Preparative chromatography

for modified oligonucleotides Method development for modified oligonucleotides, from

analytical to preparative chromatography

Preparativ kromatografi för modifierade oligonukleotider

Metodutveckling för modifierade oligonukleotider, från analytisk till

preparativ kromatografi

Rebecka Jasinski

Fakulteten för hälsa, natur- och teknikvetenskap

Examensarbete, civilingenjör kemiteknik

30 hp Handledare: Torgny Fornstedt (Karlstads Universitet) / Olof Stålberg (SGS DNA)

Examinator: Magnus Lestelius

2021-06-13

Abstract

Synthetic oligonucleotides, which are short strings of DNA or RNA, are a grooving area of

importance for the pharmaceutical industry and for companies that manufacture diagnostic

components. The manufacturing process of synthetic oligonucleotides involves many

complex processes that use separation and purification techniques like ion-exchange

chromatography, ion-pair reversed phase chromatography and ultra-performance liquid

chromatography. In this study, the focus lies on the purification process, where the main aim

is to develop a separation and purification method for modified oligonucleotides that can be

applied on different scales, from an analytical to a preparative scale.

Three modified oligonucleotides, and one unmodified with 44 bases, provided by

Scandinavian Gene Synthesis (Västerås, Sweden), were analysed and purified on an ultra-

performance liquid chromatography and on a preparative-system. Several parameters were

investigated, e.g. mobile phase composition, gradients and concentration. Practical analysis

and purification were made in two scales; analytical and semi-preparative.

The results showed that the samples contained impurities that were hard to separate from the

main sample. The scaling-up tests showed that, with increasing concentration, the impurities

become more aggregated with the main product. Fraction analysis showed that several pure

fractions were collected from the semi-preparative purification, and therefore some amount of

pure sample were collected from the semi-preparative run.

In conclusion, the method developed in this master thesis worked well as a significant amount

of samples were purified in the semi-preparative purification, and the method worked on

modified and unmodified oligonucleotides, containing different amount of modifications.

Keywords: oligonucleotides, preparative chromatography, scaling-up, ion-pair

chromatography, dibutylamine

Sammanfattning

Syntetiska oligonukleotider, vilket är korta strängar av DNA eller RNA, är ett framväxande

område i läkemedelsindustrin och för företag som tillverkar diagnostiska komponenter.

Tillverkningsprocessen för syntetiska oligonukleotider involverar många komplexa processer

som använder separation- och reningstekniker som jonbyteskromatografi,

jonparskromatografi och ultra-performance kromatografi. I denna studie ligger fokus på

reningsprocessen där det huvudsakliga syftet är att utveckla en separation- och reningsmetod

för modifierade oligonukleotider som kan appliceras på olika skalor – från analytisk till

preparativ skala.

Tre modifierade oligonukleotider, samt en omodifierad med 44 baser, tillhandahållet av

Scandinavian Gene Synthesis (Västerås, Sverige), analyserades och renades på ett ultra-

performance kromatografi system och ett preparativt reningssystem. Flertal parametrar

undersöktes, bland annat mobilfasens komposition, gradienter och koncentration. Analys och

rening utfördes i två skalor; analytisk och semi-preparativ skala.

Resultatet visade att proverna innehöll föroreningar som var svåra att separera från

huvudkomponenten. Uppskalningstesterna visade att föroreningarna blandade sig mer med

huvudkomponenten då koncentrationen ökade. Fraktionsanalyser visade att flera rena

fraktioner blev ihopsamlade från den semi-preparativa reningen, som därav visade att en

betydelsefull mängd rent prov blev renat i den semi-preparativa reningen.

Sammanfattningsvis, den metod som utvecklats i denna uppsats fungerade bra då

betydelsefulla mängder oligonukleotider kunde renas till olika grad vid den semi-preparativa

reningen, samt att metoden fungerade för både modifierade och icke-modifierade

oligonukleotider som innehöll olika mängder modifikationer.

Nyckelord: oligonukleotider, preparativ kromatografi, uppskalning, jonparskromatografi,

dibutylamin

List of abbreviations

Abbreviation

A

ACN

C

DBA

DNA

HCl

HFIP

IEX

IP-RPLC

LC

MeOH

MS

ON

RNA

RPLC

SGS DNA

T

TEA

Tris

U

UPLC

UV

Description

Adenosine

Acetonitrile

Cytidine

Dibutylamine

Deoxyribonucleic acid

Hydrochloric acid

Hexafluoro-2-propanol

Ion-exchange liquid chromatography

Ion-pair reversed phase liquid chromatography

Liquid chromatography

Methanol

Mass spectroscopy

Oligonucleotide

Ribonucleic acid

Reversed-phase liquid chromatography

Scandinavian Gene Synthesis

Thymine

Triethylamine

Trisaminomethane

Uridine

Ultra-performance liquid chromatography

Ultraviolet

Table of Contents

1. Introduction ............................................................................................................................ 1

1.1 Aim ................................................................................................................................... 2

1.2 Sustainability .................................................................................................................... 3

1.2.1 Environment .............................................................................................................. 3

1.2.2 Economic ................................................................................................................... 3

1.2.3 Social ......................................................................................................................... 3

2. Background and theory .......................................................................................................... 4

2.1 Oligonucleotides ............................................................................................................... 4

2.1.2 Modifications ............................................................................................................ 5

2.1.3 (n - 1) shortmer .......................................................................................................... 5

2.2 Chromatographic Theory and Definitions ........................................................................ 6

2.2.1 Isocratic and gradient elution .................................................................................... 6

2.2.2 Analytical Chromatography ...................................................................................... 7

2.2.2.1 Gaussian and Asymmetrical Peaks ........................................................................ 7

2.2.2.2 Retention and retention factor ................................................................................ 8

2.2.2.3 Resolution ............................................................................................................... 8

2.2.2.4 Plate Height and Theoretical Plates ...................................................................... 9

2.2.2.5 van Deemter Equation ............................................................................................ 9

2.2.2.6 Efficiency .............................................................................................................. 10

2.2.3 Preparative Chromatography ................................................................................... 11

2.2.3.1 Scaling up ............................................................................................................. 12

2.2.3.2 Langmuir Isotherm ............................................................................................... 12

2.2.3.3 Overloading .......................................................................................................... 13

2.2.4 Ion Pair Chromatography ........................................................................................ 14

2.3 Parameters Affecting Separation .................................................................................... 15

2.3.1 Mobile Phase ........................................................................................................... 15

2.3.2 Ion-pairing reagent .................................................................................................. 15

2.3.3 Column .................................................................................................................... 16

2.3.4 Detector ................................................................................................................... 16

2.3.5 Flow Rate ................................................................................................................ 16

2.3.7 Elution Mode ........................................................................................................... 16

2.4 Mass Spectroscopy ......................................................................................................... 17

3. Method and material ............................................................................................................. 18

3.1 Literature Survey ............................................................................................................ 18

3.2 Experimental .................................................................................................................. 18

3.2.2 ONs samples ............................................................................................................ 19

3.2.3 Instrumental ............................................................................................................. 19

3.2.3.1 Analytical system .................................................................................................. 19

3.2.3.2 Preparative system ............................................................................................... 20

3.2.4 Software .................................................................................................................. 21

3.2.5 Method (sample 162) .............................................................................................. 21

3.2.6 Method (sample 0206 and 2161) ............................................................................. 21

4. Results and discussion .......................................................................................................... 23

4.1 Linear and modified gradient ..................................................................................... 23

4.2 Comparison of separation capability of different mobile phases for sample 0206 .... 24

4.3 Bandwidth of zones with increasing amount of sample 162 ...................................... 25

4.4 Altering sample concentration (sample 0206)............................................................ 27

4.5 Semi-preparative purification ..................................................................................... 29

4.6 Fraction collection and analysis ................................................................................. 30

4.7 Application on unmodified ONs (sample 44) ............................................................ 33

5. Conclusion ............................................................................................................................ 34

6. References ............................................................................................................................ 35

Appendix 1 – Oligonucleotides and modifications .................................................................. 39

Appendix 2 – Gradients ............................................................................................................ 41

Appendix 3 – Chromatograms and MS spectra ....................................................................... 42

Appendix 4 – Other figures ...................................................................................................... 45

1

1. Introduction

The first dinucleotide was synthesized 65 years ago, and commercialized synthetization of

oligonucleotides (ONs) began in the 1980s when the development of useful chemical methods

was developed [1]. Even though ONs have been synthesized and analysed for years, new

methods for the purification and determination of ON sequences with modifications are

needed. ONs have many uses today; they are being used in the pharmaceutical industry in

research and as potential treatments for several diseases like cancer, AIDS and Alzheimer’s

[2]. Another grooving area for ONs are companies that manufacture diagnostic components,

like SGS DNA.

Synthesized ONs usually consists of 19-27 nucleotides [3], but can range from seven to more

than 100 bases long, with a molecular mass ranging from 2 – 20 kDa and they are

characterized by their highly negative charge on their backbone. The manufacturing process

can be divided into four main processes; synthesis, cleavage and deprotection, purification

and isolation [4]. In this master thesis, the main focus will be on purification process, which is

often performed on preparative scale. ONs are often modified to prevent nuclease digestion

and increase their stability. These modifications can be located on the backbone, the sugar

and/or the nucleobases of the ON [5].

