a 100 mM glycinebuffer, pH 2.5. Before usage, all buffers were filtered through 0.22 µm filters (Merck KGaA) and degassedin an Elmasonic S100 unit ultrasonic bath. Approximately 100 µL of each sample were filtered with aMillex®-GV sterile syringe filter with a 0.22 µm pore size hydrophilic PVDF membrane (Merck MilliporeLtd.), using a 1 mL Omnifix®-F sterile syringe, supplied by B. BraunTM. Chromacol 03-FIV snap capvials of 300 µL with 11 mm Chromacol snap closures, both supplied by Thermo Fisher Scientific, wereused. The system was run at a flow rate of 2.5 mL/min. Filtered samples of 20 µL were loaded on aPOROS A 20 µm Column (2.1 × 30 mm, 0.1 mL; Thermo Scientific). The column was equilibrated with10 column volumes of mobile phase A, eluted with a step gradient with 20 column volumes of 100%mobile phase B, and re-equilibrated with 30 column volumes of mobile phase A. The absorbance at280 nm was measured. A similar protein A purified IgG1 was used as the calibration standard. Thecalibration range was 0.1–8 mg/mL. The results were evaluated and quantified with the ChromeleonTM

7 software (Thermo Fisher Scientific).

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2.5.2 Size exclusion chromatography (SEC)

Size exclusion chromatography was used to estimate the percentage of product purity, HMWIand LMWI. A Dionex UltiMate 3000 HPLC system equipped with a diode array detector, suplied byThermo Fisher Scientific, was used. The running buffer was a 50 mM potassium phosphate buffer with150 mM NaCl, pH 7.0 (Merck KGaA). The buffer was filtered through 0.22 µm filters (Merck KGaA) anddegassed in an Elmasonic S100 unit ultrasonic bath. Approximately 100 µL of each sample were filteredwith a Millex®-GV sterile syringe filter with a 0.22 µm pore size hydrophilic PVDF membrane (Merck Milli-pore Ltd.), using a 1 mL Omnifix®-F sterile syringe, supplied by B. BraunTM. Chromacol 03-FIV snap capvials of 300 µL with 11 mm Chromacol snap closures, both supplied by Thermo Fisher Scientific, wereused. Filtered samples of 20 µL were applied to a TSKgel® G3000SWXL HPLC Column (5 µm, 7.8 × 300mm) in combination with a TSKgel SWXL Guard Column (7 µm, 6.0 × 40 mm; Tosoh, Tokyo, Japan). Theabsorbance at 280 nm was recorded, and the results were evaluated with the ChromeleonTM 7 software(Thermo Fisher Scientific). The antibody purity was calculated as the ratio of the monomer peak area(retention time between 20.96 min. to 22.04 min.) to the sum of all peak areas, based on the 280 nmsignal.

2.5.3 Bradford assay

The quantification of total protein content was determined by the Bradford assay. 25 mL of Brad-ford reagent solution was prepared by diluting 5 mL of protein assay Coomassie Blue G250 dye reagentconcentrate (Bio-Rad) in 20 mL of water. Bovine serum albumin (BSA) standards, with a concentrationrange from 12.5 µg/mL to 200 µg/mL, were used as standards for the calibration curve. 10 µL of the BSAstandards along with the 10 µL of the sample dilutions with 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA,pH 7.5, Thermo Fisher Scientific) were transfered to a Costar 96 well flat-bottom EIA plate (Bio-Rad).200 µL of the prepared Bradford reagent solution was added to each well using a multichannel pipette(Starlab). The plate was incubated in the dark cold room for 5 minutes, softly shaking. The absorbancewas measured at 595 nm in a Tecan M200 Pro Infinite (Tecan, Maennedorf, Switzerland) fluorescentplate reader.

2.5.4 Residual DNA method quantification

The determination of dsDNA content of the samples in this study was performed using thePicogreen assay (Section 2.5.4.2). Due to the continuous precipitation capture step with a concentrationof 12 mM of ZnCl2, the ZnCl2 was interfering with the quantification of the dsDNA assays. Therefore,a buffer exchange step (Section 2.5.4.1) had to be performed prior to the Picogreen assay, in orderto remove the zinc chloride. This method was only needed for the samples that did not go through aprior buffer exchange performed with a UF/DF step (see Section 2.4), which would be all the samplescorresponding to the AEX-CEX N-ADC train.

2.5.4.1 Buffer exchange step to remove the zinc chloride from the sample

When ZnCl2 was present in the sample, a prior wash step was performed in order to removeZnCl2, because the ZnCl2 seemed to interact with the dsDNA quantification assay. Briefly, 200 µL ofsample was pipetted into Amicon® Ultra 0.5 mL centrifugal filters (Merck KGaA) with a molecular weightcut-off of 50 kDa (Merck KGaA). The centrifugal filters were placed in 1.5 ml reaction tubes and cen-trifuged at 14,000 rcf for 5 min, at 4°C (Eppendorf centrifuge 5415R; Eppendorf). Then, 300 µl of 25

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mM sodium acetate buffer, pH 6.0 and conductivity 1.98 mS/cm (equilibration buffer), was added to thesample. An Heidolph REAX 2000 vortex was used and, the sample was centrifuged again. This bufferexchange procedure was repeated six times.

2.5.4.2 Picogreen assay

The concentration of dsDNA was quantified using the Quant-iTTM Picogreen® dsDNA assaykit (Life Technologies, Carlsbad, CA, USA). Two consecutive 1:10 dilutions of λ DNA standard (1000ng/mL, Thermo Fisher Scientific) in 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) were performedto prepare "dilution 2" of λ DNA standard. Serial dilutions, using multichanel pipettes (Starlab, UK) andreverse pipetting, of 150 µL of sample and λ DNA standard "dilution 2" were performed in a 96-well platewith 150 µL of 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) in each well. 100 µL of all dilutionswere transferred to a black bottom 96-well plate (Nunclon Delta Black, Thermo Scientific) and 100 µLof prediluted color Quant-iTTM Picogreen® reagent (Thermo Fisher Scientific) was added to each well.The signal intensities were measured in a Tecan M200 Pro Infinite (Tecan, Maennedorf, Switzerland)fluorescent plate reader using an excitation wavelength of 480 and an emission filter of 520 nm. Acalibration curve was recorded for concentrations ranging from 3.91 ng/mL to 500.00 ng/mL.

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3 Results and Discussion

As mentioned before in Sections 1.10 and 2, the polishing experiments after continuous ZnCl2precipitation of this dissertation were based on the flow-through chromatography method reported byIchihara et al. (2019) [11] for the anion exchange chromatography followed by cation exchange chro-matography runs (AEX-CEX train) with adjustment of the mAb loading conditions.

3.1 Initial experiments

A brief summary of the results and discussion of the initial experiments will be explained inthis Section 3.1, since due to the denaturation of the antibody product, further study was not able tobe continued with the same starting material and a new set of experiments was performed (Section3.2). The materials and methods, as well as a more detailed discussion of the results of these initialexperiments can be found in Appendix I.1 and Appendix I.2, respectively.

The starting product of these initial experiments study was obtained after continuous precipita-tion with 12 mM of ZnCl2 (Section 1.4). The starting material for the polishing experiments consisted in0.34 mg/mL of Trastuzumab (pI 8.4) with a relative monomer purity around 50%, with less than 3% ofHMWI and around 48% of LMWI. The DNA and total protein content, as well as an estimation of HCPcontent was also evaluated. The Trastuzumab starting product had 30 ± 4 ng/ml of DNA content, 387± 77 µg/mL of total protein content and an estimation of HCP content of 47 ± 77 µg/mL. However, atfirst, the DNA content was not being able to be quantified neither by the Picogreen or the qPCR assay.Later, it was found out that presence of ZnCl2 (12 mM) from the continuous precipitation was interferingwith both DNA assays. Metal ions, such as Zn2+, can interfere with the qPCR assay by causing inhibitoryeffects on DNA amplification since Taq polimerase has been reported of having no activity in the pres-ence of zinc chloride at concentrations above 1 mM [62]. Whereas, the Picogreen assay is affected bythe ZnCl2 in concentrations above 5 mM, which can lead to an 8% decrease in the signal change [48].A buffer exchange step, prior to the DNA analytics, allowed to decrease the zinc chloride to concentra-tions bellow 0.1 mM, which solved this problem of ZnCl2 interfering with the DNA assays. Nevertheless,the DNA (8.8×104 pg DNA/mg IgG) and HCP (1.4×105 ng HCP/mg IgG) content were still way abovethe levels that guarantee a medicine’s safety approval, which should be lower than 10 ppb for residualDNA and lower than 100 ppm for HCPs. Therefore, the following polishing experiments with anion andcation exchange chromatography were, indeed, necessary to remove the impurities still present in theTrastuzumab product.

The Trastuzumab starting product (0.34 mg/mL, pH 4.0 and conductivity 2.15 mS/cm) obtainedfrom the ZnCl2 precipitation, which had been stored at -20 °C, was thawed and filtered for the follow-ing flow-through chromatography experiments. Three flow-through AEX-CEX trains were studied withdifferent mAb loading conditions. The Trastuzumab loading conditions of the AEX-CEX train "1" wereadjusted to pH 6.0 and 17.00 mS/cm conductivity (ZnCl2 was still present) by adding 4 M TrisBase pH9.0 solution. The Trastuzumab loading conditions of the AEX-CEX train "2" were adjusted to pH 6.0 and4.00 mS/cm conductivity by adding 4 M TrisBase pH 9.0 solution to adjust the pH, followed by a bufferexchange with UF/DF to adjust the conductivity and remove the ZnCl2. The Trastuzumab loading condi-tions of the AEX-CEX train "3" were not adjusted, which meant that the ZnCl2 was still present and theconditions were pH 4.0 and 2.15 mS/cm conductivity.

The Picogreen assay performed to all the fractions of the AEX-CEX trains (Figure I.3 in AppendixI.2) showed that the DNA was indeed removed, preferably, in the anion exchange chromatography step

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in all of the trains studied, however due to all the DNA concentrations of the flow-through fractions beingunder the limit of detection of the assay, it was not possible to say, clearly, if there was any preferablemAb loading condition that allowed to state which final Trastuzumab product had less DNA content.However, even though there was no exact DNA quantified in the flow-through fraction, the wash andelution fractions combined of the flow-through AEX trains "1", "2" and "3" had an overall DNA removalof 77%, 38% and 16%, respectively, in relation to the DNA present in the feed. Taking this result intoconsideration, it can be concluded that the Trastuzumab loading conditions of pH 6.0 and 17 mS/cmconductivity showed more DNA removal by the AEX polishing step.

On the other hand, the CEX fractions showed some out of the ordinary results. The DNA con-centration in all the CEX feed and flow-through fractions of the three trains was bellow the assay’s limit ofdetection, however, it can be observed in Figure I.3 in Appendix I.2 that there was DNA quantified in theelution and wash fractions of the CEX of trains "1" and "3", respectively. Additionally, the DNA quantifiedin these fractions was present in a higher content than the DNA present in the respective feed, which,in theory, is an impossible result. A possible explanation is that there could still be DNA in the columnfrom previous runs which was not washed or eluted and, consequently, the CIP of the column with 2 CVof 0.5 M NaOH did not guaranteed enough incubation time. The AEX and CEX method was changedlater for the following experiments, for there to be, at least, a column CIP with 35 min. of incubationtime with NaOH. Nevertheless, the DNA present in the CEX wash and elution fractions could have beenbounded to an histone protein, and the histone consequently bounded to the cation exchanger, due tothe working pH being below the protein’s pI (making the protein positively charged). Additionally, bothtrains "1" and "3" still had ZnCl2 present in the mAb solution loaded, therefore there could be some sortof secondary interaction between the Zn2+ and the DNA molecules, resulting in the zinc precipitating theDNA and considering that the zinc is a cation, it would bind on the cation exchanger resin. Furthermore,it was observed that most of the DNA was removed in the washing step of the AEX, which means thatthese negatively charged molecules were not bound to the positively charged resin which might be dueto the exchangeable counterions of the washing buffer having stronger electrostatic interactions with thepositively charged functional groups of the resin. These suggests that there could be, indeed, a possi-bility of an interaction between DNA-Zn2+ or between DNA-histone proteins that could form complexeswith slight positive charge, resulting in these DNA molecules not binding to the anion exchanger. Ad-ditionally, the higher quantity of DNA removed in the wash fraction of the AEX train "1" might be dueto the high conductivity (17.00 mS/cm) of the Trastuzumab loading conditions, which are not favorablefor DNA binding, since it reduces the electrostatic interactions between DNA and the resin’s ligand, bypotentially reducing the effective charge of DNA [35]. Therefore, it is important to remove or decreasethe ZnCl2 concentration in the following flow-through chromatography experiments without increasingthe conductivity of the mAb solution loaded onto the columns.

The Bradford assay performed to all the fractions of the AEX-CEX trains showed that the totalprotein content was bellow the method’s limit of detection, which was an odd result, considering therewas total protein quantified in a previous analysis of the Trastuzumab starting product (AEX feed) of 387± 77 µg/mL. According with the literature, [63], the Bradford assay is not susceptible to interference bya wide range of chemicals present in samples, therefore interference by ZnCl2 was excluded. Due to somany odd results in the Bradford’s analytics performed on all the samples of the three AEX-CEX trainsstudies, the validity of the method was verified by determining the total protein concentration in a BSAstock solution and in a mAb TG5 solution of known concentrations. The method quantified total proteinconcentrations in both solutions, therefore the odd results were not related with the protein assay. AnHPLC-Protein A affinity chromatography was performed to all the fractions of the AEX-CEX trains studiedto determine antibody concentration, in which the results showed that there was no antibody present in

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any of the samples, including in the Trastuzumab starting product obtained after ZnCl2 (Figure I.4). Theseresults led to conclude that the antibody did not survive the storage conditions (pH 4.0, conductivity 2.15mS/cm, -20°C). The multiple freeze-thaws may have resulted in the loss of antibody reactivity [52].

3.2 Flow-through AEX-CEX (N-ADC and ADC) experiments with sodium acetatebuffer

The new starting Trastuzumab product was obtained after continuous precipitation with 12 mMof ZnCl2 (Section 1.4). The starting material, which consisted in 0.6 L of Trastuzumab with a mAb titre of0.29 mg/mL, was stored in the cold at 2°C to 8°C until the start of the flow-through polishing experimentson the following day and throughout the week.

The thesis study had to be adapted due to some drawbacks faced in the initial experiments(Section 3.1), such as the presence of zinc chloride after precipitation and the high conductivity of themAb solution after adjustment of the loading conditions. It has been reported in literature that pH andconductivity have been identified as parameters that may have impact on the clearance of DNA andHCP [35]. In the case of DNA clearance over AEX chromatography in flow-through mode (using also aquaternary ammonium media), Stone et al. reported that the least favorable conditions for DNA bindingare low pH and high conductivity (∼ pH 6.0 and ∼ 15.00 mS/cm conductivity) and the most favorableconditions are at high pH and low conductivity (∼ pH 8.0 and ∼ 2.00 mS/cm conductivity), nevertheless,it was concluded that even at the least favorable conditions for DNA binding, the flow-through AEXchromatography can show a confident DNA clearance capacity. Taking everything into consideration, anew set of experiments with a new approach to the adjustment of the Trastuzumab loading conditionswas performed, based on the mAb loading conditions (pH 6.0 and 4.00 mS/cm conductivity) of themethod reported by Ichihara et al. (2019).

The new aim for this thesis was to compare two trains: AEX-CEX with non-adjusted loadingconditions (N-ADC) and AEX-CEX with adjusted loading conditions (ADC) (method in Section 2.3.1). Inthe AEX-CEX N-ADC train, the Trastuzumab (precipitated material) was filtered and loaded directly ontothe AEX column with pH 3.6 and 3.47 conductivity, with ZnCl2 still present in the solution. Whereas, inthe AEX-CEX ADC train the Trastuzumab loading conditions were adjusted to pH 6.0 and 4.00 mS/cmconductivity, prior to the loading onto the AEX column by a two-step buffer exchange by UF/DF (methodin Section 2.4.1). The first step removed the zinc chloride from the antibody solution, which consisted ina five volume buffer exchange of 25 mM sodium acetate (pH 4.0, conductivity 2.02 mS/cm), while thesecond step had the purpose of adjusting the Trastuzumab solution to the loading conditions reportedin the article [11], consisting in a five volume buffer exchange with 25 mM sodium acetate (pH 6.0,conductivity 4 mS/cm).

