Development of novel biomimetic affinity adsorbents for ... · Development of novel biomimetic affinity adsorbents for plasmid DNA purification: ... elevado de estudos tem procurado

Development of novel biomimetic affinity adsorbents for

plasmid DNA purification:

Preliminary results

Cátia Isabel Pereira Jorge

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors: Professor Maria Ângela Cabral Garcia Taipa Meneses de Oliveira

Professor Duarte Miguel de França Teixeira dos Prazeres

Examination Committee

Chairperson: Professor Arsénio do Carmo Sales Mendes Fialho

Supervisor: Professor Maria Ângela Cabral Garcia Taipa Meneses de Oliveira

Member of the Committee: Professor Marília Clemente Velez Mateus

December 2014

ii

iii

Acknowledgements

First of all, I would like to acknowledge Professor Ângela Taipa for being my supervisor and

for accepting me in this Master‘s thesis, trusting in me to work on this topic and for all the support and

help that was given to me. I would also like to thank Professor Miguel Prazeres for letting me work at

MoBiol laboratory and for being my co-supervisor.

To my lab colleagues, thanks for all the support when I needed and for being there to help me.

Thank you for all the moments that we spent together. Sofia, Salomé, João Trabuco, Jorge, Pedro,

Luís Raiado and Maria thanks for all the help provided when I needed and for the constant support. I

would also like to thank my Master’s colleagues, Rita, Diana, Ana, Isabel and Liliana with whom I grew

up closer during this year. Sara A. Rosa and Raquel Santos, thanks for all the help provided in AKTA

and for being there when I had any doubts. I would also like to thank João Belchior for the help

provided in the beginning of my thesis and for teaching me most of the techniques that I would be

using. I have also to thank Filipa Gonçalves, who, during her Summer training period, helped and

assisted me during a part of my thesis work.

A big ‘thank you’ to Sara S. Rosa, Cláudia Alves and Sílvia Andrade for helping me go through

this year, for providing some discussions that helped in my work and for being there in the most

stressful times. I would also like to thank Rita Carneiro for her friendship and for listening to me when I

needed.

To my amazing friends, Andreia Pereira e Marina Machado: thank for all our conversations

and for your continuous support. Even though you were not here our conversations on the phone

helped me through a lot of stuff. Thank for being the best friends I could ask for and for being there for

me. I know that every time I need help I can talk to you.

At last, but not the least, I would like to thank my family, specially my mom. Mom, thank you

for all that you did for me and for listening every time I would ranted about something that was going

wrong. Without you I would have not made it.

iv

v

Abstract

The development of strategies to pDNA purification has become necessary for the progress of

gene therapy and DNA vaccine production processes due to the structural and chemical similarities

between pDNA and impurities present in cell extracts. Affinity chromatography plays a powerful role in

separation technology as this technique enables the purification of a biomolecule by interaction

between the target molecule and a specific ligand. A substantial number of attempts have been made

to develop affinity chromatographic matrices capable of specifically recognizing nucleic acid molecules

out of a mixture and, despite the progress made, there is room for the development of new adsorbents

with desirable properties for large-scale application. In this context, synthetic-mimic affinity ligands can

be a good alternative to generate such adsorbents. The reactivity of cyanuric chloride towards amines,

its structural rigidity and the formation of non-fissile bonds with the substituents, has been the basis for

its use as a scaffold for combinatorial synthesis and the generation of molecular diversity in synthetic

protein-mimic ligands with defined specificities and selectivities. Combinatorial solid-phase synthesis

allows the production of a large number of different compounds for random screening, in a time and

resource-effective manner. In this work, different types of chromatographic matrices were tested with

two promising ligands previously screened from a combinatorial library. It was possible to achieve

purification of pDNA with both Sepharose CL-6B and the CIM® monolithic disks derivatized with one

selected ligand. The best results were obtained under hydrophobic conditions using the CIM®

monolithic disks. The yield of pure pDNA obtained was around 91% with a purification factor of 3.78.

Keywords: plasmid purification, affinity chromatography, synthetic affinity ligands, biomimetic,

monoliths

vi

vii

Resumo

O desenvolvimento de estratégias para a purificação de pDNA tornou-se necessário para o

progresso na terapia génica e produção de vacinas de DNA devido às semelhanças entre o pDNA e

as impurezas presentes em extratos celulares. A cromatografia de afinidade desempenha um papel

importante nos processos de separação, sendo uma técnica que permite a purificação de

biomoléculas com base na interação específica entre a molécula-alvo e um ligando. Um número

elevado de estudos tem procurado desenvolver matrizes cromatográficas de afinidade capazes de

reconhecer especificamente ácidos nucleicos a partir de uma mistura e, apesar dos progressos

realizados, há espaço para o desenvolvimento de novos adsorventes com especificidade e/ou

selectividade melhoradas para aplicação em larga escala. Os ligandos sintéticos de afinidade

mímicos de proténas são uma boa alternativa para gerar novos adsorventes. A reactividade de

cloreto cianúrico em relação a aminas, a sua rigidez estrutural e a formação de ligações não-físseis

com os substituintes, levou ao seu uso como estrutura-base apropriada em síntese combinatorial e a

produção de ligandos sintéticos mímicos de proteínas, com especificidades e selectividades

definidas. O método predominantemente utilizado para gerar diversidade molecular é a síntese

combinatorial em fase sólida, pois permite a obtenção de um elevado número de compostos

diferentes para um rastreio aleatório, de um modo eficaz em termos de tempo e recursos. Neste

trabalho, testaram-se diferentes tipos de matrizes cromatográficas com dois ligandos promissores,

previamente selecionados de uma biblioteca combinatorial, e foi possível alcançar a purificação de

pDNA, tanto com Sepharose CL-6B como com discos monolíticos CIM® derivatizados com um ligando

seleccionado. O melhor resultado foi obtido em condições hidrofóbicas utilizando os discos

monolítocos CIM®. O rendimento obtido em DNA plasmídico puro foi de 91% com um factor de

purificação de 3.78.

Palavras-chave: purificação de plasmídeos, cromatografia de afinidade, ligandos sintéticos de

afinidade, biomiméticos, monólitos

Index

Acknowledgements ................................................................................................................................. iii

Abstract.....................................................................................................................................................v

Resumo .................................................................................................................................................. vii

Figure Index ............................................................................................................................................. 4

Table Index .............................................................................................................................................. 9

Abbreviations ......................................................................................................................................... 10

1. Introduction .................................................................................................................................... 11

1.1 Plasmid DNA ......................................................................................................................... 11

1.1.1 Gene therapy and DNA vaccines .................................................................................. 11

1.1.2 Plasmid Isoforms ........................................................................................................... 13

1.1.3 Cell Culture and Fermentation ....................................................................................... 13

1.1.4 Cell Lysis and Clarification ............................................................................................ 14

1.1.5 Purification by Chromatography .................................................................................... 16

1.1.5.1 Anion-Exchange Chromatography ............................................................................ 17

1.1.5.2 Hydrophobic Interaction Chromatography ................................................................. 17

1.1.5.3 Size-Exclusion Chromatography ............................................................................... 18

1.1.5.4 Affinity Chromatography ............................................................................................ 19

1.2 Affinity Ligands ...................................................................................................................... 21

1.2.1 Biological Ligands .......................................................................................................... 21

1.2.2 Synthetic Affinity Ligands .............................................................................................. 21

1.2.3 Biomimetic Ligands ....................................................................................................... 22

1.3 Monoliths as Chromatographic Matrices for Affinity Chromatography .................................. 24

1.3.1 GMA/EDMA Monoliths ................................................................................................... 24

1.3.1.1 CIM® Monolithic Columns .......................................................................................... 25

1.3.2 Agarose Monoliths ......................................................................................................... 26

1.3.3 Silica Monoliths .............................................................................................................. 26

1.3.4 Cryogels ......................................................................................................................... 27

1.3.5 Immobilization Methods for Affinity Monoliths ............................................................... 28

2

1.3.5.1 Covalent Immobilization Methods .............................................................................. 28

1.3.5.2 Non-covalent Immobilization Methods....................................................................... 31

2. Material and Methods .................................................................................................................... 32

2.1 Cell Culture ............................................................................................................................ 32

2.1.1 Pre-inoculum and inoculum ........................................................................................... 32

2.1.2 Cell lysis and Plasmid Primary Isolation ........................................................................ 33

2.2 Desalinization of a clarified E. coli lysate .............................................................................. 33

2.3 Synthesis of triazine-based adsorbents in Sepharose CL-6B ............................................... 34

2.3.1 Epoxy activation of Sepharose CL-6B ........................................................................... 34

2.3.2 Amination of previously epoxy-activated Sepharose CL-6B ......................................... 34

2.3.2.1 Determination of ammination extent in Sepharose beads ........................................ 34

2.3.3 Activation of aminated Sepharose with cyanuric chloride ............................................. 35

2.3.4 Nucleophilic substitution of the second chlorine atom of dichlorotriazinyl Sepharose (R1

substitution) ................................................................................................................................... 35

2.3.5 Nucleophilic substitution of the third chlorine atom of dichlorotriazinyl Sepharose (R2

substitution) ................................................................................................................................... 35

2.4 Chromatographic assays using ligands 5/6 and 6/5 in Sepharose CL-6B ............................ 35

2.5 Synthesis of triazine-based adsorbents in CIM® monolithic disks ......................................... 36

2.5.1 Epoxy activation of the monolithic disks ........................................................................ 36

2.5.2 Amination of previously epoxy-activated disks .............................................................. 36

2.5.3 Activation of aminated Sepharose with cyanuric chloride ............................................. 37

2.5.4 Nucleophilic substitution of the second chlorine atom of dichlorotriazinyl Sepharose (R1

substitution) ................................................................................................................................... 37


substitution) ................................................................................................................................... 37

2.5.5.1 Optimization of the R2 substitution for the monolithic supports ................................ 37

2.6 Chromatographic assays using ligands 6/5 in CIM® monolithic disks ................................... 37

2.7 Agarose gel electrophoresis .................................................................................................. 38

2.8 HPLC analysis ....................................................................................................................... 38

2.9 Adsorption of Cutinase in the matrices tested ....................................................................... 39

3. Results and Discussion ................................................................................................................. 39

3

3.1 Cell growth ............................................................................................................................. 39

3.2 Preliminary assay using selected triazine-based ligands ...................................................... 40

3.3 Chromatographic assays with triazine-based ligands ........................................................... 41

3.4 Chromatographic assays in AKTA purifier system with ligand 6/5 synthesized in Sepharose

CL-6B 46

3.5 Optimization of ligand derivatization ...................................................................................... 53

3.6 Chromatographic assays with CIM®

monolithic disks ............................................................ 54

3.6.1 Assays performed with ligand 6/5 derivatized CIM®

disk under hydrophobic conditions

54

3.6.2 Assays performed with ligand 6/5 derivatized CIM® disk under hydrophilic conditions 64

3.7 Adsorption of Cutinase in ligand 6/5 derivatized matrices .................................................... 66

4. Conclusion ..................................................................................................................................... 67

5. Further work................................................................................................................................... 68

6. References .................................................................................................................................... 70

7. Appendix ........................................................................................................................................ 74

Appendix I - Plasmid pVAX1-LacZ (Invitrogen) information .............................................................. 74

Appendix II – Plasmid pCEP4 (Invitrogen) information ..................................................................... 75

Appendix III – Plasmid pVAX1TSAGFP information ......................................................................... 76

Appendix IV - NZYDNA Ladder III (NZYTech) .................................................................................. 76

Appendix V - Pure plasmid DNA standard curve for analysis by HIC in a HPLC system ................. 77

4

Figure Index

Figure 1 – Scheme demonstrating all the process from the disease until it gets to the patient. (Adapted

from Prazeres et al 1999)8 ..................................................................................................................... 12

Figure 2 – Schematic representation of DNA structure: (a) Linear fragment, closed loop and

supercoiled topologies; (b) Plasmid DNA supercoiling. (Adapted from Ferreira et al 2005)11

.............. 13

Figure 3 - Average composition of E. coli cells. (Adapted from Ferreira 2005)11

.................................. 14

Figure 4 - Process flow sheet for the large-scale purification of sc pDNA. (Adapted from Ferreira et al

2000)9 .................................................................................................................................................... 16

Figure 5 - The Hofmeister series with anions and cations arranged in terms of their water affinity and

according to their effects on the solubility of macromolecules in aqueous solutions. (Adapted from

Freitas et al 2009)19

............................................................................................................................... 18

Figure 6 - Principle of Affinity Chromatography. NRS - non-retained substance. (Adapted from Tetala

et al 2010)21

........................................................................................................................................... 19

Figure 7 - General procedures for applying and eluting solutes from affinity columns. (Adapted from

Mallik and Hage 2006)22

........................................................................................................................ 20

Figure 8 – a) structure of a triazine-based ligand and b) examples of amine substituents mimicking the

side chains of different amino acids. (Adapted from Sousa et al 2009)33

............................................. 23

Figure 9 - Formation of a copolymer of GMA with EDMA (Adapted from Mallik et al 2006)22

.............. 25

Figure 10 – Different chemistries available in the CIM® monolithic columns

36. ................................... 26

Figure 11 - Typical reaction used for the preparation of a cryogel based on the copolymerization of

acrylamide, allyl glycidyl ether and N,N’- methylene bis-acrylamide. (Adapted from Mallik and Hage

2006)22

................................................................................................................................................... 28

Figure 12 – Covalent immobilization of ligands on GMA/EDMA monoliths by the epoxy method

(Adapted from Mallik and Hage 2006)22

................................................................................................ 29

Figure 13 - Covalent immobilization by the a) Schiff base method and the b) glutaraldehyde method

(Adapted from Mallik and Hage 2006)22

................................................................................................ 30

Figure 14 - Covalent immobilization by the CNBr method (Adapted from Mallik and Hage 2006)22

.... 31

Figure 15 - Growth curve of E.coli cells with plasmid pVAX1-LacZ, C1-TSA and pCEP4 .................... 40

Figure 16 - Resulting agarose gel from the samples collected in the washthrough process of ligand 5/6

(2ml of agarose). M – NZYDNA ladder III; 1 to 10 – Collected fractions after injecting 100 µl of the

clarified lysate (containing pVAX1-LacZ plasmid) using 0.4M Ammonium Sulphate in 20mM Tris-HCl

pH 8.0 as equilibration buffer (washthrough fractions). ......................................................................... 42

5


(2ml of agarose) using 20mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S –

Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid). ..................... 42


(2ml of agarose) using 0.4M Ammonium Sulphate in 20mM Tris-HCl pH 8.0 as equilibration buffer. L –

NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ

plasmid). The dragging marks in some lanes are due to the presence of ammonium sulphate. .......... 43


(2ml of agarose) using 20mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S –

Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid). ..................... 44


(2ml of agarose) using 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 as equilibration buffer. The

plasmids tested where pVAX1-LacZ (a), pCEP4 (b) and pVAX1TSAGFP (c). L – NZYDNA ladder III; S

– Loaded sample (100 μl clarified E. coli crude extract). The dragging marks in some lanes are due to

the presence of ammonium sulfate. ...................................................................................................... 44


(2ml of agarose) using 0.2 (a), 0.4 (b) and (c) 0.8M Sodium Citrate in 10mM Tris-HCl pH 8.0 as

equilibration buffer. L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract

containing pVAX1-LacZ plasmid). The dragging marks in some lanes are due to the presence of

ammonium sulfate. ................................................................................................................................ 45

Figure 22 - Chromatographic performance of ligand 6/5, in a 6 ml column in an AKTA purifier system.

Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in

hydrophobic conditions. The run was performed at room temperature at 1ml/min. 0.4M Ammonium

Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel

corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The

numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a), b), c) and

d) correspond to replicates of the same experiment with run d) being performed in a 2 ml column .... 47

Figure 23 - Chromatographic performance of ligand 6/5, in a 6 ml column in an AKTA purifier system.


hydrophilic conditions. The run was performed at room temperature at 1ml/min. 20mM Tris-HCl pH 8.0

was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram

presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak

correspond to the lanes in the 1% agarose gel. .................................................................................... 48

Figure 24 - Chromatographic performance of ligand 6/5 an AKTA purifier system. Sample (100μl

clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions.

The run was performed at room temperature at 1ml/min. 0.8M Ammonium Sulfate in 10mM Tris-HCl

pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the

chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on

6

top of each peak correspond to the lanes in the 1% agarose gel. a) and b) correspond to replicates of

the same experiment with a) being performed in a 6 ml column and b) in a 2 ml column. ................... 49

Figure 25 - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system.



Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a

negative linear gradient until 10 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gel

corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. ............... 50





negative linear gradient until 10 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gel

corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. a) and b)

correspond to duplicates of the same assay. ........................................................................................ 51



hydrophobic conditions. The run was performed at room temperature at (a) 1ml/min and (b) 0.5ml/min.

0.8M Sodium Citrate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer.

Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample.

The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. .......... 52


(2ml of agarose) using 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 as equilibration buffer. The

same sample was loaded in different dilutions: 1:10 (a), 1:20 (b), 1:50 (c) and 1:100 (d). L – NZYDNA

ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid).

The dragging marks in some lanes are due to the presence of ammonium sulfate. ............................ 53

Figure 29 – Percentage of derivatization of agarose with ligand 6/5 in the different conditions tested for

R2 amine substitution. ........................................................................................................................... 54

Figure 30 – Optimization of the chromatographic performance of ligand 6/5 in CIM® monolithic disks

for pDNA purification. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid)

was loaded in hydrophobic conditions. The run was performed at room temperature. 0.4M Ammonium



numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. ................. 56

Figure 31 - Chromatographic performance in a CIM® monolithic disk derivatized with ligand 6/5.