ONs can be analysed with many different methods, where ion-exchange liquid

chromatography (IEX) and ion-pair reversed phase liquid chromatography (IP-RPLC) are the

most common methods, where the separation is based on charge and hydrophobicity [6]. In

this study, the ONs will be separated and analysed using IP-RPLC on analytical and semi-

preparative scale. The ONs used in this study are focused on probes, which may contain two

or more modifications, and scorpions, that contains i.e. three modifications. Additional

purification tests were also made on an unmodified 44-bases primer. This master thesis has

been carried out in association with Scandinavian Gene Synthesis (SGS DNA, Västerås,

Sweden) and Karlstads Universitet (Karlstad, Sweden).

2

1.1 Aim

The aim of this study is to develop a separation and purification method for unmodified and

modified ONs on different scales, ranging from analytical scale to preparative scale. This

study is performed together with SGS DNA, who manufactures synthetic ONs. Different ONs

that were analysed and purified were provided by SGS DNA; the first one called “162” was a

probe, containing the modifications FAM and BHQ1. The other two, called “2161” and

“0206” were “scorpions”, containing the modifications Amine, BHQ2 and Spacer 18. Sample

2161 and 0206 were the same product but came from two different batches, which means that

they had similar properties and could be considered to be equivalent to each other. A final test

of the developed separation system was also made where the unmodified, 44 bases long

primer were purified.

When developing a purification method for ONs, it is required to begin in analytical scale.

Different parameters like buffer, concentration, column properties, gradient, sample quantity

etc. were tested until optimal conditions were achieved. Thereafter, scaling up was performed

to semi-preparative scale, using systematic methods. Two different instruments were used in

this study; an ultra-performance liquid chromatography (UPLC) instrument and a preparative

system.

3

1.2 Sustainability

1.2.1 Environment

The general manufacturing process for ONs are able to achieve a yield of about 50% and

purity of 90%. The process is flexible and can produce a variety of ONs with different

lengths, modifications and can be scaled from grams to kilograms. The negative

environmental impact of this includes chemical hazards, waste and energy supply. Particularly

waste is a big factor for the negative environmental impact due to extensive starting material,

reagents and wash solvents used. Purification with preparative methods results in large

volumes of mobile phase that are wasted [4].

One method that can be used to reduce unnecessary waste is using a PMI (process mass

intensity) calculator, that is a measurement of the quantity of raw materials required to

produce a product [4]. It is also important to have a good manufacturing process, that uses as

little material as possible.

1.2.2 Economic

Economically, it is desirable to keep the instruments and columns as long as possible, as they

are expensive. By operating the analysis and purification methods at appropriate pH, low

concentrations of salts and low column temperature, it prevents wear on the instruments.

Another important factor is to develop and use good methods that obtain good yield. Pressure

is also an important factor; when using a longer column, high pressure is required. However,

the pressure has its limits as the pump, injection devices and other parts of the system must be

able to withstand the pressure and not wear out to quickly [7].

1.2.3 Social

Synthesized ONs are used for several medical purposes, including prevention and potential

treatments for diseases like cancer, AIDS and Alzheimer’s [2]. This will increase the well-

being and health for the society overall. More recently, ONs have been used in the research

involving Covid-19, where test kits contacting ON has had a huge impact on the development

of vaccine and testing of the virus.

4

2. Background and theory

2.1 Oligonucleotides

ONs are short strings of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), whose

backbone consists of nucleotides. There are three components to each nucleotide; a base

containing nitrogen, a five-carbon sugar and a phosphate group. The five bases in ONs are

adenosine (A), guanosine (G), cytidine (C), thymine (T) and uridine (U), where DNA consists

of A, G, C and T and RNA consists of A, G, C and U. The nucleotides are connected to each

other by 3´-, 5´-phosphodiester bonds. Synthetized ONs are generally 19-27 nucleotides long,

but can range from seven to 100 bases long. Its molecular mass usually ranges between 2 – 20

kDa. The backbone is negatively charged and hydrophilic and can exist in a single-stranded

form, that is considered more flexible. It can also exist as a double-stranded form, which is

considered more rigid [3, 8, 9].

2.1.1 Manufacturing process

The manufacturing process can be divided into four different processes; synthesis, cleavage

and deprotection, purification and isolation. In the synthetic process, the ON are synthesized

with a solid-supported phosphoramidite method in four steps; detritylation, coupling,

oxidation / sulfurization and capping [4]. Once the synthesis is complete, the full-length ON is

cleaved from the solid support in the cleavage and deprotection process. In the purification

process, preparative LC is used to purify the ON, where it is most common to use IP-RPLC or

IEX. Impurities are washed out by using an eluent with an increasing strength. In the isolation

process, the obtained ON sample is desalted to remove elution buffer components [4]. In this

master thesis, the main focus lays on the purification process. The overall manufacturing

process is longer and more complex than described, and is outside the scope of this study.

5

2.1.2 Modifications

ONs are often modified to prevent nuclease digestion and increase their stability, specificity

and potency. The modifications can be located on the backbone, the sugar and/or the

nucleobases [5]. Modifications on the backbone are the foremost important area on the ON to

modify, as it is highly sensitive to nucleases. The most commonly used modification on the

backbone is phosphorothioate linkage [5].

Modifications can be divided into two categories; reporters and quenchers. The reporters are

attached to the 5´- end of the ON and the quencher is located on the 3´- end. The reporter and

quencher used in an ON should overlap in the same spectrum, where the reporter emits light

that the quencher absorbs [10]. A reporter is a fluorescent dye which contains aromatic rings.

When synthesizing ONs, the most stabile reporters absorbs around 490 nm to 515 nm. When

it absorbs, the energy emits which causes light and the absorption usually takes place at

wavelengths shorter than the remainder of the ON [11]. The fluorescent dyes effect the

retention of ONs due to their hydrophobicity [12].

ONs can contain different amount of modifications. An ON that doesn’t contain any

modifications, called primers, is often easier to purify than those containing modifications.

ONs containing two modifications are called probes [13]. An ON that contains three

modifications is called a “scorpion”.

2.1.3 (n - 1) shortmer

The amount of impurities increases as the length of the ONs increases, because longer ONs

require more synthetic steps. A common type of impurity is shortmer molecules (n - 1, n - 2,

…), where one or more nucleotides are absent in the final product. Shortmer molecules are

often formed due to failure sequences, which is a result from inefficiency in the coupling,

incomplete capping or detritylation. The n – 2, or longer shortmer, are relative easy to remove

by purification. The n – 1 however tends to remain until the final product due to lack of

selectivity in relationship to the full-length ON. These shortmers have the same activity as the

full-length ON but lower affinity and specificity [14].

6

2.2 Chromatographic Theory and Definitions

In liquid chromatography there are two phases; a mobile phase and a stationary phase. The

solute that is to be analysed enters the chromatographic system in the mobile phase, and

partitions on to the stationary phase, most commonly by adsorption. The different components

in the solute have different ability to stick on the stationary phase and therefore separation can

be made. In liquid chromatography, the stationary phase is a column that is packed with

highly porous material [15, 16].

There are several different chromatographic techniques for analysis and purification of ONs,

for example reversed-phase liquid chromatography (RPLC), IP-RPLC, IEX and UPLC [17].

In IP-RPLC, the stationary phase is non-polar, hydrophobic and the mobile phase is polar,

hydrophilic and the separation depends on the hydrophobicity of the components in the solute.

It is common to add an organic modifier when using RPLC and the effect is that the mobile

phase becomes more hydrophobic [18].

2.2.1 Isocratic and gradient elution

In isocratic elution, the composition in the mobile phase remains constant throughout the

separation. In gradient elution, the ratio of polar and non-polar compounds in the mobile

phase changes overtime during the separation. Gradient elution is preferable when the sample

that is to be separated contains components with different polarities and hydrophobicity [19].

Typically, a gradient elution starts with low percentage organic modifier. During this initial

part of the elution, hydrophobic compounds bind to the hydrophobic stationary phase whilst

the more hydrophilic compounds elutes. When the signal goes back to baseline it indicates

that most of the hydrophilic compounds has eluted, and then the %-age organic modifier is

increased. When the %-age organic modifier is increased, the hydrophobic compounds that

are bound to the stationary phase start to elute in order of increasing hydrophobicity. At the

end of the run, a wash with a high concentration of organic modifier is performed in order to

elute more hydrophobic compounds that may be retained on the column (Figure 1) [18].

Figure 1. In gradient elution, the least hydrophobic compounds elute first at low %-age organic modifier (first

blue line). Thereafter, as % organic modifier increases, compounds elute in order of increasing hydrophobicity

(green, yellow and red line). Lastly, at high %-age organic modifier, very hydrophobic compounds elute (last

blue line) [18].

7

2.2.2 Analytical Chromatography

2.2.2.1 Gaussian and Asymmetrical Peaks

Quantitative information about the solute can be obtained from areas and height of the eluted

peak. In ideal cases, the peak has the shape as a Gaussian Peak (figure 2), which means that

the peak is symmetric [15].

Figure 2. A Gaussian shaped peak [15].