Due to time limitations, a previous AEX and CEX run to determine the DNA and HCP break-through, respectively, in order to know how much of Trastuzumab solution could be loaded, was notable to be performed. Therefore, it was decided to load 225 mL of Trastuzumab solution onto the AEXcolumn and 200 mL onto the CEX column, according to the overall mAb volume loaded (200 mL) on theAEX-CEX train of the flow-through chromatography method reported by Ichihara et al. (2019), in whichthe columns were connected, contrary to this thesis study, in which the columns are not connected.

The chromatograms of the AEX and CEX runs with non-adjusted Trastuzumab loading condi-tions (N-ADC) (pH 3.6, conductivity 3.47 mS/cm) are illustrated in Figures 3.1 and 3.2, respectively.Zoomed-in chromatograms of the wash, elution and CIP steps of the AEX N-ADC and CEX N-ADC runsare illustrated in Figures I.5 and I.6 in Appendix I.3.1.

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Figure 3.1: Chromatogram of the flow-through AEX run with non-adjusted loading conditions (N-ADC) of the AEX-CEX N-ADC train with sodium acetate buffer. The AEX column was equilibrated with 25 mM sodium acetate buffer(pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min. The AEX column was loaded at the flow rate of 0.2 mL/minwith 0.29 mg/mL mAb loading at 225 mL (225 CV). The mAb loading conditions were pH 3.6 and 3.47 mS/cmconductivity. The UV was measured at wavelengths 280 nm and 214 nm.

Figure 3.2: Chromatogram of the flow-through CEX run with non-adjusted loading conditions (N-ADC) of the AEX-CEX N-ADC train with sodium acetate buffer. The CEX column was equilibrated with 25 mM sodium acetate buffer(pH 6.0, 1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min. The CEX column was loaded at the flow rate of 0.2 mL/minwith 200 mL (400 CV) of mAb flow-through solution obtained from the AEX N-ADC run. The mAb loading conditionswere pH 3.6 and 3.51 mS/cm conductivity. The UV was measured at wavelengths 280 nm and 214 nm.

The chromatograms of the AEX and CEX runs with adjusted Trastuzumab loading conditions(ADC) (pH 6.0, conductivity 4.00 mS/cm) are illustrated in Figures 3.3 and 3.4, respectively. A zoomed-in chromatogram of the sample loading of the CEX ADC flow-through run is illustrated in Figure I.7 inAppendix I.3.1. Zoomed-in chromatograms of the wash, elution and CIP steps of the AEX ADC and CEXADC runs are illustrated in Figures I.8 and I.9 in Appendix I.3.1.

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Figure 3.3: Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEXADC train with sodium acetate buffer. The AEX column was equilibrated with 25 mM sodium acetate buffer (pH 6.0,1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min. The AEX column was loaded at the flow rate of 0.2 mL/min with 0.29mg/mL mAb loading at 225 mL (225 CV). The mAb loading conditions were adjusted to pH 6.0 and 4.00 mS/cmconductivity. The UV was measured at wavelengths 280 nm and 214 nm.

Figure 3.4: Chromatogram of the flow-through CEX run with adjusted loading conditions (ADC) of the AEX-CEXADC train with sodium acetate buffer. The CEX column was equilibrated with 25 mM sodium acetate buffer (pH 6.0,1.98 mS/cm, 15 CV) at a flow-rate of 1 mL/min. The CEX column was loaded at the flow rate of 0.2 mL/min with 200mL (400 CV) of mAb flow-through solution obtained from the AEX ADC run. The mAb loading conditions were pH6.0 and 4.12 mS/cm conductivity. The UV was measured at wavelengths 280 nm and 214 nm.

Considering that both AEX and CEX runs are in flow-through mode, the antibody is not bindingto the columns and is, instead, passing through them, resulting in a flat line in the UV signals at 280nm and 214 nm during sample application, and peaks of the respective retained impurities in the washand elution fractions. It can be observed that in all chromatograms the UV absorbance signal at 214nm is higher than the UV absorbance signal at 280 nm, since at 214 nm is the absorption maximafor the peptide bond which means that proteins deficient in the aromatic amino acids, Trp, Tyr and

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phenylalanine, will also be detected. The UV signal at 254 nm or 260 nm should have been monitored,since these wavelengths are more sensitive to nucleic acids. Consequently, the purity of DNA could havebeen estimated by the ratio of absorbance at 260 nm and 280 nm [25,58].

The UV signal at 214 nm in the AEX-CEX N-ADC train (Figures 3.1 and 3.2) is at a higher valuethan in the AEX-CEX ADC train (Figures 3.3 and 3.4), which might be due to the presence of lowermolecular mAb aggregates in more quantity (Section 3.2.3). In both CEX chromatograms (Figures 3.2and 3.4), the UV signal at 280 nm decreased which could be related with less antibody passing throughthe column in comparison with the AEX chromatograms, as it will be mentioned in the following Sections3.2.3 and 3.2.4. Additionally, it can also be observed an increase in the UV signal at 214 nm in bothCEX chromatograms, which might be related with the antibody binding to the column and some flowingthrough the column.

In Figures I.5 to I.9 in Appendix I.3.1, it is possible to see that there were impurities (or evenantibody, even though it is not what should be expected) that bounded to the respective columns, byobserving the peaks in the wash and elution steps. There is even a peak in the column CIP with NaOH,which means there were impurities or antibody being removed only by sanitization of the column, andtherefore could not be quantified by any of the analytics performed. It would be expected that the peaksin the wash and elution steps of the AEX chromatograms would belong to negatively charged impuritiessuch as DNA and acidic HCPs, while the peaks in the wash and elution steps of the CEX chromatogramsshould be related to basic HCPs and mAb aggregates.

3.2.1 Residual DNA content

The DNA concentration detected in the samples collected from the fractions (feed, flow-through,wash and elution) of the AEX-CEX N-ADC and ADC trains was bellow 31.25 ng DNA/mL and bellow7.81 ng DNA/mL, respectively (Tables I.9 and I.10 in Appendix I.3.1.1). For the Picogreen and Bradfordassays, only values above the lower limit of quantification (LLOQ) were considered, since quantificationbellow LLOQ is, by definition, not acceptable. LLOQ is the lowest amount of an analyte in a sample thatcan be quantitatively determined with suitable precision and accuracy (bias). Therefore, below this valuea method can only produce semi-quantitative or qualitative data. However, it can still be important toknow the limit of detection (LOD) of the method. According with ICH [64], LOD is the lowest concentrationof an analyte in a sample which can be detected but not necessarily quantified as an exact value. Theacceptance critera for the assays’ results, in terms of precision and accuracy, was taken into accountand only concentration values within ± 20% RSD for precision and ± 20% for bias were consideredacceptable [65]. If an assay was not able to quantify a DNA or total protein concentration of a sample, itsvalue was assumed to be bellow the minimum limit of detection of the respective assay that was withinthe acceptable criteria for precision and accuracy. For both trains, the DNA concentration was bellow theLOD of the Picogreen assay that was within the acceptable criteria for precision and accuracy (± 20%RSD for precision and ± 20% for bias) [65].

Tables I.9 and I.10 in Appendix I.3.1.1 show the maximum DNA content that could exist in eachfraction sample, however, just as mentioned previously, DNA was only detected but not quantified as anexact value in any fraction. Furthermore, even though there is a difference in the DNA content of thefraction samples of the N-ADC and ADC trains, it is important to note that this reduction in the DNAcontent from the N-ADC train samples to the ADC train samples cannot be said to be dependent of thedifferent Trastuzumab loading conditions. Considering that every value was bellow the minimum LODof the respective DNA assay performed and the value of LOD, consequently, the difference observeddepends on the accuracy and precision of the analytical technique performed being within acceptable

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criteria values. In the feed and flow-through of the AEX N-ADC train it is known that the DNA contentis bellow 7031 ± 70 ng DNA (Table I.9 in Appendix I.3.1.1), while the feed and flow-through of theAEX ADC is bellow 1757 ± 70 ng DNA (Table I.10 in Appendix I.3.1.1). The concentration of DNA inppb at the end of the CEX N-ADC and ADC runs (CEX flow-through) is < 5000 ppb and < 1100 ppb,respectively. However, just as mentioned before, an exact value was not able to be determined. A typicalmAb bulk drug specification limit should be < 10 pg/mg IgG to guarantee a medicine’s safety approvalby organizations such as FDA or EMA.

3.2.2 Total protein, HCP and antibody content

Figure 3.5 shows the content in mass of total protein, antibody and HCPs present in the samplesof each fraction (feed, flow-through, wash and elution) of the AEX-CEX trains with non-adjusted andadjusted loading conditions. Tables I.11 and I.12 in Appendix I.3.1.2 show the total protein, antibodyand HCP concentrations of both trains in more detail. The slight deviations in the total protein content ofthe AEX feed in both mAb loading conditions can be explained by the relative standard deviation of theBradford assay of each data, since the starting Trastuzumab product was the same for each train.

Figure 3.5: Analysis of the total protein, mAb and HCP content of the samples collected from the AEX-CEX N-ADC(Figure A) (Trastuzumab loading conditions: pH 3.6 and 3.47 mS/cm conductivity, and ZnCl2 is still present) andAEX-CEX ADC (Figure B) (Trastuzumab loading conditions adjusted to pH 6.0 and 4.00 mS/cm conductivity, andZnCl2 has been removed).

It is possible to observe that, in almost all fractions, the antibody content is higher than the totalprotein content, which should be impossible, considering that the total protein includes the antibodyproduct in addition to the host cell protein content [52]. Even when the relative standard deviation errorof the total protein data is taken into account, the results still seem to show that the total protein is mostlyantibody, which should not be true, since there must still be HCPs present, considering that the ZnCl2precipitation did not removed them all, according with Dutra et al. [28]. It is important to note that thetotal protein was quantified using the Bradford assay, while the antibody concentration was quantifiedusing HPLC-Protein A affinity chromatography, which has a higher sensitivity, resulting in a more exactquantification of the antibody concentration values, while the total protein content determined by theBradford assay, using BSA standards, is much more sensitive to BSA than immunoglobulin G (IgG),

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thus it is more likely to underestimate the amount of total protein present [66].

It has been reported that the Bradford assay is less accurate for basic or acidic proteins, andthe majority of CHO HCPs have pIs < 7 [63]. Additionally, the sensitivity of an analytical method maybe difficult to achieve for products with lower protein concentrations [52], which in this case is quite low(mAb titre of 0.29 mg/mL). Furthermore, colorometric methods such as the Bradford assay require aBSA standard that is still not well matched to the HCP analyte. Literature advises to choose a proteinstandard that is likely to give absorbance values close to those of the protein of interest, therefore sincethe determination of the total protein concentration of an immunoglobulin is the goal, then a IgG stan-dard should have been used. However, this usually is not feasible. Generally, BSA is the most commonlyused protein standard because it is widely available in high purity and relatively inexpensive. Alterna-tively, bovine gamma globulin (BGG) could be a good standard when determining the concentration ofantibodies, because BGG produces a color response curve that is very similar to that of immunoglobulinG (IgG) [66].

Considering the low conductivity (bellow or at 4.00 mS/cm) and that the working pH of theseflow-through experiments is below the Trastuzumab’s pI of 8.4, these conditions will guarantee that themAb product is positively charged, and therefore, it will pass through the AEX column whereas negativelycharged impurities, such as acidic HCPs and DNA tend to bind to the AEX resin. In Figure 3.5, it can beobserved that in both mAb loading conditions, the total protein content remained approximately the sameafter the flow-through AEX polishing step, as it would be expected, since the impurities that could beremoved in this step should have a negative charge, which means that there were not much acidic HCPspresent in the feed. Nevertheless, there was HCP estimated in the elution fraction of AEX N-ADC andADC in a concentration bellow 25 ± 1 ng HCP/mL. These acidic HCPs (negatively charged) could havebeen associated with the mAb product and bounded to the anionic exchange resin (positively charged),while the antibody passed through the column. Due to being associated to the antibody, the Bradfordassay had more difficulty in quantifying these HCPs in the feed fraction, due to the BSA standardsunderestimating the total protein concentration of antibodies. However, when the HCPs are eluted, noantibody is present in this fraction, therefore the BSA standards can estimate a better quantification ofthese proteins.

The CEX chromatography step was responsible for far more total protein removal, includingHCPs and unfortunately, antibody product as it can be seen in Figure 3.5. The CEX N-ADC decreasedthe total protein level of the final Trastuzumab product in 2.6-fold, whereas the CEX ADC reduced thetotal protein level of the final Trastuzumab product in 1.8-fold. However, this reduction in total protein isnot associated with HCP removal, since there is no estimation of HCP content in the CEX feed. HCPscan be divided into two groups: HCPs that do not interact with the target mAb and HCPs who do. Fornon-product-associating HCPs, clearing strategies are based in its physicochemical properties (e.g.,pI, size, etc.), considering that the majority of CHO HCPs have pIs less than 7 and molecular weightless than 75 kDa [42]. Usually, these non-product-associating HCPs should be removed by the wash orelution steps of the CEX, however, there was no HCP content estimated neither in the wash or elutionfractions of both CEX N-ADC and ADC trains. On the other hand, there was HCP estimated in the CEXflow-through of both N-ADC and ADC with 18 ± 2 ng HCP/mL and 63 ± 10 ng HCP/mL, respectively.The HCPs associated with the mAb product have been reported to be more persistent and difficult tobe removed, due to formation of strong and attractive interactions, resulting in the HCPs entering theproduct fraction during chromatography purification [42, 67]. Considering these data, the AEX-CEX N-ADC train showed that the final Trastuzumab product had less HCP content than the final product of theAEX-CEX ADC train. Nevertheless, both Trastuzumab final products still had levels of HCP in a order ofmagnitude above 105 ppm, which is was above the limit of 100 ppm to guarantee a medicine’s approval

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(Tables I.11 and I.12 in Appendix I.3.1.2).

It is important to mention that the HCP content estimation used is very ambiguous, since it isbased on the fact that the total protein present in the sample is the sum of mAb product and HCPs [52],therefore, HCP can only be calculated in the samples where there is a higher content of total proteinthan mAb product, which should be true for all of the samples, but unfortunately, it was not the case,according with the results from the Bradford and HPLC-Protein A affinity chromatography representedin Figure 3.5. An ELISA assay sensitive for the detection of HCPs in CHO cell lines could, perhaps, bea more suitable method for HCP quantification, for future experiments [51,52].

The reduction of total protein in the CEX polishing step is, instead, associated with most of theantibody being present in the wash and elution fractions of both trains (Figure 3.5), which was not ex-pected to happen when operating the CEX in flow-through mode. In flow-through cation exchange chro-matography, the separation occurs in a frontal chromatography mechanism where the mAb monomerinitially binds to the column and is subsequently displaced by mAb aggregates (i.e. dimers and highermolecular weight aggregates). There is a much earlier breakthrough of the mAb monomer, due to beingthe least retained species, followed by the breakthrough of the more retained species (mAb aggre-gates) [45]. There was, in fact, antibody that passed through the column and was present in the flow-through fraction, however most of the antibody was washed away in both N-ADC and ADC trains. Figure3.6 shows that 66% and 70% of the Trastuzumab monomer present in the CEX feed of the N-ADC andADC, respectively, was in the wash fraction, whereas there were mAb breakthroughs of 27% and 29%that passed through the CEX column and were present in the final Trastuzumab product of the N-ADCand ADC trains, respectively.

Figure 3.6: Trastuzumab product quantity (%) present in the flow-through, wash and elution fractions of the cationexchange chromatography in comparison to the CEX feed, in both non-adjusted (N-ADC) and adjusted (ADC) mAbloading conditions (analysis of the antibody titre by HPLC-Protein A affinity chromatography).