7














numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) and b)

represent replicates of the same experiment. ....................................................................................... 61



hydrophobic conditions. The runs was performed at room temperature at 1ml/min. 0.4M Ammonium


negative linear gradient until 20 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gels

correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The

numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a)

corresponds to the chromatograms obtained in the experiments; b) and c) corresponds to the agarose

gels of each chromatographic run. ........................................................................................................ 63



hydrophilic conditions. The run was performed at room temperature at 1ml/min. 20mM Tris-HCl pH 8.0

was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram

presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak

correspond to the lanes in the 1% agarose gel. a) and b) represent duplicates of the same assay. ... 64



hydrophilic conditions. The runs was performed at room temperature at 1ml/min. 20mM Tris-HCl pH

8.0 was used as equilibration buffer with a linear gradient with increasing of 1M NaCl during elution.

Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded

sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

............................................................................................................................................................... 65

8

Figure 37 - Percentage of cutinase binding and not binding to the different chromatographic matrices

non-derivatized and derivatized with ligand 6/5. A volume of 1 mL of a cutinase solution (1mg/mL) was

loaded in each column. .......................................................................................................................... 66

Figure A1 - Structure and features of pVAX1/lacZ plasmid. ................................................................. 74

Figure A2 - Structure and features of pCEP4 plasmid. ......................................................................... 75

Figure A3 - Structure and features of pVAX1TSAGFP plasmid. ........................................................... 76

Figure A4 - NZYDNA Ladder III electrophoresed in a 1% (w/v) electrophoresis grade agarose gel. ... 76

Figure A5 - Calibration curve obtained by HIC in a HPLC system. Using standard plasmid

concentrations. ...................................................................................................................................... 77

9

Table Index

Table 1 - Advantages and disadvantages of pDNA vaccine. (Adapted from Ghanem et al 2013) ....... 12

Table 2 - Concentration of free amine after the amination step and after derivatization with synthetic

amines in both R1 and R2 position: corresponding ligand concentration. ............................................ 41

10

Abbreviations

AC – Affinity chromatography

AEC – Anion-exchange chromatography

AU – Absorbance units

CIM® – Convective Interaction Media

E. coli – Escherichia coli

gDNA – Genomic DNA

GFP – Green fluorescent protein

HIC – Hydrophobic interaction chromatography

HPLC – High-performance liquid chromatography

LacZ – beta-galactosidase

LB - Luria Bertany

oc- open circular

OD – Optical density

pDNA – plasmid DNA

pI – Isoelectric point

qPCR – Real-time polymerase chain reaction

sc- supercoiled

SEC – Size exclusion chromatography

SEM – Scanning electron microscopy

TNBS - 2,4,6-trinitrobenzenesulfonic acid

11

1. Introduction

1.1 Plasmid DNA

The rapid advances of plasmid DNA (pDNA) application as a viable non-viral vector for gene

therapy and for vaccination has led to an increase of the demand for efficient production and

purification methods of pDNA, a form of non-genomic DNA that makes use of cellular machinery to

express proteins or antigens1,2

. Plasmid DNA is a large molecule, normally produced from Escherichia

coli, that is employed to deliver the desired genetic information into the cells and to induce the

production of relevant proteins3,4

. It is a circular, double-stranded DNA molecule in which its length can

vary from 2 to 20 kb and has a hydrophilic backbone and an hydrophobic interior of double helix, due

to the close packing of the aromatic bases4,5

. Each DNA strand consists in linear polymer of

deoxyribonucleotides linked by phosphodiester bonds/groups which are negatively charged when pH

> 46.

1.1.1 Gene therapy and DNA vaccines

Gene therapy and DNA vaccination are promising approaches for the prevention and cure of

diseases like cancer, AIDS, and cystic fibrosis7.

The transport of therapeutic genes to the nuclei of target cells can be carried out by either viral

or non-viral vectors8. However, the use of viral vectors has raised safety and regulatory concerns

because of their toxicity and immunogenicity, as well the possible activation or deactivation of

oncogenes or tumor-suppressor genes. The insertion of therapeutic genes in non-viral vectors such as

plasmids is therefore regarded as safer8,9

.

Gene therapy is a therapeutic strategy in which nucleic acids are introduced into human cells in

order to modify their genetic information for therapeutic purposes. So, it is a process where one or

more functional genes are introduced in a patient to prevent, treat and cure certain genetic defects8,9

.

In this technique the nucleic acid, which can be pDNA, is going to encode a therapeutic, destructive or

marker protein9.

DNA vaccines are genetically engineered DNA molecules used to produce immunological

responses in organisms against diseases. pDNA-based vaccines have been genetically engineered to

produce one or two specific proteins (antigens) from a disease-causing pathogen. Plasmid DNA

vaccination mimics the natural intracellular pathogen gene expression pathways which leads to its

recognition as foreign triggering a high number of immune responses both cellular and humoral. Once

the immune system has mounted its primary immune response to destroy the pathogen, it acquires a

memorized immunity to the disease6,10

.

12

Figure 1 – Scheme demonstrating all the process from the disease until it gets to the patient. (Adapted from Prazeres et al 1999)

8

The biggest advantage of using DNA vaccines is that there is stimulation of both the humoral

and cell-mediated components of the immune system while conventional protein vaccines only

stimulate the antibody response6. Plasmid DNA-based vaccines are considered very safe due to the

lack of genetic integration and to the absence of specific immune responses to the plasmid10

.

A summary of DNA vaccines advantages and disadvantages is shown in Table 1.

Table 1 - Advantages and disadvantages of pDNA vaccine. (Adapted from Ghanem et al 2013)6

Advantages Disadvantages

DNA is inexpensive when compared to isolated

proteins or organisms used for conventional

vaccines

Testing results have been favourable in small

animals, but less impressive in larger animals

(including humans)

DNA vaccines can result in longer lasting

production of the antigenic protein; thereby booster

shots are no longer required

DNA uptake to cells apparently decreases with

increased body size

Produces stronger immune responses than

conventional vaccines

Extended immunostimulation could lead to chronic

inflammation or autoantibody production

Stability of vaccine for storage and shipping Limited to protein immunogens (not useful for non-

protein based antigens such as bacterial

polysaccharides)

Subunit vaccination with no risk for infection Risk of affecting genes controlling cell growth

Ease of development and production -

13

1.1.2 Plasmid Isoforms

Plasmids are DNA molecules in which the two ends of the DNA strands are covalently linked,

forming a closed loop (Figure 2a). When the circular DNA molecule is under or over wound around

the molecule axis, superhelix structures are formed, namely a higher order structure called

supercoiled DNA (Figure 2b)11

.

Figure 2 – Schematic representation of DNA structure: (a) Linear fragment, closed loop and supercoiled

topologies; (b) Plasmid DNA supercoiling. (Adapted from Ferreira et al 2005)11

Plasmid DNA exists in three topological forms: supercoiled, open circular and linear12

. The

biosynthesis of pDNA by E. coli results in a highly enriched supercoiled DNA extract. This is

considered the most efficient isoform at transferring gene expression. Because of this it is important to

reduce the open circular, linear and even denatured pDNA isoforms through an efficient downstream

processing13

. All these isoforms, which are less effective as delivery vectors compared to sc pDNA,

are produced during cell growth in fermentation and can also arise from damage of supercoiled

pDNA3.

Supercoiled variants are likely to be “nicked” or linearized forming the other two isoforms8. The

open circular and linear forms result from enzymatic or shear-induced breakage of the sugar–

phosphate backbone. Linear results from a single-stranded break in the supercoiled pDNA5,14,15

.

1.1.3 Cell Culture and Fermentation

Plasmids are usually produced in a recombinant E. coli host by fermentation and represent

around 3% (w/w) of the E. coli extract6. Figure 3 illustrates the average composition of E. coli cells.

14

Figure 3 - Average composition of E. coli cells. (Adapted from Ferreira 2005)11

Economic large-scale plasmid production from E. coli requires the optimization of plasmid copy

number and of biomass concentration as it might positively impact the downstream processing and

ultimately purification yields10

. The low pDNA concentration in the E. coli cell and the need to remove

large quantities of cellular debris, proteins, genomic DNA and endotoxins drive the genetic and

process strategies. For example, establishing efficient expression vectors and host-strain systems are

capable of increase the yield of the supercoiled pDNA and improve its downstream recovery and

purification3. In fermentation the main goal is to maximize the amount of supercoiled DNA that is made

and also maximize the purity at harvest. Normally, optimal purity corresponds to maximizing the

average supercoiled-plasmid copy number. Fundamental fermentation improvements often result from

genetic manipulation, being that normally the primary mechanism for certifying that E. coli cells retain

the plasmid is by growing the organisms under a selective pressure in the presence of an antibiotic for

which the plasmid contains a resistance gene14

. Strategies aimed at increasing plasmid amplification

in fermentation include the use of temperature shock, addition of chloroamphenicol and amino acid

starvation. While temperature shock is achievable on a small scale, it may be difficult to implement at

a large scale because of the time necessary to go from one temperature to another in a conventional

fermenter. These approaches have been used to provide plasmid yields that are normally acceptable

from a manufacturing viewpoint3.

1.1.4 Cell Lysis and Clarification

The pDNA extraction from E.coli cells is the major problem in a pDNA production process. In

the downstream processing the major goal is to eliminate cellular components of the host strain. Most

of the critical contaminants present in the lysate share similar characteristics of pDNA like negative

charge (RNA, gDNA, endotoxins), identical size (gDNA, endotoxins) and hydrophobicity (endotoxins).

However, the number and complexity of the processing steps in extraction, isolation, purification and

formulation of pDNA induce a kind of structural stress which can result in damage of the supercoiled

70,0%

6,5%

15,0%

5,0%

1,0% 0,5% 2,0%

Water

RNA

Proteins

gDNA

pDNA

Endotoxins

Others

15

plasmid isoform molecules. The current purification processes for pDNA includes several unit

operations after the fermentation step. These contain cell harvest, lysis, cell debris/solid separation,

precipitation, adsorption, buffer exchange and polishing/clarification and concentration steps prior to

attaining suitable for therapeutic use6.

This process starts with the recovery of cells from the broth by a step of centrifugation or

microfiltration. The next step in the downstream processing of pDNA is cell lysis, tipically an alkaline

lysis, so that all the intracellular components, including plasmid DNA, RNA, gDNA, endotoxins and

proteins, are released. This process is critical to recover large amounts of intact supercoiled pDNA in

order to obtain high overall process yields9,16

. Cells are then ressuspended and concentrated in a

appropriated buffer containing agents that will disrupt ionic and/or hydrogen bonds between lipids and

proteins3,8

. This will promote the removal of divalent cations from cell wall, outer membrane and

plasma membranes, destabilizing their structure, thus facilitating the lysis and preventing plasmid

degradation8. Following the alkaline-lysis step, a precipitate is formed that contains cell debris,

denatured proteins and nucleic acids. This precipitate must be removed by using a solid–liquid unit

operation like a centrifugation on fixed-angle rotors, which is the most common operation at the

laboratory and preparative scales. However, this type of operation is not suitable for the large-scale

production of plasmid DNA due to the centrifugal acceleration of the liquid entering the centrifuge that

can cause shearing and, consequently, break the precipitated material and DNA molecules. Filtration

is therefore the best operation to use in large-scale production processes9. Evidently, the ideal cell

lysis step would allow for all of the supercoiled pDNA to be selectively removed from the cells while all

the other macromolecular impurities remained inside an intact cell14

.

After the lysis it is necessary to have clarification and concentration steps to remove host

proteins and some host nucleic acids (such as gDNA and RNA) to reduce further the volume of the

process stream and to increase the plasmid mass fraction before chromatography. A major concern in

the clarification is the removal of high molecular weight RNA. The presence of endogenous nucleases

in plasmid preparations at the end of the alkaline-lysis step can be advantageously used to remove

high molecular weight RNA. Although the clarification and concentration steps produce a cleaner and

smaller process stream, there is evidence that these operations can be bypassed, proceeding directly

to processing by chromatography with increases in process yield8,9

.

A schematic of a general process flow sheet for the large-scale purification of pDNA is

represented in Figure 4.

16

Figure 4 - Process flow sheet for the large-scale purification of sc pDNA. (Adapted from Ferreira et al 2000)9

1.1.5 Purification by Chromatography

Liquid chromatography is central in the manufacturing of therapeutic pDNA. The overall process

must deliver a pDNA product that meets quality specifications set or recommended by international

regulatory agencies such as the FDA (Food and Drug Administration) and EMA (European Medicines

Agency). Although attempts have been made to include the chromatography step after cell lysis, it is

generally included after the impurity load and the process volume have been reduced by clarification

and concentration operations as it was explained before. The goal of chromatography is then to

remove the cellular host components like RNA, proteins, gDNA fragments, endotoxins and non-

supercoiled pDNA variants, which are virtually impossible to remove by other unit operations5.

Normally chromatography is the method of choice for the large-scale purification of supercoiled pDNA

due to the size and chemical properties of the target nucleic acid molecules (charge and

hydrophobicity), the accessibility of the nucleotide bases to ligands, and the topological constraints

imposed by supercoiling that are exploited via the interaction of nucleic acids with solid supports, with

the objective of selectively isolating and purifying plasmid DNA from impurities9. Taking in account the

properties referred before different types of chromatographic methods, such as size-exclusion (SEC),

17

anion-exchange (AEC), hydrophobic interaction (HIC), affinity (AC), and others have been integrated

into several processes for the manufacture of therapeutic pDNA5.

1.1.5.1 Anion-Exchange Chromatography

Anion-Exchange Chromatography (AEC) is a commonly used method for capture and

purification of pDNA17

. The polyanionic structure of nucleic acids can be explored in this type of

chromatography because the overall charge of nucleic acids depends on the number of bases that

make up the molecules9,11

. The retention of nucleic acids is directly proportional to charge density and

is also affected by nucleotide sequence and conformation11

.

This technique is one of the most widely used for pDNA capture, purification and quantitation

specially because of its rapid separation, no solvent requirement, easy sanitisation with sodium

hydroxide and a wide selection of process-grade stationary phases5. The major limitation of AEC is the

low selectivity of the adsorbents towards pDNA leading to the co-elution of impurities, particularly

endotoxins and high molecular weight RNA11

.

The overall interaction between the pDNA and the stationary phase is based on the local

attraction generated by opposite charges. With this, the isoforms will have different retention time in an

increasing salt gradient. With the increase of salt concentration the DNA molecules elute in the order

of the chain length which is directly related to the number of charged phosphate groups. The

supercoiled pDNA has higher charge density than the less constrained open circular form leading to

stronger electrostatic attractions to the positively charged bound ligand. Consequently, the supercoiled

pDNA will elute later than the open circular pDNA6. Base sequence and composition are also known to

affect the elution pattern of nucleic acids18

.

When using AEC to separate pDNA, the clarified lysate should always be loaded at a

sufficiently high salt concentration (typically >0.5M NaCl) to avoid the adsorption of low charge density

impurities, such as low molecular weight RNA, oligonucleotides and proteins5. Also, the selectivity

towards pDNA-based vaccines or impurities is poor due to their non-specific binding to the anion-

exchange resin stationary phase. Consequently AEC is often used in series with other purification

techniques such as SEC or agarose gel electrophoresis6.

1.1.5.2 Hydrophobic Interaction Chromatography

Hydrophobic Interaction Chromatography (HIC) is a well-established bioseparation

technique19

. The purification of pDNA by HIC takes advantage of the higher hydrophobicity of single

stranded nucleic acids and endotoxins that interact strongly with HIC media than the double stranded

nucleic acids5,11

. These interactions are promoted mainly by van der Waals interactions19

. The pDNA

molecules, which have the hydrophobic bases packed and shielded inside the double helix, tend to

have a minimal hydrophobic interaction with the HIC media. Single stranded nucleic acid impurities

show a higher exposure of the hydrophobic bases, interacting strongly with hydrophobic ligands5. The

hydrophobicity of the amphiphilic endotoxins is attributed to the lipid A portion of the molecule19

.

18

Plasmid DNA can be purified with HIC by loading feed solutions at high concentration of an

adequate salt and as a result performing step or gradient elution with low salt to remove the bound

impurities19

.

In general, to obtain success in HIC chromatography two major elements have to be

considered: the stationary phase and the mobile phase. The stationary phases can vary in the type of

ligand (phenyl, methyl and others), the ligand chain length, ligand density and on the type of matrix or

support. The most widely used ligands for HIC are linear chain alkanes with or without a terminal

amino group20

. The characteristics of the mobile phase, such as type and concentration of the salt,

pH, temperature and additives are also important. The salt type chosen is determinant for the

separation success of HIC19,20

. The interactions between ions and water, and ions and

macromolecules were initially investigated by Hofmeister being that the Hofmeister series orders ions

from strongly hydrated to weakly hydrated, mainly on the basis of their surface charge density and

water affinity (Figure 5)19

. Salts such as sodium, potassium or ammonium sulphate are the most

effective to promote interactions due to the higher ‘salting-out’ effect20

. ‘Salting-out’ is a purification

method that utilizes the reduced solubility of certain molecules in a solution of very high ionic strength

and that can control precipitation by using the different effects of various salts and their respective

concentrations. The salts ability to induce selective precipitation is dependent on many interactions

with the water and solutes.

Figure 5 - The Hofmeister series with anions and cations arranged in terms of their water affinity and according to

their effects on the solubility of macromolecules in aqueous solutions. (Adapted from Freitas et al 2009)19

1.1.5.3 Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) has been extensively used to purify pDNA5. SEC

fractionates and purifies plasmids on the basis of size and can be used alone or in sequence with

other steps, like AEC6,11

. This technique is ideal as a polishing step to remove residual contaminants

with simultaneous buffer exchange into an adequate formulation or storage buffer6.

The first experiments that used SEC for pDNA purification did not yield significant results due

to the lack of stationary phases adequate for the separation of nucleic acids with high molecular mass

and complex conformations. The introduction of other stationary phases changed this situation5. One

example is using Sephacryl S1000. This stationary phase works outstandingly well in the removal of

19

endotoxins, RNA and proteins and can be used as a single chromatographic step when purifying small

amounts of pDNA. This happens because Sephacryl S1000 is a simple, inexpensive and reproducible

matrix for pDNA purification5,9

.

In SEC the reduction of the plasmid hydrodynamic radius due to supercoiling is the basis for

the selective separation from different DNA molecules. Typically, gDNA is excluded eluting as the

peak leading edge, followed by the relaxed and then the supercoiled pDNA conformations. The

smaller molecules are easily separated from the leading DNA peak11

.