However, in almost all cases the peak obtained is asymmetrical with a small tail at the end.

When using preparative LC, where the concentrations are much higher, the peaks become

asymmetric, elongated and are strongly dependent on the adsorption isotherm, as can be seen

in figure 3 below [16].

Figure 3. Shape of asymmetrical peaks that is obtained when using preparative LC [16].

8

2.2.2.2 Retention and retention factor

The retention time is the time it takes for the sample to travel through the column, from

injection to detection. The retention factor k (1) is defined as the ratio between solute in the

stationary phase and solute in the mobile phase. tR is the retention time for the solute and t0 is

the time for the mobile phase [15]. Retention factor should lay between 2 and 9, if it is lower

the compounds are not retained and if it is higher the peaks will elute in the purge phase and

peak broadening will be observed [24].

𝑘 = 𝑡𝑅− 𝑡0

𝑡0 (1)

2.2.2.3 Resolution

When a sample move through the column it results in either a Gaussian shaped peak or an

asymmetrical peak. The longer the solute is in the column; the broader the peaks become. It is

desirable to obtain a narrow peak. The resolution describes the degree of separation between

two peaks and can be defined as the distance between two peak maxima with the average base

width. The resolution can be determined from a chromatogram, as shown in figure 4. For

quantitative analysis, a resolution value of ≥ 1,5 (baseline resolution) is desirable, as the peak

purity is 100% at this value and a full separation of two adjacent peaks has been accomplished

[20, 25]. Selectivity is the degree of separation between the peaks [18].

Figure 4. Determination of the resolution between two peaks [18].

9

2.2.2.4 Plate Height and Theoretical Plates

Theoretical plates are hypothetical divisions of columns, where each plate represents an

equilibrated partitioning of the compounds in a sample between the column and mobile phase.

The plate height H (2) is quantity that is related to the variance, which increases with the

number of steps, and the distance the solute has travelled through the column.

𝐻 = 𝜎2

𝐿 (2)

The number of theoretical plates, N (3), for a certain column length is a measurement of the

efficiency of the column [15]. tR is the retention time for the component and wh is the width at

the half of the height of the peak [26].

𝑁 = 𝐿

𝐻 = 5.545 (

𝑡𝑅

𝑤ℎ)

2

(3)

2.2.2.5 van Deemter Equation

The van Deemter equation (4) describes the three main parameters that contributes to a

broader peak; the eddy diffusion (A-term), the longitudinal diffusion (B-term) and the

resistance to mass transfer coefficient (C-term). The u describes the linear flow rate [1].

𝐻 = 𝐴 + 𝐵

𝑢+ 𝐶𝑢 (4)

It is desirable to have a small value on H, as smaller plate height results in narrower peaks.

The eddy diffusion; when molecules enter the column, they can flow through different paths

that have uneven lengths, which results in that they elute at different times because they have

different speeds depending on which path they take.

The longitudinal diffusion; longitudinal diffusion occurs when solute in the column diffuse

from high concentration to lower concentration. If the linear flow is faster, the solute spends

less time in the column and therefore less longitudinal diffusion occurs.

The resistance to mass transfer coefficient; to create equilibrium for the solute between the

mobile phase and the stationary phase, the solute must diffuse from one phase to the other.

Mass transfer describes this movement. The time is dependent on how far the solute must

travel to diffuse on the stationary phase and how fast it diffuses. The faster the linear velocity

is, the less time is available for the solute to diffuse [15, 20].

The relationship between these three coefficients can be represented as in figure 5. As can be

seen in the figure, there is an optimal flow rate which gives a minimum plate height. By using

this flow rate the maximum number of theoretical plates N is obtained [15].

10

Figure 5. There exists an optimal flow rate which obtains maximum number of theoretical plates N [15].

2.2.2.6 Efficiency

The efficiency of the column is related to the number of theoretical plates and zone

broadening, and describes the ability to elute narrow and symmetrical peaks. Good efficiency

can be obtained by minimizing the distance the compounds have to travel to diffuse. To

accomplish this, a well packed column and small particles is desirable [18].

To achieve high efficiency, small particles have been developed which require that the

separation takes place under high pressures. The pressure increases proportionally to the

square of the particle diameter and an increase in the linear velocity of the mobile phase

increases the pressure and the efficiency of the column (5). Today, a UPLC can separate

particles as small as 2 μm. Figure 6 shows how smaller particles decreases the values of the

A-term and C-term in the van Deemter curve, because the length of the irregular paths

decreases and the diffusion distance decreases. The advantages of using UPLC is that faster

separations are obtained and less solvent components are used, compared to high-performance

liquid chromatography (HPLC) [15].

11

Figure 6. Plate height decreases when using smaller particles [15].

2.2.3 Preparative Chromatography

In analytical chromatography the aim is to obtain quantitative and/or qualitative information

about several components in the solute, while the aim in preparative chromatography is to

isolate and obtain as much pure compound as possible from a mixture [15, 16]. There are two

possible ways to achieve this; by mass overloading, where the volume is kept constant and the

concentration increased, or by volume overload, where the concentration is kept constant and

the volume is increased [27]. Because the stationary phase has limited space, overloading is

often occurring and a result of this is that the eluted peak becomes distorted and

unsymmetrical [15, 16].

The chromatographic methods used in preparative LC must first be optimized for productivity

and yield. Optimizing a preparative LC system is often more difficult than analytical scale,

due to overloading, which causes non-linearity [28]. To obtain maximum efficiency in

preparative LC, the column needs to be maximized with sample and the run time must be

minimized. When increasing the load on the column it decreases the resolution, as a

consequence of peak broadening [24].

Preparative and analytical LC essentially has the same flow path, with the difference that

preparative LC has added a fraction collector. The fractions can be collected manually, based

on time or based on ultraviolet (UV) detection, where UV detection is preferable because it

decreases the number of fractions and therefore increases efficiency [24].

The void volume is the volume of the mobile phase in the column [23]. When switching from

an analytical column to a preparative or semi-preparative column, the void volume should be

approximated as it can have some effect on the retention.

12

2.2.3.1 Scaling up

When scaling up from an analytical column to a preparative column it is important to

maintain chemistries, pH conditions, particle sizes and/or column length. The first step of

scaling up is to perform analytical analyses, to confirm the presence of the target compound

and how it can be separated from the other compounds in the sample. When scaling up to

semi-preparative and preparative scale, changes in flow rates, modified gradient slopes,

injection volumes and run times require alteration, but should look similar to the analytical

gradient profile. When scaling up, equation (5) can be used to determine the flow rate in the

preparative system [24].

𝑓𝑃 = 𝑓𝐴 𝑑𝑃

2

𝑑𝐴2

𝑝𝐴

𝑝𝑃 (5)

𝑓𝑃 and 𝑓𝐴 is the flow in the preparative resp. analytical system, 𝑑𝑃 and 𝑑𝐴 is the diameter of

the preparative resp. analytical column and 𝑝𝐴 and 𝑝𝑃 is the particle sizes of the preparative

resp. analytical column [24].

Equation (6) is used when determining the injection volume in preparative system. 𝑣𝑖𝑛𝑗,𝑃 and

𝑣𝑖𝑛𝑗,𝐴 is the injection volume for preparative resp. analytical system, 𝑑𝑃 and 𝑑𝐴 is the

diameter of the preparative resp. analytical column and 𝐿𝑃 and 𝐿𝐴is the length of respective

column [24].

𝑣𝑖𝑛𝑗,𝑃 = 𝑣𝑖𝑛𝑗,𝐴 𝑑𝑃

2

𝑑𝐴2

𝐿𝑃

𝐿𝐴 (6)

2.2.3.2 Langmuir Isotherm

The Langmuir adsorption isotherm (7) is a model that describes the relationship between the

concentration of the mobile phase and stationary phase under isothermal conditions. The

subscript “e” imply equilibrium state, KL is the Langmuir equilibrium constant, qm are

adsorbed amount of solute per unit mass adsorbent, qi is the absorbed amount per unit mass of

adsorbent at equilibrium and p is the pressure [29].

𝑞𝑖 =𝑞𝑚𝐾𝐿𝑝𝑒

1+𝐾𝐿𝑝𝑒 (7)

In analytical chromatography, the concentration used is low and therefore a linear adsorption

isotherm is obtained, which means that the adsorbed concentration is proportional to the

concentration of the mobile phase and a Gaussian shaped peak is obtained. However, in

preparative LC, the high concentration affects the peak shape because the molecules in the

high concentration area tend to diffuse more as it is more difficult to find free adsorption sites.

This causes overload which results in asymmetry and bandbroadening of the obtained peak

[16].

13

2.2.3.3 Overloading

In preparative chromatography, large amounts of sample are injected, and as the column has a

finite amount of surface area it leads to overloading. As the concentration of the sample

increases, the amount of solute adsorbed at equilibrium decreases. A consequence of

overloading is tailing of the peaks, where it is most common that the front is sharp and the

rear is diffused. The retention time for the front decreases with increasing sample amount

whilst the rear has a constant retention time [30].