These results, suggest that the flow-through CEX polishing step did not operate completely inflow-through mode, considering that the antibody was mostly washed, which means that the antibodyproduct was retained in the interstitial space of the CEX matrix, but there was also antibody elutedfrom the column, which means that a small part actually remained bounded to the cation exchanger.The Eshmuno® CP-FT resin is specifically developed for the flow-through removal of mAb aggregatesunder strong binding conditions (pH 4.0-5.5, 3.00-7.00 mS/cm) that favor frontal chromatography [45].The mAb loading conditions used were pH 3.6 and 3.47 mS/cm conductivity for the AEX-CEX N-ADCtrain and pH 6.0 and 4.00 mS/cm conductivity for the AEX-CEX ADC train, which were, in both trains,outside of the pH range for strong binding conditions that favor frontal chromatography on the CEX in

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flow-through mode. Considering the fact that mAb aggregates generally have a higher surface net chargethan the antibody product due to being more hydrophobic than the antibody monomer, this characteristicwill, consequently, lead to them binding more strongly to the cation exchanger than the correspondingmonomeric form [8]. This occurrence might have been the reason why the antibody was mostly retainedin the interstitial space of the CEX resin, due to not being able to bind to the cation exchanger since theaggregates bounded more strongly and probably occupied most of the resin’s ligands due to their largersize.

Ichihara et al. (2019) used the same mAb loading conditions parameters of pH 6.0 and 4.00mS/cm conductivity and close to 100% mAb breakthrough was reported. It is important to note that thetarget mAb loading used by Ichihara et al. was above 1500 mg mAb (8.2 mg/mL) and the mAb’s pI wasof 7.7 [11], while this thesis study had a mAb loading of 65 mg mAb (0.29 mg/mL) and a mAb’s pI of8.4. Antibody recovery in the CEX has been associated with higher mAb loading (with little impact onHCP and DNA clearance) and its optimal conditions are influenced by the pI of the mAb [16]. Antibodiesare more strongly charged when the working solution pH is lower than their pI, which leads to optimalaggregate clearance on the flow-through CEX media. In these conditions, the electrostatic interactionsbetween the positively charged mAb monomer/aggregates and the negatively charged resin are stronger[68]. Since in the mAb loading conditions studied (pH 3.6 for CEX N-ADC and pH 6.0 for CEX ADC), thecharge difference between the Trastuzumab’s pI and the working pH is higher than the charge differencebetween the mAb’s pI (7.7) used in the study of Ichihara et al. (working pH 6.0), the binding of the mAbaggregates in the cation exchanger resin will be stronger in our study, difficulting the ealier breakthroughof the mAb monomer, since most of the antibody does not even bind to the ligands and is insteadretained in the interstitial space of the CEX resin due to the stronger binding of the aggregates and theirlarger size. In the study of Ichihara et al. (2019), the charge difference between the working pH (pH 6.0)and the mAb’s pI (7.7) is lower, resulting in an earlier breakthrough of the mAb monomer.

3.2.3 Antibody monomer purity and HMWI and LMWI content

The antibody (monomer) purity (%) and the HMWI (%) and LMWI (%) content in the samplescollected from both trains were calculated from the peak data of the respective size exclusion chro-matogram (method in Section 2.5.2) and are represented in Figure 3.7. The percentages values can befound in more detail in Tables I.13 and I.14 in Appendix I.3.1.3.

Figure 3.7: Size exclusion chromatography analysis of the antibody (monomer) purity (%) and HMWI (%) andLMWI (%) content in the fractions (feed, wash, elution and flow-through) of the AEX-CEX N-ADC (with non-adjustedloading conditions) and AEX-CEX ADC (with adjusted loading conditions) trains with sodium acetate buffer. Abovethe 95% dot line is the target bulk drug specification limit for monomer purity, whereas the HMWI and LMWI contentshould be lower than 1%.

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Figures 3.8 and 3.9 show the size exclusion chromatograms of the feed, flow-through, washand elution fractions of Trastuzumab in the AEX and CEX flow-through polishing steps, respectively,of the N-ADC train. Figures 3.10 and 3.11 show the size exclusion chromatograms of the feed, flow-through, wash and elution fractions of Trastuzumab in the AEX and CEX flow-through polishing steps,respectively, of the ADC train.

Figure 3.8: Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of the feed (B), flow-through (C), column wash (D) and elution (E) of the AEX N-ADC (with non-adjusted loading conditions) chromatog-raphy run. The monomer peak area is observed between 20.96 and 22.04 min. of retention time.

Figure 3.9: Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of the feed (B), flow-through (C), column wash (D) and elution (E) of the CEX N-ADC (with non-adjusted loading conditions) chromatog-raphy run. The monomer peak area is observed between 20.96 and 22.04 min. of retention time.

The mAb starting material with non-adjusted loading conditions (pH 3.6, 3.47 mS/cm) had arelative monomer purity of 10%, with 9% of HMWI and 81% of LMWI (Figure 3.8.B). The mAb startingmaterial with adjusted loading conditions (pH 6.0, 4.00 mS/cm) had a relative monomer purity of 93%,with 0% of HMWI and 7% of LMWI (Figure 3.10.B). The differences in the initial content of HMWI and

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LMWI in both trains suggest that the Biomax® membrane with a MWCO of 8 kDa, used in the bufferexchange step with UF/DF to remove the zinc chloride and adjust the loading conditions, also contributedto the HMWI and LMWI clearance, resulting in a 9.3-fold increase in antibody monomer purity. The AEXpolishing step increased the monomer purity in 1.6-fold and 1.0-fold in the Trastuzumab product N-ADCand ADC, respectively. In the wash step of the AEX ADC, there was some monomer present that waslost (Figure 3.7.A). In the CEX N-ADC flow-through fraction, there was no monomer present and therewas, instead 70% and 57% of monomer purity in the wash and elution fractions. These results supportthe hypothesis mentioned in Section 3.2.2 that the flow-through CEX polishing step did not operatecompletely in flow-through mode. In the CEX ADC flow-through fraction, the antibody that is presenthad 100% monomer purity, however there is also antibody present in the wash and elution fractions witha monomer purity of 93% and 100%, respectively. In the size exclusion chromatograms relative to theAEX-CEX N-ADC train (Figure 3.8 and 3.9), it is possible to observe a smaller fragment peak connectedto the antibody peak (20.96-22.04 min of retention time), however it is unclear whether the minor secondpeak represents a mAb aggregate. However, since most of the mAb product is in the wash fraction ofthe CEX (Figure 3.6), in the opposite of being in the flow-through, it is possible to observe that, in Figure3.9.D the antibody peak is more symmetrical, which suggests that the second peak fragment could havebeen a lower molecular weight mAb aggregate in an undesirable secondary interaction with the antibodyproduct that was separated by the CEX polishing step. This occurence may be due to the non-adjustmentof the mAb loading conditions, since in the size exclusion chromatograms of the AEX-CEX ADC train,the antibody peak is symmetrical. Perhaps, the zinc chloride from the ZnC2 continuous precipitation isinteracting with some negatively charged low molecular weight mAb aggregate, which consequently hasadsorptive properties similar to those of the antibody product, complicating the separation. It has beenreported that an increased number of metal ion binding sites on the protein surface can increase therate of aggregation [28].

Figure 3.10: Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of the feed (B), flow-through (C), column wash (D) and elution (E) of the AEX ADC (with adjusted loading conditions) chromatographyrun. The monomer peak area is observed between 20.96 and 22.04 min. of retention time.

It can be observed from Figure 3.7.B that there was no HMWI detected in the samples of theAEX-CEX ADC in comparison with the AEX-CEX N-ADC train. The HMWI of the Trastuzumab startingproduct with non-adjusted loading conditions were mostly removed in the AEX polishing step, whichleads to believe these high molecular weight impurities must have been negatively charged. Only 2%

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Figure 3.11: Overlaid UV 280 nm size exclusion chromatograms (A) of trastuzumab samples of the feed (B), flow-through (C), column wash (D) and elution (E) of the CEX ADC (with adjusted loading conditions) chromatographyrun. The monomer peak area is observed between 20.96 and 22.04 min. of retention time.

of HMWI were detected in the CEX ADC wash fraction, which could have been mAb aggregates orproduct-associated HCPs that were interacting with the mAb product, and therefore not detected in theprevious fractions analyzed, and after the frontal chromatography mechanism of the flow-through CEX,these HMWI were separated and washed.

The Trastuzumab starting material of the AEX-CEX N-ADC had a high content of LMWI of 81%,which the AEX polishing step was not quite able to reduce (Figure 3.7.C). There was LMWI removal in thewash and elution steps of the AEX, however its content in the CEX feed remained the same value of 81%.It is important to note that the volume fraction of the AEX feed is 225 mL, whereas the volume fractionof the wash and elution is 5 mL. The final Trastuzumab product (CEX flow-through fraction) consisted in43% of LMWI, which means there was a 1.9-fold reduction of lower molecular weight aggregates in theCEX N-ADC polishing step (Figure 3.9.C). The AEX-CEX ADC train demonstrated a complete removalof LMWI in the final Trastuzumab product.

The Trastuzumab final product with non-adjusted loading conditions (pH 3.6, 3.47 mS/cm) hada relative monomer purity of 0%, with 2% of HMWI and 98% of LMWI (Figure 3.9.C). The Trastuzumabfinal product with adjusted loading conditions (pH 6.0, 4.00 mS/cm) had a relative monomer purity of100%, with 0% of HMWI and 0% of LMWI (Figure 3.11.C). According with the specifications for drugdevelopment, the monomer purity should be above 95%, while aggregates should be removed to valuesbellow 1% [11, 43]. Taking this into consideration, the Trastuzumab final product of the AEX-CEX ADCtrain meets these specification criteria for a medicine’s approval.

3.2.4 Antibody monomer yield

The mAb concentration in the samples was determined by HPLC-Protein A affinity chromatog-raphy by linking the peak areas to an external calibration curve constructed. The antibody peak can beobserved between 0.84 and 0.88 min. of retention time. Figures 3.12 and 3.13 show the HPLC-Protein Aaffinity chromatograms of the feed, flow-through, wash and elution fractions of Trastuzumab in the AEXand CEX flow-through polishing steps, respectively, of the N-ADC train.

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Figure 3.12: Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samples of the feed(B), flow-through (C), column wash (D) and elution (E) of the AEX N-ADC (with non-adjusted loading conditions)chromatography run. The peak of the retained mAb is observed between 0.84 and 0.88 min. The mAb titre in eachfraction is 0.29 mg/mL (B), 0.24 mg/mL (C), 0.08 mg/mL (D) and 0.00 mg/mL (E).

Figure 3.13: Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samples of the feed(B), flow-through (C), column wash (D) and elution (E) of the CEX N-ADC (with non-adjusted loading conditions)chromatography run. The peak of the retained mAb is observed between 0.84 and 0.88 min. The mAb titre in eachfraction is 0.24 mg/mL (B), 0.07 mg/mL (C), 6.27 mg/mL (D) and 0.38 mg/mL (E).

Figures 3.14 and 3.15 show the HPLC-Protein A affinity chromatograms of the feed, flow-through, wash and elution fractions of Trastuzumab in the AEX and CEX flow-through polishing steps,respectively, of the ADC train.

From just observing the HPLC-Protein A affinity chromatograms, it is possible to conclude thatin both non-adjusted and adjusted loading conditions, the AEX polishing step allowed the antibody toflow through the column (Figures 3.12 and 3.14), with minor losses of antibody in the wash step. On

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Figure 3.14: Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samples of the feed(B), flow-through (C), column wash (D) and elution (E) of the AEX ADC (with adjusted loading conditions) chro-matography run. The peak of the retained mAb is observed between 0.84 and 0.88 min. The mAb titre in eachfraction is 0.29 mg/mL (B), 0.26 mg/mL (C), 0.07 mg/mL (D) and 0.00 mg/mL (E).

Figure 3.15: Overlaid UV 280 nm HPLC-Protein A affinity chromatograms (A) of trastuzumab samples of the feed(B), flow-through (C), column wash (D) and elution (E) of the CEX ADC (with adjusted loading conditions) chro-matography run. The peak of the retained mAb is observed between 0.84 and 0.88 min. The mAb titre in eachfraction is 0.26 mg/mL (B), 0.08 mg/mL (C), 7.23 mg/mL (D) and 0.14 mg/mL (E).

the other hand, by observing the chromatograms of the CEX polishing step (Figure 3.13 and 3.15) it ispossible to verify that there is antibody present in the flow-through, wash and elution fractions. In fact,there seems to be, in both non-adjusted and adjusted loading conditions, more Trastuzumab presentin the wash fraction than in the flow-through, which confirms the theory of the CEX polishing step nothaving functioned completely in flow-through mode mentioned in Sections 3.2.2 and 3.2.3.

In Figure 3.16, the process mAb yield (Figure 3.16.A) and the purity of the antibody monomer(Figure 3.16.B) of both AEX-CEX trains studied can be interpreted in more detail. The monomer yield

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values of the AEX-CEX N-ADC and ADC trains are presented in Tables I.13 and I.14 in Appendix I.3.1.3,respectively.

Overall, the flow-through AEX and CEX polishing steps had a higher mAb yield and antibodymonomer purity when the Trastuzumab loading conditions were adjusted to pH 6.0 and 4.00 mS/cmconductivity (Figure 3.16). The Trastuzumab product with non-adjusted loading conditions (pH 3.6, 3.47mS/cm) had a yield of 82% with 16% of antibody monomer purity after the flow-through AEX polishingstep. This results suggests that the loading conditions of pH 3.6 and 3.47 mS/cm are not optimal foraggregate removal, whereas in the adjusted loading conditions experiments, most of the low molecularweight aggregates and the few high molecular weight aggregates were removed in the buffer exchangeusing UF/DF prior to the loading onto the AEX column. The Trastuzumab product with adjusted loadingconditions (pH 6.0, 4.00 mS/cm) had a yield of 90% with 95% of antibody monomer purity after the flow-through AEX polishing step. Considering that the flow-through CEX ADC polishing step (Figure 3.15) didnot improve the yield and purity of the Trastuzumab product and the monomer purity in the AEX ADCflow-through is already at levels that guarantee a medicine’s approval, further polishing of the antibodyproduct with the flow-through CEX was unnecessary.

Figure 3.16: Analysis of the process yield (A) and antibody (monomer) purity (B) of the AEX-CEX N-ADC (withnon-adjusted loading conditions) and of the AEX-CEX ADC (with adjusted loading conditions) trains with sodiumacetate buffer (based on the AEX feed as starting product).

The differences in mAb yield between the AEX flow-through and CEX feed fractions are dueto the difference in loading volume (225 mL for AEX and 200 mL for CEX). It is important to note thatthe AEX and CEX chromatography runs were performed with a small hold time in between, since thecolumns were not connected such as the flow-through method in the article [11], and therefore aftereach chromatography run, the flow-through fractions had to be pooled and volume had to taken for theanalytical techniques, resulting in this reduction of mAb yield. The Trastuzumab final product with non-adjusted loading conditions (pH 3.6, 3.47 mS/cm) had a process mAb yield of 20% with 0% purity ofantibody, while the Trastuzumab final product with adjusted loading conditions (pH 6.0, 4.00 mS/cm)had a process mAb yield of 24% with 100% antibody purity. The antibody purity of the Trastuzumab withnon-adjusted loading conditions (pH 3.6, 3.47 mS/cm) throughout the process was already quite low inthe feed (16%) and it did not increased with the CEX polishing step. Therefore, the frontal chromatog-raphy mechanism of the flow-through CEX favored the operation conditions of pH 6.0 and 4.00 mS/cmconducticity in opposition to the conditions of pH 3.6 and 3.27 mS/cm conductivity. Taking all the resultsinto consideration, the CEX polishing step did not operate completely in flow-through mode with therebeing 66% and 70% of the antibody from the CEX feed present in the wash fraction, and only 27% and29% of the antibody was present in the flow-through of the CEX N-ADC and ADC, respectively (Figure

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3.6). The Trastuzumab present in the CEX N-ADC and ADC wash fractions had a mAb yield of 48% and56%, respectively, with a relative antibody (monomer) purity of 70% and 93%, respectively.