The main drawback is that SEC has a limited capacity (requires low volume streams with low

amounts of impurities) and selectivity for pDNA and therefore is not suitable as an initial pDNA

purification step11

. As so, SEC is commonly used in combination with other chromatographic

techniques as a final polishing step.

1.1.5.4 Affinity Chromatography

Affinity chromatography is based on the recognition of a particular structure in the target

plasmid molecule by an immobilized ligand11

. The affinity chromatography is based on the highly

specific and reversible molecular interaction of various biomolecules being that the ligand is

immobilized on a stationary phase either by covalent immobilization or physical adsorption. The

sample containing the product of interest along with other compounds is then passed through the

affinity column being that the target is captured in a highly selective manner through molecular

recognition by the ligand present in the column and other compounds pass through the column with

little or no retention. After elution the product is obtained in pure and concentrated form (Figure 6)21

.

Affinity chromatography uses natural biological processes such as molecular recognition for

the selective purification of biomolecules on the basis of their biological function or chemical structure.

These methods have the power to eliminate additional purification steps, increasing yields and

improving process economics.

Figure 6 - Principle of Affinity Chromatography. NRS - non-retained substance. (Adapted from Tetala et al

2010)21

Due to the specificity of this type of chromatography the elution of the molecules can be done

in various ways. If the compound is bound with only a weak or moderate affinity it is possible to elute

the target in the application buffer under isocratic conditions. More strongly retained substances can

20

be eluted by changing the mobile phase or column conditions. This approach is known as nonspecific

elution. A more selective elution technique known as biospecific elution can also be used. In this case

a competing agent is added so that can bind either the retained target or immobilized ligand. The

binding of this competing agent is used to prevent interactions of the target with the ligand which, in

turn, causes the target be released to the mobile phase and elute22

. These procedures are

schematically represented in Figure 7.

Figure 7 - General procedures for applying and eluting solutes from affinity columns. (Adapted from Mallik and

Hage 2006)22

The affinity ligand can consist of a wide variety of binding agents, like proteins, antibodies, etc,

and is immobilized within a column and used to selectively bind a given target or group of targets

within a sample. Because of the highly selective nature of many affinity ligands, it’s possible to isolate,

measure, or study specific targets even when they are present in complex biological samples. The

immobilized ligand is an important factor that determines the success of an affinity chromatographic

method and the type of ligand chosen can divide affinity chromatography into several categories23

.

One type of affinity chromatography that can be used for pDNA purification is the Immobilized

Metal Affinity Chromatography (IMAC). IMAC harnesses affinity interactions between metal ions

and target molecules, enabling high-efficiency separation of the target molecules from other

components present in a mixture. It has been reported that IMAC exhibited potential for removal of

21

denatured DNA and RNA from the alkaline cell lysate which bind to the IMAC column whereas the

pDNA is not retained5,6

.

Another type of affinity chromatography for pDNA purification is the Triple-helix Affinity

Chromatography (THAC). This technique is based on the formation of a triplex between an

oligonucleotide covalently linked to a chromatographic matrix, and a specific duplex sequence in the

target pDNA. The available oligonucleotides are covalently linked to the chromatographic matrix within

the stationary phase6.The best characterised triplex forms when a homopyrimidine oligonucleotide

strand binds to the major groove of a homopurine–homopyrimidine duplex DNA through the formation

of Hoogsteen hydrogen bonds between thymine (T) and adenine (A) to form TA- T triplexes, and

protonated cytosine (C+) specifically recognizing guanine (G) to form CG-C+ triplexes. These triple-

helices are only stable at acidic pH5,6

. The target DNA is then captured via an intermolecular triplex

formation with a biotinylated oligonucleotide and recovered as dsDNA when the phase is washed with

a mild alkaline buffer to destabilize the Hoogsteen H-bonds.

However, so far chromatographic operations based on affinity interactions between pDNA or

impurities with specific ligands have not been used extensively for pDNA purification5. One reason

may be the lack of effective affinity ligands with high selectivity, capacity and durability.

1.2 Affinity Ligands

Affinity technology exploits the natural specific recognition phenomena between two biological

entities forming a complex. These interactions are reversible non-covalent interactions. This

technology exploits not only the natural specific recognition phenomena but also the predictive and

rational character of the binding between the targets to purify and a complementary ligand24

. Though

most of the ligands have a natural origin, in the last decades (non-biological) ligands became viable

and safer alternatives to purify different biologic targets.

1.2.1 Biological Ligands

Most of the existing ligands, such as peptides, oligonucleotides, antibodies, and receptor

proteins are from natural sources that they aim to imitate25,26

. These ligands display high selectivity

and specificity, but suffer from high costs of production and purification, low binding capacities, limited

life cycles and low scale-up potential25,27

. Normally, these ligands require purification in due to the

possibility of contamination with host DNA and viruses. Conventional sterilization and cleaning-in-

place schedules, which are central to any production process for a biologic, cause degradation of the

immobilized ligand, leading to the shortening of the column life. They also can contaminate the final

product due to potentially toxic or immunogenic leachates. These factors have contributed to the

widespread perception that affinity chromatography based upon biological ligands has serious

drawbacks application in the large-scale purification of biopharmaceuticals25

.

1.2.2 Synthetic Affinity Ligands

Synthetic affinity ligands can circumvent the drawbacks of natural ligands by imparting

resistance to chemical and biochemical degradation and displaying ease and low cost of production27

.

22

These ligands have been well established over many decades of use and can be metal chelate and

thiophilic ligands, for example. Different synthetic affinity adsorbents are nowadays durable and

readily up-scaled25,26,28

. Additional advantages of synthetic ligands are the easy in situ sterilization at

large-scale production and the lower toxicity and immunogenicity.

The acceptance of synthetic ligands for use in large-scale chromatography led to the

development of ligands that combined the selectivity of natural ligands with the high capacity,

durability and cost-effectiveness of the synthetic systems. These ligands were designated as the

biomimetic ligands25

.

1.2.3 Biomimetic Ligands

The concept of ‘biomimetic ligands’ was introduced as an upgrade of textile dyes that were

designed to mimic the structure and binding of natural ligands24

. Some of the best known biomimetic

ligands are these textile dyes, such as Cibacron blue F3G-A, that where developed 30 years ago. A

big part of these ligands possess a triazine scaffold that is substituted with polyaromatic ring systems

solubilised with sulphonate or carboxylate functions and then decorated with electron withdrawing or

donating groups. Such triazine dyes are low-cost commodity chemicals that are easily synthesized

and immobilized onto solid phases generating high capacity adsorbents. The immobilized ligands

mimic the binding of natural anionic heterocyclic substrates such as nucleic acids, nucleotides,

coenzymes and vitamins to proteins. These ligands have a number of advantages over the use of

natural ligands making them commonly used in the research market to purify proteins like albumin,

nucleases, hydrolases, among others. However, there are some concerns over the selectivity, purity,

leakage and toxicity of the dyes limiting their use for the purification of pharmaceuticals. The need to

improve the selectivity, purity and reproducibility of these ligands led to rational molecular design

techniques25,26

.

The developments that occurred in computational technology, combinatorial synthesis and

high-throughput screening techniques allowed the extension of this concept to synthetic biomimetic

dyes and triazine non-dye ligands (de novo designed ligands), but also peptides and minimized

protein domains. The availability of crystallographic structures of proteins and complexes, together

with computer-based molecular modeling techniques, allowed the design of synthetic protein-mimic

affinity ligands. Such ligands display improved their characteristics over their natural counterparts due

to the inclusion of chemically defined and characterized groups which are easy to synthesize. They

have moderate to high specificity to the complementary targets, the which enables the use of mild

elution conditions, higher stability/resistance to sterilization and cleaning-in-place procedures,

providing higher yields of ligand utilization and lower costs and higher scalability of the processes24

.

Interactions between proteins and nucleic acids are crucial for the understanding of numerous

biological mechanisms and can be understood by atomic interactions between amino acids and

nucleotides29

. Affinity chromatography using amino acids as ligand molecules has already been used,

in the purification of plasmid DNA. Some studies using histidine, arginine and lysine as affinity ligands

were capable of purifying pDNA with sequence specificity30–32

. The atoms present in each nucleic acid

23

base allow the interaction with amino acid structures, due to the difference between nucleic acids and

between amino acids leading to a wide variety of combinations used to purify specific sequences.

Another important aspect is that some amino acids bind not only to one nucleic acid base, but to a

specific sequence, forming complexes. Not only the bases of the DNA influence binding, but also the

backbone itself can help in DNA-amino acid interactions having a stabilization effect29

.

These features of amino acid-DNA interactions can be advantageous to perform affinity

chromatography using amino acids. However the use of amino acids in this kind of process may be

expensive so the use of molecules that can mimic the properties of specific amino acids or peptides

can be of great advantage26

. As so, the use of amino acid/protein-mimic ligands might be a good

choice to purify pDNA.

An example, of the application of rational design of protein-mimic ligands was in the design of

a Protein L-mimic ligand. This research allowed to obtain a combinatorial library of 169 synthetic

affinity ligands. The library was synthesized in agarose using a well-established procedure. The affinity

ligands present in this library have a cyanuric chloride scaffold that contains two substituent groups

consisting of aliphatic and aromatic amines, each mimicking the side chain of a different amino acid.

(Figure 8)28

.

Figure 8 – a) structure of a triazine-based ligand and b) examples of amine substituents mimicking the side chains of different amino acids. (Adapted from Sousa et al 2009)

33

The Protein L combinatorial library was recently screened for binding nucleic acids and

potentially purifying plasmid DNA. As a first approach, a lysine mimetic was used in conjugation with

24

the other amino substituents to test if it would improve the binding ability towards nucleic acids. It was

proven that the synthetic mimic of lysine (1,5-diaminopentane) exhibited the same behavior as the

natural amino acid. Besides the lysine mimic, various amino acid analogues exhibited high binding

capacity towards pDNA34

.

The preliminary screening results have shown that the synthetic affinity ligand library could be

used for molecular recognition of nucleotides and, particularly, pDNA. Four ligands were selected as

leads to be further assessed for the purification of plasmid DNA from E.coli crude extracts, in either

hydrophobic or hydrophilic conditions. In the present work, one of these ligands was used for pDNA

purification.

1.3 Monoliths as Chromatographic Matrices for Affinity

Chromatography

Monoliths are supports that consist of a single, continuous piece of a porous material that is

synthesized to form a homogeneous column and that are prepared in various dimensions with

agglomeration-type or fibrous microstructures21,22

. They are prepared from monomeric precursors,

which form a skeleton with interconnected pores upon polymerization in a solvent mixture35

. These

solvents are now known as porogens and the final pore structure of the monolith is highly dependent

on the porogens used during its formation. When these solvents are removed, what remains is a

series of interconnected pores that provide routes (channels) for solvent flow through the monolith22

.

The pores in monolithic materials are classified into two types: macropores (that have diameters larger

than 50 nm) and mesopores/micropores (pore diameters in the range of 2–50 nm).These materials

can be categorized into ‘‘organic’’ and ‘‘inorganic’’, depending on the materials they are made from 21

.

Monoliths can be made in various forms and prepared inside columns, capillaries, or microfluidic

devices. Their low back-pressures allows their use at high flow rates enabling fast separations and

short analysis times and also allow rapid mass transfer to occur, helping to decrease band broadening

and providing efficient separations in work with affinity ligands. Another advantage of monoliths is that

there are various reaction schemes that can be used for their modification which is valuable when

adapting monoliths for use with a wide range of affinity ligands. All these reasons led to the growing

interest in these chromatographic matrices22

.

A variety of monoliths that have been reported for use in affinity chromatography are described

with more detail in the following sections.

1.3.1 GMA/EDMA Monoliths

Nowadays the glycidyl methacrylate (GMA)-ethylene dimethacrylate (EDMA) copolymer

system in various formats is the most frequently used system in monolith-based affinity

chromatography due to the available epoxide groups for ligand immobilization that allows a multitude

of immobilization strategies21,35

. Typically the GMA-EDMA monolith solution consists of a monomer

(GMA), crosslinker (EDMA), initiator and two porogenic solvents (Figure 9). The polymerization

mixture is poured into a mold, sealed and is then carried out either thermally or by UV depending on

25

the initiator present in the mixture. After polymerization, the seals are removed and frits are inserted

on both ends of the monolithic columns to avoid any leakage of the monolith21

. The final monolith with

epoxide functionality can be used directly for ligand (with amino group) immobilization. The ligands

can be attached via different spacers or the epoxy groups can be converted into a diol form under

acidic conditions. This diol group can be used as a precursor for various ligand coupling methods 21,22

.

Figure 9 - Formation of a copolymer of GMA with EDMA (Adapted from Mallik et al 2006)22

One advantage of using a GMA/EDMA monolith with affinity ligands is the fact that the GMA

monomer contains epoxy groups that can be used directly for covalent immobilization or as precursors

for other coupling methods. Additionally, the diol groups that can be generated on this material tend to

give a support a low nonspecific binding for many biological agents. They are also relatively easy to

prepare and have the ability to be made with a variety of surface areas and pore sizes that can be

controlled by varying the composition of the porogen. Other factors that can be varied are the

monomer-to-crosslinker ratio, the amount of each reagent and porogen, and the polymerization time,

among other items that can be used to optimize the total amount of an affinity ligand that can be

placed onto such supports. On the other hand, GMA/EDMA monoliths do tend to have low surface

areas when compared to particulate silica supports or silica monoliths, limiting the total amount of

ligand that can be immobilized onto this material and which might hamper separation efficiency. This

can be circumvented by embedding of particles or nanoparticles into the monolithic support 22,35

.

1.3.1.1 CIM® Monolithic Columns

CIM® monolithic columns, produced by BIA Separations, are a single homogeneous piece with

highly interconnected porous that can be prepared in various dimensions. These continuous stationary

phases have a matrix composed of methacrylate polymers36

.

26

CIM®

monoliths are an innovative product but are already established to be a chromatographic

media useful for biomolecules purification at any scale. These monoliths have the advantage of

operating at flow rates up to 10 times when compared to particle based supports leading to the

decrease of the time and cost of the purification process. Another advantage is the pore size that can

be adjusted to accommodate large molecules like viruses and pDNA, ensuring high binding capacities.

Such monolithic columns are supplied in different chemistries that can be contained in a single column

if necessary36,37

. The different chemistries available are shown in Figure 10.

Figure 10 – Different chemistries available in the CIM® monolithic columns

36.

1.3.2 Agarose Monoliths

Agarose in a particulate form has been a popular support for affinity separations for several

decades, so monolith supports made of agarose have also been in use for affinity chromatography.

This type of monolith is prepared by casting an agarose emulsion to generate a monolith with large

pores, with 20–200 µm in diameter. The emulsion is formed by heating a suspension of agarose in

water at 95–100°C and then adding a mixture of cyclohexane and Tween 80 while shaking. This

mixture is then poured into glass columns or forms that are fit with a plug at the bottom and kept in a

water bath at 60°C following by the decrease of the temperature to 20°C, which causes the agarose to

gel into the desired shape. These materials can be activated and used in ligand immobilization by

employing the same reaction schemes that are used for agarose particles. The main difference is that

activation of the agarose and ligand immobilization is now performed by circulating the required

solutions through the monolith rather than performing these reactions in a suspension. These

materials have basically the same advantages of traditional agarose supports including their ability to

be used with many ligands, their low nonspecific binding, and their stability over a wide pH range.

However, the large pore diameters cause a relatively low mechanical strength of these materials22

.

1.3.3 Silica Monoliths

Silica monoliths are alternative materials for the polymeric monoliths and exist in two forms for

ligand immobilization: commercially available bare silica and sol–gel entrapment method. In the case

of bare silica there are no reactive functional groups available for ligand immobilization. So, diol

groups can be created on the surface using similar methods as those described for silica particles.

27

Therefore, modification of silanol groups on the surface of the monolithic silica skeleton by silylation

reagents, such as (3-aminopropyl)trimethoxysilane or (3- glycidyloxypropyl)trimethoxysilane, is crucial.

After this activation step, ligands can be immobilized on either diol activated silica or aminopropyl

silica21,35

.

The advantages of these materials include their good efficiencies, mechanical strength and

also their high surface areas, which can be important in affinity methods that require supports with

high ligand densities. Their main disadvantage is that they are difficult to prepare directly in the

laboratory due to their shrinkage after formation. They also have the same limitations as traditional

silica particles in terms of the pH range over which they can be used (typically pH 2–8) in order to

avoid their disintegration21,22,35

. In case of the sol–gel method, the ligand can be entrapped in the

monolith in a single step, keeping the ligand activity unaltered. However, the release of alcoholic

byproducts during polymerization can lead to the denaturation of the ligands. Also, this type of

monoliths generally have limited column diameter21,22

.

1.3.4 Cryogels

Cryogels are emerging as a new class of affinity monolithic stationary phases. They have an

unique property of being hydrophilic and having macropores in the range of 10–100 µm being

relatively large compared with the macropores of GMA-EDMA (1.5 µm) and of silica monoliths (2

µm)21

. The cryogel is prepared by polymerization reactions below –10°C and using monomers

dissolved in an aqueous phase. A mixture of acrylamide, allyl glycidyl ether, and N,N’-methylene bis-

(acrylamide) is normally used to make this polymer, with TEMED and ammonium persulfate being

used as initiators. When the mixture is cooled down to 0 to -12°C there is the formation of ice crystals

forming a porous template upon and around which the polymer is formed. After polymerization, these

ice crystals are allowed to thaw and the resulting water is removed from the monolith (Figure 11). In

this approach ice crystals act as the porogen, with the shape and size of these crystals determining

the shape and size of pores in the final polymer. The main application of these monoliths are in the

purification of blood cells 22

.

28

Figure 11 - Typical reaction used for the preparation of a cryogel based on the copolymerization of acrylamide,

allyl glycidyl ether and N,N’- methylene bis-acrylamide. (Adapted from Mallik and Hage 2006)22

The main advantage of cryogel monoliths are the large pore sizes (between 10–100 µm) that

allow the free passage of large biological particles without blocking the monolith. Although this

provides cryogels with low backpressures, it also gives them much lower surface areas compared to

other chromatographic supports which can result in small amounts of immobilized ligand and low

sample capacities21,22

.