14

2.2.4 Ion Pair Chromatography

IP-RPLC is used to separate polar or ionic compounds. An ion-pairing reagent is added to the

mobile phase, which lodges to the hydrophobic stationary phase. The result of this is that the

stationary phase can be used as an ion-exchanger, where the cations are attracted to the ion-

pairing reagent attached on the stationary phase. A results of using ion-pairing reagents in the

mobile phase is that the equilibrium of the stationary phase is slow and that the separation

becomes more sensitive to temperature, pH and concentration [20, 21].

IP-RPLC is the most common method to use when analysing ONs, and is often coupled with

MS-detection. The mobile phase usually consists of a combination of alkylamines and

fluoroalcohols. Fluoroalcholos have a pKa value around 8, which causes it to ionize at high

pH and is therefore a good counter ion to alkylamines. Fluoroalcohols is a relatively new

substance to use in the mobile phase and it has improved separation, gives higher signal

intensity, higher selectivity and better resolution [14, 22].

IP-RPLC uses a reversed-phase column, where it is most common to use a C18 modified

column. A challenge when using IP-RPLC is the stability of the mobile phase. When using

alkylamines and fluoroalcholos in the mobile phase, the alkylamines act as an ion pairing

reagent, which shields the negatively charged backbone of the ON, which makes it more

hydrophobic. The fluoroalcohols improves the distribution constant between the ONs on the

stationary phase and the mobile phase. A factor that influences the retention is the

concentration alkylamine. Another factor that affects the retention is the pH value, where

higher pH gives lower retention. Therefore, it is important to find a way to easily adjust the

pH value [14, 22].

The influence of pH, temperature and content of organic modifier in eluents were studied by

evaluation of resolution, peak shape and bandbroadening effects. The ONs used in this study

have a high complexity with hydrophobic modifications or longer ONs with at least 40 bases.

The complex molecules are eluted at organic modifier concentration of 40-70% in

combination with an elevated temperature at 60˚C. The temperature and organic modifier

were used for unfolding or minimize dimerization of the ONs chains, which has been studied

by Nylander [23]. More efficient separations were obtained in this study by using the

unfolding strategy.

15

2.3 Parameters Affecting Separation

When developing new methods for liquid chromatography, there are some key parameters to

consider, for example mobile phase composition, stationary phase, detector, temperature, flow

rate, injection volume, elution mode, etc.

2.3.1 Mobile Phase

The stationary phase in RPLC is hydrophobic and for good separations, an organic, apolar,

solvent is often used. The most common organic solvent used is acetonitrile or methanol. The

effect of using organic solvents is that the mobile phase becomes more hydrophobic, which

means that the solute will eluate earlier. This occurs because when the hydrophobicity of the

mobile phase increases, the hydrophobic parts of the solute will not bind as strongly to the

stationary phase [18].

2.3.2 Ion-pairing reagent

By adding an ion-pairing reagent to the mobile phase the hydrophobicity of the charged

components can be increased. The ion-pairing reagents interacts with the charged groups in

the ON and suppresses their influence on the overall hydrophobicity. Which type and the

concentration of the ion-pairing reagent affects the behaviour of the retention and selectivity

[18].

The most common ion-pairing reagent that are used in ON manufacturing is TEA

(triethylamine). TEA has proven to achieve good results and can be combined with different

buffers [31], most commonly HFIP (hexafluoro-2-propanol) [32]. Other ion-pairing reagents

that can be used are hexylamine, N,N-dimethylbutylamine, N,N-diisopropylethylamine and

tripropylamine [31]. In this study, dibuthylamine (DBA) was chosen as ion-pairing reagent

for development purposes, and the choice was made based on the work of Goyon et al [32],

who obtained better ion-pairing efficiency with DBA.

DBA is much more hydrophobic than the commonly used TEA counter ion. Its cLogP, which

is a distribution coefficient that measures a compound’s hydrophobicity, is 2.8, compared to

1.4 for TEA. A higher value of cLogP will increase the distribution to the C18 chains on the

stationary phase. This will cause that the particle surfaces will be covered with DBA

molecules in a more pronounced degree compared with TEA. The effect of using ion-pair

reagents, also called amphipiles with different hydrophobicity was studied in capillary

electrophoresis where the capillaries were coated with C8 alkane chains. Adsorption of an

amphiphile to the surface alters the net charge of the capillary and therefore also the

electroosmotic flow [33]. The separations of the ONs are performed in a pseudo ion-exchange

mode. An electrostatic approach [33, 34] may be more useful when the understanding of ONs

interaction with the charged particle surfaces. ONs are large molecules with a length of 2 – 50

nm, containing an equal number of negatively charges as number of bases. The column

particles are covered with positively charged DBA molecules and can be treated as a charged

surface. The interaction between negatively charged ONs molecules are strong and the ONs

elutes at higher contents of organic modifier [35].

16

This is a huge advantage when the modified ONs are run in the purification systems. Analyte

with hydrophobic modifications will have a different chemical potential at higher

concentration of organic modifier. This may explain the substantial improved

chromatographic appearance with more selective interaction (charge) and improved mass

transfer or faster equilibrium interaction with the column particles. This is a possible

explanation of the observed high peak capacity of the separation and purification method

developed in this master thesis [35].

2.3.3 Column

The separation in liquid chromatography is highly dependent on the column used. The

parameters to consider when choosing a column is its length and diameter, packing material,

shape and size of particles and percent of carbon loading. The most common material for

column packing is silica [36, 37]. In preparative LC, the amount of pure substance wanted

within a given time determines which dimensions to choose on the column. If the separation

doesn’t come out well, the amount of injected ON needs to be decreased. Firstly, decrease in

the injection volume should be tested, and thereafter decrease in the concentration [24].

2.3.4 Detector

The detector that is most commonly used when analysing drug molecules is a UV/VIS

detector, because most drug molecules are aromatic or unsaturated, which absorbs in the

UV/VIS region [36].

2.3.5 Flow Rate

The flow rate is an important parameter that affects how well the peaks are separated. An

optimal flow rate gives low retention time, good shape and symmetry of the peaks and least

backpressure in the column. A risk when using too high flow rate is that the quality of the

chromatography is affected negatively, because the particles in the solute will not have

enough time to bind to the particles in the stationary phase. The optimal flow rate is related to

the van Deemter equation (4) [36].

2.3.6 Temperature

The flow rate and rate of adsorption is related to the temperature, and changes in the

temperature may affect the pressure of the column and the elution and resolution. Increasing

temperature generally improves the resolution, but it is preferable to choose ambient

temperature, as it usually minimizes the degradation the solute. In LC, the temperature usually

lies between 25˚C and 60˚C [36].

2.3.7 Elution Mode

There are two types of elution modes; isocratic elution, where all the components in the

mobile phase are mixed and pumped together as one eluent, or gradient elution, where the

ratio of components in the mobile phase changes over the course of the separation. Isocratic

elution is often easier to use but gradient elution results in better separations, as the polarity

and ionic strength changes during the run [36]. By having a shallow gradient profile focused

on the target peak, better separation and resolution can be achieved [24].

17

2.4 Mass Spectroscopy

Mass spectroscopy (MS) is an analytical method that is used to detect, quantitate and identify

molecules based on their mass-to-charge ratio in simple or complex mixtures [38]. All mass

spectrometers have three main components; an ion source, a mass separator and a detector.

These components differ from spectrometer to spectrometer, but fulfil the same purpose. In

MS, a mixture is applied in the ion source where it is ionized. Thereafter, the ions are

accelerated by an electric field and are separated based on their mass-to-charge ratio before

reaching the detector. The results are shown in a mass spectrum that displays the number of

ions detected at each value of mass-to-charge [20]. In this work, MS is used on the fractions

collected to confirm what molecular species the various peaks contained.

18

3. Method and material

3.1 Literature Survey

The background and theory, facts and data were collected through an initial literature survey,

using articles, books and fact sheets. The article Analytical and preparative separation of

phosphorothioated oligonucleotides: columns and ion-pair reagents by Enmark et al [28] was

used as basis for continued literature survey and studies. The article was closely related to the

work of this master thesis, including mass-overloading studies, fractioning and scaling up

theory. Further articles were found by using the citations in the Enmark article and searching

on search engines on the internet, e.g. Google Scholar.

3.2 Experimental

3.2.1 Mobile phase

A 0.50 M DBA (Sigma-Aldrich, USA) stock solution was prepared by mixing 100 ml Milli-Q

water and 100 ml ACN (Fisher Scientific, UK), resp. MeOH (Fisher Scientific, UK) in a

measuring glass. The pH of the solution was measured to be 8.2. 17 ml DBA was added and

37% HCl (Merck KGaA, Germany) was added dropwise until pH reached 7.9. A 50 mM Tris-

HCl buffer was prepared by weighing 30 g Trizma base powder (Sigma-Aldrich, USA) and

mixing it with 5 L Milli-Q water. 37% HCl was added until the solution reached a pH of 8.0

resp. 8.3.

Four different mobile phases were tested (table 1). Compositions A were prepared by mixing

DBA stock solution with Tris-HCl buffer. Compositions B were prepared by mixing DBA

stock solution, Tris-HCl buffer and ACN.

Table 1. Composition of the four different mobile phases that were tested.