A summary of the overall results from all the analytical techniques performed to the Trastuzumabproduct before and after each polishing step are represented in Tables I.15 and I.16 in Appendix I.3.1.4for the AEX-CEX N-ADC and AEX-CEX ADC trains, respectively.

3.3 Flow-through AEX-CEX (ADC) experiments with Tris-HCl buffer

Due to the CEX polishing steps of the previous experiments (Section 3.2) not having operatedcompletely in flow-through mode, a new set of experiments was conducted with a change in the buffersolution used and in the working pH in order to infer if the antibody would flow through the CEX columnunder new working conditions. A change of the buffer used in the flow-through chromatography wasproposed due to sodium acetate not being considered a stable buffer at pH 6.0. The pH range of sodiumacetate buffer goes from pH 3.6 to pH 5.6 [69].

Sodium acetate and Tris-HCl are buffers recommended by the Eshmuno® CP-FT resin’s userguide [41, 45], in addition to being buffers which have been used in flow-through cation exchange chro-matography studies that favor frontal separation [16, 68, 70]. Furthermore, Ichihara et al. in a previousstudy, in 2018, used 25 mM sodium acetate buffers at pH 5.0 and pH 6.0 and 25 mM Tris-HCl buffer atpH 7.0 (all buffers with 3 mS/cm conductivity) for flow-through anion and cation exchange chromatogra-phy experiments for purification of therapeutical monoclonal antibodies [16]. Loading conditions aroundpH 6.0 to pH 7.0 and conductivity bellow 4 mS/cm have been defined as optimal for mAbs with pIs of7.7 and 8.2 [16].

Three flow-through cation exchange chromatography "test" runs using Tris-HCl buffer were per-formed at pH 6.0, 7.0 and 8.0 in order to infer (without having to run the whole AEX-CEX train), whichwas the optimal pH for the antibody to flow through the CEX column without binding and eluting. Sinceat the time there was no more Trastuzumab starting material solution (antibody solution obtained afterthe ZnCl2 precipitation) left from the experiments mentioned in Section 3.2, a Trastuzumab solution, witha mAb titre of 27 mg/mL (pH 12.4, 4.40 mS/cm) obtained after Protein A affinity chromatography, thatwas stored at -20°C, was thawed and used as starting material for the flow-through CEX polishing stepproposed (method in Section 2.3.2). The loading conditions of the Trastuzumab starting material wereadjusted by buffer exchange to the conditions of the respective flow-through CEX run (method in Section2.3.2.1). Considering that the capture step of this mAb sample was Protein A affinity chromatography,there was no concerns with ZnCl2 interference.

The chromatograms of the flow-through CEX "test" runs using Tris-HCl buffer at pH 6.0, 7.0 and8.0 are represented in Figures I.10, I.11 and I.12 in Appendix I.4.1. An HPLC-Protein A affinity chro-matogram analysis was performed to the samples of the fractions (feed, flow-through, wash and elution)of the flow-through CEX "test" runs at pH 6.0, 7.0 and 8.0, in order to determine the mAb concentrationand yield (Figure I.14 in Appendix I.4.1.1). The HPLC-Protein A affinity chromatogram analysis showedthere was no antibody content in any of the sample fractions, including in the Trastuzumab starting ma-terial (27 mg/mL) that was thawed (Figure I.13 in Appendix I.4.1.1). The antibody must not have survivedthe storage conditions (pH 12.4 and -20°C), resulting in loss of antibody reactivity [52].

New antibody material was obtained from continuous ZnCl2 precipitation, which consisted inTrastuzumab (pI 8.4) with a mAb titre of 0.23 mg/mL (pH 9.8, 7.02 mS/cm) Considering the lack ofresults from the flow-through CEX "test" runs, it was proposed to study the flow-through AEX-CEX trainwith adjusted loading conditions (ADC) using Tris-HCl buffer at pH 7.0 and pH 8.0 due to the smaller

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charge difference between the antibody’s pI (8.4) and the working pH (in comparison with pH 6.0). Theseconditions would, hopefully, work better for the frontal chromatography mechanism of the flow-throughCEX, since Ichihara et al. (2019) had a charge difference between its antibody pI and the working pHof around 1.7, while in this case the charge difference is 1.4 (pH 7.0) and 0.4 (pH 8.0). The methodof the two new trains studied is mentioned in Section 2.3.3. The loading conditions of the Trastuzumabfeed solutions that were loaded onto the AEX-CEX train at pH 7.0 and onto the AEX-CEX train at pH8.0 were adjusted by a two-step buffer exchange using UF/DF to pH 7.0 and 4.00 mS/cm conductivityand to pH 8.0 and 4.00 mS/cm conductivity, respectively, while having the ZnCl2 removed (method inSections 2.4.2 and 2.4.3).

The chromatograms of the flow-through AEX and CEX runs using Tris-HCl buffer with loadingconditions of pH 8.0 and 4.00 mS/cm conductivity are represented in Section 3.3.1. The results fromthe Bradford and Picogreen assay of the AEX-CEX train at pH 8.0 are demonstrated in Section 3.3.1.1.The chromatogram of the flow-through AEX run with loading conditions of pH 7.0 and 4.00 mS/cmconductivity using Tris-HCl buffer is represented in Section 3.3.2.

Unfortunately, due to COVID-19, the flow-through CEX run at pH 7.0 using Tris-HCl buffer aswell as the rest of the respective analytical techniques (Bradford, Picogreen, SEC and HPLC-Protein Aaffinity chromatography) of the AEX-CEX trains at pH 8.0 and pH 7.0 could not be concluded, since theUniversity of Natural Resources and Life Sciences in Vienna, Austria closed its laboratories and I had toreturn to Portugal.

3.3.1 Flow-through AEX-CEX (ADC) train with Tris-HCl buffer at pH 8.0

The chromatograms of the AEX and CEX runs with loading Trastuzumab conditions of pH 8.0and 4.00 mS/cm conductivity using Tris-HCl buffer are illustrated in Figures 3.17 and 3.18, respectively. Azoomed-in chromatogram of the sample loading step of the flow-through AEX run at pH 8.0 is illustratedin Figure I.15 in Appendix I.4.2. Zoomed-in chromatograms of the wash, elution and CIP steps of theflow-through AEX and CEX runs at pH 8.0 are illustrated in Figures I.16 and I.17 in Appendix I.4.2.

It can be observed in the sample loading step of the AEX chromatogram of the AEX-CEX trainwith Tris-HCl at pH 8.0 (Figure I.15 in Appendix I.4.2) that there is an increase in the UV absorbancesat 214 nm and 280 nm around 37 column volume (CV) of mAb loading, which might be related withDNA breakthrough or even antibody breakthrough, as it will be explained in Section 3.3.1.1. Therefore,in a future flow-through AEX chromatography run with Tris-HCl at pH 8.0, a target mAb of 30 CV, forinstances, could be loaded onto the column before any DNA breaks through, instead of the target loadingof 125 CV used in this study.

Th UV absorbance at 254 nm was proposed to be monitored for the following chromatographyruns, considering that it is a wavelength more sensitive to nucleic acids, allowing to estimate the purityof DNA by the ratio of absorbance at 254 nm and 280 nm. For pure double-stranded DNA, this ratio isbetween 1.8 and 2.0 and if it is lower it may indicate the presence of impurities that absorb strongly ator near 280 nm (Section 1.8). However, the monitoring of UV absorbance at 254 nm or 260 nm onlystarted to be implemented from CEX pH 8.0 on forward, therefore only the runs CEX pH 8.0 and AEXpH 7.0 have this monitoring. In Figure 3.18, the UV absorbance at 254 nm is around 60 mAU and UVabsorbance at 280 nm is around 50 mAU, resulting in a ratio of approximately 1.2, which means that inthe CEX flow-through fraction there are species that absorb strongly at or near 280 nm, which could bethe antibody product, or mAb aggregates that were not totally washed or eluted.

In Figure I.17 in Appendix I.4.2, there was a peak in the wash step and in the sanitization of

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Figure 3.17: Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEXADC train with Tris-HCl buffer at pH 8.0. The AEX column was equilibrated with 25 mM Tris-HCl buffer (pH 8.0, 1.87mS/cm, 10 CV) at a flow-rate of 1 mL/min. The AEX column was loaded at the flow rate of 0.2 mL/min with 0.23mg/mL mAb loading at 125 mL (125 CV). The mAb loading conditions were pH 8.0 and 4.00 mS/cm conductivity.The UV was measured at wavelengths 280 nm and 214 nm.

Figure 3.18: Chromatogram of the flow-through CEX run with adjusted loading conditions (ADC) of the AEX-CEXADC train with Tris-HCl buffer at pH 8.0. The CEX column was equilibrated with 25 mM Tris-HCl buffer (pH 8.0, 1.87mS/cm, 10 CV) at a flow-rate of 1 mL/min. The CEX column was loaded at the flow rate of 0.2 mL/min with 0.23mg/mL mAb loading at 100 mL (200 CV). The mAb loading conditions were pH 8.0 and 4.00 mS/cm conductivity.The UV was measured at wavelengths 280 nm, 254 nm and 214 nm. (Note: * The chromatography run had atechnical problem and it stopped around the loading at 146 CV. The ÄKTA system had to be restarted and the mAbloading was resumed until 220 CV.)

the column with NaOH. In both peaks, the ratio between absorbance at 254 nm and 280 nm was at orbellow 1.0, which means there is no pure DNA present, as it should be expected, considering that DNAis a negatively charged impurity that is removed by the flow-through AEX polishing step. A ratio bellow1.8 also means that there are proteins present in these fractions, probably mAb aggregates, if the CEXpolishing step operated correctly in flow-through mode.

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During the flow-through CEX run at pH 8.0 on the ÄKTA pure, there were a few technical prob-lems with the column pressure. The chromatography run stopped around the loading at 146 CV (Figure3.18) due to high pressures in the delta column pressure, pre-column pressure and post-column pres-sure. Usually, pressure generated by the flow through in a column affects the packed bed and columnhardware, therefore, sometimes it is necessary to adjust the pressure limits in the chromatography sys-tem software. The pressure limits were re-adjusted, however the pre-column pressure remained at orabove two times the upper limit recommended of 0.29 MPa.

The CEX resin used in this study was individually packed into the column, and, even thoughthe calculations made to achieve good packing results were determined accurately, and the packed bedcompression was taken into account (as shown in Section 2.2.2.1), after the packing of the column, theresin’s bed height decreased from the calculated value of 2.55 cm to 2.45 cm, which resulted in a PBVof 0.48 mL, instead of 0.50 mL (Table 2.2 in Section 2.2.2.1). This decrease in the bed height might bedue to the top filter compression. The operating pressure in the CEX column being quite high might berelated to this inaccurate packed bed compression. The HETP and As values determined in the column’sperformance test (Table 2.2) after the packing of the column were both in the literature’s recommendedrange, however this test should be performed in regular intervals during the working life of the column,specially when separation performance is seen to deteriorate. A first indicator of bed deterioration dueto excessive use would be, for instances, a non symmetrical peak. Therefore, a performance test shouldbe made to the TriconTM 5 mm x 2.5 cm column packed with the Eshmuno® CP-FT resin in order to verifyif the values of HETP and As are out of the acceptable range, which would affect directly the packedcolumn’s efficiency and it would explain the higher operating pressures in the flow-through CEX run withTris-HCl at pH 8.0.

3.3.1.1 Residual DNA and total protein content

The DNA and total protein content of the Trastuzumab precipitated material (before buffer ex-change) and of the sample fractions (feed, flow-through, wash and elution) of the flow-through AEX runwith adjustment of the loading conditions to pH 8.0 and 4.00 mS/cm using UF/DF (method in Section2.4.2) are represented in Figures 3.19.A and 3.19.B, respectively. Table I.17 in Appendix I.4.2.1 and Ta-ble I.18 in Appendix I.4.2.2 show the DNA and total protein concentrations quantified by the Picogreenand Bradford assays, respectively.

The two-step buffer exchange using UF/DF to adjust the loading conditions (pH 8.0, 4.00 mS/cm)and remove the zinc chloride used a Biomax® membrane with a MWCO of 5 kDa membrane that wasresponsible for the reduction of DNA and total protein in 2.6-fold and 3.2-fold, respectively, as it can beobserved in Figure 3.19.

The DNA concentration in the Trastuzumab of the AEX feed was 488 ± 24 ng/mL and in theTrastuzumab product at the end of the AEX run was 54 ± 3 ng/mL. The flow-through AEX polishing stepreduced these negatively charged molecules content in 95% (Table I.17 in Appendix I.4.2.1).

The total protein concentration in the Trastuzumab of the AEX feed was 124 ± 16 µg/mL and inthe Trastuzumab product at the end of the AEX run was 72 ± 9 µg/mL. The flow-through AEX polishingstep reduced the total protein content in 69% (Table I.18 in Appendix I.4.2.2). This reduction of totalprotein content in the flow-through AEX step might be due to the net charge difference between theworking pH of 8.0 and the antibody’s pI (8.4) being too small, since the working pH should be at leastone unit bellow the mAb’s pI in order to guarantee the antibody would have a positive net charge [39].Therefore, some of the antibody product bounded to the anion exchange resin due to the antibody’s

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Figure 3.19: Analysis of the DNA (Figure A) and total protein (Figure B) content of the Trastuzumab precipitated ma-terial (before buffer exchange) and of the Trastuzumab feed (after buffer exchange) and of the fractions collected inthe flow-through, wash and elution from the flow-through AEX run with Tris-HCl buffer at adjusted loading conditionsof pH 8.0 and 4.00 mS/cm conductivity.

surface net charge having less positive charges because of the higher working pH. There could bethe possibility of both the DNA and mAb monomer having been competing for the binding to the anionexchange resin’s ligands. Meanwhile, there was DNA in the flow-through that was not removed by theAEX step, which might be the breakthrough observed in the chromatogram (Figure 3.18). However, thisbreakthrough could also be antibody that was passing through the column without binding to the anionexchanger. Further analysis by HPLC-Protein A affinity chromatography would be also able to confirmif this hypothesis is correct. The monitoring of absorbance at 254 nm could have helped in verifying ifthere was pure dsDNA in the peak of wash and elution fractions.

3.3.2 Flow-through AEX (ADC) run with Tris-HCl buffer at pH 7.0

The chromatogram of the flow-through AEX run with adjusted Trastuzumab loading conditionsat pH 7.0 (and 4.00 mS/cm conductivity) using Tris-HCl buffer is illustrated in Figure 3.20. A zoomed-inchromatogram of the sample loading step of the flow-through AEX ADC run at pH 7.0 with Tris-HCl bufferis illustrated in Figure I.18 in Appendix I.4.3. A zoomed-in chromatogram of the wash, elution and CIPsteps of the flow-through AEX ADC run at pH 7.0 is illustrated in Figures I.19 in Appendix I.4.3.

It can be observed in the sample loading step of AEX chromatogram of the AEX-CEX train withTris-HCl at pH 7.0 (Figure I.18 in Appendix I.4.3) that there is an increase in the UV absorbance signalsat around 60 CV of mAb loading, which might be related with DNA breakthrough. Therefore, in a futureflow-through AEX chromatography run with Tris-HCl at pH 7.0, a target mAb of 50 CV, for instances,could be loaded onto the column before any DNA breaks through, instead of the target loading of 125CV used in this study.

The purity of DNA can be estimated by the ratio of absorbance at 260 nm and 280 nm. In themAb loading step (Figure I.18 in Appendix I.4.3), it can be observed that the UV absorbances at 260nm and 280 nm are very similar, therefore there in no pure double-stranded DNA present in the flow-through fraction, there is however species that absorb strongly near 280 nm, which is most likely the

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Figure 3.20: Chromatogram of the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEXADC train with Tris-HCl buffer at pH 7.0. The AEX column was equilibrated with 25 mM Tris-HCl buffer (pH 7.0, 2.29mS/cm, 10 CV) at a flow-rate of 1 mL/min. The AEX column was loaded at the flow rate of 0.2 mL/min with 0.23mg/mL mAb loading at 125 mL (125 CV). The mAb loading conditions were pH 7.0 and 4.00 mS/cm conductivity.The UV was measured at wavelengths 280 nm, 260 nm and 214 nm.

target antibody protein.