1.3.5 Immobilization Methods for Affinity Monoliths

Like it was referred before several approaches have been reported for placing ligands within

monolithic supports for chromatography. Some examples including covalent immobilization methods,

biospecific adsorption and entrapment are discussed below.

1.3.5.1 Covalent Immobilization Methods

Covalent immobilization is one of the approaches that can be used to bind affinity ligands to

monolith supports, being one of the most used techniques. The immobilization of the ligand has to be

performed after the monolith column has been prepared. Immobilization can be achieved by

circulating a solution of the ligand through the column or by dipping the column in a solution containing

the ligand. A disadvantage of circulating the ligand through a monolith is that a larger amount of ligand

is generally required in the circulation method to compensate for the additional volume of ligand

solution that is employed22

.

1.3.5.1.1 The Epoxy Method

Some common covalent immobilization methods have already been adapted for work with

monolithic columns. One of these is the epoxy-based method. This method involves nucleophilic

attack of an epoxy group on the monolith by amine groups on a protein or ligand, leading to formation

of a stable secondary amine linkage (Figure 12). This approach can be used directly with GMA/

EDMA monoliths, since epoxy groups are present as the functional groups of the GMA monomers.

The method can be performed in a single step but it has a slower reaction rate than other available

29

methods, which can result in low amounts of immobilized ligand or long immobilization times.

Depending on the reaction conditions, this method can be used for ligands that contain amine, sulf-

hydryl, or hydroxyl groups21,22

.

Figure 12 – Covalent immobilization of ligands on GMA/EDMA monoliths by the epoxy method (Adapted from

Mallik and Hage 2006)22

1.3.5.1.2 The Schiff base and Glutaraldehyde Methods

Another technique that has been adapted for the covalent immobilization of ligands in

monoliths is the Schiff base method. This is an amine-based coupling method that can be used with

GMA/EDMA monoliths by first converting their epoxy groups into diols. These diol groups are then

oxidized with periodic acid to give aldehyde groups that will react with primary amines on proteins and

other ligands to form a Schiff base. Since this is a reversible reaction, the Schiff base is converted

upon its formation by reducing it with sodium cyanoborohydride to give a secondary amine. This

method has a faster rate of reaction than the epoxy method and allows higher ligand densities than

many other amine-based coupling methods. The main disadvantage of the Schiff base method is the

need to use reducing agents that may affect the immobilized ligand (eg. its biological activity). A

method closely related with the Schiff base technique is the glutaraldehyde method. In this

immobilization approach, an epoxy group is first converted to an amine form by reacting the epoxy

groups on the monolith surface with reagents such as ethylenediamine or hexanediamine. This amine-

activated support is next reacted with a dialdehyde producing an aldehyde-activated monolith. This

method has many of the advantages of the Schiff base method but involves more steps for the

preparation of the activated support. However, it does result in a longer spacer being placed between

the support and ligand, which can be useful in avoiding steric hindrance effects when dealing with

small ligands and binding of large bioentities (such as proteins or plasmids)22

. The Schiff base and

glutaraldehyde methods are illustrated in Figure 13.

30

Figure 13 - Covalent immobilization by the a) Schiff base method and the b) glutaraldehyde method (Adapted

from Mallik and Hage 2006)22

1.3.5.1.3 Other Methods

An alternative technique for covalent immobilization of ligands onto monoliths is the

carbonyldiimidazole (CDI) method. This process begins by converting epoxy groups in the monolith

into diol groups. These diols are reacted with 1,1’-carbonyldiimidazole to produce imidazolyl

carbamate groups. This activated support is then used for ligand immobilization by a nucleophilic

substitution that can occur between the activated sites and primary amines of the ligand, resulting in a

stable amide linkage. This method is faster than the epoxy method and involves fewer steps than the

Schiff base or glutaraldehyde methods but gives rise to lower ligand densities than the Schiff base

technique22

.

Covalent immobilization of ligands on monoliths can also be achieved by the disuccinimidyl

carbonate (DSC) method, the hydrazide method and the cyanogen bromide (CNBr) method described

in the literature. The disuccinimidyl carbonate (DSC) method also begins by converting epoxy groups

on a monolith like GMA/EDMA into diol groups. These diol groups are next reacted with DSC to place

succinimidyl carbonate groups on the monoliths surface. The activated form of the monolith is then

reacted with a ligand such as a protein that contains primary amine groups to form a stable carbamate

linkage. This method is fast and has been reported to be complete within 10 hours, however the

stability of activated monolith is low, requiring that proper care be taken to avoid side reactions due to

hydrolysis22

.

31

The hydrazide method is an example of a coupling technique that can be used with

glycoproteins and carbohydrate-containing ligands. This begins by producing an aldehyde-activated

monolith, in the same manner as described earlier for the Schiff base method, which is activated with

a reagent such as adipic dihydrazide, with any remaining aldehyde groups later being reduced to

alcohols with sodium borohydride. Although this method involves more steps than many of the other

techniques that have been discussed, the ability to immobilize through carbohydrate chains is an

attractive means for the site-selective attachment of antibodies and other glycoproteins to solid

supports resulting in a higher activity for such ligands when compared to amine-based coupling

methods22

The last method and the most common immobilization method in traditional affinity

chromatography is the cyanogen bromide (CNBr) method (Figure 14). This method is performed by

combining an ice cold, basic solution of CNBr with agarose or a polysaccharide-based support. This

immobilization technique is relatively simple and easy to perform. However, it does involve the use of

CNBr, which is toxic and a chemical hazard. In addition, ligands immobilized by the CNBr method are

not as stable as those produced by many other amine-based coupling techniques and have a

tendency to generate ion-exchange sites on the support that can lead to nonspecific binding22

.

Figure 14 - Covalent immobilization by the CNBr method (Adapted from Mallik and Hage 2006)22

1.3.5.2 Non-covalent Immobilization Methods

1.3.5.2.1 Biospecific Adsorption

A technique to immobilize the ligand without using covalent immobilization is to adsorb it to a

support through noncovalent interactions (biospecific adsorption). Normally, to accomplish this it is

necessary to covalently immobilize another substance to the support, a secondary ligand, which can

bind the ligand of interest in a way that does not interfere with the ligand's ability to bind its target. If

necessary, the ligand can later be cross-linked with the secondary ligand to provide a more stable

stationary phase22

.

1.3.5.2.2 Entrapment

A second approach by which an affinity ligand can be immobilized noncovalently is through

entrapment. In this process, the affinity ligand is incorporated as part of the polymerization mixture.

During polymerization, the support grows around the ligand and entraps or encapsulates it within the

support. This procedure is attractive for use with sol-gel materials as they are formed in an aqueous

solvent, allowing the ligand to be entrapped in a compatible solvent that should not lead to any

significant denaturation. The sol-gel entrapment process can be divided into several steps. When

32

working with silicates, the process involves hydrolysis of alkoxysilanes, followed by condensation of

hydrated silica to form siloxane bonds. Next, there is polycondensation of the additional silanol groups

to form cyclic oligomers. During the growing of the silica network the ligand gets entrapped. The main

advantage of this technique is that all recognition sites of the ligand will remain accessible and active if

appropriate conditions for sol-gel formation are selected. However, there are some difficulties

associated with controlling the pore size of the resulting support, the high degree of shrinkage of the

sol-gels, and the loss of protein activity that can occur if improper silanes are used for sol-gel

formation21,22

.

2. Material and Methods

2.1 Cell Culture

2.1.1 Pre-inoculum and inoculum

The plasmid pVAX1-LacZ, with 6050 bp, was used with Escherichia coli DH5α (Invitrogen) as

host cells. The cells where stored in autoclaved 20%(v/v) glycerol at -80°C. Then, to start the cell

culture, the pre-inoculum was grown in 100 ml shake flasks with 30 ml of LB (Luria Bertani from Sigma

Aldrich) and 30µL of 30µg/ml kanamycin. The growth was performed overnight at 37°C and 250 rpm in

an orbital shaker (AGITORB 200) until an optical density of at least 1(at 600nm) was obtained, so that

exponential growth was guaranteed upon cell collection. After this the inoculum was performed. The

first thing to do was to determine the volume of pre-inoculum necessary to start the inoculums with an

O.D. equal to 0.2 at 600 nm. To do this it was necessary to use Equation 1, where O.D.i and O.D.f

correspond respectively to the O.D. from the pre-inoculum (which is the initial O.D.) and the O.D.

required for the inoculum, which is 0.2; Vi a Vf are the volume of pre-inoculum necessary to start the

inoculum and the volume of inoculums, respectively.

𝑂𝐷𝑖 × 𝑉𝑖 = 𝑂𝐷𝑓 × 𝑉𝑓 (1)

The inoculum was grown in 2000 ml Erlenmeyers with 250 ml LB medium previously autoclaved

and 250 µL of 30µg/ml kanamycin. The cells were grown until an O.D. at 600 nm of around 3 was

obtained, which indicated that stationary phase was reached. The cells were then harvested by

centrifugation at 6000xg for 15 minutes, at 4°C, with a SLA 3000 rotor in a Sorvall RC6 centrifuge. The

supernatant was discarded and the pellet was then used for the alkaline lysis. If the alkaline lysis was

not performed right after the centrifugation the pellet was stored at 4°C for further processing7.

For comparative studies in the chromatographic assays, plasmids pCEP4 (Invitrogen) and

pVAX1TSAGFP, with 10410 and 5112 bp, respectively were also used. These plasmids had E. coli

DH5α as host cells and their growth was promoted in the same way as explained before with the

exception of the antibiotic used in pCEP4 that was ampicillin.

33

2.1.2 Cell lysis and Plasmid Primary Isolation

The recovered pellet was then suspended in a volume of P1 solution (50 mM glucose, 25 mM

Tris-HCl pH 8.0, 10 mM EDTA pH 8.0) determined by using Equation 2. To ressuspend the cells a

vortex was used. After this the total volume of solution was passed to two smaller centrifuge tubes (45

ml). In these tubes solution P2 (0.2 M NaOH, 1% (m/v) SDS) was added. The volume necessary of P2

was half the volume of solution P1. The addition of this solution was necessary to start the alkaline

lysis. This was followed by gentle homogenization and rest at room temperature for 10 min. To stop

the lysis solution P3 (5M potassium acetate, 6.8 M glacial acetic acid) was used in the same volume of

P2. This was followed by gentle homogenization and rest on ice for 10 min. The resulting suspension

was then centrifuged in a SS-34 rotor for 30 minutes at 20000xg and 4°C in a Sorvall RC6 centrifuge.

This centrifugation was performed to remove proteins, precipitated gDNA and cell debris. The

supernatant was recovered and centrifuged again in the same conditions. The lysate was then stored

at -20°C or the primary isolation was performed right after this step.

VP1 =O.D.×Inoculum Volume

60 (2)

The first step of the primary isolation was the addition of 99.6% (v/v) isopropanol to the

alkaline lysate. A volume corresponding to 70% of the lysate total volume was added7. The mixture

was then gently mixed and left at -20°C at least 2 hours. In this step all the nucleic acids present in the

lysate are precipitated to promote their recovery. The mixture was the centrifuged in the SS-34 rotor

using the same settings as in the cell lysis. The supernatant was discharged and the tubes with the

resulting pellet were inverted on the top of absorbent paper to remove the remaining isopropanol. After

this, 500 µL of Tris-HCl 20 mM pH 8.0 was added to each tube and the pellet was ressuspended.

The clarification of the lysate was then performed by adding 0.165g of ammonium sulphate

right before the injection in a column for the chromatographic assays7. This step was necessary to

remove traces of impurities left in the lysate such as proteins and high molecular weight RNA. The

mixture was homogenized and left on ice for 15 minutes followed by centrifugation for 30 minutes at

20000xg and at 4°C. The supernatant was then transferred to new eppendorf tubes and stored at -

20°C until future processing.

2.2 Desalinization of a clarified E. coli lysate

To promote the hydrophilic environment necessary for some binding assays it was necessary

to proceed to the desalinization of the clarified E. coli lysate. The protocol applied followed the

manufacturer’s instructions. Amicon Ultra-0.5 mL Centrifugal Filters for DNA purification and

concentration were used with a 3K filter in which molecules smaller than 3000 Da, like the salt

particles, are filtered and exit the main solution while the nucleic acids are retained.

Samples of 1 ml of clarified lysate were divided into two aliquots of 500μl and each one was

transferred to a 3K filter. The two eppendorfs were centrifuged (Eppendorf centrifuge 5417R) at room

temperature for 20 min at 14000xg. The content of the eppendorf was discarded and the volume of

34

sample contained in the filter was passed to a new eppendorf followed by the addition of 800μl of

20mM Tris-HCl pH 8.0.

2.3 Synthesis of triazine-based adsorbents in Sepharose CL-6B

The synthesis of triazine-based ligands was performed in Sepharose CL-6B using a well-

established described methodology38

.

2.3.1 Epoxy activation of Sepharose CL-6B

The epoxy activation was performed according to a method described previously39

. The first

step was to wash the Sepharose CL-6B on a sinter funnel with distilled water to remove the ethanol

solution used to store the gel. The gel was then suspended in 0.8 ml of 1M NaOH per gram of moist

gel and 0.1 ml of epichlorohydrin was added per gram of gel. The mixture was incubated overnight

with gentle agitation in an orbital agitator AGITORB 200 with a 170rpm agitation,at 30°C. The

activated gel was washed thoroughly with distilled water and used for the amination step.

2.3.2 Amination of previously epoxy-activated Sepharose CL-6B

The epoxy-activated agarose was aminated according to a protocol previously described28

.

The epoxy-activated gel was suspended in 1.5 ml of ammonia per gram of moist gel. The slurry was

incubated overnight with gentle agitation in an orbital agitator AGITORB 200, at 30°C. The aminated

gel was then washed with distilled water to remove the remaining ammonia. Washing was performed

until the pH decreased to the pH of distilled water and no ammonia odour could be detected. The

aminated support was either used immediatly for activation with cyanuric chloride or stored in 20%

(v/v) ethanol at 0-4°C.

2.3.2.1 Determination of primary amine groups in Sepharose beads

The density of the primary amine groups on the aminated gel was determined with a 2,4,6-

trinitrobenzenesulphonic acid (TNBS) based method40

. This method is based on the reaction of the

matrix with an excess of TNBS and the spectrophotometric analysis of the remaining TNBS by

reaction with glycine, after the removal of the solid phase. The first step is the addition of 4.5 ml of

0.1M sodium tetraborate (Na2B4O7) and 0.5 ml of 0.01 M TNBS to an amount of aminated gel

containing not more than 2-2.5 µmol of amino groups. A reference sample was also prepared but

without the gel. This mixture was incubated for 2 hours in an orbital agitator AGITORB 200, at 37°C.

After the incubation, the gel was centrifuged for 5 minutes at 2655xg and 0.5 ml of supernatant was

diluted with 2.5 ml of 0.1M Na2B4O7 and 0.25 ml of 0.03M glycine. For each sample, a blank was

prepared with 0.5 ml of supernatant, 2.5 ml of Na2B4O7 and 0.25 ml of water instead of glycine. The

mixture was left for 25 minutes at room temperature and in the end 5 ml of cold methanol was added.

The absorbance of each sample was then determined against its own blank at 340 nm. The

concentration of amino groups was determined from the difference between absorbances of each

sample and the reference sample and with a molar absorption coefficient ε (trinitophenyl derivative of

glycine) equal to 1.24x104 M

-1.cm

-1.

35

2.3.3 Activation of aminated Sepharose with cyanuric chloride

The aminated gel was suspended in 1 ml of acetone/water 50% (v/v) per gram of gel. The

slurry was incubated at 0°C, in an ice bath, in an Aralab AGITORB 200 shaker and an amount

corresponding to 5 molar equivalent relative to the extent of amination of cyanuric chloride was

dissolved in acetone (8.6 ml per gram of cyanuric chloride) and divided in four aliquots. Each aliquot

was added with a space of 30 minutes to the slurry while maintaining the mixture at 0°C and with

agitation. While this process was occuring the pH was monitored and maintained neutral by addition of

1M NaOH. The gel was then washed with 2x10 gel volumes of each acetone/water mixture (v/v) – 1:1,

1:3, 0:1, 1:1, 3:1, 1:0 – and then with abundant water to remove the remaining cyanuric chloride. The

activated gel was immediately used for the substitution of R138,41,42

.

2.3.4 Nucleophilic substitution of the second chlorine atom of dichlorotriazinyl Sepharose

(R1 substitution)

For the substitution of the second chlorine atom in the triazine ring, an amount corresponding

to 2 molar equivalent (relative to the determined density of amine groups in the support) of

phenethylamine, for ligand 5/6, and isomylamine, for ligand 6/5, was dissolved in distilled water (1 ml

of mixture per gram of gel). The slurry was then incubated at 30°C for 24 hours in a rotary shaker

AGITORB 200. After this period the gel was washed with distilled water in a sintered funnel. The gel

was either stored in 20% (v/v) ethanol at 0-4°C or used immediately for the substitution with an amine

compound at R2 position.


substitution)

The R2 substitution was performed with a 5 molar equivalent (relative to the determined

density of amine groups in the support) of isomylamine, for ligand 5/6, and phenethylamine, for ligand

6/5, dissolved in distilled water (3 ml per gram of gel). The slurry was then incubated in a rotary oven

from Amersham Pharmacia Biotech at 83°C, for 72 hours. After this period the gel was washed with

water and stored in 20% (v/v) ethanol at 0-4°C.