Composition A Composition B

Mobile phase A Tris-HCl pH 8.0

50 mM DBA / 5% ACN

Tris-HCl pH 8.0

50 mM DBA / 80% ACN

Mobile phase B Tris-HCl pH 8.0

75 mM DBA / 7.5% ACN

Tris-HCl pH 8.0

75 mM DBA / 80% ACN

Mobile phase C Tris-HCl pH 8.0

100 mM DBA / 5% MeOH

Tris-HCl pH 8.0

100 mM DBA / 80% MeOH

Mobile phase D Tris-HCl pH 8.3

75 mM DBA / 7.5% ACN

Tris-HCl pH 8.3

75 mM DBA / 80% ACN

19

3.2.2 ONs samples

The ONs samples 126, 0206 and 2161, that were similar to each other, were provided by the

synthetic department of SGS DNA. The samples that were provided were “crashed batches”,

which means they were too impure for the company’s standards. Both sample 0206 and 2161

had the same three modifications and similar length. Sample 2161 had a concentration of 192

μM and its molecular weight was 14510 g/mol. Sample 0206 had a concentration of 186 μM

and its molecular weight was also 14510 g/mol. Sample 126 contained two modifications and

had a concentration of 100 μM. Also, an unmodified primer containing 44 bases was used to

investigate if the methods developed could be used on an unmodified ON. The sequences of

the ONs are shown in table 2 below.

Table 2. Sequences of the four ONs used.

3.2.3 Instrumental

The mass spectrometer used was a XEVO G2-XS QTof Quadrupole Time of Flight mass

spectrometer from Waters.

3.2.3.1 Analytical system

An ACQUITY UPLC system from Waters was used for analytical studies of sample 2161 and

fraction analysis. The system was equipped with an ACQUITY UPLC H-Class Binary pump,

a sample manager-fixed loop, a column manager and an ACQUITY UPLC PDA eλ Detector,

which can detect and quantify low concentrations of samples and compare spectra over a

broad wavelength range. The system was coupled with the software Empower. The column

temperature was set to 60˚C and the detector was set to detect at 260 nm for all tests.

The columns used in the UPLC system was an ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x

100 mm and ACQUITY PREMIER Oligonucleotide BEH C18, 1.7 μm, 2.1 x 50 mm.

Sample Sequence

162 FAM – 28 mer – BHQ1

0206 Amine – 26 mer – BHQ2 - Spacer 18 – 17 mer

2161 Amine – 26 mer – BHQ2 – Spacer 18 – 17 mer

44 44 mer

20

Figure 7. An ACQUITY UPLC system from Waters was used for analytical studies.

3.2.3.2 Preparative system

A preparative system, which consisted of different parts from Waters, was used for analytical

and semi-preparative studies on sample 0206. The pump was a 2535 low pressure mixing

gradient pump and the detector was a 2998 PDA detector. The system also had a fraction

collector, and had the opportunity to use columns in different sizes; analytical columns to

preparative columns.

Two different silica based columns were used in the preparative system; an analytical column

and a semi-preparative column. The analytical column was an XBridge C18, 3.5 μm, 4.6 x

100 mm and the semi-preparative column was an XBridge C18, 5 μm, 19 x 100 mm.

Figure 8. Preparative system used for analytical and semi-preparative studies.

21

The void volume of the two columns used in the preparative system were calculated by using

the following formula [24]:

𝑉0 = 𝑡𝑉 × 𝑓 (8)

where V0 is the void volume, tV is the void time and f is the flow rate. The void time when

using XBridge C18, 3.5 μm, 4.6 x 100 mm was 0.86 minutes and the flow rate was 1.5

ml/min, which resulted in a void volume of 1.29 ml. The void time when using XBridge C18,

5 μm, 19 x 100 mm was 1.33 minutes and the flow rate was 18 ml/min, which resulted in a

void volume of 23.94 ml.

3.2.4 Software

The software used to control the systems and process data in both the analytical and

preparative system was Empower Chromatography Data System 3, feature release 5, from

Waters.

To achieve an equivalent, optimal gradient when scaling up to bigger columns, a preparative

HPLC column method transfer tool from Thermo Fisher was used (figure 36, appendix 4). In

this tool, the properties of the analytical column and gradient are entered in the columns to the

left, and the properties of the semi-preparative column are entered in the columns to the right,

and an appropriate gradient is calculated.

3.2.5 Method (sample 162)

Sample 162 was analysed on the UPLC system, firstly analytically and then overloading

testing was carried out. Overloading testing with concentration and volume was analysed on

the UPLC system with mobile phase A (table 1), column XBridge C18, 3.5 μm, 4.6 x 100 mm

and gradient shown in table 6 (appendix 2). For the test with concentration, these parameters

and volume, were kept constant. The concentrations that were tested were 2, 4, 6 and 8 mM

and were obtained by diluting the sample with different amount of mobile phase A. For the

test with volume, the concentration was kept constant. The volumes that were tested were 20,

40, 60 and 80 μl. Investigation of the absorption spectra was used to confirm if the peaks

contained impurities or pure sample.

3.2.6 Method (sample 0206 and 2161)

Sample 2161 was diluted with mobile phase A to achieve concentration 96 μM and a volume

of 200 μl. The sample was analysed in the UPLC system with a linear gradient (table 7,

Appendix 2) to get an overview of its retention behaviour. The injection volume was 4 μl. The

result from this injection are shown in figure 9.

Sample 0206 was analysed with a linear gradient (table 8, appendix 2) in the preparative

system to get an overview of its retention behaviour. The injection volume was 20 μl, as it

was the smallest injection loop that was available.

22

By applying a linear gradient, it could be calculated at which %-mobile phase the main peak

and impurities eluted. The gradient was adjusted to achieve optimal separation of the

impurities and main peak by changing the flow rate and ratio % A och % B overtime. The

results of the modified gradient are shown in table 3 below.

Table 3. Modified gradient for sample 0206 in the preparative system, using mobile phase D.

When the gradient was set, four different mobile phases were tested (table 1) for sample 0206

on the preparative system.

Overloading attempts were made in the preparative system with sample 0206 and the

analytical XBridge column. As the injection loop was more difficult to change, the volume

was kept constant at 20 μl and the concentration was altered. The concentrations of the

samples were altered by diluting them with different ratio mobile phase A. Eight different

concentrations were tested; 0.1, 0.2, 0.5, 1, 2, 3, 5 and 7 mM.

Semi-preparative studies were made in the preparative system, with the semi-preparative

XBridge column on sample 2161, which had a concentration of 192 μM, and sample 0206,

which had a concentration of 1 mM. The injection volume of 1.7 ml and flow rate of 18

ml/min were calculated using equation (5) and (6). An equivalent gradient was produced by

using the preparative transfer tool from Thermo Fischer.

The fraction collector was set to collect 1.5 ml fractions between 7 and 12 minutes, at a

threshold of 4% and a slope of 2 V/min. A total of 30 fractions were collected for sample

2161 and 46 fractions were collected for sample 0206. Selected fractions around the main

peak and impurity peaks were analysed in the UPLC, using the gradient shown in table 9

(appendix 2), column ACQUITY PREMIER Oligonucleotide BEH C18, 1.7 μm, 2.1 x 50 mm

and an injection volume of 4 μl. Selected fractions around the main peak were collected and

were analysed on MS to confirm what the peaks contained.

Time (min) Flow (ml/min) % A % B

initial 1.5 95 5

7.00 1.5 39 61

15.00 1.5 37 63

19.00 1.5 20 80

23

4. Results and discussion

The result of sample 2161 when it was analysed on the UPLC system to get an overview over

the retention behaviour are shown in figure 9 below. The chromatogram showed that the

sample contained several impurity peaks that eluted close to the main peak. The UPLC system

gave quite good separation between the peaks, but as the sample was meant to be used in

preparative chromatography, it was expected that it would become difficult to get a good

separation in the preparative system.

Figure 9. Chromatogram of sample 2161, with concentration 96 μM, injection volume of 4 μl and mobile phase

D (table 1), when analysed in the UPLC system to get an overview of its retention behaviour. The gradient used

are shown in table 7, Appendix 2. The column used was ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 100 mm.

4.1 Linear and modified gradient

The linear gradient used on sample 0206 in the preparative system showed that the main peak

and impurity peaks eluted between 13 and 17 minutes, which was equivalent to 58% B to

70% B (figure 10). The gradient was altered several times before a modified gradient (table 3)

was constructed, where the slope was decreased between 7 and 15 minutes (figure 11). The

results showed that the impurities had similar characteristics as the main peak, as it was

difficult to get a good separation between the main peak and the impurities. It was assumed

that the main peak was the highest peak, i.e. the peak eluting at 15 minutes in figure 10 and

just before 10 minutes in figure 11, and the other peaks were impurities. This was confirmed

with fraction analysis.

Theoretically, a decreasing slope of % B should make the peaks elute further apart from each

other, but as can be seen in the chromatograms below that is not true in this case. In the linear

gradient, the difference in retention time between the first and last peak are 1.570 minutes,

while in the modified gradient the difference in retention time is 0.876 minutes. This is most

likely due to a delay time in the preparative system due to a dead volume that was not taken

into account. This causes the gradient to elute later than what it is set to do, which causes the

peaks to elute closer to each other. However, it was decided to keep the modified gradient

because faster elution was desirable. As samples 0206 and 2161 had similar properties, the

modified gradient were used for both samples.