In Figure I.19 in Appendix I.4.3, the wash peak has a UV absorbance at 260 nm around 2400mAU and a UV absorbance at 280 nm around 1350 mAU, which means that the ratio between ab-sorbance at 260 nm and 280 nm is around 1.8, meaning that the wash peak is mostly pure double-stranded DNA. The ratio between absorbance at 260 nm and 280 nm in the elution peak is approximately2.0, therefore in the wash and elution fractions pure double-stranded DNA was removed. The peak inthe CIP fraction has a ratio between absorbance at 260 nm and 280 nm of 1.0, which means there werealso proteins (i.e. acidic HCPs) that absorb strongly or near 280 nm still bounded in the column that weresanitized with NaOH. Nevertheless, further technical analytics would have to be performed in order toquantify DNA, HCP and mAb content to confirm this interpretation of the chromatogram.

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4 Conclusions and Future Perspectives

This study allowed to demonstrate that the success of a flow-through polishing process of re-combinant antibodies depends on the working conditions (pH and conductivity) chosen. The principle ofthe flow-through polishing reported here consisted in the following steps: AEX, for the removal of neg-atively charged impurities, including DNA and acidic HCPs; followed by CEX, for the removal of largerbasic species including product related aggregates and basic HCPs. The polishing experiments of thisstudy were performed after continuous ZnCl2 precipitation. It was found out that the zinc chloride was in-terfering with the DNA quantification assays, due to Zn2+ causing inhibitory effects on DNA amplification.Furthermore, it could be also possible that the Zn2+ was forming secondary interactions with the DNAmolecules, which consequently could be forming complexes with histone proteins, and mAb aggregates,affecting the clearance of DNA by the AEX step, due to the non-binding of these impurities to the anionexchanger resin in those cases.

The post-continuous ZnCl2 precipitation purification of Trastuzumab reported in this thesis studywas achieved using two different flow-through ion exchange chromatography trains: AEX-CEX with non-adjusted conditions (N-ADC) (pH 3.6, 3.47 mS/cm) and AEX-CEX with adjusted loading conditions(ADC) (pH 6.0, 4.00 mS/cm). The flow-through chromatography method followed was based on thestudy reported by Ichihara et al. (2019) with the same adjusted loading conditions and using sodiumacetate buffer. The optimal adjustment of the mAb loading conditions found to work the best was toperform a prior two-step buffer exchange by UF/DF in order to remove the zinc chloride and to adjust themAb loading conditions before loading onto the AEX column. In the DNA assay analytics of the AEX-CEX N-ADC train, a buffer exchange would have to be performed to decrease the ZnCl2 concentrationto bellow 0.1 mM, in order to allow DNA quantification by the Picogreen assay.

The DNA clearance in the AEX-CEX N-ADC and AEX-CEX ADC trains was inconclusive. Boththe Trastuzumab starting product (AEX feed) and final product (CEX flow-through) in both trains wereunder the LOD of the DNA assay, therefore quantification of an exact DNA value was not able to bedetermined. Considering the DNA concentration at LOD, it is only known that the concentration of DNAin the final Trastuzumab product is bellow 5000 ppb and bellow 1100 ppb for the AEX-CEX N-ADC andADC trains, respectively. Therefore, it is not possible to state if the DNA content is bellow the typical mAbbulk drug specification limit of 10 ppb.

The HCP content method estimation performed was quite ambiguous and no HCP content wasestimated for the Trastuzumab starting product, although there was HCP estimated for the Trastuzumabfinal product, in both trains. In the final Trastuzumab product, the mAb concentration is quite lower thanin the starting product, therefore the Bradford assay is able to quantify more amount of total proteinusing the BSA standards which, usually leads to an underestimation of the total protein content whena higher immunoglobulin G (IgG) content is present. In both trains, the levels of HCP estimated in theTrastuzumab final product were way above the limit of 100 ppm to guarantee a medicine’s approval. TheHCP clearance is usually more challenging than, for instances, DNA removal, due to the heterogenityin both size and charge of these impurities. HCPs can be associated with the mAb product throughstrong and attractive interactions, resulting in the HCPs entering the product fraction during chromatog-raphy purification. Nevertheless, the ELISA assay could, perhaps, be a more suitable method for HCPquantification, for future experiments.

The adjustment of the mAb loading conditions using a two-step buffer exchange with UF/DFperformed a complementary impurity reduction. The Trastuzumab starting product of the AEX-CEX ADCtrain had an increase of 83% in antibody monomer purity, while the HMWI content decreased 9%,

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removing all HMWI present, and the LMWI content decreased 74%, compared with the Trastuzumabstarting product of the AEX-CEX N-ADC train.

The flow-through AEX polishing had process mAb yields of 82% and 90% for the non-adjustedand adjusted loading conditions, respectively, which means that the working pH used guaranteed thatthe antibody product was positively charged and therefore did not bound to the anion exchanger. Theflow-through CEX polishing step did not, however, operated completely in flow-through mode, consider-ing that the antibody was retained in the interstitial space of the CEX matrix and was, mostly, presentin the wash fractions in both trains, due to the mAb aggregates binding more strongly to the cationexchanger, resulting in the mAb monomer having more difficulty in binding to the resin’s ligands, consid-ering the aggregates’ larger size. The final process mAb yield was 20% and 24% for the non-adjustedand adjusted loading conditions. The differences in the pH of the loading conditions in the AEX-CEXN-ADC and AEX-CEX ADC will affect more the CEX polishing step, since its optimal working conditionsare influenced by the pI of the mAb. Since the Trastuzumab has a pI of 8.4, the mAb loading conditionsof pH 3.6 in the N-ADC train will probably lead to the antibody and mAb aggregates having more affinitywith the cation exchanger resin, since the charge difference (4.8) is higher than in the ADC train, inwhich the loading conditions were of pH 6.0, which meant the net charge difference (2.4) was lower.There was, indeed, more antibody in the flow-through fraction of the CEX ADC, however a differenceof 2% in comparison with the CEX N-ADC. It is important to note, that the working pH of the sampleloading in the N-ADC train was not the same pH of the equilibration, wash and elution buffers, and itshould have been. The starting conditions (pH and ionic strength) should be the same as the sampleloading conditions to guarantee binding of all charged target impurities. However, this did not happen inthe AEX-CEX N-ADC train, while in the AEX-CEX ADC train, the ionic strength of the starting buffer wasnot the same as the one in the sample loading. In addition to that, the Trastuzumab starting product ofall the experiments in this thesis study had a low titre and perhaps, the flow-through mode in the CEXworks better when there is overloading of the resin’s capacity, which leads to a faster mAb monomerbreakthrough [11,16].

It can be concluded that the adjustment of the mAb loading conditions to pH 6.0 and 4.00 mS/cmconductivity gave better results in terms of mAb yield, mAb purity and HMWI and LMWI removal, with thetwo-step buffer exchange using UF/DF being the step most responsible for these results. Additionally,since the flow-through CEX ADC polishing step did not improve the yield and purity of the Trastuzumabproduct and, considering that the monomer purity in the AEX ADC flow-through is already at levels thatguarantee a medicine’s approval (> 95%), further polishing of the antibody product with the flow-throughCEX was unnecessary.

A change of the buffer as well as the working pH of the flow-through chromatography experi-ments was proposed. However, due to COVID-19, the flow-through experiments of the AEX-CEX trainswith loading conditions adjusted to pH 7.0 and pH 8.0 (and 4.00 mS/cm conductivity) using Tris-HClbuffer were not able to be concluded. Nevertheless, from the gathered results it was possible to observethat at mAb loading conditions adjusted to pH 8.0, some of the antibody must have bounded to the anionexchange resin, due to the working pH not being at least one unit bellow the Trastuzumab’s pI of 8.4,which resulted in the antibody’s surface net charge having less positive charges due to this higher work-ing pH. It is possible that at mAb loading conditions adjusted to pH 7.0, the flow-through CEX polishingstep would work better, since the net charge difference between the working pH and the mAb’s pI ismore similar to the one reported in the Ichihara et al. (2019) study. A previous study by Ichihara et al.(2018) reported several flow-through polishing experiments with two mAbs (with pIs of 7.7 and 8.2) usingworking conditions with pH ranging from pH 5.0 to pH 7.0 and conductivity ranging from 3.00 mS/cmto 7.00 mS/cm, using sodium acetate and Tris-HCl buffers. A possible future experiment would be the

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AEX-CEX train with adjusted loading conditions to pH 6.0 and conductivity 4.00 mS/cm using Tris-HClbuffer to compare how the different buffer would directly affect the results in the flow-through CEX pol-ishing step and overall monomer yield and purity. In the Eshmuno® CP-FT resin’s guide, it is reportedthat this cation exchange resin is specifically developed for the flow-through removal of mAb aggregatesunder strong binding conditions that favor frontal chromatography with pH ranging from pH 4.0-5.5 andconductivity ranging from 3.00-7.00 mS/cm [45]. Therefore, future studies with working conditions be-tween those ranges to verify the flow-through CEX polishing step performance would be a hypothesis totake into consideration.

The loading conditions of the mAb solution (i.e. pH and conductivity) as well as the mass mAbloading will have a direct effect on mAb recovery, monomer purity and the reduction of HCP and DNA,and consequently, in the optimization of the flow-through chromatography steps. Differences in thebreakthrough concentrations of the mAb monomer and impurities is also an important factor to takeinto account, in order to know how much mAb solution can be loaded before DNA or even HCP breaksthrough, while guaranteeing maximum antibody monomer breakthrough. Therefore, depending on themAb being purified, an optimal operating window regarding the loading conditions should be studied inorder to maximize the performance of a complete flow-through process.

At first, this thesis included the step of activated carbon (AC) prior to the AEX step, since itpromotes a better removal of LMWI, such as HCP and DNA, according to several studies [11, 16], thanonly the flow-through AEX and CEX polishing train (Section 1.6.2). Considering that lower molecularweight aggregates were the impurities most present in the final product of this study, the introduction ofthe AC polishing step would contribute, this way, to a better clearance of LMWI. Soluble impurities of DNAand HCP that may precipitate upon pH and conductivity adjustment can be subsequently captured by theactivated carbon, therefore the phenomena of precipitation of impurities might be helpful to flow-throughpolishing in this case. Perhaps, it is possible that the introduction of the AC step, prior to the AEX, couldremove the zinc chloride from the continuous ZnCl2 precipitation capture step as well. If this hypothesiswas possible, then the two-step buffer exchange using UF/DF would not be needed or, perhaps, onlya buffer exchange step would be necessary for the adjustment of the loading conditions, which wouldresult in a reduction of buffer consumption. Nevertheless, further studies had to be performed about thishypothesis.

This thesis was based on a polishing study in continuous mode [11], however it is important tonote that the flow-through purification performed in this thesis was not done in fully connected AEX andCEX columns, since the goal was to recreate the flow-through chromatography experiments and verifythe separation of impurities and purification of the antibody using a flow-through operation. In this thesisexperiences, there were hold up times between the AEX and CEX steps for the analytical techniquesand for the adjustment of the mAb loading conditions prior to the AEX. There were off-line adjustmentsof the loading conditions of the AEX feed, however the CEX feed was not adjusted, therefore there couldbe some slight deviations of the loading conditions previously adjusted. An on-line titration system couldhelp implement a more continuous process to the one performed in this thesis, by being able to controland regulate the pH in the chromatography runs [18]. Nevertheless, it is known that a multi-column setup, such as the approach of the connected flow-through chromatography of the article published byIchihara et al. (2019), is possible for further studies, which includes some advantages to continuousmanufacturing such as the reduction of buffer consumption and shorter processing times, since, forinstances, both polishing steps, AEX and CEX, would share the same equilibration, wash and elutionsteps.

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I Appendix

I.1 Appendix I.1 - Materials and Methods of the Initial experiments

I.1.1 qPCR

Host cell DNA was quantified using the resDNASEQ® Quantitative CHO DNA kit (Thermo FisherScientific). Sample preparation and analysis were carried out according with the manufacturer’s instruc-tions mentioned in the resDNASEQ® Quantitive CHO DNA kit user guide [47]. Subsequent quantitationof residual DNA was carried out on a Bio-Rad MiniOpticon real-time qPCR system.

I.1.1.1 qPCR standard curve spiked with zinc chloride

A standard curve prepared from dilutions with constant DNA concentration of 30 pg CHO DNA/mLspiked with zinc chloride concentrations varying from 0.1 mM to 12 mM was created. Zinc chloride so-lutions of 0.1 mM, 2 mM, 4 mM, 6 mM, 10 mM and 12 mM were prepared. In order to follow the sameconditions of the serial dilutions of the standard curve of the method [47], the dilutions of the zinc chlo-ride standard curve were prepared from a 1:10 dilution of the serial dilution 4 (SD4) (3 pg CHO DNA/10µL dilution) of the method’s standard curve [47]. The dilutions prepared for the zinc chloride standardcurve are represented in Table I.1. The standard curve tubes SC1, SC2, SC3, SC4, SC5 and SC6 wereprepared by adding 10 µL of the respective dilution (D1, D2, D3, D4, D5, D6) and 20 µL of PCR mix.The rest of the method was the same described in Section I.1.1.

Table I.1: Dilutions for the zinc chloride standard curve.

Tube label Dilution

D1 450 µL of 0.1 mM ZnCl2 + 50 µL SD4

D2 450 µL of 2 mM ZnCl2 + 50 µL SD4





I.1.2 Ultrafiltration/Diafiltration (UF/DF) to adjust the the loading conditions to pH 6.0 and 1.98mS/cm conductivity (sodium acetate buffer)

The ultrafiltration cassette used in this study was a Biomax® Pellicon® 2 "mini" filter membranewith a molecular weight cut-off of 8 kDa from EDM Millipore Corporation (USA). The membrane wascomposed of polyethersulfone (PES) and the filtration area of the Biomax® 8 kDa was 0.10 m2. Themembrane was placed with the two die-cut silicon gaskets, in the holder which was tightened with aQuatrolit 2179-60 torque wrench from Elora (10-60 N.m; 9-45 Lb.ft) by setting the torque wrench settingsto 20.3-22.6 N.m prior to use. The UF/DF run was performed on an ÄKTA flux system (GE Healthcare).

The feed tank was filled with 250 mL of mAb supernatant solution (titre 0.34 mg/mL, pH 4.0, con-ductivity 2.15 mS/cm) and the retentate was continuously stirred (30-60 rpm) and recirculated throughthe Biomax® 8 kDa membrane using a peristaltic pump at a maximum transfer flow rate of 50 mL/min.

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The run was operated at a maximum operating pressure (feed to permeate) of 2.0 bar. The mAb su-pernatant solution was buffer exchanged for 3 volumes with 25 mM sodium acetate buffer pH 6.0 andconductivity 1.98 mS/cm (starting buffer) to guarantee the zinc chloride removal at a constant volumeby continuously feeding diafiltration buffer into the retentate vessel. The system and membrane werewashed with 0.1 M NaOH, by recirculating the solution for 30 minutes. The system was stored in 20%ethanol and the membrane was stored in 0.1M NaOH.

I.1.3 Flow-through chromatography method

The AEX and CEX flow-through chromatography methods of the AEX-CEX trains of the initialexperiments are presented in Tables I.2 and I.3, respectively. Three flow-through AEX-CEX trains wereperformed: AEX-CEX train "1" with loading conditions adjusted to pH 6.0 and 17 mS/cm conductivity, byadding 4 M TrisBase pH 9 solution; AEX-CEX train "2" with loading conditions adjusted to pH 6.0 and1.98 mS/cm conductivity, by buffer exchange using UF/DF (Section I.1.2), and AEX-CEX "3" train withno adjustment of the loading conditions (pH 4.0 and 2.15 mS/cm conductivity).