2.4 Chromatographic assays using ligands 5/6 and 6/5 in Sepharose

CL-6B

For the first assays, 2 ml of the different resins containing the affinity ligands 5/6

(phenethylamine/isomylamine) and 6/5 (isomylamine/phenethylamine) were packed in a 4ml (0.8 x

6cm) PD-10 column from Amersham-Pharmacia Biosciences. For each ligand two binding conditions

were tested: a hydrophobic environment, with the equilibration buffer being 0.4 M ammonium sulphate

in 20 mM Tris-HCl pH 8.0, and a hydrophilic environment, with 20 mM Tris-HCl pH 8.0 as equilibration

buffer. To perform the binding assay, 8 ml of regeneration buffer (NaOH (0,1M) in 30% (v/v)

isopropanol in distilled water) was passed through the column followed by 8 ml of water and 10 ml of

equilibration buffer. Then 100 µl of clarified lysate was injected in the column. After the flow passed

through, 2 ml of elution buffer was added and 200 µl fractions were collected. The columns were

36

washed with water and stored in 20% (v/v) ethanol at 4°C. The samples were analyzed by agarose gel

electrophoresis.

The synthesized resin 6/5 was chosen for further testing and packed in a TRICORN 10/50 (GE

HEALTHCARE) column to test in the AKTA purifier.

The clarified lysate extracted from E.coli was injected in the columns to test the binding in

different conditions. To test these conditions different buffers were used.

The columns were firstly equilibrated with 5 CVs of equilibration buffer. The sample loop used

(100μl) was emptied by passing equilibration buffer in the amount of three times its volume. After

equilibration and sample injection, the bound material was eluted by a simple washthrough process,

where the elution buffer was the same as the equilibration buffer. The eluted and washthrough

fractions were collected using a Frac-920 fraction collector and analyzed by agarose gel

electrophoresis. Each fraction collected had a volume of 500 µL. Selected samples with purified

plasmid were further quantified by HPLC analysis. In some assays different salt gradients were tested

to elute the bound material.

2.5 Synthesis of triazine-based adsorbents in CIM® monolithic disk

The synthesis of triazine-based ligands was performed in CIM® EDA monolithic disks (BIA

Separations, Ljubljana, Slovenia) that contain free amino groups that are required for the ligands

immobilization using the protocol described before38,42

. Because the monolith is a solid support it was

important to use quantities of reagents during the procedure enough to cover the entire monolith to

avoid dried spots. The quantities of the reagents where increased but the proportions used were

maintained as in the protocol with the Sepharose CL-6B.

2.5.1 Epoxy activation of the CIM® monolithic disk

In the epoxy activation step the monolith was first washed with distilled water to remove the

ethanol solution used in storage. Then, the monolith was placed in a 20 ml capped vial whit 2.67 mL of

1M NaOH and 0.33 ml of epichlorohydrin. The mixture was incubated overnight with gentle agitation in

a rotary shaker, at 30°C. The monolithic disk was then washed thoroughly with distilled water and

used for the amination step.

2.5.2 Amination of previously epoxy-activated disk

The epoxy-activated monolith was aminated with 5 ml of ammonia. The mixture was incubated

overnight with gentle agitation in a rotary shaker at 30°C. The monolith was then washed with distilled

water to remove the remaining ammonia until the pH decreased to the pH of distilled water and no

ammonia odour could be detected. The aminated disk was either used in the moment for activation

with cyanuric chloride or stored in 20% (v/v) ethanol at 0-4°C.

37

2.5.3 Activation of aminated CIM® disk with cyanuric chloride

The monolithic disk was suspended in 5 ml of acetone/water 50% (v/v) and incubated at 0°C

in an ice bath, on a shaker, with 0.83 g of cyanuric chloride dissolved in 7.17 mL of acetone and

divided in four aliquots. Each aliquot was added with a space of 30 minutes to the flask while

maintaining the mixture at 0°C and with agitation. While this process was occuring the pH was

monitored and maintained neutral by addition of 1M NaOH. The disk was then washed with 100 mL of

each acetone/water mixture (v/v) – 1:1, 1:3, 0:1, 1:1, 3:1, 1:0 – and then with abundant water to

remove the remaining cyanuric chloride. The substitution of R1 was immediately performed.

2.5.4 Nucleophilic substitution of the second chlorine atom of dichlorotriazinyl CIM® disk (R1

substitution)

For the substitution of the second chlorine atom in the triazine ring, 8.5µL of isomylamine was

dissolved in 3 mL of distilled water. The mixture was then incubated at 30°C, for 24 hours, in a rotary

shaker. After this period the monolith was washed with distilled water. The disk was either stored in

20% (v/v) ethanol at 0-4°C or used immediately for the R2 substitution.

2.5.5 Nucleophilic substitution of the third chlorine atom of dichlorotriazinyl CIM® disk (R2

substitution)

The R2 substitution was performed with a 21.3 µL of phenethylamine dissolved in 9 mL of

distilled water. The slurry was then incubated in a rotary oven (Amersham Pharmacia Biotech) at 80°C

for 72 hours. After this period the monolith was washed with water and stored in 20% (vv/v) ethanol at

0-4°C.

2.5.5.1 Optimization of the R2 substitution for the monolithic support

The CIM® disks chosen for future testing with the ligands have a drawback, the stability to

temperature. According to the manufacturer’s instructions these disks should not be subjected to

temperatures above 40°C. Because of this different temperatures and incubation times were tested for

the R2 substitution during the matrix derivatization. According to the protocol described before in 1.4.5

different incubation conditions were tested in Sepharose CL-6B for R2 substitution.

2.6 Chromatographic assays using ligands 6/5 in CIM® monolithic

disk

Monolithic disks, with a mean pore size of 657 nm, (CIM® EDA Disks, BIA Separations,

Ljubljana, Slovenia) were used as a solid phase. The disks were synthesized with the ligands 6/5

(isomylamine/phenethylamine) and then installed into specially designed cartridges from the same

manufacturer to perform some assays in the AKTA purifier. The clarified lysate was injected in the

column to test binding and elution profiles in different conditions.

The fractions were collected using a Frac-920 fraction collector and analyzed by agarose gel

electrophoresis. Selected samples, with purified plasmid, were further quantified by HPLC analysis.

38

2.7 Agarose gel electrophoresis

Electrophoresis was performed using GE Healthcare/Amersham Pharmacia Electrophoresis

EPS 3501 XL Power Supply and three submarine electrophoresis units: for 20cm gels a Hoefer HE

99, and for 10cm gels a Hoefer HE 33. The fractions recovered from the chromatographic steps were

analyzed in 1% agarose horizontal gel, where 20 µL of each sample was loaded. With the differents

submarines used, different voltages and times were applied: 100V for 1 hour for the 10cm gels and

120V for 1:30h for the 20cm gels. NZYDNA DNA Ladder III (NZYTech) was used as DNA weight

marker when plasmid samples were applied in the gels. After the runs, the gels were stained with

0.5mg/ml ethidium bromide and analyzed using Stratagene EagleEye II Video Imaging System.

2.8 HPLC analysis

The plasmid DNA collected during chromatographic elution experiments was quantified by

HPLC analysis43

. The column used was a 15 PHE-PE column (4.6mmx10cm) (GE Healthcare)

connected to an AKTA purifier system to accomplish a rapid analytical hydrophobic interaction

chromatography. The column was firstly equilibrated with 10mM Tris-HCl pH 8.0 buffer with 1.5M

ammonium sulphate. Then 100μl of each sample to be analyzed was injected. Elution of pDNA

isoforms was performed at 1ml/min for 0.8 min with equilibration buffer. After this, the ammonium

sulphate concentration was decreased to 0.0 M for 0.7 min, to elute bound species and in the end the

column was re-equilibrated with 1.5M ammonium sulphate in 10mM Tris-HCl pH 8.0 during 5.5 min.

The absorbance throughout the process was recorded at 260 nm. A calibration curve was constructed

with standard plasmid concentrations. The samples used for the different plasmid concentrations were

obtained by purifying pDNA using the High Pure Plasmid Isolation Kit (Roche), and by preparing

solutions of pure plasmids with concentrations between 0 and 70ng/µL. The peak areas of pDNA were

quantified and plasmid recovery yield (pDNA yield) was calculated according to Equation 3:

pDNA yield (%) =Mass of collected plasmid

Mass of injected plasmid× 100 (3)

The mass of injected plasmid is the mass of plasmid in the lysate solution that is injected in

the column and the mass of collected plasmid is the mass of plasmid that is collected from the

synthetic ligand column after the elution process. After determining the pDNA yield, the HPLC purity (ζ

(%)) was calculated with Equation 4, where Area (pDNA), Area total (lysate) and Area total (blank)

can be obtained from the chromatograms.

𝜁 (%) =𝐴𝑟𝑒𝑎 (𝑝𝐷𝑁𝐴)

𝐴𝑟𝑒𝑎 𝑇𝑜𝑡𝑎𝑙 (𝑙𝑦𝑠𝑎𝑡𝑒)−𝐴𝑟𝑒𝑎 𝑇𝑜𝑡𝑎𝑙 (𝑏𝑙𝑎𝑛𝑘)× 100 (4)

The purification factor (PF) was calculated by the reason between the final HPLC purity, after

the synthetic affinity ligand chromatographic run, and the initial DNA purity in the injected clarified

sample (Equation 5).

39

𝑃𝐹 =𝜁𝑓𝑖𝑛𝑎𝑙(%)

𝜁𝑖𝑛𝑖𝑡𝑖𝑎𝑙(%) (5)

2.9 Adsorption of Cutinase in the matrices tested

To prove that the derivatization of the ligands was successful in the CIM® monolithic columns a

control test solution with cutinase of 1 mg/mL was performed. This protocol followed a method

previously described that assessed that the ligand tested presented a binding capacity of 20-50%33

.

This assay was performed both in derivatized and non-derivatized monolithic disks in an AKTA purifier

system. To start the process towards cutinase the disks were washed with a regeneration solution

(0.1M NaOH in 30% (v/v) isopropanol), followed by distilled water and then by equilibration buffer

(20mM Tris-HCl, pH 8.0). A solution of 1 ml of cutinase (1mg/ml) was then injected. Protein was

measured by absorbance of the different fractions at 280 nm. The fractions were collected using a

Frac-920 fraction collector and quantified using a BCA™ Protein Assay Kit and the percentage of

protein realesed was determined. Samples were prepared with the addition and mixture of 25 µL of

sample solution in 200 µL of Pierce reagents (50:1 of reagent A in B) at the microplate wells. After

mixture the samples were incubated for 30 minutes at 37ºC. Then, absorbance was measured at

562nm in a microplate reader from Molecular Devices (Sunnyvale, CA, USA). The protein standard

used was bovine serum albumin (BSA) with a concentration range from 0 µg/mL to 2000 µg/mL. For

each sample, triplicates were made. The Micro BCATM

Protein Assay kit (for a protein concentration

range from 2 µg/mL to 40 µg/mL) was also used, when necessary. When using the Micro BCATM

Protein Assay, the sample volume was 150 µL and the detection reagent volume was 150 µL and the

microplate well was thoroughly mixed for some seconds and then incubated for 2 hours at 37ºC. This

assay was also performed using columns containing 1 mL of gel with both aminated and derivatized

Sepharose CL-6B to compare with the results obtained with the monolithic matrix.

3. Results and Discussion

3.1 Cell growth

E. coli cell hosting pVAX1-LacZ, pCEP4 and pVAX1TSAGFP plasmids were grown as pre-

inoculum overnight in 100 ml erlenmeyers containing 30 ml of LB medium at 37°C and 250rpm. A

sample of this pre-inoculum was then transferred to a 2000 ml Erlenmeyer with 250 ml of LB medium

to grow as an inoculum being necessary to determine the volume of pre-inoculum needed to achieve a

starting absorbance of 0.2.

After inoculation, the absorbance at 600 nm was read at specific times to allow following the

growth of the cells. Figure 15 illustrates the growth curves for the different recombinant E. coli cells.

40

Time (min)

0 100 200 300 400 500

O. D

. 600 n

m

0

1

2

3

4

5

6

pVAX1-LacZ

pVAX1TSAGFP

pCEP4

Figure 15 - Growth curve of E.coli cells with plasmid pVAX1-LacZ, C1-TSA and pCEP4

The maximum absorbance for pVAX1-LacZ was obtained at 375 minutes. After that, a slight

decline occurred. In the case of pCEP4 and pVAX1TSAGFP the maximum absorbances were

obtained at minute 330. To know in which phase the cells were it was necessary to follow the growth

for more hours. Because the growth of these strains was already well known it was possible to predict

that the cells were in the exponential phase, the phase where the cells have to be recovered to obtain

the biomass necessary to proceed to alkaline lysis and plasmid DNA recovery.

3.2 Preliminary assay using selected triazine-based ligands

The combination of ligands used in the present work was chosen after a preliminary screening

of a triazine-based ligand library. The ligands chosen were the phenethylamine/isoamylamine (5/6)

and its symmetric isoamylamine/phenethylamine (6/5). While phenethylamine is mimetic of

phenylalanine, isoamylamine is a mimetic of leucine. Both ligands were shown to exhibit a strong

binding to nucleic acids in both hydrophobic and hydrophilic environments34

.

Ligands 5/6 and 6/5 have been synthesized in Sepharose CL-6B following a well-established

procedure38

. During solid-phase synthesis it was important to determine the extension of the amination

in the support. This determination is useful not only to continue the synthesis but also to know if in the

end of the process all the amines have been substituted by the amine mimic compounds. The values

of amine density determined after amination and after R1 and R2 substituition of chlorines at the

triazine ring (see Figure 8) are shown in Table 2.

41

Table 2 - Concentration of free amine after the amination step and after derivatization with synthetic amines in

both R1 and R2 position: corresponding ligand concentration.

Ligands

Free Amines after Amination

(µmol/g gel)

Free Amines after Derivatization

(µmol/g gel)

Ligand

Concentration

(µmol/g gel)

5/6 25 0 25

6/5

1) 25 9E-07 25

2) 18.3 1.6 17

For assays with ligand 6/5 it was necessary to perform twice the solid-phase synthesis of the

ligand-adsorbent. The ligand density on the support was, however, slightly different as both the

amines after the amination step and the residual amines had distinct values in the second synthesis.

The concentration of free amines after amination was lower than the values obtained in the first

synthesis (25 vs 18.3 µmol/ g gel) leading to a lower density of ligands in the gel matrix, a result that

could affect the assays performed with this resin. However, some reports talk about the advantage of

using relatively low ligand density because a dense layer of ligand would obstruct the access of an

isolated ligand molecule onto the buried binding sites of pDNA44,45

.

3.3 Chromatographic assays with triazine-based ligands

In the chromatographic assays with the two selected triazine-based ligands (5/6 and 6/5) the

sample injected was a clarified lysate. These extracts contain plasmid DNA (supercoiled and open

circular isoforms), RNA, traces of genomic DNA and proteins. When such extracts are applied in the

column containing these ligands it is likely that the different types of nucleic acids can interact with the

immobilized ligand. The double-stranded plasmid has the hydrophobic bases shielded inside the

double helix affecting the retention times of the different pDNA isoforms present in the lysate46

. The

first chromatographic assay was performed with ligand 5/6 in hydrophobic conditions (20 mM Tris-HCl

buffer pH 8.0 with 0.4M ammonium sulfate). The profile obtained is shown in Figure 16 after

electrophoresis analysis.

By the gel analysis is possible to see that, starting in fraction 5 purified supercoiled pDNA was

obtained which means that in a hydrophobic environment pDNA is not retained in the column. As

opposite, RNA is retained in the column which means that it binds to the ligands likely due to the

exposition of the hydrophobic regions of RNA. This means that this molecule interacts strongly with

ligand 5/6. Not only the binding of RNA to the ligand affects its retention time but it is also necessary to

consider the size of the molecules. The smaller size of RNA molecules compared to pDNA can also

explain its retention in the column.

42

Figure 16 - Resulting agarose gel from the samples collected in the washthrough process of ligand 5/6 (2ml of

agarose). M – NZYDNA ladder III; 1 to 10 – Collected fractions after injecting 100 µl of the clarified lysate (containing pVAX1-LacZ plasmid) using 0.4M Ammonium Sulphate in 20mM Tris-HCl pH 8.0 as equilibration buffer (washthrough fractions).

When the same column was tested in a hydrophilic environment (20 mM Tris-HCl pH 8.0) the

profile obtained was different, as shown in Figure 17.


agarose) using 20mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid).

In these conditions it was not possible to obtain purified supercoiled DNA. All fractions present

oc pDNA and also RNA which is less retained and co-eluted with pDNA. This might be because in a

hydrophobic environment the hydrophobic residues of RNA are no longer exposed as they were and

ligand 5/6, which is a mimic of a hydrophobic dipeptide (Phe-Leu), can no longer interact with it.

An interesting fact observed in a previous screening was that ligand 5/6 presented symmetry

with ligand 6/5 with both exhibiting strong binding to nucleic acids in hydrophobic conditions. Based in

this previous result, it was also important to test ligand 6/5. This ligand was firstly tested in

hydrophobic conditions (20 mM Tris-HCl buffer pH 8.0 with 0.4 ammonium sulfate) (Figure 18).

43


agarose) using 0.4M Ammonium Sulphate in 20mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid). The dragging marks in some lanes are due to the presence of ammonium sulphate.

The profile obtained in these conditions is very similar to the one obtained with ligand 5/6 in

hydrophobic conditions. This was expected due to symmetry previously observed34

. So, with this

column in this environment it is also possible to purify supercoiled pDNA. Like it was explained before,

the exposed hydrophobic regions of RNA will interact with the ligand. This behavior is very similar to

the one normally obtained with HIC in which RNA is also retained by the hydrophobic resin while the

more hydrophilic plasmid DNA is excluded7. With this ligand it was apparently possible to separate sc

pDNA from other pDNA isoforms, in some of the washthrough fractions (fraction 4 – 9).

In hydrophilic conditions (20 mM Tris-HCl buffer pH 8.0) the profile obtained with ligand 6/5

was again very similar to the one obtained before under hydrophobic conditions (Figure 19).

Because of the symmetry previously reported for the two ligands it was expected a result

similar to that represented in Figure 17. This did not happen and in hydrophilic environment with

ligand 6/5 it was also possible to remove the RNA and separate the different pDNA isoforms. This

might show that even though ligands 5/6 and 6/5 are strong binders 6/5 is stronger than 5/6 what

allowed to retain the RNA longer in these conditions.

44


agarose) using 20mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid).