24

Figure 10. Linear gradient for sample 0206, with concentration 186 μM, injection volume 20 μl and mobile

phase D (table 1), in the preparative system with the main peak eluting at 15 minutes. The gradient used are

shown in table 8, Appendix 2. The column used was XBridge C18, 3.5 μm, 4.6 x 100 mm.

Figure 11. Modified gradient for sample 0206, with concentration 186 μM, injection volume 20 μl and mobile

phase D (table 1), in the preparative system, with the main peak eluting just before 10 minutes. The gradient

used are shown in table 3. The column used was XBridge C18, 3.5 μm, 4.6 x 100 mm.

4.2 Comparison of separation capability of different mobile phases for sample 0206

Four different mobile phases were tested for sample 0206 in the preparative system, using the

modified gradient (table 3) and column XBridge C18, 3.5 μm, 4.6 x 100 mm. The mobile

phases differed in that they had different concentrations of DBA, mobile phase C contained

MeOH instead of ACN and mobile phase D had a pH of 8.3 instead of 8.0. The results

showed that difference between the tested mobile phases had a limited effect on separation of

the complex sample 0206. The results of the separations with different mobile phases are

shown in figure 29, 30, 31 and 32 in appendix 3. It was decided to keep mobile phase D for

further analyses, as it had somewhat better separation and for developing purpose, based on

the work from Anacleto et al [13].

25

4.3 Bandwidth of zones with increasing amount of sample 162

The result of the analytical testing for sample 162 on the UPLC system are shown in figure 12

below. It showed that there were two large peaks eluting quite close to each other. The peak to

the right contained the main product and the peak to the left was an impurity peak, that was

confirmed with the study of absorption spectra.

Figure 12. Analytical chromatogram of sample 162, with concentration 1 mM, injection volume 4 μl and mobile

phase A (table 1) in the UPLC system. The column used was XBridge C18, 3.5 μm, 4.6 x 100 mm.

The result of altering concentrations and injection volume for sample 162 are shown in figure

13 resp. 14 below, where the complex sample 162 had an impurity peak before the main

sample. The sample came from a test run on a new synthesis system where the amount of the

fluorophore FAM was low in the synthesis. This resulted in a substantial amount of the

sample without FAM, but still containing the quencher BHQ at the 5´-end. This sample was

used to study the effect when the same sample amount was added at different concentrations

or with increasing sample volume. The overloading of the particle surface is dependent of the

area that each molecule occupies and its amount as describes in the Langmuir isotherm. The

number of moles injected are the same for 2 mM and 20 μl, 4 mM and 40 μl, 6 mM and 60 μl

and for 8 mM and 80 μl. It is shown that an increase in the concentration of the sample gives

broader zones, where the rear zone is kept stable and the front zone is pushed forward. It also

shows that the impurity peak, that elutes before the main peak, becomes more focused with

increasing sample concentration. However, when a larger volume is injected there will cause a

bandspread due to that the injected volume will move molecules longer into the column. This

will contribute to a spread of the sample zone and this will contribute to a wider zone at

detection. This is shown in figure 14 below. The conclusion of these experiments is that there

may be a substantial decrease in bandwidth of sample zones if the sample concentration is

increased compared to an increase in sample volume.

26

Figure 13. Injection with 2, 4, 6 resp. 8 mM of sample 162, with injection volume 10 μl and mobile phase A

(table 1), when testing overloading with concentration in the UPLC system. The column used was XBridge C18,

3.5 μm, 4.6 x 100 mm.

Figure 14. Injection with 20, 40, 60 and 80 μl of sample 162, with concentration 1 mM and mobile phase A

(table 1), when testing overloading with the volume in the UPLC system. The column used was XBridge C18,

3.5 μm, 4.6 x 100 mm.

Absorption spectra was used to confirm which peak contained main product and which peak

contained impurities. The absorption spectra of the first eluting peak are shown in figure 15,

and the absorption spectra of the second eluting peak are shown in figure 16. The main

product contained the modifications FAM and BHQ1. FAM is a fluorescent that is attached to

the 5´-end of the ON [39] and absorbs at 495 nm. As can be seen in the absorptions spectra,

the first peak is missing the FAM modification, whilst the second peak contains FAM and is

therefore the main product.

Figure 15. Absorption spectra of the first eluting peak of sample 162, showing that the peak did not contain the

modification FAM.

27

Figure 16. Absorption spectra of the second eluting peak of sample 162, showing that the peak contained the

modification FAM.

4.4 Altering sample concentration (sample 0206)

Alteration of the concentration for sample 0206 in the preparative system showed that the

main peak and the closest impurity peak ensembles towards each other as the concentration

increases. The experimental conditions for this test was the same as when developing the

modified gradient (section 4.1 Linear and modified gradient), where all parameters except

concentration was kept constant. From the chromatogram shown in figure 17, its appears as a

maximum of the detector is reached at 3 mM and therefore it appears that all concentrations

above that have the same area.

Figure 17. Overlay plot of different concentrations of sample 0206, ranging from 0.1 – 7 mM, when testing

overloading with concentration in the preparative system. The volume was kept constant at 20 μl; mobile phase

D (table 1) and the gradient shown in table 7 were used. The column used was XBridge C18, 3.5 μm, 4.6 x 100

mm.

28

The concentrations were plotted against the number of theoretical plates to illustrate how the

column efficiency decreases as the concentration increases (figure 18) [40]. The number of

theoretical plates were calculated using equation (3). The retention time and width were

obtained from Empower. The plot shows a relative large decrease in number of theoretical

plates between 1 and 3 mM. The flattening of the curve between 3 and 7 mM could be due to

the overload of the detector signal.

Figure 18. A plot showing decreasing number of theoretical plates as concentration of sample 0206 increases,

when testing overloading by concentration in the preparative system. The corresponding chromatogram of the

test are shown in figure 17 above.

29

4.5 Semi-preparative purification

Semi-preparative purifications were made on sample 0206 and 2161 on the preparative

system. The injection volume and flow rate were calculated for hand whilst the gradient was

produced with the help from Thermo Fisher Scientific preparative calculation tool. The reason

for this is that the calculation tool gave a different result when calculating the injection

volume and therefore the hand-calculated results were used. The scaling-up, for both sample

0206 and 2161, was based on the analytical separation that were made for sample 0206

(figure 11).

The semi-preparative purification gave rather good results, as shown in figure 19 and 20

below. The samples were very impure to begin with, but with the purification on sample

0206, several pure fractions were isolated and obtained.

Figure 19. Semi-preparative purification of sample 2161 with a concentration of 192 μM, injection volume 1.7

ml and flow rate of 18 ml/min. Mobile phase D (table 1) was used and the XBridge C18, 5 μm, 19 x 100 mm

column.

Figure 20. Semi-preparative purification of sample 0206 with a concentration of 1 mM, injection volume 1.7 ml

and flow rate of 18 ml/min. Mobile phase D (table 1) was used and the XBridge C18, 5 μm, 19 x 100 mm

column. The analytical equivalently chromatogram can be seen in figure 11.

30

4.6 Fraction collection and analysis

Fraction collection on the semi-preparative purification on sample 0206 were made, where 46

fractions were collected (figure 21). Fraction collection were also made for sample 0206 when

the concentration was 7 mM (figure 22), where 22 fractions were collected.

Figure 21. Fraction collection around the main peak and impurities for sample 0206, with concentration 1 mM,

injection volume 1.7 ml and flow rate 18 ml/min, in the semi-preparative purification, on the preparative system.

Mobile phase D (table 1) was used and the XBridge C18, 5 μm, 19 x 100 mm column. Fractions 24 – 33, that

were analysed in the UPLC system, lies within the thick lines.

Figure 22. Fraction collection around the main peak and impurities for sample 0206, concentration 7 mM, in the

preparative system. The volume was kept constant at 20 μl; mobile phase D (table 1) and the gradient shown in

table 7 were used. The column used was XBridge C18, 3.5 μm, 4.6 x 100 mm. Fraction 19, that lies within the

thick lines, were analysed on the UPLC system.

31

Fractions 24 – 33 from the semi-preparative purification were selected to be analysed on the

UPLC system to decide their purities. The results of the analysis are shown in figure 24 and

table 4. Percentage area is equivalent to the purity of the fraction. A purity of 90% is

considered acceptable and therefore fractions 28, 29, 30 and 31 are considered pure. This

result is consistent with what is shown in figure 21, that the pure fractions are taken from the

main peak. Fraction 25 also has an area over 90%, but as can be seen in figure 21, the fraction

is not taken from the main product peak. Fraction 19 were collected from the 7 mM

purification and analysed on the UPLC. The results showed an area of 68.5% and are shown

in picture 23 below.

Figure 23. Fraction 19 from sample 0206, concentration 7 mM, was analysed in the UPLC system using the

ACQUITY PREMIER Oligonucleotide BEH C18, 1.7 μm, 2.1 x 50 mm column, injection volume 4 μl and

mobile phase D (table 1).