Table I.2: AEX flow-through method for the AEX-CEX train "1", "2" and "3" of the initial experiments with aTrastuzumab titre of 0.34 mg/mL. The mAb loading conditions for AEX "1" were adjusted to pH 6.0 and 17. mS/cmconductivity with 4 M TrisBase pH 9. The mAb loading conditions for the train AEX "2" were adjusted to pH 6.0 and1.98 mS/cm conductivity by buffer exchange using UF/DF. The mAb loading conditions for the train AEX "3" werepH 4.0 and conductivity 2.15 mS/cm. The method flow-rate was 1 mL/min. The UV was measured at wavelengths280 nm and 214 nm. The AEX column was loaded at the flow rate of 0.2 mL/min with a target of mAb loading at 133mL (133 CV).






CIP 1 M NaOH 4



CIP 20% ethanol 2

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Table I.3: CEX flow-through method for the AEX-CEX train "1", "2" and "3" of the initial experiments with aTrastuzumab titre of 0.34 mg/mL. The mAb loading conditions for CEX "1" were pH 6.0 and 17 mS/cm conduc-tivity. The mAb loading conditions for the train CEX "2" were pH 6.0 and 1.98 mS/cm conductivity. The mAb loadingconditions for the train CEX "3" were pH 4.0 and conductivity 2.15 mS/cm. The method flow-rate was 1 mL/min. TheUV was measured at wavelengths 280 nm and 214 nm. The CEX column was loaded at the flow rate of 0.2 mL/minwith a target of mAb loading at 80 mL (160 CV).






CIP 0.5 M NaOH 2



CIP 20% ethanol 2

I.2 Appendix I.2 - Results and Discussion of the Initial experiments

I.2.1 Analysis of the precipitated trastuzumab

The starting mAb product for the polishing experiments of this study was obtained after contin-uous precipitation with 12 mM of ZnCl2. The starting material consisted in 2.8 L of Trastuzumab (pI 8.4)with a concentration of 0.34 mg/mL, which was measured by HPLC-Protein A affinity chromatography(Figure I.1).

Figure I.1: UV 280 nm HPLC-Protein A affinity chromatogram of the Trastuzumab supernatant solution precipitatedwith ZnCl2 (mAb feed solution of the AEX). The peak of the retained mAb is observed between 0.84 and 0.88 min.This mAb sample was analyzed the same day (25th Nov 2019) of the ZnCl2 precipitation.

A SEC analysis was performed (Figure I.2) to the Trastuzumab supernatant precipitated withzinc chloride which showed a relative monomer purity around 50%, with less than 3% of HMWI andaround 48% of LMWI.

Additionally, the DNA content was also evaluated. At first, the Picogreen assay was not quanti-fying any DNA content of the Trastuzumab sample precipitated. At the time, it was thought that perhapsthe Picogreen detection limit (3.91 ng/mL) was not low enough, therefore, a qPCR was performed for thequantification of dsDNA, which has a detection limit of 0.003 ng/mL. However, the qPCR assay (Sec-tion I.1.1 in Appendix I.1) also did not show any results of DNA quantified in the sample after ZnCl2

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Figure I.2: UV 280 nm size exclusion chromatogram (SEC) of the Trastuzumab supernatant solution precipitatedwith ZnCl2, which is the mAb feed solution of the AEX. This mAb sample was analyzed the same day (25th Nov2019) of the ZnCl2 precipitation.

precipitation. Later, it was found out that the presence of ZnCl2 (12 mM) from the continuous precip-itation might have been interfering with the DNA assays. Metal ions, such as Zn2+, can interfere withthe qPCR assay by causing inhibitory effects on DNA amplification. Taq polimerase has been reportedof having no activity in the presence of zinc chloride at concentrations above 1 mM [62]. On the otherhand, the Quant-iTTM PicoGreen® assay remains linear in the presence of several compounds that com-monly contaminate nucleic acid preparations, although the signal intensity may be affected. In terms ofZnCl2, the maximum acceptable concentration for the Picogreen assay is 5 mM, which leads to an 8%decrease in the signal change after that concentration [48]. Furthermore, other compounds might affectthe Picogreen method signal, such as sodium acetate that has a maximum acceptable concentration of30 mM which results in a 3% increase in the signal [48].

To verify if the zinc chloride was interfering with the qPCR assay, a standard DNA curve preparedfrom dilutions with a constant DNA concentration of 30 pg CHO DNA/mL spiked with zinc chloride inconcentrations ranging from 0.1 mM to 12 mM ZnCl2 was created (see method in Section I.1.1.1 inAppendix I.1). The Trastuzumab sample obtained after ZnCl2 precipitation was buffer exchanged sixtimes (method in Section 2.5.4.1) prior to the qPCR DNA quantification, in order to decrease the zincchloride to lower than 0.1 mM ZnCl2. An undiluted and buffer exchanged Trastuzumab samples afterZnCl2 precipitation were quantified for DNA content using the qPCR assay with the zinc chloride standardcurve created and the "normal" standard DNA curve of the method [47]. Table I.4 shows that in the zincchloride standard curve, only the standard curve (SC) dilution 1 presented a quantified value of 0.01 pgDNA/mL. No DNA was quantified for the undiluted trastuzumab sample after precipitation, whereas, aconcentration of 1.27 pg DNA/mL (3.75 pg/mg IgG) was quantified for the buffer exchanged trastuzumabsample after precipitation, which is under the limit of 10 ppb requested for a medicine’s safety approval.Therefore, the zinc chloride must indeed be interfering with the DNA assay at concentrations above0.1 mM ZnCl2, since every zinc chloride standard dilution had a concentration of 30 pg DNA/mL. Inaddition to that, there was only DNA quantified in the Trastuzumab sample which was previously bufferexchanged, a step which guaranteed a decrease of ZnCl2 to a concentration below below 0.1 mM,allowing DNA quantification.

Due to the Picogreen assay being a more affordable method for DNA quantification than qPCR,the rest of the DNA concentration analysis of this thesis study were performed using the Picogreenassay, with a prior buffer exchange of the samples to remove/decrease the zinc chloride concentrationoriginated from the precipitation step. Additionally, a Bradford assay was performed to quantify the totalprotein content in the samples. The DNA, total protein and HCP content are represented in Table I.5.

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Table I.4: qPCR DNA quantification using a standard curve spiked with concentrations of ZnCl2 ranging from 0.1mM to 12 mM and a qPCR "normal" DNA standard curve as standard control curve.

Method standards pg DNA/mLZnCl2 standards

with 30 pg DNA/mLpg DNA/mL

SC1 300000 SC1 with 0.1 mM ZnCl2 0.01

SC2 30000 SC2 with 2 mM ZnCl2 0.00





To guarantee a medicine’s safety approval, the residual DNA and HCP content must be lower than 10ppb (10 ng/mg IgG) and 100 ppm (100 ng/mg IgG), respectively. After continuous ZnCl2 precipitation,the DNA (quantified by the Picogreen assay) and HCP content are still way above those limits, thereforeit is necessary to continue the Trastuzumab purification.

Table I.5: DNA (ng/mL) and total protein (µg/mL) content of the trastuzumab supernatant after ZnCl2 precipitationusing the Picogreen and qPCR assay, and the Bradford method, respectively. An estimation of the HCP content(Total protein = mAb titer + HCP) of the starting product was determined considering the mAb titre of 0.34 mg/mL.

DNA

(ng/mL)

DNA in ppb

(pg DNA/mg IgG)

Total protein

(µg/mL)

HCP

(µg/mL)

HCP in ppm

(ng HCP/mg IgG)

Trastuzumab

(starting product)

Picogreen qPCR Picogreen qPCR387 ± 77 47 ± 77 1.4×105

30 ± 4 1.27×10-3 8.8×104 3.75

When comparing the DNA quantified in the Trastuzumab starting product using both DNA assays(Table I.5), it is possible to observe that the values were not at all similar, considering that the Picogreenis determining a DNA concentration more than 104 times the concentration determined by the qPCR. TheqPCR assay usually offers a comparable or even higher sensitivity for DNA concentration measurementsthan the Picogreen assay [71] and it also allows the detection of dsDNA in a final concentration as lowas 0.003 ng/mL, meanwhile, the Picogreen method’s lowest detection of dsDNA is 3.91 ng/mL. However,as mentioned before, it has been reported that Taq polimerase has no activity above 1 mM ZnCl2, whileZnCl2 interferes with the Picogreen assay above concentrations of 5 mM, and even though the zincchloride concentration was reduced to bellow 0.1 mM with the buffer exchange, it might be possible thatthe qPCR might be more sensitive to the zinc chloride interference than the Picogreen assay, resultingin the qPCR underestimating the DNA concentration present in the sample. Additionally, it has beenreported [71] that the Picogreen assay can sometimes overestimate the measured concentrations ofsamples by almost 10 times compared to other DNA quantification methods, such as the qPCR. Suchdifference in the measured concentration can be caused by the use of the λ DNA standard. The λ DNAwas used to create the calibration curve because it was supplied with the Quant-iTTM PicoGreen® kitand recommended by the manufacturer. Futhermore, as mentioned previously, the maximum acceptableconcentration of sodium acetate for the Picogreen assay is 30 mM and above that results in a 3%increase in the signal intensity. The use of 100 mM sodium acetate pH 5.0 buffer for the resolubilizationafter precipitation might have caused the increase of the fluorescence signal intensity which may haveled to the higher values of DNA quantified by the Picogreen compared with the qPCR assay.

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I.2.2 Flow-through chromatography experiments

In this thesis study, the antibody will pass through the column, meanwhile impurities will bind.The antibody will be present in the columns’ flow-through effluent, and no other impurity is expectedto be there (such as residual host cell DNA and HCP) until the dynamic binding capacity (DBC) of thecolumn is reached and DNA or HCP begins flowing through the respective column without binding. Thedefinition of DBC, in this case, would be the amount of target impurity that binds to the resin under givenflow conditions before a significant breakthrough of unbound impurity occurs. It is important to find wherethe breakthrough of the column for each of the impurities occurs (DNA and HCP), in order to know howmuch can be loaded of antibody solution onto the column. The flow-through effluent from the columnswill be collected in fractions, that can be pooled, and assayed for DNA and HCP breakthrough, since itis expected that this effluent only contains antibody while the other impurities bind to the columns. Thecolumns should be overloaded to ensure that the breakthrough of impurities occurs, however most timesthe breakthrough is difficult to be observed since there is no real time data and the analytical methodsto determine where it occurred are slow and off-line [72].

The idea was to run an AEX to determine the breakthrough of DNA in order to know howmuch to load of Trastuzumab solution. DNA is negatively charged, therefore it is supposed to bind tothe AEX column. The breakthrough of HCP is more difficult to determine using the CEX, consideringboth the monomer and the non-product proteins should be charged positively (working pH is belowthe Tratuzumab’s pI (8.4)), which means that both bind to the CEX column. The cation exchange resin(Eshmuno® CP-FT) used in this study is specifically developed for the flow-through removal of mAbaggregates under strong binding conditions that favor frontal chromatography, where the least retainedmAb monomer (antibody) is obtained from a fast breakthrough followed by the breakthrough of the moreretained species, such as HCP. Therefore, the monomer breakthrough happens in first place, followedby the HCP breakthrough and mAb aggregates. Furthermore, in order to estimate the HCP content, twooff-line assays (Bradford, which quantifies the total protein, and HPLC-Protein A affinity chromatography,which determines the mAb titre) had to be performed by analyzing the flow-through effluent fractions.Additionally, the Bradford assay does not distinguish between HCP or mAb monomer.

Considering the 2.8 L of Trastuzumab starting product with a low titre of 0.34 mg/mL, and theconcentration of DNA content determined (Table I.5), it was difficult to load a great amount of mAbsolution in the AEX column in order to guarantee a breakthrough of DNA due to the high dynamicbinding capacity of the HiTrap® Capto QTM column (1 mL) [44]. Furthermore, the mAb loading flow-rateof 0.2 mL/min, combined with a larger loading volume, would be too much time consuming without, mostprobably, any observation of breakthrough. Therefore, the volume of Trastuzumab solution to be loadedonto the AEX and CEX columns was decided to be 133 CV and 160 CV, respectively, according to thesimilar column volume loaded reported in the flow-through chromatography method (AEX-CEX) used byIchihara et al. (2019).

The 2.8 L of Trastuzumab solution (0.34 mg/mL, pH 4.0, conductivity 2.15 mS/cm) obtainedafter ZnCl2 precipitation had been stored at -20 °C, after the capture step. For the following flow-throughchromatography experiments, the Trastuzumab solution was thawed, adjusted depending on the loadingconditions of the experiment train, and filtered before being loaded onto the AEX column.

I.2.2.1 Adjustment of the loading conditions

In order to follow the conditions of the flow-through chromatography experiments of the publishedarticle by Ichihara et al. (2019) as close as possible, the loading conditions had to be adjusted to pH 6.0

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and 4.0 mS/cm conductivity. Therefore, for the first AEX-CEX chromatography (train "1"), the antibodysolution was thawed and the pH was adjusted until pH 6.0 by adding around 20 mL of 4 M TrisBasesolution at pH 9.0. However, after having adjusted the pH, the conductivity had risen until 17 mS/cm,which was already a different loading condition compared with the article, which had a conductivity of4.00 mS/cm. This pH adjustment was not ideal due to the zinc chloride still being present in the antibodysolution and also because of the increase of conductivity in 4.3-fold. An increase in conductivity hasbeen shown to not only decrease the HCP clearance due to the reduction of the binding capacity ofHCPs on the cation exchanger, but also to have a negative impact on monomer purity [16].

A 15 mL Trastuzumab sample (0.34 mg/mL, pH 4.0, conductivity 2.15 mS/cm) was thawed inorder to see if the pH could be adjusted with 100 mM NaOH without a big increase in the conductivity.However, 16 mL were needed to be added to increase the pH from 4.0 to 6.0, while the conductivityincreased from 2.15 mS/cm to 7.00 mS/cm, which means that the antibody solution was diluted 1:2 toobtain loading conditions similar to those wanted. The Trastuzumab solution started to become a bitcloudy after the adjustment with 100 mM NaOH and the antibody started to precipitate, perhaps due tothe presence of zinc chloride.

Due to the previous adjustments of the Trastuzumab’s loading conditions not being the idealones, another AEX-CEX (train "2") run was performed to improve the adjustment of the loading con-ditions of the AEX-CEX train "1". The pH of the Trastuzumab feed solution of the AEX-CEX train "2"was adjusted to pH 6.0 with 4 M TrisBase pH 9.0 and then the antibody solution was buffer exchangedwith three volumes of equilibration buffer (25 mM sodium acetate, pH 6.0 and 1.98 mS/cm conductivity)to remove the zinc chloride and adjust the conductivity, using UF/DF (method in Section I.1.2 in Ap-pendix I.1). The Trastuzumab loading conditions of the AEX-CEX train "2" were pH 6.0 and 1.98 mS/cmconductivity.

A third AEX-CEX (train "3") was performed with no adjustment of the loading conditions, (mean-ing that the loading conditions were pH 4.0 and 2.15 mS/cm conductivity), in order to compare the threeapproaches (trains "1", "2" and "3"). The AEX and CEX flow-through chromatography methods of theAEX-CEX trains "1", "2" and "3" are mentioned in Section I.1.3 in Appendix I.1.

I.2.3 Residual DNA content

The DNA content of the samples collected from the AEX-CEX trains "1" (mAb loading conditionsadjusted to pH 6.0 and 17 mS/cm conductivity and ZnCl2 is still present) , "2" (mAb loading conditionsadjusted to pH 6.0 and 1.98 mS/cm conductivity and ZnCl2 has been removed) and "3" (mAb loadingconditions not adjusted - pH 4.0 and conductivity 2.15 mS - and ZnCl2 is still present) was determinedusing the Picogreen assay (Section 2.5.4.2). A buffer exchange (Section 2.5.4.1) had to be performedprior to the quantification of DNA content, in order to decrease the ZnCl2 concentration of the samplescollected in the AEX-CEX train "1" and "3", since the zinc chloride interferes with the DNA assay, asit was mentioned previously. The DNA content of all the fraction steps (feed, flow-through, wash andelution) of the AEX-CEX runs of the three trains is represented in Figure I.3. Tables I.6, I.7 and I.8show the DNA concentration (ng/mL) of all the fraction steps of the AEX-CEX runs, as well as the exactrelative standard associated to each concentration and the exact DNA mass value (ng) for the trains"1", "2" and "3", respectively. The acceptance critera for the assays’ results, in terms of precision andaccuracy, was taken into account and only concentration values within ± 20% RSD for precision and± 20% for bias were considered acceptable [65]. If an assay was not able to quantify a DNA or totalprotein concentration of a sample, its value was assumed to be bellow the minimum limit of detection ofthe respective assay that was within the acceptable criteria for precision and accuracy.