To confirm the results, the same assay was performed at the bench scale, using columns with

2 mL of gel, with different plasmids to ensure that the different plasmid sizes would not affect the

results obtained. The assays were performed with the same resin and buffers to avoid errors related

with small changes that could have occurred. The results are illustrated in Figure 20.


agarose) using 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 as equilibration buffer. The plasmids tested where pVAX1-LacZ (a), pCEP4 (b) and pVAX1TSAGFP (c). L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract). The dragging marks in some lanes are due to the presence of ammonium sulfate.

45

In these assays it was proven that the plasmid size does not affect the separation pattern. It is

possible to observe that it is possible to obtain purified pDNA with lysates containing different

plasmids, normally around fractions 4 to 7. In Figure 20a it was observed a fraction with apparently

only sc pDNA (fraction 8). However, the band was too faded and in this fraction traces of RNA were

present as contaminants. In Figure 20b all the pDNA collected was in the supercoiled isoform. This

result might be related to the low quantity of oc pDNA that was present in the initial sample. In all the

circumstances the RNA was no longer totally retained in the column in the final washthrough fractions.

This result is similar to other studies where it was possible to separate plasmids with different sizes

using the same chromatographic process47

.

Another important aspect was to test different types of buffers. To this purpose sodium citrate

was tested in different concentrations and the retention pattern was assessed. These results are

illustrated in Figure 21.


agarose) using 0.2 (a), 0.4 (b) and (c) 0.8M Sodium Citrate in 10mM Tris-HCl pH 8.0 as equilibration buffer. L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid). The dragging marks in some lanes are due to the presence of ammonium sulfate.

These results show that even using sodium citrate as elution buffer it is also possible to obtain

purified pDNA. In all concentrations tested RNA was no co-eluted with pDNA, being released only in

the column regeneration. However some differences could be observed with the increase of the salt

concentration. The major difference occured with the higher salt concentration tested, 0.8M sodium

citrate. In Figure 21c it is observed that the concentration of the initial extract in DNA and RNA was

46

much higher than the others. However, at this salt concentration, the pDNA samples collected

presented a much lower concentration which means that the DNA was retained in the column. This

could be related to the interaction of the molecules with the ligands. It is possible that at higher salt

concentrations the pDNA interacts strongly with the ligands by hydrophobic interaction, being

necessary to optimize the method to promote the molecules elution44

3.4 Chromatographic assays in AKTA purifier system with ligand 6/5

synthesized in Sepharose CL-6B

After performing the studies by gravity flow affinity chromatography columns experiments with

ligand 6/5 was also carried out in a more controlled system, the AKTA purifier. This system provides a

more controlled view of binding and elution processes and allows the control of the pressure and the

flow rate in order to optimize the elution process. Due to the results obtained previously with ligand

6/5, this ligand was chosen for further studies and was tested in this system. The conditions tested in

the AKTA purifier were the same as before, both in hydrophobic and hydrophilic conditions.

In the first experiments performed using ligand 6/5 in the AKTA purifier system, the clarified crude

extract from E. coli was injected into the column in hydrophobic conditions (0.4M Ammonium Sulfate in

20mM Tris-HCl pH 8.0) and pDNA was eluted in the washthrough. The results are illustrated in Figure

22.

Figure 22 - Chromatographic performance of ligand 6/5, in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The

run was performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a), b), c) and d) correspond to replicates of the same experiment with run d) being performed in a 2 ml column

a)

b)

c)

d)

oc pDNA

sc pDNA

oc pDNA

sc pDNA

fr. 2 - 7

fr. 8 - 14

fr. 2 - 5

fr. 6 - 14

fr. 2 - 4

fr. 5 - 14

fr. 2 - 3

fr. 4 - 14

47

Figure 22 (cont.) - Chromatographic performance of ligand 6/5, in a 6 ml column in an AKTA purifier system.

Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a), b), c) and d) correspond to replicates of the same experiment with run d) being performed in a 2 ml column

In these studies it is possible to notice that only in Figure 22c it was possible to obtain one

fraction with the sc pDNA isolated from other isoforms (fraction 5). However, the quantity of pDNA

obtained very low and contamination with RNA was observed after fraction 6. This result could be

connected to an insufficient column regeneration in between assays. In the other assays the RNA was

retained in the column and it was possible to obtain pure pDNA. Nevertheless, it was not possible to

isolate sc pDNA. One curious aspect is that in Figure 22a the concentration of RNA present in the

initial E.coli extract was very low, not being possible however to confirm that the collected fractions

were RNA-free since this could be very diluted and not detectable in the agarose gel. Another aspect

to note is the impossibility to get a better resolution in the chromatograms with a better separation of

the two peaks in the chromatogram. Once again, this was a result similar to HIC where the plasmid

molecules do not interact with the column being eluted in the flow through. This behavior is due to the

fact that hydrophobic bases of the double-stranded plasmid molecules are packed and shielded inside

the helix, leading to a minimal interaction with the chromatographic matrix46,48

The results obtained in the AKTA purifier system conditions were not similar to the ones

achieved in the same conditions in the gravity flow assays (Figure 18). One of the reasons might be

the initial concentration of DNA applied in the columns. In the chromatographic assays the initial

concentration of plasmid DNA applied in the column was between 170.1 and 875.4 ng/µL which are

high values, possibly affecting the results obtained. The yield of recovery of pure pDNA was 76% with

a)

b)

c)

d)

oc pDNA

sc pDNA

oc pDNA

sc pDNA

fr. 2 - 7

fr. 8 - 14

fr. 2 - 5

fr. 6 - 14

fr. 2 - 4

fr. 5 - 14

fr. 2 - 3

fr. 4 - 14

48

a purification factor of 11. The yield was higher than the reported value for experiments using HIC

(70%) but lower than the yield obtained in monolith membranes (100%)49,50

.

The next step was to perform a similar assay but in hydrophilic conditions. In this assay the

sample was loaded in 20 mM Tris-HCl pH 8.0 buffer. The results are illustrated in Figure 23.

The profiles obtained while performing this method were similar to those obtained in the

gravity flow assays. It is important to notice that the peak correspondent to the RNA is much bigger

than the other ones which means that the sample applied contained a higher RNA concentration.

Another important aspect is the fact that with the AKTA purifier only fraction 7 has the sc pDNA

isolated from the other isoforms when compared to the results obtained in the same environment in

the gravity flow assays (see Figure 19).

Figure 23 - Chromatographic performance of ligand 6/5, in a 6 ml column in an AKTA purifier system. Sample

(100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophilic conditions. The run was performed at room temperature at 1ml/min. 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

In the HPLC analysis, a pDNA yield of 47% was obtained which is much lower than the yield

obtained for experiments with the same ligand in a hydrophobic environment. The purification factor

obtained for this condition was 3.

Another study performed with ligand 6/5-adsorbent was the separation in a hydrophobic

environment with increased ionic strength in the buffer to understand how it would affect the

separation pattern. To perform this, the concentration of ammonium sulfate in the equilibration/elution

buffer was increased to 0.8M. The results are presented in Figure 24.

In Figure 24a the initial sample presents a very low concentration of plasmid DNA and no

detectable RNA, with only three fractions containing pDNA after elution. In fraction 5 apparently only

sc pDNA was present. However, like it was explained before, it is not possible to confirm if RNA was

retained in the column because the E.coli extract has no RNA or it is present in extremely low

concentration. In Figure 24b the isolation of sc pDNA was not accomplished but the RNA was

retained in the column so it was possible to obtain pure pDNA. These results are similar to the results

obtained with 0.4M ammonium sulfate, so the increase of the ionic strength seems not to affect the

pattern of elution. Further HPLC analysis was performed to access the yield achieved. The result

oc pDNA

sc pDNA

L S 3 4 5 6 7 8 9 10

fr. 3

fr. 4 - 6

fr. 7 - 10

49

obtained was around 65% with a purification factor of 3. Both these values are lower than the results

obtained with 0.4M ammonium sulfate. This might explained by a stronger interaction of the nucleic

acids with the ligand, leading to a partial retention of plasmid molecules and a lower exclusion of

pDNA.

Figure 24 - Chromatographic performance of ligand 6/5 an AKTA purifier system. Sample (100μl clarified E. coli

crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 0.8M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) and b) correspond to replicates of the same experiment with a) being performed in a 6 ml column and b) in a 2 ml column.

As so, after performing assays with an isocratic elution it was necessary to execute some

assays with a linear elution gradient to understand if it was possible to obtain sc pDNA with the

decrease of salt concentration. These assays were performed with a starting concentration of 1.5M

ammonium sulfate in 10mM of Tris-HCl pH 8.0 and ending elution with only 10mM Tris-HCl pH 8.0,

mimicking an environment similar to the one used in HIC 7 (Figure 25).

In the first run performed (Figure 25) the separation pattern was very different from all the

results obtained before. In the beginning of elution, where the salt concentration was higher, the oc

and sc pDNA were eluted. Still, with the decrease of salt concentration it was possible to elute more sc

pDNA. This happens because with the decreasing of the ionic strength the environment becomes

a)

b)

oc pDNA

sc pDNA

oc pDNA

sc pDNA

fr. 2 - 5

fr. 6 - 15

fr. 2 - 3

fr. 4 - 14

50

similar to hydrophilic conditions. It was already proven that ligand 6/5 presents low binding in

hydrophilic conditions, what would cause the elution of the sc pDNA 34

.

Figure 25 - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 1.5M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a negative linear gradient until 10 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample.

While this was an important result it was essential to evaluate its reproducibility, being

necessary to complete more runs in the same conditions (Figure 26).

Figure 26 - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 1.5M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a negative linear gradient until 10 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. a) and b) correspond to duplicates of the same assay.

fr. 2 - 6

fr. 7 - 14

fr. 15 - 28

oc pDNA

sc pDNA

sc pDNA

a)

b)

oc pDNA

sc pDNA

fr. 2 - 6

fr. 7 - 14

oc pDNA

sc pDNA fr. 2 - 4

fr. 5 - 10

51

Figure 26 (cont.) - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic

conditions. The run was performed at room temperature at 1ml/min. 1.5M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a negative linear gradient until 10 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. a) and b) correspond to duplicates of the same assay.

In these runs the results obtained were different from those represented in Figure 25. These

results were similar to all the results obtained in all the other conditions tested where it was possible to

remove all the RNA but it was not possible to separate the pDNA isoforms.

At the bench gravity flow assays using a column with 2 mL of gel it was performed an assay

with sodium citrate. So, it was also important to see the molecules behavior in the same conditions in

a more controlled system. For that effect, 0.8M sodium citrate was used as elution buffer when

performing some runs in the AKTA purifier. The runs were performed at 1 and 0.5 mL/min. The results

are illustrated in Figure 27.

Figure 27 - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The

run was performed at room temperature at (a) 1ml/min and (b) 0.5ml/min. 0.8M Sodium Citrate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

a)

b)

oc pDNA

sc pDNA

fr. 2 - 6

fr. 7 - 14

oc pDNA

sc pDNA fr. 2 - 4

fr. 5 - 10

a)

b)

oc pDNA

sc pDNA

oc pDNA

sc pDNA

fr. 2 - 5

fr. 6 - 14

fr. 2 - 5

fr. 6 - 14

52

Figure 27 (cont.) - Chromatographic performance of ligand 6/5 in a 6 ml column in an AKTA purifier system. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic

conditions. The run was performed at room temperature at (a) 1ml/min and (b) 0.5ml/min. 0.8M Sodium Citrate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

The results obtained in these conditions where very similar to those obtained in the gravity

flow assays. The first test was performed with a flow of 1 ml/min (Figure 27a). In these conditions the

purification of sc pDNA was obtained starting in fraction 5. This was a good result however it was

important to try to separate the peaks obtained to get a better resolution. To achieve this, the flow rate

was decreased to 0.5 ml/min (Figure 27b). The results were similar and the separation of the peaks

was not achieved meaning that the flow rate decrease was not enough to obtain a good resolution.

The yield in pure pDNA obtained in these conditions was 78% with a purification factor of 2. This yield

achieved was higher than prior studies where sodium citrate was used in replacement of ammonium

sulfate in HIC (59%) but the purification factor presented a lower value (6.8)51

.

The analysis performed before had demonstrated that, in hydrophobic conditions RNA was

retained in the column, while a large portion of pDNA was excluded in the washthrought fractions.

Some assays were carried out by gravity flow assays to confirm that the pDNA exclusion was related

with the molecules interaction with the ligand and not with the matrix binding capacity. Samples in

different decreasing dilutions were therefore loaded in the column under identical buffer conditions

(Figure 28).

The results presented above showed that the separation of pDNA from RNA was due to a

stronger interaction of RNA with the ligand and not because the binding capacity of the matrix was

exceeded. This can be confirmed because if it were a case of capacity the pDNA would not be

excluded when the injected sample was too diluted. In Figure 28 it is possible to observe that the DNA

is excluded in the first fractions in all the dilutions tested, even when the lysate was loaded with a

dilution of 1:100 (Figure 28d) where the sample is so diluted that the RNA is no longer detected in the

agarose gel. Another important aspect to notice is that the separation pattern is maintained with the

constant sample dilutions.

a)

b)

oc pDNA

sc pDNA

oc pDNA

sc pDNA

fr. 2 - 5

fr. 6 - 14

fr. 2 - 5

fr. 6 - 14

53


agarose) using 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 as equilibration buffer. The same sample was loaded in different dilutions: 1:10 (a), 1:20 (b), 1:50 (c) and 1:100 (d). L – NZYDNA ladder III; S – Loaded sample (100 μl clarified E. coli crude extract containing pVAX1-LacZ plasmid). The dragging marks in some lanes are due

to the presence of ammonium sulfate.

3.5 Optimization of ligand derivatization

After all the assays were performed in Sepharase CL-6B it was important to test the selected

ligand (6/5) in other supports. The support selected in the present work was the CIM® monolithic disk.

According to the manufacturer’s instructions CIM® monolithic disks should not be exposed to

temperatures higher than 40°C. The protocol for solid-phase ligand synthesis on CIM required a step

at 82°C for 3 days (see Materials and Methods, section 2.6.5). It was therefore attempted to make the

R2 amine substitution on Sepharose CL-6B at lower temperatures and different incubation times in

order to access the degree of derivatization. The results are illustrated in Figure 29.

In Figure 29 it is possible to observe that the optimal conditions found for R2 substitution were

80°C for 3 days, these being the conditions already stated in the well-established Sepharose

derivatization38

. Under these conditions a higher percentage of derivatization (92.2%) was obtained

with a lower standard deviation (1.4%). In all the other conditions tested presented a lower degree of

derivatization (between 26.4 and 62.2%) with higher standard deviations which would make the

derivatization process less efficient and trustable. Based upon this study, it was decided to proceed

a) b)

c) d)

54

with the derivatization of CIM® disk at 80°C during 3 days (for R2 substitution). During the 3 days the

monolith was observed several times a day to ensure that there was no damage at the outside of the

disk. While this was accomplished it was not possible to know if there were changes internally and if

the ligands were really synthesized in the CIM® monolithic disk. For this it would have been necessary

to visualize the internal surface of the monolith (possibly using methods like SEM, for example37

).

Figure 29 – Percentage of derivatization of agarose with ligand 6/5 in the different conditions tested for R2 amine

substitution.

3.6 Chromatographic assays with CIM® monolithic disks

3.6.1 Assays performed with ligand 6/5 derivatized CIM®

disk under hydrophobic conditions

The first assays performed with the monoliths derivatized with ligand 6/5 aimed at the

optimization of the chromatographic run. Monolithic columns have been previously described as a

good option to achieve pDNA purification due to their high binding capacity, excellent mass transfer

properties and high number of accessible binding sites for large biomolecules. In addition, with

monolithic columns the target molecule can be eluted in a concentrated form with a reduced

degradation due to the short contact times with the chromatographic matrix52,53

. To optimize the

chromatographic procedure in ligand 6/5-derivatized CIM® monolith all the different steps of the

procedure were performed in different ranges of column volumes (CV) until an optimal process was

reached. In the equilibration and elution steps 20 mM Tris-HCl buffer pH 8.0 with 0.4M ammonium

sulfate was used. The results of these experiments are shown in Figure 30.

The first assay was performed with 20 CV of equilibration buffer, 5 CV of elution buffer, 5 CV

of regeneration buffer and 5 CV of water at a flow rate of 1 ml/min (Figure 30a). In the chromatogram

it is possible to observe that the volumes of elution, regeneration and cleaning of the column were not

Conditions Used

50ºC, 3 days 50ºC, 6 days 60ºC, 3 days 65ºC, 3 days 65ºC, 6 days 70ºC, 3 days 80ºC, 1 day 80ºC, 3 days

De

riva

tiza

tio

n P

erc

en

tag

e

0

20

40

60

80

100

55

enough to have an efficient separation method. However, in the agarose gel it is possible to notice that

from the few fractions collected two of them presented purified sc pDNA (fractions 3 and 4). It is also

observed that the RNA was retained in the column. Because monoliths have bigger pores than

Sepharose it is confirmed that the pDNA is excluded from the column because it does not interact with

the immobilized ligand and not because of a size exclusion mechanism.

Figure 30 – Optimization of the chromatographic performance of ligand 6/5 in CIM® monolithic disks for pDNA

purification. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in

hydrophobic conditions. The run was performed at room temperature. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

56

Figure 30 (cont.) – Optimization of the chromatographic performance of ligand 6/5 in CIM® monolithic disks for

pDNA purification. Sample (100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

57

The second assay was performed with 30 CV of equilibration buffer, 10 CV of elution buffer,

10 CV of regeneration buffer and 30 CV of water to clean the column (Figure 30b). The

chromatogram obtained in these conditions presented worst resolution than in the first conditions

tested. Not only the resolution was poor but the elution was also too short and did not allow the

washthrough of unbound molecules. However, the agarose gel presented the same pattern as most of

the gels obtained with Sepharose. All the RNA was retained in the column but it was not possible to

isolate sc pDNA from other isoforms.

The elution was then increased to 20 CV maintaining the other steps as in experiment b).