Figure 24. Analysis of fractions 24 – 33, analysed in the UPLC system, using the ACQUITY PREMIER

Oligonucleotide BEH C18, 1.7 μm, 2.1 x 50 mm column, injection volume 4 μl and mobile phase D (table 1).

32

Table 4. Summary of results from the fraction analysis for the semi-preparative purification, showing the purity

of each fraction.

Fractions 24 and 29 from the semi-preparative purification and fraction 19 from the 7 mM

purification were analysed on MS. The results from the MS analysis confirmed that fraction

24 was taken from an impurity peak that did not contain any main product (figure 33,

Appendix 3). The MS analysis showed that fraction 29, that was taken from the main peak,

contained main product as it showed a peak at 14511 Da (figure 34, appendix 3). It also

showed a peak at 14466 Da, which was due to a fault in the synthesis and the sample was

therefore discarded. The peaks appearing after 14511 Da comes from the MS system, and

does not come from the sample. It was desirable that fraction 19 would show a combination of

fractions 24 och 29, to show that the impurities were pushed further in the main peak as the

concentration increased (figure 35, appendix 3). However, MS analysis of fraction 19 was

similar to that of fraction 29, i.e. it contained main product with a peak at 14511 Da and

14466 Da.

RT (min) SampleName Inj. ID Vial Height (μV) % Area

1 3.826 0206 1mM frac.24 4031 2:A,2 540680 89.65

2 3.801 0206 1mM frac.25 4034 2:A,3 299567 93.64

3 3.852 0206 1mM frac.26 4039 2:A,4 204748 53.91

4 3.883 0206 1mM frac.27 4042 2:A,5 2161297 86.99

5 3.877 0206 1mM frac.28 4045 2:A,6 2794974 90.86

6 3.879 0206 1mM frac.29 4048 2:A,7 2738977 95.20

7 3.885 0206 1mM frac.30 4051 2:A,8 2498000 96.43

8 3.895 0206 1mM frac.31 4054 2:B,1 1660067 96.47

9 3.903 0206 1mM frac.32 4057 2:B,2 732617 63.31

10 3.951 0206 1mM frac.33 4060 2:B,3 520177 74.70

Mean 3.875

% RSD 1.07

Min 204748

33

4.7 Application on unmodified ONs (sample 44)

A primer containing 44 bases, with a purity of 45% were analysed in the UPLC system

(Figure 25) and purified in the preparative-system by the company with the method developed

in this master thesis. The purification was successful, where the sample was purified in one

step, from 45% to 99% (figure 37 resp. 38, Appendix 4), which shows that the method

developed can be applied on both modified and unmodified ONs with different complexities.

This method may contribute to substantial improvement when applied in production. Further

tests will be performed where one desalting step may be removed.

Figure 25. Analysis in the UPLC system showed a purity of 45%. Purification were made in the preparative-

system and fraction analysis showed that the sample had increased the purity to 99%.

34

5. Conclusion

The main aim for this master thesis was to develop a method that would improve the

separation and purification of modified ONs. The method developed in this master thesis

includes the choice of instruments, gradient, flow rate, fraction collection and analysis and

concentration of sample and mobile phase composition, where the choice of the ion-pairing

reagent DBA was of most importance. The method was developed using two different

systems; a UPLC system and a preparative-system and analyses were made in both analytical

and semi-preparative scales. By investigating different parameters, a general method was

developed that worked for the purification of modified and unmodified primers, probes and

scorpions. The results of using DBA for the separation and purification of primers is very

promising. The inherent properties of DBA contribute to a higher surface charge of the

stationary phase, as it is a more hydrophobic ion-pairing agent than the more commonly used

TEA. This will result to a change in percentage of organic modifier at elution for the different

ONs. It is believed that the company will save a significant amount of money and resources

by using the method developed in this master thesis. Continued work includes validation of

the method developed so that it can be used in production. Also further investigation

regarding mobile phase and how DBA contributes to better purification is desirable.

35

6. References

[1] Bonilla J.V, Srivatsa G.S. Handbook of Analysis of Oligonucleotides and Related

Products. Taylor and Francis Group, LLC; 2011

[2] Studzinska S. Review on investigations of antisense oligonucleotides with the use of mass

spectrometry. Talanta. Volume 176, 1 January 2018, Pages 329-343

[3] McGinnis A.C, Chen B, Bartlett M.G. Chromatographic methods for the determination of

therapeutic oligonucleotides. Journal of Chromatography B. Volumes 883–884, 1 February

2012, Pages 76-94

[4] Andrews B.I, Antia F.D, Brueggemeier S.B, Diorazio L.J, Koenig S.G, Kopach M.E, et al.

Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing. J. Org. Chem.

2021; 86(1): 49-61

[5] Sharma V.K, Watts J.K. Oligonucleotide therapeutics: chemistry, delivery and clinical

progress. Future Medicinal Chemistry. 2015; 7(16). https://doi.org/10.4155/fmc.15.144

[6] Biba M, Welch C.J, Mao B, Foley J.P. Liquid Chromatography Methods for the

Separation of Short RNA Oligonucleotides. J. Chromatogr. A 2013, vol. 1304, p. 69-77

[7] Guiochon G. The limits of the separation power of unidimensional column liquid

chromatography. Journal of Chromatography A, 1126 (2006) 6–49.

https://doi.org/10.1016/j.chroma.2006.07.032

[8] Noll B, Roehl I, authors of Chapter 3 “Separation of Oligonucleotides and Related

Substances” in the book “Analysis of Oligonucleotides and their Related Substances”, editors:

Okafo G, Elder D, Webb M

[9] Tymoczko J.L, Berg J.M, Stryer L. Biochemistry: A Short Course. New York, NY: W.H.

Freeman & Company, 2015

[10] Butler J.M. Advanced Topics in Forensic DNA Typing: Methodology. Chapter 3 - DNA

Quantitation. Academic Press, 2012, Pages 49-67. https://doi.org/10.1016/B978-0-12-374513-

2.00003-8

[11] Müller-Taubenberger A, Ishikawa-Ankerhold H.C. Fluorescent Reporters and Methods

to Analyze Fluorescent Signals. Methods Mol. Biol., 983 (2013), pp. 93-112.

[12] Oefner P.J, Huber C.G, Umlauft F, Berti G.N, Stimpf E, Bonn G.K. High-Resolution

Liquid Chromatography of Fluorescent Dye-Labeled Nucleic Acids. Anal. Biochem., 223

(1994), p. 39

[13] Anacleto C, Ouye R, Schoenbrunner N. Orthogonal ion pairing reversed phase liquid

chromatography purification of oligonucleotides with bulky fluorophores. 2014; 1329: 78-82.

https://doi.org/10.1016/j.chroma.2013.12.072

[14] El Zahar N.M., Magdy N., El-Kosasy A.M., Bartlett M.G. Chromatographic approaches

for the characterization andquality control of therapeutic oligonucleotide impurities.

Biomedical Chromatography 2018, vol. 32(1). https://doi.org/10.1002/bmc.4088

36

[15] Fornstedt T, Forssén P, Westerlund D, Basic HPLC Theory and Definitions: Retention,

Thermodynamics, Selectivity, Zone Spreading, Kinetics, and Resolution, in: V. Pino, J.L.

Anderson, A. Berthod, A.M. Stalcup (Eds.), Anal. Sep. Sci., Wiley-VCH Verlag GmbH &

Co. KGaA, Weinheim, Germany, 2015: pp. 1–24.

http://doi.wiley.com/10.1002/9783527678129.assep001 (accessed January 23, 2017).

[16] Fornstedt T, Forssén P and Samuelsson J, authors of Chapter 18 “Modeling of

Preparative Liquid Chromatography of Small Molecules” in the book “Liquid

Chromatography: Fundamentals and Instrumentation” (Handbooks in Separation Science

series 2013, 9780124158078; pages 407-425), editors: Salvatore Fanali, Paul R. Haddad,

Colin Poole, Peter Schoenmakers, David K. Lloyd

[17] Q. Zhang, H. Lv, L. Wang, M. Chen, F. Li, C. Liang, Y. Yu, F. Jiang, A. Lu, G. Zhang

Recent methods for purification and structure determination of oligonucleotides Int. J. Mol.

Sci., 17 (2016), p. 2134

[18] GE Healthcare Bio-Sciences AB. Hydrophobic Interaction and Reversed Phase

Chromatography, Principles and Methods [Handbook].

[19] LibreTexts. High Performance Liquid Chromatography [Internet]. [updated 2020-08-16;

cited 2021-05-19]. Available from:

https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Anal

ytical_Chemistry)/Instrumental_Analysis/Chromatography/High_Performance_Liquid_Chro

matography

[20] Harris D.C. Quantitative chemical analysis. Ninth edition. New York, NY: W.H.

Freeman and Company, [2016]

[21] Tilly-Melin A, Askemark Y, Wahlund K.G, Schill G. Retention Behavior of Carboxylic

Acids and Their Quaternary Ammonium Ion Pairs in Reversed Phase Chromatography with

Acetonitrile as Organic Modifier in the Mobile Phase. Analytical Chemistry. 1979; 51(7):

976-983

[22] Sutton JM, Guimaraes GJ, Annavarapu V, van Dongen WD, Bartlett MG. Current State

of Oligonucleotide Characterization Using Liquid Chromatography−Mass Spectrometry:

Insight into Critical Issues. J. Am. Soc. Mass Spectrom. 2020, 31, 9, 1775–1782.

https://doi.org/10.1021/jasms.0c00179

[23] Nylander J. The dependence of chromatographic conditions for separation of

oligonucleotides in different AEX-HPLC columns [master’s thesis in the Internet].