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Figure I.3: Analysis of the DNA content of the samples collected from the AEX-CEX trains "1" (TrastuzumAb loadingconditions adjusted to pH 6.0 and 17 mS/cm conductivity and ZnCl2 is still present) , "2" (Trastuzumab loadingconditions adjusted to pH 6.0 and 1.98 mS/cm conductivity and ZnCl2 has been removed) and "3" (Trastuzumabloading conditions not adjusted - pH 4.0 and conductiviy 2.15 mS - and ZnCl2 is still present), using the Picogreenassay. (The red asterisk (*) means that the DNA content in the collected fractions was bellow the limit of detection ofthe DNA assay (Picogreen assay), therefore the DNA content of these fractions cannot be exactly determined and itcan be any value bellow the maximum range of the respective column graph plotted. The darker coloured columnscorrespond to the feed and flow-through fractions and the lighter coloured columns correspond to the wash andelution fractions.)

The starting product (AEX feed) was the same for all three AEX-CEX train experiments witha DNA content of 3990 ± 479 ng of DNA. The concentration of DNA (pg DNA/mg IgG) in the feed is8.8×104 ppb, which is way above the limit of approval of a medicine that should be bellow 10 ppb ofDNA.

In the AEX-CEX train "1" (Table I.6) and in the AEX-CEX train "3" (Table I.8), the anion exchangechromatography reduced the DNA content in 7.7-fold (Figure I.3). Whereas in the AEX-CEX train "2" (Ta-ble I.7), the anion exchange chromatography reduced the DNA content in 3.8-fold (Figure I.3). However,this difference in the reduction of DNA cannot be said to be dependent of the differences in the ad-justment of the mAb loading conditions, since every value was bellow the minimum limit of detectionof the respective DNA assay, which depends on the accuracy and precision of the analytical techniqueperformed, which in the case of the AEX-CEX train "2", there was a higher relative standard deviationerror for the minimum detection limit of 3.91 ng/mL, so the 7.81 ng/mL was considered to be the mostcorrect.

Since the residual DNA is a negatively charged impurity, it was expected to be removed by theanion exchange chromatography polishing step, by binding to the positively charged resin. However, itwas not expected to see DNA being removed in the elution fraction of the cation exchange chromatogra-phy in train "1", because the cation exchanger is a positively charged resin. It is an odd result, moreover,it is also possible to observe that there is more DNA content in the CEX elution fraction than there is inthe CEX feed fraction, which initially does not make sense. Nevertheless, perhaps the DNA was boundto an histone protein, and the histone consequently binds to the cation exchanger, due to the working

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pH being below the protein’s pI (making the protein positively charged), explaining the presence of DNAin the elution fraction of the CEX. Additionally, the zinc could be precipitating the DNA and consideringthat the zinc is a cation, it will bind on the cation exchanger resin. However, the zinc should be freely insuspension and, besides, the interaction between zinc and DNA should not be a very stable interaction,but, perhaps, it could be some sort of secondary interaction.

All of the CEX fractions (feed, flow-through, wash and elution) of the AEX-CEX train "2" werebellow the minimum limit of detection of Picogreen’s considered acceptable (< 7.91 ng/mL), therefore itis not possible to state whether or not there was any removal of DNA, however it is not expected to therebe any, since DNA alone should not bind to a cation exchanger.

There was DNA removed in the wash fraction of the CEX of train "3", which it was not expectedconsidering it was DNA that did not bound to the cation exchanger resin and it was not present in theCEX feed either. Besides the amount of DNA present in the wash fraction is higher than the CEX feed,which cannot be possible. There could still be some DNA in the column from previous runs which wasnot washed or eluted and considering that the CIP with NaOH of this method was only during 2 CV(which was what the resin’s guide recommended [41]), there might not have been enough incubationtime with NaOH to sanitize the column. The method was changed later for the following experiments, forthere to be at least a CIP with a 35 min. of incubation time with NaOH.

In the end of the CEX, the purified Trastuzumab product (CEX flow-through) had bellow 313 ±13 ng of DNA for both train "1" and "3" and bellow 625 ± 37 ng of DNA for train "2". The flow-throughAEX-CEX trains with mAb loading conditions adjusted with 4 M TrisBase pH 9.0 to pH 6.0 and 17 mS/cmconductivity (train "1") and mAb loading conditions not adjusted (pH 4.0, conductivity 2.15 mS/cm) (train"3") reduced the DNA content in, at least, 12.7-fold. Whereas, the flow-through AEX-CEX train with mAbloading conditions adjusted to pH 6.0 and 1.98 mS/cm conductivity with 4 M TrisBase pH 9.0, followedby buffer exchange with three volumes exchanges with 25 mM sodium acetate (pH 6, conductivity 1.98mS/cm) reduced the DNA content in, at least, 6.4-fold. Although, just as mentioned before this differencedepends on the accuracy and precision of the DNA assay performed.

Most of the DNA was removed in the washing step of the AEX, which means that most of theDNA did not end up binding to the negatively charged resin. Perhaps there was in fact some sort ofinteraction between the DNA and the zinc or between the DNA and an histone protein, which wouldform complexes with positive charges and, therefore would not bind to the anion exchanger.

The AEX-CEX (train "1") with mAb loading conditions (pH 6.0, 17 mS/cm conductivity) adjustedwith 4 M TrisBase was able to removed more DNA in the AEX washing and elution steps than the othertrains. The AEX washing step of train "1" removed 2.3-fold more DNA than of the AEX washing step oftrain "2" and removed 3.9-fold more DNA than the washing step of train "3". The AEX elution step oftrain "1" removed more 1.5-fold more DNA than of the AEX elution step of train "2" and more 18.5-foldmore DNA than of the AEX elution step of train "3". The wash and elution steps combined of the flow-through AEX trains "1", "2" and "3" had an overall DNA removal of 77%, 38% and 16%, respectively,which means that the Trastuzumab loading conditions of pH 6.0 and 17 mS/cm conductivity allowedmore DNA removal by the AEX polishing step.

In none of the AEX polishing steps performed of each train ("1", "2" and "3") experiments, it wasable to determine a DNA breakthrough due to all the flow-through fractions assayed being bellow thelimit of detection of the Picogreen assay.

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Table I.6: Results of the DNA content (quantified by the Picogreen assay) of the samples collected from the AEX-CEX train "1" with mAb loading conditions adjusted to pH 6.0 and 17 mS/cm conductivity.

Volume (mL) DNA (ng/mL) RSD (%) DNA (ng)

AEX feed 133 30 12% 3990 ± 479

AEX flow-through 133 < 3.91 4% < (520 ± 21)

AEX wash 10 236 12% 2360 ± 283

AEX elution 10 72 8% 720 ± 58

CEX feed 80 < 3.91 4% < (313 ± 13)

CEX flow-through 80 < 3.91 4% < (313 ± 13)

CEX wash 5 < 3.91 4% < (20 ± 1)

CEX elution 5 284 6% 1420 ± 85

Table I.7: Results of the DNA content (quantified by the Picogreen assay) of the samples collected from the AEX-CEX train "2" with mAb loading conditions adjusted to pH 6.0 and 1.98 mS/cm conductivity.


AEX feed 133 30 12% 3990 ± 479

AEX flow-through 133 < 7.81 6% < (1039 ± 62)

AEX wash 10 104 11% 1040 ± 114

AEX elution 10 48 13% 480 ± 62

CEX feed 80 < 7.81 6% < (625 ± 37)

CEX flow-through 80 < 7.81 6% 625 ± 37

CEX wash 5 < 7.81 6% < (39 ± 2)

CEX elution 5 < 7.81 6% < (39 ± 2)

Table I.8: Results of the DNA content (quantified by the Picogreen assay) of the samples collected from the AEX-CEX train "3" with mAb loading conditions of pH 4.0 and 2.15 mS/cm conductivity.


AEX feed 133 30 12 3990 ± 479

AEX flow-through 133 < 3.91 16 < (520 ± 83)

AEX wash 10 61 18 610 ± 110

AEX elution 10 < 3.91 16 < (39 ± 6)

CEX feed 80 < 3.91 16 < (313 ± 50)

CEX flow-through 80 < 3.91 16 < (313 ± 50)

CEX wash 5 157 17 785 ± 133

CEX elution 5 < 3.91 16 < (20 ± 3)

I.2.4 Total protein content

The Bradford assay performed to the samples collected of every AEX-CEX trains ("1", "2" and"3") did not quantified any exact total protein content, considering every value was bellow the minimumlimit of detection of the Bradford assay (< 12.5 µg/mL). There was total protein quantified in the start-

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ing product (AEX feed) and HCP content estimated, as it can be seen in Table I.5 (from a previousBradford assay). Therefore, there was at least antibody in the feed of the AEX (0.34 mg/mL), as it canbe observed from the HPLC-Protein A affinity chromatogram of the Trastuzumab obtained after ZnCl2precipitation (Figure I.1), which should be in a concentration way above the minimum limit of detectionof the Bradford assay. Furthermore, there must still be HCP in the feed material of this study, since theHCP content results reported by Dutra et al. after the ZnCl2 precipitation show that there should stillbe HCP to be removed, after the capture step [28]. It has been reported that, occasionally, HCPs canprecipitate during freezing or when the sample pH is adjusted from acidic to neutral, which was the case.In addition, storage at 2°C to 8°C or multiple freeze-thaws may result in loss of reactivity [52]. Moreover,the sensitivity of an analytical method may be difficult to achieve for products with lower protein con-centrations [52], and the starting material of these experiments had an antibody titre of 0.34 mg/mL.Additionally, literature has shown that the Bradford assay is not susceptible to interference by a widevariety of chemicals present in samples [63], therefore interference of zinc chloride with the total proteinquantification method can be excluded.

I.2.4.1 Verification of the Bradford method’s validity

A bovine serum albumin (BSA) stock solution with 500 µg/mL of total protein content and amAb TG5 solution with a known concentration of 4600 µg/mL were quantified, using the frozen BSAstandards prepared for the Bradford assay (and used for every Bradford assay performed in this study),in order to verify the method’s validity. A total protein content of 772 ± 85 µg/mL and 3381 ± 231 µg/mLwere quantified for the BSA stock solution and the mAb TG5 solution, respectively. Since the Bradfordassay standard curve was calibrated with BSA standards, the method is much more sensitive to BSAthan immunoglobulin G (IgG), thus it is more likely to underestimate the amount of protein concentrationin a BSA solution, whereas for a IgG sample, the total protein content is likely to be overestimated [63].However, what was verified from the experiment was the complete opposite. The protein concentrationof the BSA stock solution was overestimated, while the protein concentration of the mAb TG5 solutionwas underestimated. Nevertheless, the Bradford assay did quantify the protein’s concentration of bothsamples, even if overestimated and underestimated with already taking the relative standard deviationerrors into account. Therefore, the Bradford assay is a valid method for determination of total proteinconcentration.

I.2.5 Antibody titre determination

An HPLC-Protein A affinity chromatography was performed to all of the samples collected fromthe AEX-CEX trains "1", "2" and "3" to determine the antibody concentration. The results did not quantifyany antibody in any of the samples, including in the Trastuzumab obtained after ZnCl2 precipitation(Figure I.4), which had been quantified with a mAb titre of 0.34 mg/mL after the precipitation experiment,with the HPLC-Protein A affinity chromatogram showing an antibody peak between 0.84 and 0.88 min.of retention time in Figure I.1. All the results from the analytical methods that did not detect any antibodyor any total protein in all of the Trastuzumab samples, led to conclude that the antibody solution mighthave denatured and it did not survive the storage conditions (pH 4.0, 2.15 mS/cm conductivity andtemperature -20°C). The multiple freeze-thaws with storage in the cold at 2°C to 8°C may have resultedin the loss of antibody reactivity [52].

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Figure I.4: UV 280 nm HPLC-Protein A affinity chromatogram of the Trastuzumab supernatant solution precipitatedwith ZnCl2, which is the mAb feed solution of the AEX. The peak of the retained mAb is observed between 0.84 and0.88 min. This mAb sample was analyzed two months and half (8th Fev 2020) after the ZnCl2 precipitation (25th Nov2019). During this time, the samples were stored at pH 4.0 and -20°C.

I.3 Appendix I.3 - Results and Discussion of the Flow-through AEX-CEX (N-ADCand ADC) experiments with sodium acetate buffer

I.3.1 Flow-through AEX-CEX (N-ADC and ADC) trains with sodium acetate buffer

Zoomed-in chromatograms of the wash, elution and CIP steps of the AEX and CEX runs withnon-adjusted conditions (N-ADC) are illustrated in Figures I.5 and I.6.

Figure I.5: Zoomed-in chromatogram of the wash (225-235 CV), elution (235-245 CV) and CIP (245-252 CV) stepsof the flow-through AEX run with non-adjusted loading conditions (N-ADC) of the AEX-CEX N-ADC train. The AEXcolumn was washed with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using25 mM sodium acetate buffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. Thecolumn was sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min.

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Figure I.6: Zoomed-in chromatogram of the wash (400-410 CV), elution (410-420 CV) and CIP (420-434 CV) stepsof the flow-through CEX run with non-adjusted loading conditions (N-ADC) of the AEX-CEX N-ADC train. The CEXcolumn was washed with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using25 mM sodium acetate buffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. Thecolumn was sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2 mL/min.

A zoomed-in chromatogram of the sample loading step of the CEX ADC flow-through run isillustrated in Figure I.7. Zoomed-in chromatograms of the wash, elution and CIP steps of the AEX ADCand CEX ADC runs are illustrated in Figures I.8 and I.9.

Figure I.7: Zoomed-in chromatogram of the mAb loading step at 200 mL (400 CV) of the flow-through CEX run withadjusted loading conditions (ADC) of the AEX-CEX ADC train with sodium acetate buffer at pH 6.0.

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Figure I.8: Zoomed-in chromatogram of the wash (225-235 CV), elution (235-245 CV) and CIP (245-252 CV) stepsof the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train. The AEX columnwas washed with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mMsodium acetate buffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. The columnwas sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min.

Figure I.9: Zoomed-in chromatogram of the wash (400-410 CV), elution (410-420 CV) and CIP (420-434 CV) stepsof the flow-through CEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train. The CEX columnwas washed with 25 mM sodium acetate buffer (pH 6.0, 1.98 mS/cm conductivity, 10 CV) and eluted using 25 mMsodium acetate buffer with 1 M NaCl (pH 6.0, 85.1 mS/cm, 10 CV), both steps at 1 mL/min of flow rate. The columnwas sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2 mL/min.

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I.3.1.1 Residual DNA content

Tables I.9 and I.10 show the DNA content of the samples collected from the fractions of theAEX-CEX N-ADC and ADC trains, respectively, quantified by the Picogreen assay.

Table I.9: DNA content of the samples collected from the AEX-CEX N-ADC train with non-adjusted mAb loadingconditions (pH 3.6 and 3.47 mS/cm conductivity).

Volume (mL) DNA (ng/mL) RSD (%) DNA (ng) DNA (pg/mg IgG)

AEX N-ADC feed 225

< 31.25 1

< (7031 ± 70) < 1.1×105

AEX N-ADC flow-through 225 < (7031 ± 70) < 3.8×105

AEX N-ADC wash 10 < (313 ± 3) 0

AEX N-ADC elution 10 < (313 ± 3) < 1.3×105

CEX N-ADC feed 200

< 31.25 1

< (6250 ± 63) < 1.3×105

CEX N-ADC flow-through 200 < (6250 ± 63) < 5.0×103

CEX N-ADC wash 5 < (156 ± 2) < 8.3×104

CEX N-ADC elution 5 < (156 ± 2) < 4.8×105

Table I.10: DNA content of the samples collected from the AEX-CEX ADC train with adjusted mAb loading conditions(pH 6.0 and 4.00 mS/cm conductivity).