(Figure 30c).The profile obtained in this assay was totally different from the previous ones. Two peaks

were obtained, the first one during elution and the second one during the cleaning of the column with

water. However, during this assay only fractions of the first peak were collected, and it was necessary

to repeat this assay to perform a complete analysis (see Figure 31). In the chromatogram it is

observed that only fraction 2 and 7 present DNA. In fraction 2 it was obtained only sc pDNA but in very

low concentration. Fraction 7 presented pDNA in the different isoforms. Once again the RNA was

retained in the column.

The following assay performed used conditions similar to those of HIC but using a

equilibration/elution buffer with lower ionic strenght7. In this assay the equilibration and the elution

were performed with 0.4M ammonium sulfate followed by 10 CV of 20 mM Tris-HCl pH 8.0 and 30 CV

of water (Figure 30d). In the chromatogram it can be observed that in the first peak eluted only oc

pDNA could be detected in the agarose gel and pDNA was present in very low quantity. Further

analysis by HPLC confirmed that the yield in pDNA was around 7%, a low result when compared to

other studies53–56

.

Based upon the physical and chemical composition of the monolithic columns it was expected

that the different flow rates used would not affect the separation selectivity, being flow-rate

independent53

. As so, the following assays were performed in the same conditions as before (Figure

30d) but with a flow rate of 2 ml/min (Figure 30e and 30f). In these assays two peaks of unbound

molecules were eluted in the beginning of the chromatographic separation. However, it is possible to

perceive that the elution step should be longer because when it ended there were still some molecules

being eluted. Nevertheless, in both assays it was possible to obtain purified pDNA with some RNA

being released in the end of elution. The chromatograms obtained in these conditions were similar to

the one in Figure 30d which demonstrates that the molecules separation is flow-independent as

expected.

After the optimization assays operational conditions were established as 30 CV of equilibration

buffer, 20 CV of elution buffer, 10 CV of regeneration buffer and 30 CV of water to clean the column

This method was performed a few more times to test its reproducibility. The results are shown in

Figure 31.

The first thing to observe in these assays are the chromatograms obtained (Figure 31a). It is

possible to understand that with different samples and in different days the chromatographic profile is

58

always similar, only varying the peak intensity that is related with the sample concentration. These

results are the first step to confirm the method reproducibility. Then it was necessary to evaluate the

agarose gels made for each chromatographic run. In the first chromatographic run, presented in

Figure 31b, it is shown that the first peak corresponds to the oc pDNA. The open circular isoform is

slightly more hydrophobic than the supercoiled and it is then likely that the sc pDNA is retained in the

column under hydrophobic conditions. The sc pDNA isoform was eluted on the step where water was

passed through the column (fractions 29-33). However, it is possible to see that in the fractions with sc

pDNA a low quantity of oc pDNA was also co-eluted. The RNA was retained in the matrix being eluted

only during the cleaning of the column.

The second chromatographic run (Figure 31c) showed a similar result to the prior one. The

main difference is related with the first 10 fractions collected. Despite the fact that in the previous

assay there was no pDNA visible, in fractions 1-10, in this assay it is possible to see both oc and sc

pDNA. This result might be related to the concentration of plasmid DNA loaded in the column. The

remaining fractions also feature some difference in the results because, oppositely to the previous run,

from fraction 13 to the last one there is predominantly sc pDNA with very low quantities of oc pDNA.

Figure 31 - Chromatographic performance in a CIM® monolithic disk derivatized with ligand 6/5. Sample (100μl

clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The runs

were performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) corresponds to the chromatograms obtained in the experiments; b), c), d) and e) corresponds to the agarose gels of each chromatographic run

59

Figure 31 (cont.) - Chromatographic performance in a CIM® monolithic disk derivatized with ligand 6/5. Sample

(100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The runs were performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) corresponds to the chromatograms obtained in the experiments; b), c), d) and e) corresponds to the agarose gels of each chromatographic run.

In the third chromatographic run (Figure 31d) it was possible to obtain pure oc pDNA in the

first eleven fractions due to this isoform lower hydrophobicity. In the next fractions it was obtained pure

pDNA but always with the oc and sc pDNA present. This result is somewhat different from the

previous chromatographic runs. This may be related with the sample loaded in the column, in which

the third run presented a much higher concentration in DNA and RNA than the previously used

samples.

c) Chromatographic Run 2

d) Chromatographic Run 3

e) Chromatographic Run 4

Washthrough Washing with water

oc pDNA

sc pDNA


oc pDNA

sc pDNA

sc pDNA

oc pDNA

oc pDNA

sc pDNA

60

The last chromatographic run performed (Figure 31e) shows a behavior similar to the third

one. The major difference is related with the first fractions collected, in which both isoforms are

excluded in the washthrough and water-eluted fractions. In this assay it was not possible to obtain

pDNA isoforms isolated from each other but, as shown in the previous results, it was possible to

remove all the RNA that remained retained in the monolith. While there were some differences in the

several assays performed under hydrophobic conditions they apparent to be related with the initial

extract loaded in the column. Similarly to what occurred in the Sepharose CL-6B assays, in a

hydrophobic environment the DNA is not retained in the column, so the ligand 6/5 do not bind to DNA,

while RNA is retained in the due to the higher exposition of hydrophobic regions in RNA34

. Further

HPLC quantitative analyses have shown that the yields in pDNA obtained were between 84 and

99.5% which is a good result when compared with other studies performed54,57

. The purification factor

obtained was around 4. When compared with the tests performed in the agarose matrix derivatized

with ligand 6/5 the main difference is the peak that shows up when water is passed through the

monolith. In this peak it is possible to elute remaining pDNA absorbed in the matrix, which leads to a

higher pDNA yield when using the CIM® monolithic disks (in agarose the pDNA yield was 76%).

To assess the effect of ammonium sulfate concentration in the binding profile, an experiment

was realized with an isocratic elution using 0.8M ammonium sulfate as equilibration and elution buffer.

The results are presented in Figure 32.

The chromatogram shows a similar profile to the ones obtained in the conditions represented

in Figure 31. However, when analyzing the agarose gel some important differences were observed.

The first peak eluted (fractions 2–20) contained mostly pDNA as in previous conditions. But, in the

fractions collected in the second peak eluted (fractions 30-33) RNA was present, affecting the purity of

the pDNA excluded. When compared to the results obtained with ligand 6/5 derivatized Sepharose

(see Figure 24) it is observed that in that matrix it was possible to obtain pure pDNA but with a slightly

lower yield. By HPLC analysis yields of 84% and 12% of pure and impure pDNA, respectively, were

obtained. Therefore, from these studies it was concluded that higher ionic strengths were not

favorable for the purification process in derivatized CIM® monoliths.


clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 0.8M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

(30 – 33)

sc pDNA

oc pDNA


(2 – 20)

61


(100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was performed at room temperature at 1ml/min. 0.8M Ammonium Sulfate in 10mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

An experiment was also performed in hydrophobic conditions with an isocratic elution with

1.5M ammonium sulfate in 20 mM Tris-HCl buffer pH 8.0. The results are shown in Figure 33.


clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The run was

performed at room temperature at 1ml/min. 1.5M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) and b) represent replicates of the same experiment.

(30 – 33)

sc pDNA

oc pDNA


(2 – 20)

62

The chromatograms obtained in these conditions showed two defined peaks with very good

resolution. However, in the agarose gel in the fractions correspondent to each peak neither pDNA nor

RNA were detected out. A SDS-PAGE gel analysis was performed to evaluate if the high intensity

observed could be due to the presence of remaining proteins (even though protein absorb light at a

maximum wavelength of 280 nm) but no proteins could be detected (results not shown). The only

exception occured in the second peak in Figure 33b where the fractions collected contained pure

pDNA with a yield of 63%. Similar results were obtained in the derivatized Sepharose CL-6B, in which

at high concentration of ammonium sulfate the pDNA and RNA were absorbed in the column, being

only eluted during the cleaning of the column (results not shown). This is comparable to other reports

in the literature for the purification of plasmids with other hydrophobic ligands44

.

After conducting assays with an isocratic elution it was also important to perform some tests

with a gradient elution. In this case the initial ammonium sulfate concentration, of 0.4M, was

decreased gradually during elution until there was 0M ammonium sulfate in the matrix. The results are

illustrated in Figure 34.

Similar chromatograms were obtained in the runs performed (Figure 34a). In the first

chromatographic run the first four fractions collected were shown to contain oc, sc, and also linear

pDNA (fractions 2-4), while the following 3 fractions contained only oc pDNA (Figure 34b). Again, the

presence of several isoforms in the first fractions might be associated with the quantity of pDNA

present in the initial sample injected in the column. One the other hand, in the agarose gel it was not

possible to understand what was eluted in the second peak. In a second chromatographic run (Figure

34c). Only oc pDNA could be detected in the first seven fractions, while in the second peak the elution

of both isoforms was achieved in some fractions. Overall, this process was apparently similar to the

assays performed with isocratic elution, reaching a yield of pDNA around 89% but with a purification

factor of 8.


clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The runs was performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a negative linear gradient until 20 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) corresponds to the chromatograms obtained in the experiments; b) and c) corresponds to the agarose gels of each chromatographic run.

63


(100μl clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophobic conditions. The runs was performed at room temperature at 1ml/min. 0.4M Ammonium Sulfate in 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution was performed with a negative linear gradient until 20 mM Tris-HCl pH 8.0 (0M Ammonium Sulfate). Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) corresponds to the chromatograms obtained in the experiments; b) and c) corresponds to the agarose gels of each chromatographic run.

So far, all the assays performed with the derivatized monolithic disks in hydrophobic

conditions indicated that the most efficient way to obtain pure pDNA was to perform an isocratic

elution with 0.4M ammonium sulfate in 20 mM Tris-HCl pH 8.0. But, the main objective of this work,

which was to purify sc pDNA, was not accomplished in these assays.

Some studies reported in the literature performed in CIM® monolithic disks (non-grafted CIM

®

CDI disks) have shown that it is possible to obtain purified sc pDNA53

. Another study, performed with

CIM® IDA monolithic disks has shown a removal of impurities with a yield of oc and sc pDNA around

90%54

. A comparable result could not be achieved in the assays performed with higher concentrations

of ammonium sulfate in the CIM®

disk derivatized with ligand 6/5. However in the assays performed

with 0.4M ammonium sulfate it was possible to achieve yields in oc and sc pDNA higher than 90%. A

drawback of these tests in hydrophobic conditions is the use of salts to achieve the purification.

64

3.6.2 Assays performed with ligand 6/5 derivatized CIM® disk under hydrophilic conditions

The CIM® monolithic disk derivatized with ligand 6/5 was also tested using hydrophilic

conditions. The first assays where realized with an isocratic elution in 20 mM Tris-HCl buffer pH 8.0

using 20 CV for washthrough elution and are represented in Figure 35.


clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophilic conditions. The run was

performed at room temperature at 1ml/min. 20mM Tris-HCl pH 8.0 was used as equilibration buffer and elution buffer. Agarose gel corresponds to the chromatogram presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel. a) and b) represent duplicates of the same assay.

The first assay performed in these conditions, illustrated in Figure 35a, showed a similar

profile to the one obtained when using 1.5M ammonium sulfate as elution buffer (isocratic). In the

agarose gel no molecules could, however, be detected in the first peak due to the low quantity of

pDNA eluted in this step. In the second peak, the pDNA was co-eluted with RNA. The second assay

performed in these conditions showed a similar chromatogram (Figure 35b). The agarose gel shows

the elution of oc and sc pDNA in the first peak but with a higher quantity of oc pDNA and two fractions

containing pure oc pDNA (fractions 7 to 9). In the second peak pDNA was elute with a small quantity

of contaminating RNA. Plasmid DNA is highly negatively charged molecule and due to its size and

charge plasmid molecules interact with the positively charged resins through several binding sites58

. In

this case, as the ligand on the matrix has a predominant hydrophobic nature, electrostatic interactions

can only take place between negatively charged phosphate groups and remaining (non-substituted)

a) b)

(2 – 8) (31 – 34)


oc pDNA

sc pDNA

(2 – 9) (30 – 32)

Washing with water Washthrough

oc pDNA

sc pDNA

65

native amine groups in the monolith. The HPLC analysis has shown that it was possible to obtain an

average yield of 62% with a purification factor of 2.

In the following assays, sample was loaded in 20 mM Tris-HCl buffer pH 8.0 and a gradient

between 0-1 M of sodium chloride was applied in 20 CV, following by washing with water and column

regeneration. The results are shown in Figure 36.


clarified E. coli crude extract containing pVAX1-LacZ plasmid) was loaded in hydrophilic conditions. The runs was

performed at room temperature at 1ml/min. 20mM Tris-HCl pH 8.0 was used as equilibration buffer with a linear gradient with increasing of 1M NaCl during elution. Agarose gels correspondent to the chromatograms presented. L – NZYDNA ladder III; S – Loaded sample. The numbers in brackets on top of each peak correspond to the lanes in the 1% agarose gel.

When compared to the previous assay, a difference can be observed during the elution with

NaCl where two peaks were eluted (Figure 36). In the agarose gel correspondent to the

chromatogram it is observed that in the first peak a big part of the oc pDNA is eluted. Then, with the

decreasing of the electrostatic interactions the elution of both oc and sc pDNA occurs. Due to the low

quantity of DNA eluted in the third peak no molecules were detected in the gel. However, it is possible

that only remaining pDNA was eluted in these fractions. Like it was expected, with the increase of salt

concentration the remaining pDNA was eluted, even with low concentrations of NaCl45

. This assay

presented a yield of 95% of the pDNA recovered.

When analyzing all the results achieved when using ligand 6/5 derivatized monolithic support it

can be concluded that it was not possible to obtain the pure sc pDNA. Yet, it was possible to remove

all RNA from loaded samples. Further processing steps are required to separate the pDNA isoforms

prior to obtain therapeutic grade sc pDNA. However, this work proved that a monolith-based

chromatographic support derivatized with a biomimetic ligand is more advantageous than a

(18 – 22)

8 – 13)

Washing with water Washthrough

oc pDNA

sc pDNA

66

conventional particle-based support derivatized with the same ligand for purification of pDNA leading

to higher pDNA yields54

. Still, it shall be necessary to assess the presence of other impurities in the

collected fractions of pDNA.

3.7 Adsorption of Cutinase in ligand 6/5 derivatized matrices

The CIM® monolithic disks could not be subjected to the TNBS method, used in Sepharose

CL-6B to quantify initial and residual amine groups upon derivatization and ligand concentration on the

support (see Materials and Methods section 2.3.2.1). This is an invasive method that would destroy

the monolithic disk. As so, and despite the results obtained above that indicated the presence of

ligand 6/5 in the CIM®

derivatized monolith a further test was performed to compare derivatized and

non-derivatized CIM® disks. A previous screening of ligand 6/5 synthesized in agarose with cutinase

from F. solani pisi showed that this ligand tend to bind 50% of loaded protein under defined conditions

(see Materials and Methods section 2.10)33

. Therefore, a control test using cutinase was performed in

both non-derivatized and derivatized Sepharose and CIM® monolithic disks. The results are shown in

Figure 37.

Figure 37 - Percentage of cutinase binding and not binding to the different chromatographic matrices non-

derivatized and derivatized with ligand 6/5. A volume of 1 mL of a cutinase solution (1mg/mL) was loaded in each column.

The graph represented above shows that cutinase does not bind to aminated Sepharose, a

result that was already expected33

. In the Sepharose derivatized with ligand 6/5 a percentage of

46.1% of bound cutinase was obtained. This result is in accordance with the results achieved in

previous studies33

. In the non-derivatized CIM® monolithic disk the percentage of bound cutinase was

18.6%, which is a higher percentage than that obtained in the aminated Sepharose and may be

explained by unspecific interactions that take place with the monolith backbone. The CIM® EDA disk

has a weak type of anion exchange group that is fully charged between pH 3-959

. Another peculiar

aspect of the CIM® EDA disk is that during production of the matrix only 50-60% of the epoxy groups

67

are converted with the remaining epoxy groups being ended capped with OH groups (information

provided by BIA Separations). These groups available in the disk can have some capacity to interact

with cutinase, explaining the higher value of enzyme that was bound to the matrix. The derivatized

monolith presented a value of 68.1% of cutinase bound to the matrix. The difference (49.8%) is

comparable to the result obtained with Sepharose.

These results could confirm the presence of ligand 6/5 in the CIM® derivatized monolith,

although its density in the matrix was not determined in the present work.

4. Conclusion

The assessment of a large combinatorial library of triazine-scaffolded synthetic affinity ligands to

bind nucleic acids and potentially purify plasmid DNA from E. coli crude extracts was performed in a

previous work. From this previous screening, two symmetric ligands (5/6 and 6/5), mimicking a

dipeptide Phe-Leu, were selected and their capability to purify pDNA was studied.

Sepharose CL-6B derivatized with ligands 5/6 and 6/5 was used in gravity flow assays to purify a

plasmid (pVAX1-LacZ) in both hydrophobic and hydrophilic conditions. Under hydrophobic binding

conditions (20 mM Tris HCl buffer pH 8.0 with 0.4 M ammonium sulphate) both ligands have shown

that it was possible to purify sc pDNA in some fractions excluded from the columns. pDNA (oc pDNA

and sc pDNA) was separated from RNA that was totally retained in the column. With a hydrophilic

binding environment (20 mM Tris HCl buffer pH 8.0) the behavior of solid-phase synthesized ligands

was less symmetric. With ligand 5/6, the RNA was less retained in the column, and co-eluted partially

with pDNA. Oppositely, when performing the assays with ligand 6/5 the removal of RNA was

accomplished and isolation of sc pDNA was obtained in some fractions.

Ligand 6/5 was also shown to perform similarly with three plasmids of different sizes (pVAX1-

LacZ (6.1 kb), pCEP4 (10.4 kb) and pVAX1TSAGFP (5.1 kb)) under hydrophobic conditions.

Based on these results, ligand 6/5 was chosen as the lead to perform chromatographic assays in

the AKTA purifier system.