Stockholm: Kungliga Tekniska högskolan (KTH); 2020 [cited 2021-05-26]. Available from:

https://www.diva-portal.org/smash/get/diva2:1456097/FULLTEXT01.pdf

[24] Agilent InfinityLab. Principles and practical aspects of preparative liquid

chromatography. © Agilent Technologies, Inc. 2019 [cited 2021-05-27]. Available from:

https://www.agilent.com/cs/library/primers/public/primer-preparative-liquid-chromatography-

5994-1016EN-agilent.pdf

[25] GE Healthcare Bio-Sciences AB. Ion Exchange Chromatography & Chromatofocusing,

Principles and Methods [2010]. Available from:

37

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-

Aldrich/General_Information/1/ge-ion-exchange-chromatography.pdf

[26] Stauffer E, Dolan J.A, Newman R. Fire Debris Analysis. 1 ed. © Academic Press 2007.

Chapter 8, Gas Chromatography and Gas Chromatography—Mass Spectrometry.

https://doi.org/10.1016/B978-012663971-1.50012-9

[27] Cretier G, Rocca J.L. Mass Overload in Preparative Liquid Chromatography: Band

Broadening and Maximum Sample Load. Chromatographia. 1985; 20: 461-465.

https://doi.org/10.1007/BF02344786

[28] Enmark M, Bagge J, Samuelsson J, Thunberg L, Örnskov E, Leek H, Limé F, Fornstedt.

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and

ion-pair reagents. Analytical and Bioanalytical Chemistry (2020) 412:299–309.

https://doi.org/10.1007/s00216-019-02236-9

[29] Azizian S, Eris S, Wilson L.D. Re-evaluation of the century-old Langmuir isotherm for

modeling adsorption phenomena in solution. Chemical Physics. 2018; 513: 99-104.

https://doi.org/10.1016/j.chemphys.2018.06.022

[30] Fornstedt T, Guiochon G. Nonlinear Effects in LC and Chiral LC. Anal. Chem. 2001, 73,

21, 608 A–617 A. https://doi.org/10.1021/ac012533b

[31] Gong L, McCullagh J.S.O. Comparing ion‐pairing reagents and sample dissolution

solvents for ion‐pairing reversed‐phase liquid chromatography/electrospray ionization mass

spectrometry analysis of oligonucleotides. Rapid Communications in Mass Spectrometry.

2013; 28(4): 339-350. https://doi.org/10.1002/rcm.6773

[32] Goyon A, Zhang K. Characterization of Antisense Oligonucleotide Impurities by Ion-

Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic

Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional

Liquid Chromatography Setup. Anal. Chem. 2020, 92, 8, 5944–5951.

https://doi.org/10.1021/acs.analchem.0c00114

[33] Ståhlberg J. Retention models for ions in chromatography. Journal of Chromatography

A. 1999; 855(1): 3-55. https://doi.org/10.1016/S0021-9673(99)00176-4

[34] Stålberg O*, Ståhlberg J. Theoretical aspects on the regulation of electroosmotic flow in

1 capillary electrophoresis by adding charged amphiphiles. Journal of Chromatography A,

776 (1997) 311–318. https://doi.org/10.1016/S0021-9673(97)00243-4

[35] Israelachvili J. Intermolecular & Surface Forces. 2nd ed. New York: Academic Press;

1991

[36] N. S. Lakka and C Kuppan (October 26th 2019). Principles of Chromatography Method

Development, Biochemical Analysis Tools - Methods for Bio-Molecules Studies, Oana-Maria

Boldura, Cornel Baltă and Nasser Sayed Awwad, IntechOpen, DOI: 10.5772/intechopen.89501.

Available from: https://www.intechopen.com/books/biochemical-analysis-tools-methods-for-bio-

molecules-studies/principles-of-chromatography-method-development

38

[37] Guiochon G. The limits of the separation power of unidimensional column liquid

chromatography. Journal of Chromatography A. Volume 1126, Issues 1–2, 8 September 2006,

Pages 6-49. https://doi.org/10.1016/j.chroma.2006.07.032

[38] ThermoFisher Scientific. Overview of Mass Spectrometry for Protein Analysis [Internet].

[cited 2021-05-19]. Available from: https://www.thermofisher.com/de/de/home/life-

science/protein-biology/protein-biology-learning-center/protein-biology-resource-

library/pierce-protein-methods/overview-mass-spectrometry.html

[39] LGC Biosearch Technologies. FAM; 5' Modification [Internet]. [cited 2021-05-20].

Available from: https://www.biosearchtech.com/support/resources/oligo-modifications/fam-5-

modification

[40] McCalley D.V. Overload for Ionized Solutes in Reversed-Phase High-Performance

Liquid Chromatography. Anal. Chem. 2006, 78, 2532-2538.

https://doi.org/10.1021/ac052098b

[41] LGC Biosearch Technologies. BHQ-1 CPG; Glycolate, 1000 Å [Internet]. [cited 2021-

05-20]. Available from: https://www.biosearchtech.com/products/synthesis-

reagents/cpg/bhq1-cpg-glycolate44-1000-

[42] LGC Biosearch Technologies. BHQ-2 CPG; Glycolate, 1000 Å [Internet]. [cited 2021-

05-20]. Available from: https://www.biosearchtech.com/products/synthesis-

reagents/cpg/bhq2-cpg-glycolate44-1000-

[43] LGC Biosearch Technologies. Spacer 18 Amidite (DMT-Hexa(ethylene glycol))

[Internet]. [cited 2021-05-20]. Available from:

https://www.biosearchtech.com/products/synthesis-reagents/amidites/spacer-18-amidite-

dmthexaethylene-glycol

39

Appendix 1 – Oligonucleotides and modifications

Table 5. Oligonucleotides used in this master thesis.

Amine

An amine is located at the 5´-end of samples 0206 and 2161. In the final product, the

modification “Quasar 670” would have been attached to the amine, but as the product failed

during manufacturing, the quasar modification was never attached.

BHQ1

BHQ1 is a modification that is attached to the 3´-end of ONs and absorbs between 480 and

580 nm [41].

Figure 26. Structure of the modification BHQ1. Figure provided by SGS DNA.

BHQ2

BHQ2 is a modification that is attached to the 3´-end of ONs and absorbs between 560 to 670

nm. [42].

Sample Sequence Molecular weight (g/mol)

162 FAM – 28 mer – BHQ1 9629

0206 Amine – 26 mer – BHQ2 - Spacer 18 – 17 mer 14510

2161 Amine – 26 mer – BHQ2 – Spacer 18 – 17 mer 14510

44 44 mer -

40

Figure 27. Structure of the modification BHQ2. Figure provided by SGS DNA.

FAM

FAM is a modification at is attached to the 5´-end of ONs and fluoresces in the green region

of the visible spectrum. It is common to use FAM in the combination with BHQ1 [39].

Figure 28. Structure of the modification FAM. Figure provided by SGS DNA

Spacer 18

The spacer 18 modification is used to incorporate spacer arms into ONs and is added to create

longer spacer arms and increase stability [43].

41

Appendix 2 – Gradients

Table 6. Modified gradient for sample 162 in the UPLC system, using mobile phase A.

Table 7. Gradient used for initial testing on sample 2161 in the UPLC system, using mobile phase A.

Table 8. Gradient used for initial testing on sample 0206 in the preparative system, using mobile phase D.

Table 9. Gradient used for analysis of the selected fractions in the UPLC, using mobile phase D.

Time (min) Flow (ml/min) % A % B

initial 1.5 90 10

1.50 1.5 90 10

19.00 1.5 55 45

Time (min) Flow (ml/min) % A % B

initial 0.4 95 5

19.00 0.4 20 80

Time (min) Flow (ml/min) % A % B

initial 1.5 95 5

19.00 1.5 10 90

Time (min) Flow (ml/min) % A % B

initial 0.4 95 5

1.00 0.4 75 25

7.00 0.4 20 80

42

Appendix 3 – Chromatograms and MS spectra

Figure 29. Separation of sample 0206 with mobile phase A.

Figure 30. Separation of sample 0206 with mobile phase B.

43

Figure 31. Separation of sample 0206 with mobile phase C.

Figure 32. Separation of sample 0206 with mobile phase D.

Figure 33. MS spectrum of fraction 24, sample 0206, semi-preparative purification.

44

Figure 34. MS spectrum of fraction 29, sample 0206, semi-preparative purification.

Figure 35. MS spectrum of fraction 19, sample 0206, 7 mM purification.

45

Appendix 4 – Other figures

Figure 36. A preparative HPLC column method transfer tool from ThermoFisher was used to calculate an

equivalent gradient for the semi-preparative column.

46

Figure 37. Fraction collection of sample 44 made in the semi-preparative system.

Figure 38. Fraction collection of sample 44 in the preparative system showed that several fractions had a purity

of 99%.