Volume (mL) DNA (ng/mL) RSD (%) DNA (ng) DNA (pg/mg IgG)

AEX ADC feed 225

< 7.81 1

< (1757 ± 70) < 2.7×104

AEX ADC flow-through 225 < (1757 ± 70) < 1.2×105

AEX ADC wash 10 < (78 ± 3) 0

AEX ADC elution 10 < (78 ± 3) < 3.0×104

CEX ADC feed 200

< 7.81 1

< (1562 ± 62) < 3.0×104

CEX ADC flow-through 200 < (1562 ± 62) < 1.1×103

CEX ADC wash 5 < (39 ± 2) < 5.7×104

CEX ADC elution 5 < (39 ± 2) < 1.0×105

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I.3.1.2 Total protein, HCP and antibody content

Tables I.11 and I.12 show the total protein quantified by the Bradford assay, the antibody titredetermined by HPLC-Protein A affinity chromatography and an estimation of the HCP content of thesamples collected from the fractions of the AEX-CEX N-ADC and ADC trains, respectively.

Table I.11: Total protein content, mAb titre and HCP estimation of the samples collected from the AEX-CEX N-ADCtrain with non-adjusted mAb loading conditions (pH 3.6 and 3.47 mS/cm conductivity).

Volume

(mL)

Total protein

(µg/mL)

RSD

(%)

Total protein

(mg)

mAb titre

(mg/mL)

HCP

(µg/mL)

HCP

(ng/mg IgG)

AEX N-ADC feed 225 221 12 49.7 ± 6.0 0.29 ∼ 0 ∼ 0

AEX N-ADC flow-through 225 224 16 50.4 ± 2.5 0.24 ∼ 0 ∼ 0

AEX N-ADC wash 10 < 25 5 < (0.3 ± 0.0) 0.08 ∼ 0 ∼ 0

AEX N-ADC elution 10 < 25 5 < (0.3 ± 0.0) 0.00 < (25 ± 1) 0

CEX N-ADC feed 200 224 16 44.8 ± 7.2 0.24 ∼ 0 ∼ 0

CEX N-ADC flow-through 200 83 9 16.6 ± 1.0 0.07 18 ± 2 2.8×105

CEX N-ADC wash 5 5110 6 25.6 ± 1.6 6.27 ∼ 0 ∼ 0

CEX N-ADC elution 5 252 6 1.3 ± 0.1 0.38 ∼ 0 ∼ 0

Table I.12: Total protein content, mAb titre and HCP estimation of the samples collected from the AEX-CEX ADCtrain with adjusted mAb loading conditions (pH 6.0 and 4.00 mS/cm conductivity).

Volume

(mL)

Total protein

(µg/mL)

RSD

(%)

Total protein

(mg)

mAb titre

(mg/mL)

HCP

(µg/mL)

HCP

(ng/mg IgG)

AEX ADC feed 225 248 15 55.8 ± 8.4 0.29 ∼ 0 ∼ 0

AEX ADC flow-through 225 253 14 56.9 ± 4.6 0.26 ∼ 0 ∼ 0

AEX ADC wash 10 54 8 0.5 ± 0.0 0.07 ∼ 0 ∼ 0

AEX ADC elution 10 < 25 4 < (0.3 ± 0.0) 0.00 < (25 ± 1) 0

CEX ADC feed 200 253 14 50.6 ± 7.1 0.26 ∼ 0 ∼ 0

CEX ADC flow-through 200 139 16 27.8 ± 5.6 0.08 63 ± 10 8.3×105

CEX ADC wash 5 5534 20 27.7 ± 2.0 7.23 ∼ 0 ∼ 0

CEX ADC elution 5 < 50 7 < (0.3 ± 0.0) 0.14 ∼ 0 ∼ 0

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I.3.1.3 Monomer yield, monomer purity, HMWI and LMWI content

The monomer yield and purity, as well as the high molecular weigh impurities (HMWI) and lowmolecular weight impurities (LMWI) content of the AEX-CEX N-ADC and AEX-CEX ADC trains arerepresented in Tables I.13 and I.14, respectively.

Table I.13: Monomer yield, monomer purity and HMWI and LMWI content of the fractions (feed, flow-through, washand elution) of the AEX-CEX N-ADC (with non-adjusted loading conditions) train.

Monomer yield (%) Monomer purity (%) HMWI (%) LMWI (%)

AEX N-ADC feed - 10 9 81

AEX N-ADC flow-through 82 16 3 81

AEX N-ADC wash 1 0 25 75

AEX N-ADC elution 0 0 27 73

CEX N-ADC feed 73 16 3 81

CEX N-ADC flow-through 20 0 2 98

CEX N-ADC wash 48 70 0 30

CEX N-ADC elution 3 57 0 43

Table I.14: Monomer yield, monomer purity and HMWI and LMWI content of the fractions (feed, flow-through, washand elution) of the AEX-CEX ADC (with adjusted loading conditions) train.

Monomer yield (%) Monomer purity (%) HMWI (%) LMWI (%)

AEX ADC feed - 93 0 7

AEX ADC flow-through 90 95 0 5

AEX ADC wash 1 25 0 75

AEX ADC elution 0 0 0 0

CEX ADC feed 80 95 0 5

CEX ADC flow-through 24 100 0 0

CEX ADC wash 56 93 2 5

CEX ADC elution 1 100 0 0

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I.3.1.4 Overall results

A summary of the overall results from all the analytical techniques performed to the Trastuzumabproduct before and after each polishing step are represented in Tables I.15 and I.16 for the AEX-CEXN-ADC and AEX-CEX ADC trains, respectively.

Table I.15: Overall results of the analytics (DNA, total protein, HCP, mAb yield, mAb purity, HMWI and LMWI) ofthe AEX-CEX train with non-adjusted loading conditions (N-ADC) at pH 3.6 and 3.47 mS/cm). The mAb yield of theAEX N-ADC flow-through was 82%, however the CEX feed has a mab yield of 73% due to the differences in themAb loading volume.

DNA

(ng/mL)

Total protein

(µg/mL)

HCP

(µg/mL)

mAb titer

(mg/mL)

mAb yield

(%)

mAb purity

(%)

HMWI

(%)

LMWI

(%)

Trastuzumab starting product

(AEX N-ADC feed)< 31.25 ± 0 221 ± 27 ∼ 0 0.29 - 10 9 81

Trastuzumab intermediate product

(CEX N-ADC feed)< 31.25 ± 0 224 ± 36 ∼ 0 0.24 73 16 3 81

Trastuzumab final product

(CEX N-ADC flow-through)< 31.25 ± 0 83 ± 7 18 ± 2 0.07 20 0 2 98

Table I.16: Overall results of the analytics (DNA, total protein, HCP, mAb yield, mAb purity, HMWI and LMWI) of theAEX-CEX train with adjusted loading conditions (ADC) at pH 6.0 and 4.00 mS/cm). The mAb yield of the AEX ADCflow-through was 90%, however the CEX feed has a mab yield of 80% due to the differences in the mAb loadingvolume.

DNA

(ng/mL)

Total protein

(µg/mL)

HCP

(µg/mL)

mAb titer

(mg/mL)

mAb yield

(%)

mAb purity

(%)

HMWI

(%)

LMWI

(%)

Trastuzumab starting product

(AEX ADC feed)< 7.81 ± 0 248 ± 37 ∼ 0 0.29 - 93 0 7

Trastuzumab intermediate product

(CEX ADC feed)< 7.81 ± 0 253 ± 35 ∼ 0 0.26 80 95 0 5

Trastuzumab final product

(CEX ADC flow-through)< 7.81 ± 0 139 ± 22 63 ± 10 0.08 24 100 0 0

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I.4 Appendix I.4 - Results and Discussion of the Flow-though AEX-CEX (ADC)experiments with Tris-HCl buffer

I.4.1 CEX (ADC) "test" runs with Tris-HCl buffer at pH 6.0, 7.0 and 8.0

The chromatograms of the CEX "test" runs with adjusted Trastuzumab loading conditions at pH6.0, 7.0 and 8.0 (and 4.00 mS/cm conductivity) using Tris-HCl buffer are illustrated in Figures I.10, I.11and I.12, respectively.

Figure I.10: Chromatogram of the flow-through CEX test run with adjusted loading conditions (ADC) with Tris-HClbuffer at pH 6.0. The CEX column was equilibrated with 25 mM Tris-HCl buffer (pH 6.0, 2.24 mS/cm, 10 CV) at aflow-rate of 1 mL/min. The CEX column was loaded at the flow rate of 0.2 mL/min with 2.25 mg/mL mAb loadingat 5 mL (10 CV). The mAb loading conditions were pH 6.0 and 4.00 mS/cm conductivity. The UV was measured atwavelengths 280 nm and 214 nm. The CEX column was washed (10-20 CV) with 25 mM Tris-HCl buffer (pH 6.0,2.24 mS/cm conductivity, 10 CV) and eluted (20-30 CV) using 25 mM Tris-HCl buffer with 1 M NaCl (pH 6.0, 86.2mS/cm, 10 CV), both steps at 1 mL/min of flow rate. The column was sanitized (30-44 CV) with 0.5 M NaOH for 14CV at the flow rate of 0.2 mL/min.

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I.4.1.1 Antibody titre determination

The HPLC-Protein A affinity chromatogram of the Trastuzumab starting material solution thawed(mAb titer 27 mg/mL, pH 12.4, 4.40 mS/cm) obtained after Protein A affinity chromatography is rep-resented in Figure I.13. The HPLC-Protein A affinity chromatograms of the Trastuzumab feed, flow-through, wash and elution fractions of the flow-through CEX "test" runs at pH 6.0, 7.0 and 8.0 usingTris-HCl buffer are represented in Figure I.14.

Figure I.13: UV 280 nm HPLC-Protein A affinity chromatogram of a trastuzumab thawed solution sample with anantibody titre of 27 mg/mL obtained after Protein A affinity chromatography (capture step) used for the CEX ADC(with adjusted loading conditions) test runs with Tris-HCl buffer at pH 6.0, 7.0 and 8.0. The peak of the retained mAbis observed between 0.84 and 0.88 min.

Figure I.14: Overlaid UV 280 nm HPLC-Protein A affinity chromatograms of trastuzumab samples of the feed, flow-through, column wash and elution of the CEX ADC (with adjusted loading conditions) chromatography test runs withTris-HCl buffer at pH 6.0 (A), 7.0 (C), 8.0 (E). The CEX ADC trastuzumab feed solution (2.25 mg/mL) for the testruns at pH 6.0 (B), 7.0 (D) and 8.0 (E) originated from a dilution of a trastuzumab thawed solution with an antibodytitre of 27 mg/mL. The peak of the retained mAb is observed between 0.84 and 0.88 min.

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I.4.2 Flow-through AEX-CEX (ADC) train with Tris-HCl buffer at pH 8.0

A zoomed-in chromatogram of the sample loading step of the flow-through AEX ADC run at pH8.0 with Tris-HCl buffer is illustrated in Figure I.15. Zoomed-in chromatograms of the wash, elution andCIP steps of the flow-through AEX ADC and CEX ADC runs at pH 8.0 are illustrated in Figures I.16 andI.17.

Figure I.15: Zoomed-in chromatogram of the mAb loading step at 125 mL (125 CV) of the flow-through AEX runwith adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HCl buffer at pH 8.0.

Figure I.16: Zoomed-in chromatogram of the wash (125-135 CV), elution (135-145 CV) and CIP (145-152 CV) stepsof the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HCl bufferat pH 8.0. The AEX column was washed with 25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm conductivity, 10 CV) andeluted using 25 mM sodium acetate buffer with 1 M NaCl (pH 8.0, 85.5 mS/cm, 10 CV), both steps at 1 mL/min offlow rate. The column was sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min.

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Figure I.17: Zoomed-in chromatogram of the wash (220-230 CV), elution (230-240 CV) and CIP (240-254 CV) stepsof the flow-through CEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HCl bufferat pH 8.0. The CEX column was washed with 25 mM Tris-HCl buffer (pH 8.0, 1.87 mS/cm conductivity, 10 CV) andeluted using 25 mM sodium acetate buffer with 1 M NaCl (pH 8.0, 85.5 mS/cm, 10 CV), both steps at 1 mL/min offlow rate. The column was sanitized with 0.5 M NaOH for 14 CV at the flow rate of 0.2 mL/min.

I.4.2.1 Residual DNA content

Table I.17 shows the DNA content, quantified by the Picogreen assay, of the Trastuzumab start-ing material (before buffer exchange) as well as the Trastuzumab of the flow-through AEX run withadjusted mAb loading conditions of pH 8.0 and 4.00 mS/cm conductivity with Tris-HCl buffer.

Table I.17: DNA content of the Trastuzumab starting material precipitated (before buffer exchange), Trastuzumabfeed (after buffer exchange) and of the samples collected from the flow-through AEX run with Tris-HCl at adjustedloading conditions of pH 8.0 and 4.00 mS/cm conductivity.

Volume (mL) DNA (ng/mL) RSD (%) DNA (µg) DNA (pg/mg IgG)

Starting material precipitated

(before buffer exchange)125 1256 11 157 ± 17 5.5 × 106

AEX feed (after buffer exchange) 125 488 5 61 ± 3 2.1 × 106

AEX flow-throughfractions 1A1-1A3 45 < 7.81 2

3 ± 1 -fractions 1A4-1B1 80 54 6

AEX wash 10 1762 10 18 ± 2 -

AEX elution 10 829 5 8 ± 0 -

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I.4.2.2 Total protein content

Table I.18 shows the total protein content, quantified by the Bradford assay, of the Trastuzumabstarting material (before buffer exchange) as well as the Trastuzumab of the flow-through AEX run withadjusted mAb loading conditions of pH 8.0 and 4.00 mS/cm conductivity with Tris-HCl buffer.

Table I.18: Total protein content of the Trastuzumab starting material precipitated (before buffer exchange),Trastuzumab feed (after buffer exchange) and of the samples collected from the flow-through AEX run with Tris-HCl at adjusted loading conditions of pH 8.0 and 4.00 mS/cm conductivity.

Volume (mL) Total protein (µg/mL) RSD (%) Total protein (mg) HCP (µg/mL HCP (ng/mg IgG)

Starting material precipitated

(before buffer exchange)125 406 25 51 ± 8 176 7.8 ×10^5

AEX feed (after buffer exchange) 125 124 13 16 ± 2 ∼0 ∼0

AEX flow-throughfractions 1A1-1A2 30 <25 13

5 ± 1 - -fractions 1A3-1B1 95 72 13

AEX wash 10 119 13 1 ± 0 - -

AEX elution 10 163 14 2 ± 0 - -

I.4.3 Flow-through AEX (ADC) run with Tris-HCl buffer at pH 7.0

A zoomed-in chromatogram of the sample loading step of the flow-through AEX ADC run at pH7.0 with Tris-HCl buffer is illustrated in Figure I.18. A zoomed-in chromatogram of the wash, elution andCIP steps of the flow-through AEX ADC run at pH 7.0 is illustrated in Figures I.19.

Figure I.18: Zoomed-in chromatogram of the mAb loading step at 125 mL (125 CV) of the flow-through AEX runwith adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HCl buffer at pH 7.0.

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Figure I.19: Zoomed-in chromatogram of the wash (125-135 CV), elution (135-145 CV) and CIP (145-152 CV) stepsof the flow-through AEX run with adjusted loading conditions (ADC) of the AEX-CEX ADC train with Tris-HCl bufferat pH 7.0. The AEX column was washed with 25 mM Tris-HCl buffer (pH 7.0, 2.29 mS/cm conductivity, 10 CV) andeluted using 25 mM sodium acetate buffer with 1 M NaCl (pH 7.0, 85.7 mS/cm, 10 CV), both steps at 1 mL/min offlow rate. The column was sanitized with 1 M NaOH for 7 CV at the flow rate of 0.2 mL/min.

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