Different elution buffers were studied for separation of RNA from plasmid DNA isoforms using

Sepharose CL-6B derivatized with ligand 6/5 in the AKTA purifier system. Assays were performed with

0.4M, 0.8M and 1.5M of ammonium sulfate in the binding buffer. Similar purification results could be

obtained in these assays, although the reproducibility in some cases was not fully proven.

Nevertheless, in most assays, RNA was retained in the column while pDNA was excluded in the flow

through fractions. With 20 mM Tris HCl buffer pH 8.0 containing 0.8M sodium citrate, purification of sc

pDNA was also obtained in some fractions and pDNA was equally isolated from RNA.

The derivatization of CIM® monoliths with triazine-scaffold synthetic affinity ligands has been

attempted and successfully achieved.

68

CIM® monolithic disks were derivatized with ligand 6/5 following a solid-phase protocol well

established for agarose. It was demonstrated that, despite the manufacturer’s instructions, CIM®

monolithic disks stand stable at 82°C, for 72 hours. These conditions were established to be the

minimal required to ensure a value of 90% of derivatization in a systematic study of solid-phase

synthesis of ligand 6/5 in Sepharose CL-6B, at different temperatures and incubation times.

Although a quantitative assessment of ligand 6/5 density on the CIM® support was not possible

with the methodology used for Sepharose CL-6B (TNBS test) the presence of the ligand in CIM®-

derivatized disks was assessed and confirmed by the binding profile towards cutinase from Fusarium

solani pisi, which was similar to the one reported in previous studies with ligand 6/5-agarose

adsorbent.

Chromatographic assays were performed with the CIM® monolithic disk derivatized with ligand

6/5 in conditions previously tested with ligand 6/5 derivatized Sepharose CL-6B. Similar elution

patterns for pDNA and RNA were obtained when comparing with Sepharose CL-6B under identical

binding/elution conditions. In hydrophobic (best) conditions pDNA was excluded in the wash through

fractions while RNA was mostly retained in the column.

With CIM® monolithic disks, pure plasmid DNA was isolated from E. coli lysate extracts with

higher yield (91%) as compared to Sepharose (76.2) under best tested conditions (20 mM Tris HCl pH

8.0 with 0.4 M ammonium sulphate).

5. Further work

Further work will include to try to further optimize the binding and elution procedures in CIM®

derivatized monoliths with different concentrations of ammonium sulfate in the binding buffer, to

attempt the separation of sc pDNA (more hydrophobic) from oc DNA isoform (less hydrophobic). This

would include the use ammonium sulfate substitutes to promote hydrophobic interaction with the

ligand. Sodium citrate was used in the present work at 0.8 M concentration. However, some assays

could be performed in different concentrations. All tests were carried out in Tris-HCl buffer. Another

salt/buffer described in the literature as an effective promoter of hydrophobic binding with related

ligands is potassium phosphate51

. The binding temperature in hydrophobic conditions could also be

changed as it could affect ligand 6/5 affinity/binding towards pDNA.

The work for this dissertation was based in one ligand (6/5, mimic of a Phe-Leu dipeptide). Other

ligands already described as potential good ligands in the screening of a large combinatorial ligand

library could also be tested in CIM monolithic disks. Ligands such as 1/8 (mimic of Ala-Gln), 3/5 (mimic

of Tyr-Phe), 4/3 (mimic of Lys-Tyr), 5/8 (mimic of Phe-Gln) and 5/11 (mimic of Phe-Ile) that also

exhibited different affinities towards different DNA sequences could be exposed to the same tests

performed with ligand 6/5 in Sepharose CL-6B and, depending on the results, also in CIM® monolithic

disks34

.

69

In order to obtain a known density of ligands in the CIM® monolithic disks it is important to

perform a non-invasive methodology to quantify amine groups on the surface of the support. A

possible method is to monitor the initial and post-modification residual amine groups with a ionic

capacity measurement60

.

Finally, it would also be important to perform a purity assessment to quantify overall contaminants

removal. Only RNA removal was assessed in the present work. However, it would be necessary to

ensure the elimination of other contaminants like proteins, endotoxins and gDNA by performing

appropriate assays required for this evaluation44,55

.

70

6. References

1. Sousa, A., Sousa, F. and Queiroz, J.A. Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices. J. Sep. Sci. 33, 2610–8 (2010).

2. Han, Y., Liu, S., Ho, J., Danquah, M.K. and Forde, G.M. Using DNA as a drug—Bioprocessing and delivery strategies. Chem. Eng. Res. Des. 87, 343–348 (2009).

3. Shamlou, P.A. Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA. Biotechnol. Appl. Biochem. 37, 207–18 (2003).

4. Sousa, F., Prazeres, D.M.F. and Queiroz, J.A. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA. Trends Biotechnol. 26, 518–25 (2008).

5. Diogo, M.., Queiroz, J.A. and Prazeres, D.M.F. Chromatography of plasmid DNA. J. Chromatogr. A 1069, 3–22 (2005).

6. Ghanem, A., Healey, R. and Adly, F.G. Current trends in separation of plasmid DNA vaccines: a review. Anal. Chim. Acta 760, 1–15 (2013).

7. Diogo, M.M. Queiroz, J.A, Monteiro, G.A., Martins, S.A.M., Ferreira, G.N.M. and Prazeres, D.M.F. Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography. Biotechnol. Bioeng. 68, 576–83 (2000).

8. Prazeres, D.M.F., Ferreira, G.N.M., Monteiro, G.A., Cooney, C.L. and Cabral, J.M.S. Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy : problems and bottlenecks. 17, 169–174 (1999).

9. Ferreira, G.N.M., Monteiro, G.A., Prazeres, D.M.F. and Cabral, J.M.S. Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. 18, 380–388 (2000).

10. Prather, K.J., Sagar, S., Murphy, J. and Chartrain, M. Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification. Enzyme Microb. Technol. 33, 865–883 (2003).

11. Ferreira, G.N.M. Chromatographic Approaches in the Purification of Plasmid DNA for Therapy and Vaccination. Chem. Eng. Technol. 28, 1285–1294 (2005).

12. Balagurumoorthy, P., Adelstein, S.J. and Kassis, A.I. Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA. Anal. Biochem. 381, 172–4 (2008).

13. Sousa, A., Sousa, F. and Queiroz, J.A. Biorecognition of supercoiled plasmid DNA isoform in lysine-affinity chromatography. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 877, 3257–60 (2009).

14. Kelly, W.J. Perspectives on plasmid-based gene therapy: challenges for the product and the process. Biotechnol. Appl. Biochem. 37, 219–23 (2003).

15. Remaut, K., Sanders, N.N., Fayazpour, F., Demeester, J. and De Smedt, S.C. Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes. J. Control. Release 115, 335–43 (2006).

16. Hernández, O.R., Fernández, M.L., Hernández, J.V., Peña, M.P. and Díaz, E.M.. Scalable Technology to Produce Pharmaceutical Grade Plasmid DNA for Gene Therapy, Gene Therapy

71

- Developments and Future Perspectives, Prof. Chunsheng Kang (Ed.), ISBN: 978-953-307-617-1, InTech (2011)

17. Stadler, J., Lemmens, R. and Nyhammar, T. Plasmid DNA purification. J. Gene Med. 6 Suppl 1, S54–66 (2004).

18. Prazeres, D.M.F., Schluep, T. and Cooney, C. Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. J. Chromatogr. A 806, 31–45 (1998).

19. Freitas, S.S., Santos, J.A.L. and Prazeres, D.M.F. Plasmid purification by hydrophobic interaction chromatography using sodium citrate in the mobile phase. Sep. Purif. Technol. 65, 95–104 (2009).

20. Queiroz, J.A., Tomaz, C.T. and Cabral, J.M.S. Hydrophobic interaction chromatography of proteins. J. Biotechnol. 87, 143–59 (2001).

21. Tetala, K.K.R. and van Beek, T.A. Bioaffinity chromatography on monolithic supports. J. Sep. Sci. 33, 422–38 (2010).

22. Mallik, R. and Hage, D.S. Affinity monolith chromatography. J. Sep. Sci. 29, 1686–1704 (2006).

23. Hage, D.S., Anguizola, J.A., Bi, C., Li, R., Matsuda, R., Papastavros, E., Pfaunmiller, E., Vargas, J., Zheng, X. Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments. J. Pharm. Biomed. Anal. 69, 93–105 (2012).

24. Roque, A.C.A. and Lowe, C.R. Advances and applications of de novo designed affinity ligands in proteomics. Biotechnol. Adv. 24, 17–26 (2006).

25. Lowe, C.R., Lowe, A.R. and Gupta, G. New developments in affinity chromatography with potential application in the production of biopharmaceuticals. J. Biochem. Biophys. Methods 49, 561–74 (2001).

26. Lowe, C.R. Combinatorial approaches to affinity chromatography. Curr. Opin. Chem. Biol. 5, 248–56 (2001).

27. Roque, A.C.A., Taipa, M.A. and Lowe, C.R. A new method for the screening of solid-phase combinatorial libraries for affinity chromatography. J. Mol. Recognit. 17, 262–7 (2004).

28. Roque, A.C.A., Taipa, M.A. and Lowe, C.R. Synthesis and screening of a rationally designed combinatorial library of affinity ligands mimicking protein L from Peptostreptococcus magnus. J. Mol. Recognit. 18, 213–24 (2005).

29. Sousa, F., Cruz, C. and Queiroz, J.A. Amino acids-nucleotides biomolecular recognition: from biological occurrence to affinity chromatography. J. Mol. Recognit. 23, 505–18 (2010).

30. Sousa, F., Tomaz, C.T., Prazeres, D.M.F. and Queiroz, J.A. Separation of supercoiled and open circular plasmid DNA isoforms by chromatography with a histidine-agarose support. Anal. Biochem. 343, 183–5 (2005).

31. Sousa, F., Matos, T., Prazeres, D.M.F. and Queiroz, J.A. Specific recognition of supercoiled plasmid DNA in arginine affinity chromatography. Anal. Biochem. 374, 432–4 (2008).

32. Sousa, A., Sousa, F., Prazeres, D.M.F. and Queiroz, J.A. Histidine affinity chromatography of homo-oligonucleotides. Role of multiple interactions on retention. Biomed. Chromatogr. 23, 745–53 (2009).

72

33. Sousa, I.T., Ruiu, L., Lowe, C.R. and Taipa, M.A. Synthetic affinity ligands as a novel tool to improve protein stability. J. Mol. Recognit. 22, 83–90 (2009).

34. Belchior, J.F.R. Screening and evaluation of synthetic protein-mimic affinity ligands for the purification of plasmid DNA. M.Sc Diss. Biotechnol. Inst. Super. Técnico, Univ. Lisboa (2013).

35. Sproß, J. and Sinz, A. Monolithic media for applications in affinity chromatography. J. Sep. Sci. 1958–1973 (2011). doi:10.1002/jssc.201100400

36. CIM Convective Interaction Media. at <http://www.biaseparations.com/education/brochures/product/download/file_id-730>

37. Brne, P. Convective Interaction Media short monolithic columns : Enabling chromatographic supports for the separation and purification of large biomolecules Review. 1876–1892 (2005).

38. Palanisamy, U.D., Hussain, A., Iqbal, S., Sproule, K. and Lowe, C.R. Design, synthesis and characterisation of affinity ligands for glycoproteins. J. Mol. Recognit. 12, 57–66 (1999).

39. Filippusson, H., Erlendsson, L.S. and Lowe, C.R. Design, synthesis and evaluation of biomimetic affinity ligands for elastases. J. Mol. Recognit. 13, 370–381 (2000).

40. Antoni, G., Presentini, R. and Neri, P. A simple method for the estimation of amino groups on insoluble matrix beads. Anal. Biochem. 129, 60–3 (1983).

41. Teng, S.F., Sproule, K., Hussain, A. and Lowe, C.R. A strategy for the generation of biomimetic ligands for affinity chromatography . Combinatorial synthesis and biological evaluation of an IgG binding ligand. 67–75 (1999).

42. Roque, A.C.A., Taipa, M.A. and Lowe, C.R. An artificial protein L for the purification of immunoglobulins and Fab fragments by affinity chromatography. J. Chromatogr. A 1064, 157–167 (2005).

43. Diogo, M.M., Queiroz, J.A, Prazeres, D.M.F.. Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography. J. Chromatogr. A 998, 109–117 (2003).

44. Caramelo-Nunes, C., Gabriel, M.F., Almeida, P., Marcos, J.C. and Tomaz, C.T. Negative pseudo-affinity chromatography for plasmid DNA purification using berenil as ligand. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 944, 39–42 (2014).

45. Benčina, M., Podgornik, A. and Štrancar, A. Characterization of methacrylate monoliths for purification of DNA molecules. J. Sep. Sci. 27, 801–810 (2004).

46. Diogo, M.M., Queiroz, J.A., Monteiro, G.A. and Prazeres, D.M.F. Separation and analysis of plasmid denatured forms using hydrophobic interaction chromatography. Anal. Biochem. 275, 122–4 (1999).

47. Bicho, D., Sousa, Â., Sousa, F., Queiroz, J.A. and Tomaz, C.T. Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support. J. Sep. Sci. 37, 2284–92 (2014).

48. Moreira, K.A., Diogo, M.M., Prazeres, D.M.F., Filho, J.L.L. and Porto, A.L.F. Purification of plasmid ( pVaxLacZ ) by hydrophobic interaction chromatography. Braz. Arch.Biol. Technol. 48, 113–117 (2005).

73

49. Pereira, L.R., Prazeres, D.M.F. and Mateus, M. Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization. J. Sep. Sci. 33, 1175–84 (2010).

50. Urthaler, J., Schlegl, R., Podgornik, A., Strancar, A., Jungbauer, A. and Necina, R. Application of monoliths for plasmid DNA purification. J. Chromatogr. A 1065, 93–106 (2005).

51. Bonturi, N., Radke, V.S.C.O., Bueno, S.M.A., Freitas, S., Azzoni, A.R. and Miranda, E.A.Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate. J. Chromatogr. B. 919-920, 67–74 (2013).

52. Mota, É., Sousa, A., Černigoj, U., Queiroz, J.A., Tomaz, C.T. and Sousa, F. Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger. J. Chromatogr. A 1291, 114–21 (2013).

53. Sousa, A., Bicho, D., Tomaz, C.T., Sousa, F. and Queiroz, J.A. Performance of a non-grafted

monolithic support for purification of supercoiled plasmid DNA ଝ. J. Chromatogr. A 1218,

1701–1706 (2011).

54. Shin, M.J., Tan, L., Jeong, M.H., Kim, J.H. and Choe, W.S. Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification. J. Chromatogr. A 1218, 5273–8 (2011).

55. Sousa, A., Tomaz, C.T., Sousa, F. and Queiroz, J.A. Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications. J. Chromatogr. A 1218, 8333–43 (2011).

56. Han, Y., You, G., Pattenden, L.K. and Forde, G.M. The harnessing of peptide–monolith constructs for single step plasmid DNA purification. Process Biochem. 45, 203–209 (2010).

57. Smrekar, F., Podgornik, A., Ciringer, M., Kontrec, S., Raspor, P., Strancar, A. and Peterka, M. Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns. Vaccine 28, 2039–45 (2010).

58. Danquah, M.K. and Forde, G.M. The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 853, 38–46 (2007).

59. EDA for Ligand Immobilization. at <http://www.biaseparations.com/interactions/category/32-activated/category/118-eda-for-ligand-immobil>

60. Podgornik, A., Vidič, J., Jančar, J., Lendero, N., Frankovič, V. and Štrancar, A. Noninvasive Methods for Characterization of Large-Volume Monolithic Chromatographic Columns. Chem. Eng. Technol. 28, 1435–1441 (2005).

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7. Appendix

Appendix I - Plasmid pVAX1-LacZ (Invitrogen) information

Figure A1 - Structure and features of pVAX1/lacZ plasmid.

Weight: 6050 bp

CMV promoter: bases 137-724

T7 promoter/priming site: bases 664-683

LacZ ORF: bases 773-3829

BGH reverse priming site: bases 3874-3891

BGH polyadenylation signal: bases 3880-4104

Kanamycin resistance gene: bases 4277-5071

pUC origin: bases 5371-6044

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Appendix II – Plasmid pCEP4 (Invitrogen) information

Figure A2 - Structure and features of pCEP4 plasmid.

Weight: 10186 bp

CMV promoter: bases 1-588

Multiple cloning site: bases 619-676

SV40 polyadenylation signal: bases 685-926

OriP: bases 1349-3319

EBNA-1 gene (complementary strand): bases 3620-5545

Ampicillin resistance gene: bases 6171-7031

pUC origin: bases 7040-7815

TK promoter: bases 8183-8345

Hygromycin resistance gene: bases 8409-9419

TK polyadenylation signal: bases 9431-9708

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Appendix III – Plasmid pVAX1TSAGFP information

Figure A3 - Structure and features of pVAX1TSAGFP plasmid.

Weight: 5112 bp

Appendix IV - NZYDNA Ladder III (NZYTech)

Figure A4 - NZYDNA Ladder III electrophoresed in a 1% (w/v) electrophoresis grade agarose gel.

NZYDNA Ladder III

Catalogue numbers:

MB04401, 200 lanes

MB04402, 500 lanes

Storage conditions: NZYDNA Ladder III should be stored at -20 °C until first use. Thereafter, the product can be

stored at 4 °C for up to 6 months. Avoid multiple freeze thaw cycles, as these can damage the product.

Shipping conditions: Room temperature

Product life: Three years

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Description: NZYDNA Ladder III is a ready-to-use molecular weight marker, specially designed for easy size

determination and DNA quantification. For best results using our ladder range we recommend using NZYTech

agaroses.

Sizing: NZYDNA Ladder III produces a pattern of 14 regularly spaced bands, ranging from 200 to 10000 bp

Quantification: When using the standard loading of 5 µL per lane (714 ng of DNA) each band corresponds to a

precise quantity of DNA

Appendix V - Pure plasmid DNA standard curve for analysis by HIC in a

HPLC system

Figure A5 - Calibration curve obtained by HIC in a HPLC system, using standard plasmid concentrations.

Linear equation obtained from the pDNA calibration curve:

𝑦 = 0,3833𝑥 − 0,5291

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